Review ArticleMode of Action of Lactoperoxidase as Related to ItsAntimicrobial Activity A Review
F Bafort1 O Parisi1 J-P Perraudin2 and M H Jijakli1
1 Plant Pathology Laboratory Liege University Gembloux Agro-Bio Tech Passage des Deportes 2 5030 Gembloux Belgium2 Taradon Laboratory Avenue Leon Champagne 2 1480 Tubize Belgium
Correspondence should be addressed to F Bafort francoisebafortulgacbe
Received 17 June 2014 Revised 19 August 2014 Accepted 19 August 2014 Published 16 September 2014
Academic Editor Qi-Zhuang Ye
Copyright copy 2014 F Bafort et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Lactoperoxidase is a member of the family of the mammalian heme peroxidases which have a broad spectrum of activity Theirbest known effect is their antimicrobial activity that arouses much interest in in vivo and in vitro applications In this context theproper use of lactoperoxidase needs a good understanding of its mode of action of the factors that favor or limit its activity andof the features and properties of the active molecules The first part of this review describes briefly the classification of mammalianperoxidases and their role in the human immune system and in host cell damage The second part summarizes present knowledgeon the mode of action of lactoperoxidase with special focus on the characteristics to be taken into account for in vitro or in vivoantimicrobial use The last part looks upon the characteristics of the active molecule produced by lactoperoxidase in the presenceof thiocyanate andor iodide with implication(s) on its antimicrobial activity
1 Introduction
Mammalian peroxidases are distinct from plant peroxidasesin size amino acid homologies nature of the prostheticgroup and binding of the prosthetic group to the proteinPlant peroxidases consist of approximately 300 amino acidswith a noncovalently bound heme moiety while mammalianperoxidases have 576 to 738 amino acids with a cova-lently bound heme moiety [1] Animalsrsquo peroxidases displayhigh sequence homology compared to plant peroxidases [23] Mammalian peroxidases can detoxify peroxide protectagainst pathogens and induce the production of thyroidhormones while plant peroxidases trigger defense reactionsagainst pathogens and stress remove hydrogen peroxide andare involved in the metabolism of lignin and auxin and in theoxidation of toxic reductors [1]
Based on amino acids homologies peroxidases are nowclassified into two superfamilies The first superfamily clus-ters peroxidases from plant archea bacteria and fungi and isclassified into three classes Class I is composed of intracellu-lar peroxidases such as yeast cytochrome 119888 peroxidase ascor-bate peroxidase and catalase peroxidase Class II is formed
by secretory fungal peroxidases such as manganese andlignin peroxidases and Class III consists in secretory plantperoxidases including one of the most studied peroxidasesthe horseradish peroxidase [4 5]The second one is called theperoxidase-cyclooxygenase superfamily and clusters mam-malian peroxidases together with protein from invertebratebacterial plant and fungal species and other metalloproteinssuch as cyclooxygenase [5] This latter superfamily originallycalled ldquothemyeloperoxidase familyrdquo shares a domain of about500 amino acids corresponding to the catalytic site [6] and isclassified into several subfamilies one of them is the chordataperoxidases in which the mammalian peroxidases are found[5] The main clades are the myeloperoxidases (MPO) eos-inophil peroxidases (EPO) and the lactoperoxidase (LPO)branch Another clade consists in thyroid peroxidase (TPO)which is distantly related to MPO EPO and LPO [5]
MPO is a lysosomal constituent in neutrophils andmacrophages and displays antimicrobial activity during thepostinfection inflammatory process but is also involved inacute inflammatory diseases and in other pathologies such asatherosclerosis [7ndash10] EPO is secreted in eosinophils and dis-plays cytotoxic activity against parasites bacteria and fungi
Hindawi Publishing CorporationEnzyme ResearchVolume 2014 Article ID 517164 13 pageshttpdxdoiorg1011552014517164
2 Enzyme Research
It could be associated to allergic eosinophilic inflammatorydisease pathologies [1 10 11] LPO and salivary peroxidaseare found in secretions of exocrine glands and are associatedto antibacterial and antifungal activity [12ndash14] Finally TPOis a membrane enzyme localized in thyroid follicle cells thattakes part in the synthesis of thyroid hormones [1]
This review summarizes present knowledge on the modeof action of lactoperoxidase which can be extended to themammalian peroxidases mode of action and on the specificinteraction of LPO with thiocyanate and iodide together oralone with implications on its antimicrobial activity
2 Mode of Action of Lactoperoxidase
LPO is a calcium- and iron-containing glycoprotein arrangedin a single polypeptide chain of about 80 kDa [15 18 19]Human LPO is moderately cationic with a pI of ca 75 butbovine LPO is more cationic with a pI of ca 96 [18 19]MPO is a highly cationic (pI of ca 10) dimeric protein of146 kDa eachmonomer containing one calcium and iron andjoined together by a disulfide bridge [18] The ion calciumplays an important role in the stability of both enzymes[19 20] The active site in LPO MPO EPO and TPO is theheme which consists in a protoporphyrin IX derivative fixedcovalently through two ester linkages via a conserved distalaspartate and glutamate residues a third link being presentin MPO and consisting in a thioether sulfonium bond witha methionine [4 15 19 21] These covalent bonds which arespecific for vertebrate peroxidases result in a distortion of thesymmetry and planarity of the prosthetic group and give theunique spectroscopic and redox properties of these proteins[4 20 21] The proximal heme ligand is a highly conservedhistidine residue which is hydrogen bonded to a conservedasparagine residue that acts as hydrogen-bond acceptor[20 22] On the distal heme site a conserved histidine-arginine couple plays a role in the proton transfer duringthe formation of Compound I and a conserved glutamineresidue and several conserved water molecules are involvedin a hydrogen-bond network acting in halide delivery andbinding [4 18 20] A conserved asparagine residue in LPOEPO and MPO located close to the distal histidine seemsalso critical for the catalysismechanism [18 20]The substratechannel in LPO is narrower longer and more hydrophobiccompared toMPOwith the consequence that the LPO-activesite seems to be less exposed to surrounding media [22 23]
Heme peroxidases are oxidoreductase enzymes that actthrough different reactionmechanisms Although some char-acteristics are specific to a member of the family the sameglobal procedure is followed by all members The cyclebegins with the transformation of the native enzyme intoCompound I Afterwards and dependingmainly on substrateconcentrations Compound I enters the halogenation cycle orthe peroxidase cycle which both end by the enzyme returningto its native state (see Figure 1)
21 Formation of Compound I The first reaction of thenative enzyme starts in the presence of hydrogen peroxidewhich acts as a relatively specific electron acceptor [24]
Native enzyme
Compound IOxidation state +2
Compound IIOxidation state +1
(give +
AHAH
Halogenation cycle
Peroxidase cycle
Oxidation state of compounds I or II with regard to the native enzyme a positive value reflects an oxidation
Figure 1 Halogenation or peroxidase cycle of peroxidases Com-pound I
Other substrates have been described such as ethyl hydroper-oxide peroxyacetic acid cumene hydroperoxide and 3-chloroperoxybenzoic acid [20 25] Compound I is formed asfollows
Peroxidase (native form) +H2O2997888rarr Compound I +H
2O(1)
The native enzyme undergoes a two-electron oxidationTwo electrons are transferred from the enzyme to hydrogenperoxide which is reduced into water Compound I is twooxidizing equivalents above the native enzyme one is in theoxyferryl heme center and the other is present as an organiccation located on the porphyrin ring [4 26 27] CompoundI is not specific regarding the electron donor [24] and thecomposition of the medium determines its subsequent cycle(see Figure 1) As Compound I is very unstable an aminoacid residue of the apoprotein is oxidized in the absenceof an exogenous electron donor [14] This reaction yieldsCompound I isomer that is very similar to Compound IIregarding its iron redox state and its incapability to react withhalogens [20]
22 The Halogenation Cycle In the presence of a halogen(Clminus Brminus or Iminus) or a pseudohalogen (SCNminus) Compound Iis reduced back to its native enzymatic form through a two-electron transfer while the (pseudo)halogen is oxidized intoa hypo(pseudo)halide (see Figure 1) Hypo(pseudo)halides(OXminus) are powerful oxidants with antimicrobial activity [1428ndash32] The halogenation cycle is described by the followingreaction
Compound I + Xminus (halogen or pseudo-halogen)
997888rarr Native enzyme +OXminus(2)
The oxidation rate of halogens by peroxidase-derivedCompound I depends on various factors One of these factors
MPO Compound I apparent second-order rate constant at
LPO Compound I apparent second-order rate constant at
Clminus chloride Brminus bromide Iminus iodide and SCNminus thiocyanate
pH 7 from [16]
pH 7 from [15]
Figure 2 Apparent second-order rate constant at pH 7 (times104Mminus1 sminus1) of the reaction between myeloperoxidase Compound I or lactoper-oxidase Compound I with (pseudo)halides [15 16]
is the standard reduction potential of the enzyme whichdiffers among peroxidases and plays a role in their capacityto oxidize specific (pseudo)halides The redox reaction canoccur only if the reduction potential of the enzyme is equalor superior to the reduction potential of the substrate Thestandard reduction potential at pH 7 of Compound I perox-idases and the couple of two-electron reduction HOXXminus isranking in the following ascending rank LPOCompound I ltEPO Compound I lt MPO Compound I HOSCNSCNminus ltHOIIminus ≪ HOBrBrminus lt HOClminusClminus [17 18 33 34] Thisinvolves that only the MPO Compound I is able to oxidizeClminus with appropriate rates LPO being able to oxidize withhigh rates Iminus and SCNminus and slowly Brminus [16 17 20 33 34]Interestingly although LPO Compound I has the lowestreduction potential compared to EPOCompound I andMPOCompound I it catalyzes the oxidation of Iminus and SCNminus withthe highest rates (Figure 2) [15ndash17 20] This suggests thatother factors play a role such as anion size anion access andanion binding as well as structural and amino acid differencesin the active and binding site between enzymes [17 18 20]
The reduction potential of the Compound Inativeenzyme and HOXXminus redox couples depends on reactantconcentrations and pH values At a specific reactant concen-tration it decreases with increasing pH values but slopesdiffer (Figure 3) [17] This means that there exists a thresholdpH value above which the oxidation of halides becomesthermodynamically unfavorable especially for halides withhigh reduction potential such as Clminus and Brminus [17]
Concentrations of (pseudo)halogens also affect theiraffinity to Compound I (Figure 4)
Although plasma Clminus concentrations are 1000-foldhigher than Brminus and SCNminus MPO Compound I oxidizessimilar amounts of SCNminus and Clminus [35] EPO Compound I
Compound Inative enzyme
Threshold pH value
Redu
ctio
n po
tent
ial (
V)
pH
HOXOXminus H2O
Figure 3 Illustration (according to [17]) of the pH thresholdvalue above which the oxidation of the halogen by mammalianheme peroxidase will be thermodically unfavorable The reductionpotential of the couple Compound Inative enzyme and the couplehalogen (X = chloride bromide) HOXOXminus is expressed with anillustrative function of the pH at a specific concentration of enzymeand substrates
preferentially oxidizes SCNminus in the presence of physiologicalconcentrations of SCNminus Brminus and Iminus [36] In the salivaof healthy adults where Clminus concentrations are only about25-fold higher than SCNminus SPO Compound I and MPOCompound I primarily generate hypothiocyanite [37 38]The levels of Iminus in human milk saliva blood and tissuesexcept the thyroid gland are below 1120583M and its in vivooxidation by Compound I is negligible [17 38] In humanmilk peroxidase activity is only derived from leucocytes AsMPO is able to oxidize Clminus and Clminus milk concentration is
Iodide 2120583MThiocyanate 120120583MChloride 13 mM
Figure 4 Illustration of the interaction between the biodisponibility of a peroxidase the (pseudo)halogen concentration in plasma in salivaand in milk and the production of oxidant molecules MPO myeloperoxidase SPO salivary peroxidase LPO bovine lactoperoxidaseOClminus hypochlorite and OSCNminus hypothiocyanite Although chloride is the most available substrate compared to thiocyanate bromide andiodide thiocyanate is the most effective substrate for the Compound I and hypothiocyanite could be produced at equal or superior levelscompared to hypohalides
high oxidation of Clminus is possible although it has never beenreported [14] In bovine milk lactoperoxidase is an abundantenzyme and with mean concentrations of Iminus and SCNminus of310 120583gkg and 02ndash15mgkg respectively oxidation is possi-ble [19 39] Nevertheless the relative abundance of SCNminus inall secretions blood and tissues and its better capacity as anelectron donor make it one of the main in vivo substratesof Compound I lactoperoxidase and myeloperoxidase for2-electron oxidation compared to halides [15] In in vitroapplications the ratio between (pseudo)halides regulates theratio of hypohalides generated by the reaction However asSCNminus is the most effective substrate for Compound I itspresence even in small quantities enhances its oxidation[14 35 36]
23 The Peroxidase Cycle Alternatively Compound I canshift to the peroxidase cycle which consists of two sequen-tial one-electron transfers back to the enzyme that yield(i) Compound II and (ii) the native enzyme while thesubstrate is oxidized into a radical (Figure 1) [40ndash42] Theperoxidase cycle is summarized in the following equations
Compound I + AH 997888rarr Compound II + A∙
Compound II + AH 997888rarr Native enzyme + A∙(3)
Compound I is not specific regarding the one-electrondonor it can be exogenous or endogenous and a lot of candi-dates have been described [18 20 43]Hydrogen peroxide canundergo a one-electron oxidation onlywithMPOCompoundI with the formation of superoxide [16 20 44 45]
During the first step of the peroxidase cycle the cationlocated in the porphyrin ring undergoes a one-electronreduction with formation of Compound II and concomitantoxidizing of one one-electron substrate [4 24] Compound II
maintains one oxidizing equivalent in the oxyferryl center[4 24] Finally this latter is reduced back to the native enzymewith the oxidation of a second one-electron donor
The standard reduction potential of the couple Com-pound ICompound II is high and allowed the one-electronoxidation by Compound I of a wide range of substrates[18 20] In contrast the standard reduction potential of thecouple Compound IInative enzyme is low and restrains thenumbers of possible substrates for Compound II [18 20]With the result that (i) the Compound IInative enzymestandard reduction potential is too low to react with halogensand (ii) the nature of substrates strongly influenced theirability to be oxidized by mammalian peroxidase compoundII [2 14 20] therefore when the enzyme is in this state it hasto be first reduced to the ground state before possibly partici-pating to the halogenation cycle and producing antimicrobialmolecules [14] Moreover the reduction of Compound II tothe ground state is the rate-limiting step [45 46] that is theperoxidase cycle interferes with the halogenation cycle andslows down antimicrobial activity [47]
The peroxidase cycle has been described as a possiblecatalytic sink for nitric oxide (NO) [46] but also for hydrogenperoxide in the case of a moderate excess of H
2O2relative
to LPO [24] Increase of NO removal from media evenin presence of Clminus after addition of MPO EPO or LPOand accelerated rates of Compound I and Compound IIreduction in presence of NO show that peroxidases mayregulate the bioavailability of NO [46] In conditions ofhigh excess of hydrogen peroxide relative to LPO and inthe absence of an exogenous electron donor CompoundII is transformed into Compound III which is 3 oxidativeequivalents above the native enzyme In moderate excessconditions Compound III can be partially reconverted intoCompound II and can reenter the peroxidase cycle [24 40]
Enzyme Research 5
Otherwise the enzyme is irreversibly inactivated the hemefraction is cleaved and iron is released [48] In the presenceof an exogenous two-electron donor the enzyme is largelyprotected from hydrogen peroxide because the halogenationcycle is favored Furthermore protection is higher withiodide because oxidized iodide consumes H
2O2to produce
oxygen and iodide in a reaction called the pseudocatalyticactivity of peroxidase [24 40 49]
However thiocyanate can act as a one-electron donor andbe part of the peroxidase cycle with the sequential formationof two thiocyanate radicals [47] With 200120583M SCNminus LPOis predominantly in its native form this indicates that thehalogenation cycle prevails [47]
In the presence of both one- and two-electron donorscompetition for oxidation can occur and favor the halogena-tion or the peroxidase cycle The presence of EDTA inhibitsthe oxidation of iodide due to competition for binding toCompound I [50] The standard reduction potential betweenthe donors favors the molecule with the lowest reductionpotential Thereby the respective reduction potentials of theone- and two-electron oxidation of thiocyanate at very lowpH are 165V and 082V and promote the halogenationcycle [51] In the case of low concentrations of halides orthiocyanate below 10 120583M Iminus or 3 120583M SCNminus CompoundI reacts with any suitable exogenous or endogenous one-electron donor with the subsequent formation of CompoundII and a negligible oxidation rate of halides and thiocyanate[14]
24 Inhibition of the Function of Mammalian Heme Peroxi-dase The function of heme peroxidases can be inhibited inseveral ways that could be classified into three categoriesThe first one could represent an inhibition of the enzymeby (i) molecules or proteins and (ii) external conditionssuch as pH and temperature For example cyanide azidenitrite mercaptomethylimidazole thiourea superoxide highlevels of nitric oxide and high levels of thiocyanate bindto the native enzyme and alter Compound I formation [2046 47 52ndash54] With thiocyanate inhibition is linked to therestriction of the binding site to hydrogen peroxide andthe interaction of SCNminus with a water molecule [23] Highconcentration of H
2O2or Iminus will inactivate irreversibly LPO
with liberation of free iron [48 55] Temperature between73∘C and 83∘C depending on the heating time results inunfolding and inactivation of LPO [19] Extreme pH isinactivating enzymes and at low pH an amino acid groupprobably histidine is protonated which prevents the bindingof H2O2[56] Some proteases such as pepsin and pronase are
able to inactivate LPO by proteolysis but chymotrypsin did itvery slowly and trypsin and thermolysin are not active againstLPO [19]
The second group of inhibitors could concern substancesor proteins which are able to interfere with the catalyticmechanism For example catalase consumes H
2O2and will
stop the formation of Compound I [30 52] Competitionbetween substrates can also interfere with the reaction cyclesuch as SCNminus which competes very effectively with Clminus Brminusand Iminus [52 53] HOCl has the capacity to bind to LPO native
enzyme and convert it into Compound I Above 100 120583MHOCl mediates the destruction of the LPO heme center[57]
The third class could be related to substances or pro-teins which are buffering active molecules produced duringthe catalytic reaction For example presence of thiosulfatethioglycolate glutathione dithiothreitol cysteineNAD(P)Hand tyrosine will reduce the antimicrobial activity throughreacting with OClminus OBrminus OIminus or OSCNminus [52 53 58 59]The enzyme NADH-OSCN oxidoreductase is able to reduceOSCNminus in SCNminus [60]
3 Activity of Lactoperoxidase withThiocyanate andor Iodide
LPO concentrations in cowrsquos milk are around 30mg Lminus1depending on season diet and calving and breeding season[61] LPO extraction from whey or milk is based on awell-developed industrial process [62] Compared to MPOand EPO LPO is easily isolated and manufactured in largequantities As a result cowrsquos milk peroxidase is the favoritemolecule for in vitro or in vivo applications such as con-servation of raw and pasteurized milk storage of emulsionsand cosmetics moisturizing gel and toothpaste in human drymouth veterinary products and preservation of foodstuffs[19 61 63 64]
31 Activity of LPO Related to Hypothiocyanite
311 Mode of Action of Hypothiocyanite Thiocyanate is oxi-dized in a two-electron reaction that yields hypothiocyaniteHypothiocyanite has a pKa of 53 [65] It is more acidic thanhypohalides that have pKas of 75 (HOCl) 86 (HOBr) and106 (HOI) [14 66] All hypo(pseudo)halides (OXminus) are in anacid-base equilibrium association with their correspondingacid hypo(pseudo)halide (HOX) For example in the case ofhypothiocyanite
HOSCN 999447999472 OSCNminus +H+ (4)
The acid form has a higher oxidation potential and ismore soluble in nonpolar media so that it passes throughhydrophobic barriers such as cell membranes more easilybut it is less stable than the basic form (OXminus) [14 66]Hypohalide acids are predominant in acidic to neutral mediaand even in basic conditions for HOBr and HOI whereashypothiocyanite needs a pH below 53 to be predominant inthe acid form [66 67]
SCNminus is the two-electron donorwith the lowest reductionpotential and therefore forms the hypothiocyanite acid withthe lowest oxidative power compared to hypohalous acidsHypohalous acids rank as follows with increasing oxidativestrength OSCNminus lt OIminus lt OBrminus lt OClminus [28 66]These characteristicsmake hypothiocyanite relatively specificregarding its molecular target (Figure 5) that is a thiolmoiety [28 59 68]
6 Enzyme Research
- SH group- NAD(P)H
- SH group- NAD(P)H- Reduced pyridine nucleotide- R-S-R (thioether group)
HOSCNOSCNminus
HOIOIminusI2
- NH2 group
Figure 5 Target group of hypothiocyanite hypoiodite and iodineDue to its low oxidation power hypothiocyanite is relatively specificand is not reactive against all thiols In vivo hypoiodite seems tobe selectively directed against reduced pyridine nucleotide becauseeven the presence of excess glutathione and methionine doesnot thoroughly inhibit their oxidation HOSCNOSCNminus acidic orbasic form of hypothiocyanite HOIOIminus acidic or basic form ofhypoiodite and I
2 iodine
R-S-SCN or R-S-I
LPO R-SH
+ R-S-OHOSCNminusSCNminus SCNminus
or Iminus or Iminusor minusOI
H2O
H2O
2
Figure 6 Illustration of the cofactor role of SCNminus or Iminus Whenthe necessary conditions are fulfilled that is (i) no substratecompetitor for SCNminus or Iminus for binding to lactoperoxidase (ii)enough peroxidase H
2O2and SCNminus or Iminus (iii) enough R-SH
and (iv) no incorporation of SCNminus or Iminus in stable byproducts thequantity of OSCNminus or OIminus produced depends only on the amountof H2O2 SCNminus thiocyanate Iminus iodide H
2O2 hydrogen peroxide
LPO lactoperoxidase R-SH peptide or protein with a thiol moietyR-S-SCN or R-S-I sulfenyl thiocyanate or iodide R-SOH sulfenicacid OSCNminus hypothiocyanite and OIminus hypoiodite
Sulfhydryl oxidation by OSCNminus generates sulfenyl thio-cyanate in equilibrium with sulfenic acid [68]
SCNminus +H2O2+ LPO 997888rarr OSCNminus + LPO
R-SH +OSCNminus 997888rarr R-S-SCN +OHminus
R-S-SCN +H2O 997888rarr R-S-OH + SCNminus +H+
(5)
The cycle of reactions shows that thiocyanate acts likea cofactor for LPO (Figure 6) so that the total number ofoxidized sulfhydryls is independent of SCNminus as long as(i) thiocyanate is not exhausted (ii) thiocyanate is not incompetition with other substrates for the binding to Com-pound I (iii) thiocyanate is not incorporated into an aromaticamino acid (iv) enoughH
2O2is present and (v) thiol moiety
is still available [68 69]Although the target of OSCNminus is a thiol moiety not
all sulfhydryls are equally sensitive to OSCNminus albumincysteine mercaptoethanol dithiothreitol glutathione and 5-thio-2-nitrobenzoic acid are all oxidized but 120573-lactoglobulinis poorly oxidized probably due to a limited accessibility ofsulfhydryls to OSCNminus [68] In some conditions that is thejoint presence of LPO enough H
2O2and SCNminus and after the
oxidation of available sulfhydryls modification of tyrosinetryptophan and histidine protein residues can occur and that
could be linked to the formation of a labile powerful oxidantsuch as sulfur dicyanide [68]
Some authors suggest that (SCN)2is formed during the
enzymatic reaction and then chemically hydrolyzed intohypothiocyanite [14 69 70] However a recent publicationdemonstrates that (SCN)
2cannot be a precursor during the
enzymatic oxidation of SCNminus at neutral pH inmammals [71]Hypothiocyanite is less stable in acid conditions with
high concentrations of SCNminus and in the presence of (SCN)2
and it is thought to break down via the following net reaction[14]
4HOSCN +H2O 997888rarr 3SCNminus + CNOminus + SO
4
2minus+ 6H+ (6)
A recent study based notably on spectroscopic and chro-matographic methods proposes the following net equationwithin the 4ndash7 pH range
3HOSCN +H2O 997888rarr XSO
4
2minus+ XHCN
+ (1 minus X) SO3
2minus+ (1 minus X)CNOminus
+ 2SCNminus + (5 minus X)H+
(7)
The proportions of end anions were different at pH 4 andpH 7 at pH 7 the proportion of CNOminus was higher SCNminusformation was slower and no CNminus was detected [71]
It might seem easier to produce hypothiocyanite chemi-cally in in vitro applications but producing hypothiocyanitechemically from the oxidation of SCNminus by a halogen (Cl
2or
Br2) or by a hypohalous acid (HOCl or HOBr) in basic media
is tricky due to overoxidation of SCNminus [66] The referencemethod in the literature to produce 1- to 2-day stable OSCNminusis by hydrolyzing (SCN)
2in basic conditions [72ndash74]
Hypothiocyanite inhibitors have been described Forexample CNminus a weak acid buffer dissolved carbonate excesshydrogen peroxide hydrofluoric acid metallic ions glyc-erol or ammonium sulfate accelerates the decomposition ofOSCNminus whereas sulfonamide stabilizes it [67 72]
Appropriate concentrations of substrates induce en-hanced activity [75]
312 Biological Activity of Hypothiocyanite The biologicalactivity of hypothiocyanite is summarized in Figure 7
The sulfhydryl moiety is essential for the activityof numerous enzymes and proteins Inhibition of bacte-rial glycolysis through the oxidation of hexokinase gly-ceraldehyde-3-phosphate dehydrogenase (GAPDH) aldo-lase and glucose-6-phosphate dehydrogenase has beenobserved [14 51 65 70 76] Inhibition of respiration andglucose transport is associated with the alteration of cellmembranes or transporters [14 51 65 77] Irreversible inhi-bition is linked to long periods of incubation and bacterialsensitivity depends on the bacterial species and on hypothio-cyanite concentrations [14 51 59] Increased concentrationsof reducing agents such as glutathione and cysteine canreverse the inhibition through buffering hypothiocyaniteand converting the reduced thiol back into sulfhydryl [1478] This defense mechanism is used by Escherichia coli itinduces the CysJ promoter during the stress response to the
Enzyme Research 7
Bacteria
Inhibition of glycolysis Inhibition of respirationInhibition of glucose transport
Possible defense mechanism of bacteria- Increased GSH and cysteine content
Peptide proteinwith SH moiety
HOSCNOSCNminus
- NAD(P)H-dependent reduction of OSCNminus
Figure 7 Biological activity of hypothiocyanite on bacteria and possible defensemechanism of the bacteria Reversible inhibition is observedin that (i) hypothiocyanite is not reactive against all thiols and (ii) if hypothiocyanite is removed or diluted the pathogen recovers Irreversibleinhibition is linked to (i) long period of incubation (ii) the bacterial species and (iii) hypothiocyanite concentration HOSCNOSCNminus acidicor basic form of hypothiocyanite and GSH glutathione
pH lt 6
Influence of iodide concentration
Influence of the pH
1 I2 and high Iminus
I5minusI6
minus
I2I3minus
6 lt pH lt 9
HOII2OHI2I3minus
Iminus + H2O2 + LPO rarr active molecules
I2 (without Iminus)HOIOIminusI2I3
minusHI2Ominus
Figure 8 Illustration of the molecules that can be present after oxidation of iodide by lactoperoxidase in presence of H2O2The active species
depend mainly on the concentration of iodide (upper part) and the pH (lower part) The species with an oxidant power are represented inbold
lactoperoxidase system [79] Another resistance mechanismcould be the NAD(P)H-dependent reduction of OSCNminuswithout any loss of the sulfhydryl compound [14 72 78]Alteration of the bacterial membrane increases the efficacyof hypothiocyanite [80]
Furthermore the activity of the entire system (enzyme +substrates) is known to be more effective than hypothiocyan-ite alone whether enzymatically or chemically producedThis has been explained by the production of short-livedhighly reactive intermediates such as O
2SCNminus and O
3SCNminus
by the enzyme or by the oxidation of OSCNminus in conditionsof excess H
2O2[65 73 81] The activity of hypothiocyanite
has been described against bacteria such as Actinomyces sppBacillus cereus Lactobacillus spp Staphylococcus albus Saureus Streptococcus spp Escherichia coli Legionella pneu-mophila Salmonella typhimurium Pseudomonas fluorescensP aeruginosa Campylobacter jejuni C coli and Listeriamonocytogenes [14 32] Reversible inhibition is observed
when cells recover after OSCNminus is depleted [14 59] Irre-versible inhibition is obtained with long-term incubation andhigh level of OSCNminus [59] Higher concentration of SCNminuscompared to Iminus is necessary to obtain inhibition against E coliand accumulation of OSCNminus is observed as it is not reactiveagainst all thiols [59]Therefore the activity of the SCNminus-LPOsystem appears to be more bacteriostatic than bactericidal
32 Activity of LPO Related to Oxidized Iodide
321 Chemistry of Oxidized Iodide Iodide is oxidized byCompound I through a single two-electron transfer thatyields oxidized Iminus in the form of I
2or HOI [14 24 82ndash85]
The active agent is composed of a mixture of species that arenot yet formally detailed due to the very complex behaviorand stability of I
2and HOI in aqueous environments that
strongly depend on pH values and iodide concentrations[66 82 83 86]
8 Enzyme Research
Based on the inorganic chemistry of iodine in waterand literature on enzymatic oxidation of iodide the activemolecules have been described as follows (Figure 8)
(i) Under pH 6 and in the presence of iodide only I2
Iminus and I3
minus are present and the only active moleculeis I2 I2concentrations decrease with increasing
concentrations of Iminus At an initial 1mM I2 with Iminus
concentrations ranging from 1mM to 100mM I2
concentrations fall from almost 1mM to 001mM asdescribed by the following association reaction [2482 83 86]
I2+ Iminus 999447999472 I
3
minus (8)
(ii) In solution within a 6ndash9 pH range and with a max-imum 1mM iodide a mixture of HOII
2OHI2I3
minus
is formed in which I3
minus is not active and I2OH is
probably less reactive than HOI or I2[86 87] If
Iminus concentrations are above 10mM I3
minus representsthe main species formed and the concentration ofactive molecules relatively drops The mechanism issummarized in the following net equations
HOI + Iminus +H+ 999447999472 I2OHminus +H+
999447999472 I2+H2O 999447999472 I
2+ Iminus 999447999472 I
3
minus
(9)
(iii) In iodine solution without iodide or when availableiodide has been oxidized the number of I
2-derived
molecules decreases with decreasing I2concentra-
tions At 1000 120583M I2 with pH-related ratios five
relevant species are observed (I2 HOI I
3
minus HI2Ominus
and OIminus) At 10 120583M I2 the main species are only I
2
HOI and OIminus and HOI could represent up to 90 ofthe active oxidant molecules at pH 8-9 [86] Below apH of 106 the following reactions are involved
I2+H2O 999447999472 HOI + Iminus +H+ (hydrolysis of I
2)
I2+ Iminus 999447999472 I
3
minus(triiodide formation independent of pH)
(10)
(iv) At high Iminus and 1 I2concentrations as in Lugol
solution I5
minus and I6
minus are formed and represent 82of the active oxidative agents [86] after the followingreaction
I3
minus+ I2999447999472 I5
minus(pentaiodide formation)
2I3
minus999447999472 I6
2minus(dimerization of I
3
minus)
(11)
The stability of HOI and I2is linked to their dispro-
portionation in iodate which has no oxidative activity inneutral and basic pH conditions [86]The disproportionationreactions read as follows
3HOI 999447999472 IO3
minus+ 2Iminus + 3H+ (disproportionation of HOI)
3I2999447999472 IO
3
minus+ 5Iminus + 6H+ (disproportionation of I
2)
(12)
I2stability increases at higher pHvalues andhigher iodide
concentrations [86] In drinking water HOI disproportion-ation is slow and varies substantially HOI has a half-life of4 days to 35 years depending on (i) the initial level of HOIthat speeds its decomposition and (ii) the presence of boratephosphate or carbonate that catalyzes its decomposition [8889]
322 Mode of Action of Oxidized Iodide The oxidativestrength of I
2is between that of the corresponding hypo-
halous acid HOI and the hypoiodite ion OIminus and ranks asfollows 0485V (OIminus) lt 0536V (I
2) lt 0987V (HOI) [66]
HOI reacts through very rapid oxidation of thiolgroups oxidation of NAD(P)H oxidation of 120573-nicotinamidemononucleotide direct reaction with thioether groupsthrough sulfoxidation and slow oxidation of the aminemoiety (Figure 5) [87 90 91] At low Iminus concentrationsiodination of tyrosine residues is catalyzed by the enzyme[14] In a cellular environment HOI seems to be more selec-tively directed against the degradation of reduced pyridinenucleotides thanHOCL andHOBr because even the presenceof excess glutathione methionine or oxidized glutathionedoes not thoroughly inhibit their oxidation [87]
In some conditions that is (i) enough iodide H2O2 and
peroxidase (ii) no accumulation of oxidized iodide and (iii)no incorporation of iodide into stable byproducts such astyrosine residues iodide acts as a cofactor (Figure 6) andthe proportion of oxidized sulfhydryls is proportional to theamount of H
2O2as described below [85 92]
2Iminus +H2O2+ LPO (native enzyme)
997888rarr I2+ 2H2O + LPO (native enzyme)
R-SH + I2997888rarr R-S-I + Iminus +H+
R-S-I+H2O 997888rarr R-S-OH + Iminus +H+
(13)
In the case of high concentrations of Iminus andor H2O2
inhibition of tyrosine iodation has been observed [83] andrelated to the pseudocatalytic redox degradation of H
2O2
with formation of O2when excessive H
2O2is present (reac-
tion 1) and production of I3
minus when excessive amounts of Iminusare present (reaction 2)
I2+H2O 997888rarr O
2+ 2Iminus + 2H+ (reaction 1)
I2+ Iminus 999447999472 I
3
minus(reaction 2)
(14)
Both reactions deplete the amount of the active oxidizingagent I
2 In the absence of tyrosine oxidized iodide reacts
with nucleophilic molecules such as Iminus Clminus or OHminus to formI2 I3
minus ICl ICl2 IOH and I
2OH [82] Some anions such as
Clminus HPO4
minus or OHminus reduce the amount of I2I3
minus but thiseffect is inversely proportional to the concentration of Iminusabove pH 9 I
2is hydrolyzed and IO
3
minus is formed [82]HOI can be produced chemically through oxidation of Iminus
by Cl2or O3 with a short half-life due to overoxidation of
HOI byCl2andO
3[89] and through oxidation of Iminus byHOCl
HOBr or NH2Cl with a longer half-life [87 89]
Enzyme Research 9
Bacteria
Inhibition of glycolysis Inhibition of respirationInhibition of glucose transportInhibition of the pentose phosphate pathway
- Peptide protein with- SH moiety- thioether moiety- NAD(P)H
HOIOIminus
I2
Figure 9 Biological activity of hypoiodite or iodine on bacteria Irreversible inhibition is observed and could be linked to (i) oxidation ofthiol groups NAD(P)H and thioether groups (ii) high reactivity of HOII
2against thiol and reduced nicotinamide nucleotides and (iii) the
incorporation of iodide in tyrosine residue of protein (iodination of protein) HOIOIminus acid or basic form of hypoiodite and I2 iodine
323 Biological Action of Oxidized Iodide The biologicalaction of oxidized iodide (Figure 9) is similar to that ofhypothiocyanite but differs in that (i) the reactivity of oxi-dized iodide is complete against thiol group and (ii) cells didnot recover after removing of oxidized iodide [59]
Due to the cofactor role of Iminus inhibition of respirationin Escherichia coli in the presence of LPO H
2O2 and Iminus is
complete with only 10120583M NaI whereas 100 120583M of solely I2
is necessary to obtain complete inhibition This is directlyrelated to the oxidation of sulfhydryls not to the percentageof iodine incorporation [92 93]
E coli seems to be more sensitive if the bacteria areincubated together with the entire system (enzyme H
2O2
and iodide) rather than adding several minutes after mixingthe enzyme with its substrates This could be linked to theformation of an unstable reactive intermediate [52]
The activity of the Iminus peroxidase system is more effectiveagainst E coli than the SCNminus system in that lower Iminusconcentrations are necessary all sulfhydryls are oxidized andcells do not recover even if the amount of I
2is not sufficient
to oxidize all SH groups [59 80] Against L acidophilushigh non physiological amounts of Iminus are necessary to obtaininhibitionwhereas small concentrations of SCNminus are effective[70]
CNminus azide EDTA and SCNminus inhibit the formation ofoxidized iodide [50 52] Increased pH values and increasedamounts of thiol and NAD(P)H compounds reduce theactivity of the iodide peroxidase system [52]
LPO-H2O2-Iminus in presence of Streptococcus mitis is active
against Staphylococcus aureus and E coli [94] LPO-H2O2-
Iminus is active against Micrococcus S aureus Listeria monocy-togenes Bacillus cereus E coli and Candida albicans [12 1980] In the presence of other peroxidases the Iminus peroxidasesystem is active against Schistosoma mansoni Fusariumnucleatum andActinobacillus actinomycetemcomitans [31 9596] Compared to SCNminus Iminus-LPO shows bactericidal activities[14 19 80]
33 Activity of LPO Related to Hypoiodite and Hypothiocyan-ite The combination of SCNminus with Iminus in the lactoperoxidasesystem has been poorly studied Tackling the enzymaticmechanism is tricky and contradictory results have been
found about microbial activity in the concomitant presenceof SCNminus and Iminus
In the presence of SCNminus and Iminus there is competitionbetween the two substrates for oxidation by lactoperoxidase[14 36] Iminus alone exhibits bactericidal activity but an SCNminusIminusratio of 01 inhibits that bactericidal effect and an SCNminusIminusratio of 1 antagonizes it due to competition for oxidation andfaster decomposition of HOSCN in the presence of Iminus [14]Against A actinomycetemcomitans the peroxidase systemwith Iminus Clminus or a combination of Iminus and Clminus is effective butaddition of SCNminus cancels the antibacterial effect [96] On theother hand a synergistic or unaffected effect of iodide in theSCNminus-H
2O2-LPO system has been shown against Candida
albicans E coli S aureusAspergillus niger and Pseudomonasaeruginosa [19 97]
4 Conclusion
Themolecular evolution of heme peroxidases and the preser-vation of their catalytic domain [6] show that the productionof strong oxidants is a powerful part of the nonimmunedefense mechanisms against pathogenic bacteria fungi orparasite which made the use of those enzymes in practicalapplications worthwhile
The enzymatic reactions involving mammalian peroxi-dases are complex and various molecules can promote orreduce dramatically the antibacterial activity of the per-oxidase system In order to favor the halogenation cyclerequired in in vitro and in vivo antimicrobial applicationsseveral points have to be taken into account (i) to avoid thepresence of competitors to iodide or thiocyanate for bindingto Compound I and to avoid the presence of inhibitorsof the enzyme or of active molecules (ii) to avoid excessH2O2concentration which is able to destruct the enzyme
and to react with iodine or hypoiodite with loosing of activemolecules (iii) to favor the presence of hypoiodite instead ofiodine due to the association reaction of iodine with iodide(iv) to avoid excess concentration of thiocyanate which caninhibit formation of Compound I (v) to use the entiresystem (enzyme + substrates) instead of active moleculesalone (vi) to favor moderate acid pH when hypothiocyaniteis the active molecule (vii) for bactericidal fungicidal or
10 Enzyme Research
parasitical applications the use of iodide has to be preferred(viii) the use of combined presence of iodide and thiocyanatehas to be checked carefully for efficacy and (ix) to favor thecofactor role of iodide or thiocyanate
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] P J OrsquoBrien ldquoPeroxidasesrdquoChemico-Biological Interactions vol129 no 1-2 pp 113ndash139 2000
[2] W Jantschko P G Furtmuller M Allegra et al ldquoRedoxintermediates of plant and mammalian peroxidases a compar-ative transient-kinetic study of their reactivity toward indolederivativesrdquo Archives of Biochemistry and Biophysics vol 398no 1 pp 12ndash22 2002
[3] S Kimura and M Ikeda-Saito ldquoHuman myeloperoxidase andthyroid peroxidase two enzymes with separate and distinctphysiological functions are evolutionarily related membersof the same gene familyrdquo Proteins Structure Function andGenetics vol 3 no 2 pp 113ndash120 1988
[4] G Battistuzzi M Bellei C A Bortolotti and M Sola ldquoRedoxproperties of heme peroxidasesrdquo Archives of Biochemistry andBiophysics vol 500 no 1 pp 21ndash36 2010
[5] M Zamocky C Jakopitsch P G Furtmuller C Dunand and CObinger ldquoThe peroxidase-cyclooxygenase superfamily recon-structed evolution of critical enzymes of the innate immunesystemrdquo Proteins Structure Function and Genetics vol 72 no2 pp 589ndash605 2008
[6] H Daiyasu and H Toh ldquoMolecular evolution of the myeloper-oxidase familyrdquo Journal of Molecular Evolution vol 51 no 5 pp433ndash445 2000
[7] D Serteyn S Grulke T Franck A Mouithys-Mickalad andG Deby-Dupont ldquoNeutrophile myeloperoxidase protectiveenzyme with strong oxidative activitiesrdquo Annales de MedecineVeterinaire vol 147 no 2 pp 79ndash93 2003
[8] S C Whitman S L Hazen D B Miller R A HegeleJ W Heinecke and M W Huff ldquoModification of type IIIVLDL their remnants and VLDL from apoE- knockout miceby p-hydroxyphenylacetaldehyde a product of myeloperox-idase activity causes marked cholesteryl ester accumulationin macrophagesrdquo Arteriosclerosis Thrombosis and VascularBiology vol 19 no 5 pp 1238ndash1249 1999
[9] T J Barrett and C L Hawkins ldquoHypothiocyanous acid benignor deadlyrdquo Chemical Research in Toxicology vol 25 no 2 pp263ndash273 2012
[10] M M Lloyd D M van Reyk M J Davies and C L HawkinsldquoHypothiocyanous acid is a more potent inducer of apoptosisand protein thiol depletion in murine macrophage cells thanhypochlorous acid or hypobromous acidrdquo Biochemical Journalvol 414 no 2 pp 271ndash280 2008
[11] J Wang and A Slungaard ldquoRole of eosinophil peroxidase inhost defense and disease pathologyrdquo Archives of Biochemistryand Biophysics vol 445 no 2 pp 256ndash260 2006
[12] M Ahariz and P Courtois ldquoCandida albicans susceptibility tolactoperoxidase-generated hypoioditerdquo Clinical Cosmetic andInvestigational Dentistry vol 2 pp 69ndash78 2010
[13] A Welk C Meller R Schubert C Schwahn A Kramerand H Below ldquoEffect of lactoperoxidase on the antimicrobialeffectiveness of the thiocyanate hydrogen peroxide combinationin a quantitative suspension testrdquo BMC Microbiology vol 9article 134 2009
[14] K M Pruitt and J O Tenovuo Eds The Lactoperoxidase Sys-tem Chemistry and Biological Significance vol 27 of Immunol-ogy Series Marcel Dekker New York NY USA 1985
[15] P G Furtmuller W Jantschko G Regelsberger C JakopitschJ Arnhold and C Obinger ldquoReaction of lactoperoxidasecompound I with halides and thiocyanaterdquo Biochemistry vol41 no 39 pp 11895ndash11900 2002
[16] P G Furtmuller U Burner and C Obinger ldquoReaction ofmyeloperoxidase compound I with chloride bromide iodideand thiocyanaterdquo Biochemistry vol 37 no 51 pp 17923ndash179301998
[17] J Arnhold E Monzani P G Furtmuller M Zederbauer LCasella and C Obinger ldquoKinetics and thermodynamics ofhalide and nitrite oxidation by mammalian heme peroxidasesrdquoEuropean Journal of Inorganic Chemistry no 19 pp 3801ndash38112006
[18] M J Davies C L Hawkins D I Pattison and M D ReesldquoMammalian heme peroxidases from molecular mechanismsto health implicationsrdquo Antioxidants and Redox Signaling vol10 no 7 pp 1199ndash1234 2008
[19] J N de Wit and A C M van Hooydonk ldquoStructure functionsand applications of lactoperoxidase in natural antimicrobialsystemsrdquo Nederlands melk en Zuiveltijdschrift vol 50 no 2 pp227ndash244 1996
[20] P G Furtmuller M Zederbauer W Jantschko et al ldquoActivesite structure and catalytic mechanisms of human peroxidasesrdquoArchives of Biochemistry and Biophysics vol 445 no 2 pp 199ndash213 2006
[21] M Zederbauer P G Furtmuller S Brogioni C JakopitschG Smulevich and C Obinger ldquoHeme to protein linkages inmammalian peroxidases impact on spectroscopic redox andcatalytic propertiesrdquo Natural Product Reports vol 24 no 3 pp571ndash584 2007
[22] G Battistuzzi M Bellei J Vlasits et al ldquoRedox thermodynam-ics of lactoperoxidase and eosinophil peroxidaserdquo Archives ofBiochemistry and Biophysics vol 494 no 1 pp 72ndash77 2010
[23] I A Sheikh A Singh N Singh et al ldquoStructural evidence ofsubstrate specificity inmammalian peroxidases structure of thethiocyanate complex with lactoperoxidase and its interactionsat 24 a 24 A resolutionrdquo The Journal of Biological Chemistryvol 284 no 22 pp 14849ndash14856 2009
[24] H Kohler and H Jenzer ldquoInteraction of lactoperoxidase withhydrogen peroxide Formation of enzyme intermediates andgeneration of free radicalsrdquo Free Radical Biology and Medicinevol 6 no 3 pp 323ndash339 1989
[25] P G Furtmuller U Burner W Jantschko G Regelsberger andC Obinger ldquoTwo-electron reduction and one-electron oxida-tion of organic hydroperoxides by human myeloperoxidaserdquoFEBS Letters vol 484 no 2 pp 139ndash143 2000
[26] A Taurog M L Dorris and D R Doerge ldquoMechanism ofsimultaneous iodination and coupling catalyzed by thyroidperoxidaserdquo Archives of Biochemistry and Biophysics vol 330no 1 pp 24ndash32 1996
[27] J E Erman L B Vitello J Matthew Mauro and J KrautldquoDetection of an oxyferryl porphyrin 120587-cation-radical interme-diate in the reaction between hydrogen peroxide and a mutant
Enzyme Research 11
yeast cytochrome c peroxidase Evidence for tryptophan-191involvement in the radical site of compound Irdquo Biochemistryvol 28 no 20 pp 7992ndash7995 1989
[28] M T Ashby ldquoInorganic chemistry of defensive peroxidases inthe human oral cavityrdquo Journal of Dental Research vol 87 no10 pp 900ndash914 2008
[29] J D Chandler and B J Day ldquoThiocyanate a potentially usefultherapeutic agent with host defense and antioxidant propertiesrdquoBiochemical Pharmacology vol 84 no 11 pp 1381ndash1387 2012
[30] E C Jong W R Henderson and S J Klebanoff ldquoBactericidalactivity of eosinophil peroxidaserdquo Journal of Immunology vol124 no 3 pp 1378ndash1382 1980
[31] E C Jong A A F Mahmoud and S J Kelbanoff ldquoPeroxidase-mediated toxicity to schistosomula of Schistosoma mansonirdquoJournal of Immunology vol 126 no 2 pp 468ndash471 1981
[32] L M Wolfson and S S Sumner ldquoAntibacterial activity of thelactoperoxidase system a reviewrdquo Journal of Food Protectionvol 56 no 10 pp 887ndash892 1993
[33] J Arnhold P G Furtmuller G Regelsberger and C ObingerldquoRedox properties of the couple compound Inative enzyme ofmyeloperoxidase and eosinophil peroxidaserdquo European Journalof Biochemistry vol 268 no 19 pp 5142ndash5148 2001
[34] P G Furtmuller J Arnhold W Jantschko M Zederbauer CJakopitsch and C Obinger ldquoStandard reduction potentials ofall couples of the peroxidase cycle of lactoperoxidaserdquo Journalof Inorganic Biochemistry vol 99 no 5 pp 1220ndash1229 2005
[35] C J van Dalen M W Whitehouse C C Winterbourn and AJ Kettle ldquoThiocyanate and chloride as competing substrates formyeloperoxidaserdquo Biochemical Journal vol 327 no 2 pp 487ndash492 1997
[36] A Slungaard and J R Mahoney Jr ldquoThiocyanate is the majorsubstrate for eosinophil peroxidase in physiologic fluids impli-cations for cytotoxicityrdquoThe Journal of Biological Chemistry vol266 no 8 pp 4903ndash4910 1991
[37] J Tenovuo ldquoAntimicrobial function of human salivamdashhowimportant is it for oral healthrdquoActaOdontologica Scandinavicavol 56 no 5 pp 250ndash256 1998
[38] R Ihalin V Loimaranta and J Tenovuo ldquoOrigin structure andbiological activities of peroxidases in human salivardquo Archives ofBiochemistry and Biophysics vol 445 no 2 pp 261ndash268 2006
[39] J A Rooke J F Flockhart and N H Sparks ldquoThe potentialfor increasing the concentrations of micro-nutrients relevant tohuman nutrition inmeat milk and eggsrdquo Journal of AgriculturalScience vol 148 no 5 pp 603ndash614 2010
[40] H Kohler A Taurog and H B Dunford ldquoSpectral studieswith lactoperoxidase and thyroid peroxidase interconversionsbetween native enzyme compound II and compound IIIrdquoArchives of Biochemistry and Biophysics vol 264 no 2 pp 438ndash449 1988
[41] I Yamazaki H S Mason and L Piette ldquoIdentification byelectron paramagnetic resonance spectroscopy of free radicalsgenerated from substrates by peroxidaserdquoThe Journal of Biolog-ical Chemistry vol 235 pp 2444ndash2449 1960
[42] B Chance ldquoThe kinetics and stoichiometry of the transitionfrom the primary to the secondary peroxidase peroxide com-plexesrdquo Archives of Biochemistry and Biophysics vol 41 no 2pp 416ndash424 1952
[43] K M Pruitt B Mansson-Rahemtulla D C Baldone andF Rahemtulla ldquoSteady-state kinetics of thiocyanate oxidationcatalyzed by human salivary peroxidaserdquo Biochemistry vol 27no 1 pp 240ndash245 1988
[44] B G J M Bolscher and R Wever ldquoA kinetic study of thereaction between humanmyeloperoxidase hydroperoxides andcyanide inhibition by chloride and thiocyanaterdquo Biochimica etBiophysica Acta Protein Structure and Molecular Enzymologyvol 788 no 1 pp 1ndash10 1984
[45] L A Marquez J T Huang and H Brian Dunford ldquoSpectraland kinetic studies on the formation of myeloperoxidase com-pounds I and II roles of hydrogen peroxide and superoxiderdquoBiochemistry vol 33 no 6 pp 1447ndash1454 1994
[46] HMAbu-Soud and S LHazen ldquoNitric oxide is a physiologicalsubstrate for mammalian peroxidasesrdquoThe Journal of BiologicalChemistry vol 275 no 48 pp 37524ndash37532 2000
[47] Y R Tahboub S Galijasevic M P Diamond and H MAbu-Soud ldquoThiocyanate modulates the catalytic activity ofmammalian peroxidasesrdquo Journal of Biological Chemistry vol280 no 28 pp 26129ndash26136 2005
[48] H Jenzer W Jones and H Kohler ldquoOn the molecularmechanismof lactoperoxidase-catalyzedH
2O2metabolism and
irreversible enzyme inactivationrdquo The Journal of BiologicalChemistry vol 261 no 33 pp 15550ndash15556 1986
[49] R P Magnusson A Taurog and M L Dorris ldquoMechanism ofiodide-dependent catalatic activity of thyroid peroxidase andlactoperoxidaserdquo The Journal of Biological Chemistry vol 259no 1 pp 197ndash205 1984
[50] D K Bhattacharyya U Bandyopadhyay and R K BanerjeeldquoEDTA inhibits lactoperoxidase-catalyzed iodide oxidation byacting as an electron-donor and interacting near the iodidebinding siterdquoMolecular and Cellular Biochemistry vol 162 no2 pp 105ndash111 1996
[51] C L Hawkins ldquoThe role of hypothiocyanous acid (HOSCN) inbiological systems HOSCN in biological systemsrdquo Free RadicalResearch vol 43 no 12 pp 1147ndash1158 2009
[52] S J Klebanoff ldquoIodination of bacteria a bactericidal mecha-nismrdquo Journal of Experimental Medicine vol 126 no 6 pp1063ndash1078 1967
[53] S J Klebanoff ldquoMyeloperoxidase-halide-hydrogen peroxideantibacterial systemrdquo Journal of Bacteriology vol 95 no 6 pp2131ndash2138 1968
[54] R K Banerjee and A G Datta ldquoSalivary peroxidasesrdquoMolecu-lar and Cellular Biochemistry vol 70 no 1 pp 21ndash29 1986
[55] MHuwiler H Jenzer andHKohler ldquoThe role of compound IIIin reversible and irreversible inactivation of lactoperoxidaserdquoEuropean Journal of Biochemistry vol 158 no 3 pp 609ndash6141986
[56] R Wever W M Kast J H Kasinoedin and R Boelens ldquoTheperoxidation of thiocyanate catalysed by myeloperoxidase andlactoperoxidaserdquo Biochimica et Biophysica Acta (BBA)ProteinStructure and Molecular vol 709 no 2 pp 212ndash219 1982
[57] C E A Souza D Maitra G M Saed et al ldquoHypochlorousacid-induced heme degradation from lactoperoxidase as anovel mechanism of free iron release and tissue injury ininflammatory diseasesrdquo PLoS ONE vol 6 no 11 Article IDe27641 2011
[58] J Carlsson ldquoBactericidal effect of hydrogen peroxide is pre-vented by the lactoperoxidase-thiocyanate system under anaer-obic conditionsrdquo Infection and Immunity vol 29 no 3 pp 1190ndash1192 1980
[59] E L Thomas and T M Aune ldquoLactoperoxidase peroxidethiocyanate antimicrobial system correlation of sulfhydryloxidation with antimicrobial actionrdquo Infection and Immunityvol 20 no 2 pp 456ndash463 1978
12 Enzyme Research
[60] J Carlsson Y Iwami and T Yamada ldquoHydrogen peroxideexcretion by oral streptococci and effect of lactoperoxidase-thiocyanate-hydrogen peroxiderdquo Infection and Immunity vol40 no 1 pp 70ndash80 1983
[61] K D Kussendrager and A C M van Hooijdonk ldquoLactoperox-idase physico-chemical properties occurrence mechanism ofaction and applicationsrdquoTheBritish Journal of Nutrition vol 84supplement 1 pp S19ndashS25 2000
[62] J P Perraudin ldquoProteines a activites biologiques lactoferrineet lactoperoxydase Connaissances recemment acquises et tech-nologies drsquoobtentionrdquo Lait vol 71 no 2 pp 191ndash211 1991
[63] J-W Boots and R Floris ldquoLactoperoxidase From catalyticmechanism to practical applicationsrdquo International Dairy Jour-nal vol 16 no 11 pp 1272ndash1276 2006
[64] A C M van Hooijdonk K D Kussendrager and J M SteijnsldquoIn vivo antimicrobial and antiviral activity of components inbovine milk and colostrum involved in non-specific defencerdquoBritish Journal of Nutrition vol 84 supplement 1 pp S127ndashS1342000
[65] D M Hogg and G R Jago ldquoThe antibacterial action of lac-toperoxidaseThe nature of the bacterial inhibitorrdquo BiochemicalJournal vol 117 no 4 pp 779ndash790 1970
[66] M T Ashby ldquoHypothiocyaniterdquo in Advances in InorganicChemistry R van Eldik and I-B Ivana Eds chapter 8 pp 263ndash303 Academic Press New York NY USA 2012
[67] E L Thomas ldquoLactoperoxidase-catalyzed oxidation of thio-cyanate equilibria between oxidized forms of thiocyanaterdquoBiochemistry vol 20 no 11 pp 3273ndash3280 1981
[68] T M Aune and E LThomas ldquoOxidation of protein sulfhydrylsby products of peroxidase-catalyzed oxidation of thiocyanateionrdquo Biochemistry vol 17 no 6 pp 1005ndash1010 1978
[69] T M Aune and E L Thomas ldquoAccumulation of hypothiocyan-ite ion during peroxidase-catalyzed oxidation of thiocyanateionrdquo European Journal of Biochemistry vol 80 no 1 pp 209ndash214 1977
[70] J D Oram and B Reiter ldquoThe inhibition of streptococci bylactoperoxidase thiocyanate and hydrogen peroxideThe effectof the inhibitory system on susceptible and resistant strains ofgroup N streptococcirdquo Biochemical Journal vol 100 no 2 pp373ndash381 1966
[71] J Kalmar K L Woldegiorgis B Biri and M T AshbyldquoMechanism of decomposition of the human defense factorhypothiocyanite near physiological pHrdquo Journal of the Ameri-can Chemical Society vol 133 no 49 pp 19911ndash19921 2011
[72] H Hoogendoorn J P PiessensW Scholtes and L A StoddardldquoHypothiocyanite ion the inhibitor formed by the system lac-toperoxidase thiocyanate hydrogen peroxide I Identification ofthe inhibiting compoundrdquoCaries Research vol 11 no 2 pp 77ndash84 1977
[73] L Bjorck and O Claesson ldquoCorrelation between concentrationof hypothiocyanate and antibacterial effect of the lactoperoxi-dase system against Escherichia colirdquo Journal of Dairy Sciencevol 63 no 6 pp 919ndash922 1980
[74] P Nagy S S Alguindigue and M T Ashby ldquoLactoperoxidase-catalyzed oxidation of thiocyanate by hydrogen peroxide areinvestigation of hypothiocyanite by nuclear magnetic reso-nance and optical spectroscopyrdquo Biochemistry vol 45 no 41pp 12610ndash12616 2006
[75] Y Adolphe M Jacquot M Linder A-M Revol-Junelles andJ-B Milliere ldquoOptimization of the components concentrationsof the lactoperoxidase system by RSMrdquo Journal of AppliedMicrobiology vol 100 no 5 pp 1034ndash1042 2006
[76] M Adamson and K M Pruitt ldquoLactoperoxidase-catalyzedinactivation of hexokinaserdquo Biochimica et Biophysica Acta vol658 no 2 pp 238ndash247 1981
[77] M N Mickelson ldquoGlucose transport in Streptococcus agalac-tiae and its inhibition by lactoperoxidase-thiocyanate-hydrogenperoxiderdquo Journal of Bacteriology vol 132 no 2 pp 541ndash5481977
[78] E L Thomas K A Pera K W Smith and A K ChwangldquoInhibition of Streptococcus mutans by the lactoperoxidaseantimicrobial systemrdquo Infection and Immunity vol 39 no 2 pp767ndash778 1983
[79] J Sermon K Vanoirbeek P De Spiegeleer R Van Houdt AAertsen and C W Michiels ldquoUnique stress response to thelactoperoxidase-thiocyanate enzyme system in Escherichia colirdquoResearch in Microbiology vol 156 no 2 pp 225ndash232 2005
[80] E L Thomas and T M Aune ldquoSusceptibility of Escherichia colito bactericidal action of lactoperoxidase peroxide and iodideor thiocyanaterdquoAntimicrobial Agents andChemotherapy vol 13no 2 pp 261ndash265 1978
[81] K M Pruitt J Tenovuo R W Andrews and T McKaneldquoLactoperoxidase-catalyzed oxidation of thiocyanate polaro-graphic study of the oxidation productsrdquo Biochemistry vol 21no 3 pp 562ndash567 1982
[82] M Huwiler and H Kohler ldquoPseudo-catalytic degradation ofhydrogen peroxide in the lactoperoxidaseH
2O2iodide sys-
temrdquo European Journal of Biochemistry vol 141 no 1 pp 69ndash741984
[83] M Huwiler U Burgi and H Kohler ldquoMechanism of enzymaticand non-enzymatic tyrosine iodination Inhibition by excesshydrogen peroxide andor iodiderdquo European Journal of Bio-chemistry vol 147 no 3 pp 469ndash476 1985
[84] M Morrison G S Bayse and A W Michaels ldquoDeterminationof spectral properties of aqueous I2 and I3- and the equilibriumconstantrdquo Analytical Biochemistry vol 42 no 1 pp 195ndash2011971
[85] E L Thomas and T M Aune ldquoPeroxidase catalyzed oxidationof protein sulfhydrylsmediated by iodinerdquoBiochemistry vol 16no 16 pp 3581ndash3586 1977
[86] W Gottardi ldquoIodine and disinfection theoretical study onmode of action efficiency stability and analytical aspects in theaqueous systemrdquo Archiv der Pharmazie vol 332 no 5 pp 151ndash157 1999
[87] W A Prutz R Kissner W H Koppenol and H RueggerldquoOn the irreversible destruction of reduced nicotinamidenucleotides by hypohalous acidsrdquo Archives of Biochemistry andBiophysics vol 380 no 1 pp 181ndash191 2000
[88] Y Bichsel and U Von Gunten ldquoHypoiodous acid kinetics ofthe buffer-catalyzed disproportionationrdquo Water Research vol34 no 12 pp 3197ndash3203 2000
[89] Y Bichsel and U von Gunten ldquoOxidation of iodide andhypoiodous acid in the disinfection of natural watersrdquo Environ-mental Science and Technology vol 33 no 22 pp 4040ndash40451999
[90] W A Prutz R Kissner T Nauser and W H Koppenol ldquoOnthe oxidation of cytochrome c by hypohalous acidsrdquo Archives ofBiochemistry and Biophysics vol 389 no 1 pp 110ndash122 2001
[91] A Virion J L Michot D Deme and J Pommier ldquoNADPHoxidation catalyzed by the peroxidaseH
2O2system Iodide-
mediated oxidation of NADPH to iodinated NADPrdquo EuropeanJournal of Biochemistry vol 148 no 2 pp 239ndash243 1985
Enzyme Research 13
[92] E L Thomas and T M Aune ldquoCofactor role of iodide in per-oxidase antimicrobial action against Escherichia colirdquo Antimic-robial Agents and Chemotherapy vol 13 no 6 pp 1000ndash10051978
[93] E L Thomas and T M Aune ldquoOxidation of Escherichiacoli sulfhydryl components by the peroxidase-hydrogenperoxide-iodide antimicrobial systemrdquo Antimicrobial Agentsand Chemotherapy vol 13 no 6 pp 1006ndash1010 1978
[94] C B Hamon and S J Klebanoff ldquoA peroxidase-mediatedstreptococcus mitis-dependent antimicrobial system in salivardquoJournal of Experimental Medicine vol 137 no 2 pp 438ndash4501973
[95] R Ihalin J Nuutila V Loimaranta M Lenander J Tenovuoand E-M Lilius ldquoSusceptibility of Fusobacterium nucleatum tokilling by peroxidase-iodide-hydrogen peroxide combinationin buffer solution and in human whole salivardquo Anaerobe vol9 no 1 pp 23ndash30 2003
[96] R Ihalin V Loimaranta M Lenander-Lumikari and J Ten-ovuo ldquoThe effects of different (pseudo)halide substrates onperoxidase-mediated killing of Actinobacillus actinomycetem-comitansrdquo Journal of Periodontal Research vol 33 no 7 pp 421ndash427 1998
[97] E H Bosch H van doorne and S de Vries ldquoThe lactoper-oxidase system the influence of iodide and the chemical andantimicrobial stability over the period of about 18 monthsrdquoJournal of AppliedMicrobiology vol 89 no 2 pp 215ndash224 2000
It could be associated to allergic eosinophilic inflammatorydisease pathologies [1 10 11] LPO and salivary peroxidaseare found in secretions of exocrine glands and are associatedto antibacterial and antifungal activity [12ndash14] Finally TPOis a membrane enzyme localized in thyroid follicle cells thattakes part in the synthesis of thyroid hormones [1]
This review summarizes present knowledge on the modeof action of lactoperoxidase which can be extended to themammalian peroxidases mode of action and on the specificinteraction of LPO with thiocyanate and iodide together oralone with implications on its antimicrobial activity
2 Mode of Action of Lactoperoxidase
LPO is a calcium- and iron-containing glycoprotein arrangedin a single polypeptide chain of about 80 kDa [15 18 19]Human LPO is moderately cationic with a pI of ca 75 butbovine LPO is more cationic with a pI of ca 96 [18 19]MPO is a highly cationic (pI of ca 10) dimeric protein of146 kDa eachmonomer containing one calcium and iron andjoined together by a disulfide bridge [18] The ion calciumplays an important role in the stability of both enzymes[19 20] The active site in LPO MPO EPO and TPO is theheme which consists in a protoporphyrin IX derivative fixedcovalently through two ester linkages via a conserved distalaspartate and glutamate residues a third link being presentin MPO and consisting in a thioether sulfonium bond witha methionine [4 15 19 21] These covalent bonds which arespecific for vertebrate peroxidases result in a distortion of thesymmetry and planarity of the prosthetic group and give theunique spectroscopic and redox properties of these proteins[4 20 21] The proximal heme ligand is a highly conservedhistidine residue which is hydrogen bonded to a conservedasparagine residue that acts as hydrogen-bond acceptor[20 22] On the distal heme site a conserved histidine-arginine couple plays a role in the proton transfer duringthe formation of Compound I and a conserved glutamineresidue and several conserved water molecules are involvedin a hydrogen-bond network acting in halide delivery andbinding [4 18 20] A conserved asparagine residue in LPOEPO and MPO located close to the distal histidine seemsalso critical for the catalysismechanism [18 20]The substratechannel in LPO is narrower longer and more hydrophobiccompared toMPOwith the consequence that the LPO-activesite seems to be less exposed to surrounding media [22 23]
Heme peroxidases are oxidoreductase enzymes that actthrough different reactionmechanisms Although some char-acteristics are specific to a member of the family the sameglobal procedure is followed by all members The cyclebegins with the transformation of the native enzyme intoCompound I Afterwards and dependingmainly on substrateconcentrations Compound I enters the halogenation cycle orthe peroxidase cycle which both end by the enzyme returningto its native state (see Figure 1)
21 Formation of Compound I The first reaction of thenative enzyme starts in the presence of hydrogen peroxidewhich acts as a relatively specific electron acceptor [24]
Native enzyme
Compound IOxidation state +2
Compound IIOxidation state +1
(give +
AHAH
Halogenation cycle
Peroxidase cycle
Oxidation state of compounds I or II with regard to the native enzyme a positive value reflects an oxidation
Figure 1 Halogenation or peroxidase cycle of peroxidases Com-pound I
Other substrates have been described such as ethyl hydroper-oxide peroxyacetic acid cumene hydroperoxide and 3-chloroperoxybenzoic acid [20 25] Compound I is formed asfollows
Peroxidase (native form) +H2O2997888rarr Compound I +H
2O(1)
The native enzyme undergoes a two-electron oxidationTwo electrons are transferred from the enzyme to hydrogenperoxide which is reduced into water Compound I is twooxidizing equivalents above the native enzyme one is in theoxyferryl heme center and the other is present as an organiccation located on the porphyrin ring [4 26 27] CompoundI is not specific regarding the electron donor [24] and thecomposition of the medium determines its subsequent cycle(see Figure 1) As Compound I is very unstable an aminoacid residue of the apoprotein is oxidized in the absenceof an exogenous electron donor [14] This reaction yieldsCompound I isomer that is very similar to Compound IIregarding its iron redox state and its incapability to react withhalogens [20]
22 The Halogenation Cycle In the presence of a halogen(Clminus Brminus or Iminus) or a pseudohalogen (SCNminus) Compound Iis reduced back to its native enzymatic form through a two-electron transfer while the (pseudo)halogen is oxidized intoa hypo(pseudo)halide (see Figure 1) Hypo(pseudo)halides(OXminus) are powerful oxidants with antimicrobial activity [1428ndash32] The halogenation cycle is described by the followingreaction
Compound I + Xminus (halogen or pseudo-halogen)
997888rarr Native enzyme +OXminus(2)
The oxidation rate of halogens by peroxidase-derivedCompound I depends on various factors One of these factors
MPO Compound I apparent second-order rate constant at
LPO Compound I apparent second-order rate constant at
Clminus chloride Brminus bromide Iminus iodide and SCNminus thiocyanate
pH 7 from [16]
pH 7 from [15]
Figure 2 Apparent second-order rate constant at pH 7 (times104Mminus1 sminus1) of the reaction between myeloperoxidase Compound I or lactoper-oxidase Compound I with (pseudo)halides [15 16]
is the standard reduction potential of the enzyme whichdiffers among peroxidases and plays a role in their capacityto oxidize specific (pseudo)halides The redox reaction canoccur only if the reduction potential of the enzyme is equalor superior to the reduction potential of the substrate Thestandard reduction potential at pH 7 of Compound I perox-idases and the couple of two-electron reduction HOXXminus isranking in the following ascending rank LPOCompound I ltEPO Compound I lt MPO Compound I HOSCNSCNminus ltHOIIminus ≪ HOBrBrminus lt HOClminusClminus [17 18 33 34] Thisinvolves that only the MPO Compound I is able to oxidizeClminus with appropriate rates LPO being able to oxidize withhigh rates Iminus and SCNminus and slowly Brminus [16 17 20 33 34]Interestingly although LPO Compound I has the lowestreduction potential compared to EPOCompound I andMPOCompound I it catalyzes the oxidation of Iminus and SCNminus withthe highest rates (Figure 2) [15ndash17 20] This suggests thatother factors play a role such as anion size anion access andanion binding as well as structural and amino acid differencesin the active and binding site between enzymes [17 18 20]
The reduction potential of the Compound Inativeenzyme and HOXXminus redox couples depends on reactantconcentrations and pH values At a specific reactant concen-tration it decreases with increasing pH values but slopesdiffer (Figure 3) [17] This means that there exists a thresholdpH value above which the oxidation of halides becomesthermodynamically unfavorable especially for halides withhigh reduction potential such as Clminus and Brminus [17]
Concentrations of (pseudo)halogens also affect theiraffinity to Compound I (Figure 4)
Although plasma Clminus concentrations are 1000-foldhigher than Brminus and SCNminus MPO Compound I oxidizessimilar amounts of SCNminus and Clminus [35] EPO Compound I
Compound Inative enzyme
Threshold pH value
Redu
ctio
n po
tent
ial (
V)
pH
HOXOXminus H2O
Figure 3 Illustration (according to [17]) of the pH thresholdvalue above which the oxidation of the halogen by mammalianheme peroxidase will be thermodically unfavorable The reductionpotential of the couple Compound Inative enzyme and the couplehalogen (X = chloride bromide) HOXOXminus is expressed with anillustrative function of the pH at a specific concentration of enzymeand substrates
preferentially oxidizes SCNminus in the presence of physiologicalconcentrations of SCNminus Brminus and Iminus [36] In the salivaof healthy adults where Clminus concentrations are only about25-fold higher than SCNminus SPO Compound I and MPOCompound I primarily generate hypothiocyanite [37 38]The levels of Iminus in human milk saliva blood and tissuesexcept the thyroid gland are below 1120583M and its in vivooxidation by Compound I is negligible [17 38] In humanmilk peroxidase activity is only derived from leucocytes AsMPO is able to oxidize Clminus and Clminus milk concentration is
Iodide 2120583MThiocyanate 120120583MChloride 13 mM
Figure 4 Illustration of the interaction between the biodisponibility of a peroxidase the (pseudo)halogen concentration in plasma in salivaand in milk and the production of oxidant molecules MPO myeloperoxidase SPO salivary peroxidase LPO bovine lactoperoxidaseOClminus hypochlorite and OSCNminus hypothiocyanite Although chloride is the most available substrate compared to thiocyanate bromide andiodide thiocyanate is the most effective substrate for the Compound I and hypothiocyanite could be produced at equal or superior levelscompared to hypohalides
high oxidation of Clminus is possible although it has never beenreported [14] In bovine milk lactoperoxidase is an abundantenzyme and with mean concentrations of Iminus and SCNminus of310 120583gkg and 02ndash15mgkg respectively oxidation is possi-ble [19 39] Nevertheless the relative abundance of SCNminus inall secretions blood and tissues and its better capacity as anelectron donor make it one of the main in vivo substratesof Compound I lactoperoxidase and myeloperoxidase for2-electron oxidation compared to halides [15] In in vitroapplications the ratio between (pseudo)halides regulates theratio of hypohalides generated by the reaction However asSCNminus is the most effective substrate for Compound I itspresence even in small quantities enhances its oxidation[14 35 36]
23 The Peroxidase Cycle Alternatively Compound I canshift to the peroxidase cycle which consists of two sequen-tial one-electron transfers back to the enzyme that yield(i) Compound II and (ii) the native enzyme while thesubstrate is oxidized into a radical (Figure 1) [40ndash42] Theperoxidase cycle is summarized in the following equations
Compound I + AH 997888rarr Compound II + A∙
Compound II + AH 997888rarr Native enzyme + A∙(3)
Compound I is not specific regarding the one-electrondonor it can be exogenous or endogenous and a lot of candi-dates have been described [18 20 43]Hydrogen peroxide canundergo a one-electron oxidation onlywithMPOCompoundI with the formation of superoxide [16 20 44 45]
During the first step of the peroxidase cycle the cationlocated in the porphyrin ring undergoes a one-electronreduction with formation of Compound II and concomitantoxidizing of one one-electron substrate [4 24] Compound II
maintains one oxidizing equivalent in the oxyferryl center[4 24] Finally this latter is reduced back to the native enzymewith the oxidation of a second one-electron donor
The standard reduction potential of the couple Com-pound ICompound II is high and allowed the one-electronoxidation by Compound I of a wide range of substrates[18 20] In contrast the standard reduction potential of thecouple Compound IInative enzyme is low and restrains thenumbers of possible substrates for Compound II [18 20]With the result that (i) the Compound IInative enzymestandard reduction potential is too low to react with halogensand (ii) the nature of substrates strongly influenced theirability to be oxidized by mammalian peroxidase compoundII [2 14 20] therefore when the enzyme is in this state it hasto be first reduced to the ground state before possibly partici-pating to the halogenation cycle and producing antimicrobialmolecules [14] Moreover the reduction of Compound II tothe ground state is the rate-limiting step [45 46] that is theperoxidase cycle interferes with the halogenation cycle andslows down antimicrobial activity [47]
The peroxidase cycle has been described as a possiblecatalytic sink for nitric oxide (NO) [46] but also for hydrogenperoxide in the case of a moderate excess of H
2O2relative
to LPO [24] Increase of NO removal from media evenin presence of Clminus after addition of MPO EPO or LPOand accelerated rates of Compound I and Compound IIreduction in presence of NO show that peroxidases mayregulate the bioavailability of NO [46] In conditions ofhigh excess of hydrogen peroxide relative to LPO and inthe absence of an exogenous electron donor CompoundII is transformed into Compound III which is 3 oxidativeequivalents above the native enzyme In moderate excessconditions Compound III can be partially reconverted intoCompound II and can reenter the peroxidase cycle [24 40]
Enzyme Research 5
Otherwise the enzyme is irreversibly inactivated the hemefraction is cleaved and iron is released [48] In the presenceof an exogenous two-electron donor the enzyme is largelyprotected from hydrogen peroxide because the halogenationcycle is favored Furthermore protection is higher withiodide because oxidized iodide consumes H
2O2to produce
oxygen and iodide in a reaction called the pseudocatalyticactivity of peroxidase [24 40 49]
However thiocyanate can act as a one-electron donor andbe part of the peroxidase cycle with the sequential formationof two thiocyanate radicals [47] With 200120583M SCNminus LPOis predominantly in its native form this indicates that thehalogenation cycle prevails [47]
In the presence of both one- and two-electron donorscompetition for oxidation can occur and favor the halogena-tion or the peroxidase cycle The presence of EDTA inhibitsthe oxidation of iodide due to competition for binding toCompound I [50] The standard reduction potential betweenthe donors favors the molecule with the lowest reductionpotential Thereby the respective reduction potentials of theone- and two-electron oxidation of thiocyanate at very lowpH are 165V and 082V and promote the halogenationcycle [51] In the case of low concentrations of halides orthiocyanate below 10 120583M Iminus or 3 120583M SCNminus CompoundI reacts with any suitable exogenous or endogenous one-electron donor with the subsequent formation of CompoundII and a negligible oxidation rate of halides and thiocyanate[14]
24 Inhibition of the Function of Mammalian Heme Peroxi-dase The function of heme peroxidases can be inhibited inseveral ways that could be classified into three categoriesThe first one could represent an inhibition of the enzymeby (i) molecules or proteins and (ii) external conditionssuch as pH and temperature For example cyanide azidenitrite mercaptomethylimidazole thiourea superoxide highlevels of nitric oxide and high levels of thiocyanate bindto the native enzyme and alter Compound I formation [2046 47 52ndash54] With thiocyanate inhibition is linked to therestriction of the binding site to hydrogen peroxide andthe interaction of SCNminus with a water molecule [23] Highconcentration of H
2O2or Iminus will inactivate irreversibly LPO
with liberation of free iron [48 55] Temperature between73∘C and 83∘C depending on the heating time results inunfolding and inactivation of LPO [19] Extreme pH isinactivating enzymes and at low pH an amino acid groupprobably histidine is protonated which prevents the bindingof H2O2[56] Some proteases such as pepsin and pronase are
able to inactivate LPO by proteolysis but chymotrypsin did itvery slowly and trypsin and thermolysin are not active againstLPO [19]
The second group of inhibitors could concern substancesor proteins which are able to interfere with the catalyticmechanism For example catalase consumes H
2O2and will
stop the formation of Compound I [30 52] Competitionbetween substrates can also interfere with the reaction cyclesuch as SCNminus which competes very effectively with Clminus Brminusand Iminus [52 53] HOCl has the capacity to bind to LPO native
enzyme and convert it into Compound I Above 100 120583MHOCl mediates the destruction of the LPO heme center[57]
The third class could be related to substances or pro-teins which are buffering active molecules produced duringthe catalytic reaction For example presence of thiosulfatethioglycolate glutathione dithiothreitol cysteineNAD(P)Hand tyrosine will reduce the antimicrobial activity throughreacting with OClminus OBrminus OIminus or OSCNminus [52 53 58 59]The enzyme NADH-OSCN oxidoreductase is able to reduceOSCNminus in SCNminus [60]
3 Activity of Lactoperoxidase withThiocyanate andor Iodide
LPO concentrations in cowrsquos milk are around 30mg Lminus1depending on season diet and calving and breeding season[61] LPO extraction from whey or milk is based on awell-developed industrial process [62] Compared to MPOand EPO LPO is easily isolated and manufactured in largequantities As a result cowrsquos milk peroxidase is the favoritemolecule for in vitro or in vivo applications such as con-servation of raw and pasteurized milk storage of emulsionsand cosmetics moisturizing gel and toothpaste in human drymouth veterinary products and preservation of foodstuffs[19 61 63 64]
31 Activity of LPO Related to Hypothiocyanite
311 Mode of Action of Hypothiocyanite Thiocyanate is oxi-dized in a two-electron reaction that yields hypothiocyaniteHypothiocyanite has a pKa of 53 [65] It is more acidic thanhypohalides that have pKas of 75 (HOCl) 86 (HOBr) and106 (HOI) [14 66] All hypo(pseudo)halides (OXminus) are in anacid-base equilibrium association with their correspondingacid hypo(pseudo)halide (HOX) For example in the case ofhypothiocyanite
HOSCN 999447999472 OSCNminus +H+ (4)
The acid form has a higher oxidation potential and ismore soluble in nonpolar media so that it passes throughhydrophobic barriers such as cell membranes more easilybut it is less stable than the basic form (OXminus) [14 66]Hypohalide acids are predominant in acidic to neutral mediaand even in basic conditions for HOBr and HOI whereashypothiocyanite needs a pH below 53 to be predominant inthe acid form [66 67]
SCNminus is the two-electron donorwith the lowest reductionpotential and therefore forms the hypothiocyanite acid withthe lowest oxidative power compared to hypohalous acidsHypohalous acids rank as follows with increasing oxidativestrength OSCNminus lt OIminus lt OBrminus lt OClminus [28 66]These characteristicsmake hypothiocyanite relatively specificregarding its molecular target (Figure 5) that is a thiolmoiety [28 59 68]
6 Enzyme Research
- SH group- NAD(P)H
- SH group- NAD(P)H- Reduced pyridine nucleotide- R-S-R (thioether group)
HOSCNOSCNminus
HOIOIminusI2
- NH2 group
Figure 5 Target group of hypothiocyanite hypoiodite and iodineDue to its low oxidation power hypothiocyanite is relatively specificand is not reactive against all thiols In vivo hypoiodite seems tobe selectively directed against reduced pyridine nucleotide becauseeven the presence of excess glutathione and methionine doesnot thoroughly inhibit their oxidation HOSCNOSCNminus acidic orbasic form of hypothiocyanite HOIOIminus acidic or basic form ofhypoiodite and I
2 iodine
R-S-SCN or R-S-I
LPO R-SH
+ R-S-OHOSCNminusSCNminus SCNminus
or Iminus or Iminusor minusOI
H2O
H2O
2
Figure 6 Illustration of the cofactor role of SCNminus or Iminus Whenthe necessary conditions are fulfilled that is (i) no substratecompetitor for SCNminus or Iminus for binding to lactoperoxidase (ii)enough peroxidase H
2O2and SCNminus or Iminus (iii) enough R-SH
and (iv) no incorporation of SCNminus or Iminus in stable byproducts thequantity of OSCNminus or OIminus produced depends only on the amountof H2O2 SCNminus thiocyanate Iminus iodide H
2O2 hydrogen peroxide
LPO lactoperoxidase R-SH peptide or protein with a thiol moietyR-S-SCN or R-S-I sulfenyl thiocyanate or iodide R-SOH sulfenicacid OSCNminus hypothiocyanite and OIminus hypoiodite
Sulfhydryl oxidation by OSCNminus generates sulfenyl thio-cyanate in equilibrium with sulfenic acid [68]
SCNminus +H2O2+ LPO 997888rarr OSCNminus + LPO
R-SH +OSCNminus 997888rarr R-S-SCN +OHminus
R-S-SCN +H2O 997888rarr R-S-OH + SCNminus +H+
(5)
The cycle of reactions shows that thiocyanate acts likea cofactor for LPO (Figure 6) so that the total number ofoxidized sulfhydryls is independent of SCNminus as long as(i) thiocyanate is not exhausted (ii) thiocyanate is not incompetition with other substrates for the binding to Com-pound I (iii) thiocyanate is not incorporated into an aromaticamino acid (iv) enoughH
2O2is present and (v) thiol moiety
is still available [68 69]Although the target of OSCNminus is a thiol moiety not
all sulfhydryls are equally sensitive to OSCNminus albumincysteine mercaptoethanol dithiothreitol glutathione and 5-thio-2-nitrobenzoic acid are all oxidized but 120573-lactoglobulinis poorly oxidized probably due to a limited accessibility ofsulfhydryls to OSCNminus [68] In some conditions that is thejoint presence of LPO enough H
2O2and SCNminus and after the
oxidation of available sulfhydryls modification of tyrosinetryptophan and histidine protein residues can occur and that
could be linked to the formation of a labile powerful oxidantsuch as sulfur dicyanide [68]
Some authors suggest that (SCN)2is formed during the
enzymatic reaction and then chemically hydrolyzed intohypothiocyanite [14 69 70] However a recent publicationdemonstrates that (SCN)
2cannot be a precursor during the
enzymatic oxidation of SCNminus at neutral pH inmammals [71]Hypothiocyanite is less stable in acid conditions with
high concentrations of SCNminus and in the presence of (SCN)2
and it is thought to break down via the following net reaction[14]
4HOSCN +H2O 997888rarr 3SCNminus + CNOminus + SO
4
2minus+ 6H+ (6)
A recent study based notably on spectroscopic and chro-matographic methods proposes the following net equationwithin the 4ndash7 pH range
3HOSCN +H2O 997888rarr XSO
4
2minus+ XHCN
+ (1 minus X) SO3
2minus+ (1 minus X)CNOminus
+ 2SCNminus + (5 minus X)H+
(7)
The proportions of end anions were different at pH 4 andpH 7 at pH 7 the proportion of CNOminus was higher SCNminusformation was slower and no CNminus was detected [71]
It might seem easier to produce hypothiocyanite chemi-cally in in vitro applications but producing hypothiocyanitechemically from the oxidation of SCNminus by a halogen (Cl
2or
Br2) or by a hypohalous acid (HOCl or HOBr) in basic media
is tricky due to overoxidation of SCNminus [66] The referencemethod in the literature to produce 1- to 2-day stable OSCNminusis by hydrolyzing (SCN)
2in basic conditions [72ndash74]
Hypothiocyanite inhibitors have been described Forexample CNminus a weak acid buffer dissolved carbonate excesshydrogen peroxide hydrofluoric acid metallic ions glyc-erol or ammonium sulfate accelerates the decomposition ofOSCNminus whereas sulfonamide stabilizes it [67 72]
Appropriate concentrations of substrates induce en-hanced activity [75]
312 Biological Activity of Hypothiocyanite The biologicalactivity of hypothiocyanite is summarized in Figure 7
The sulfhydryl moiety is essential for the activityof numerous enzymes and proteins Inhibition of bacte-rial glycolysis through the oxidation of hexokinase gly-ceraldehyde-3-phosphate dehydrogenase (GAPDH) aldo-lase and glucose-6-phosphate dehydrogenase has beenobserved [14 51 65 70 76] Inhibition of respiration andglucose transport is associated with the alteration of cellmembranes or transporters [14 51 65 77] Irreversible inhi-bition is linked to long periods of incubation and bacterialsensitivity depends on the bacterial species and on hypothio-cyanite concentrations [14 51 59] Increased concentrationsof reducing agents such as glutathione and cysteine canreverse the inhibition through buffering hypothiocyaniteand converting the reduced thiol back into sulfhydryl [1478] This defense mechanism is used by Escherichia coli itinduces the CysJ promoter during the stress response to the
Enzyme Research 7
Bacteria
Inhibition of glycolysis Inhibition of respirationInhibition of glucose transport
Possible defense mechanism of bacteria- Increased GSH and cysteine content
Peptide proteinwith SH moiety
HOSCNOSCNminus
- NAD(P)H-dependent reduction of OSCNminus
Figure 7 Biological activity of hypothiocyanite on bacteria and possible defensemechanism of the bacteria Reversible inhibition is observedin that (i) hypothiocyanite is not reactive against all thiols and (ii) if hypothiocyanite is removed or diluted the pathogen recovers Irreversibleinhibition is linked to (i) long period of incubation (ii) the bacterial species and (iii) hypothiocyanite concentration HOSCNOSCNminus acidicor basic form of hypothiocyanite and GSH glutathione
pH lt 6
Influence of iodide concentration
Influence of the pH
1 I2 and high Iminus
I5minusI6
minus
I2I3minus
6 lt pH lt 9
HOII2OHI2I3minus
Iminus + H2O2 + LPO rarr active molecules
I2 (without Iminus)HOIOIminusI2I3
minusHI2Ominus
Figure 8 Illustration of the molecules that can be present after oxidation of iodide by lactoperoxidase in presence of H2O2The active species
depend mainly on the concentration of iodide (upper part) and the pH (lower part) The species with an oxidant power are represented inbold
lactoperoxidase system [79] Another resistance mechanismcould be the NAD(P)H-dependent reduction of OSCNminuswithout any loss of the sulfhydryl compound [14 72 78]Alteration of the bacterial membrane increases the efficacyof hypothiocyanite [80]
Furthermore the activity of the entire system (enzyme +substrates) is known to be more effective than hypothiocyan-ite alone whether enzymatically or chemically producedThis has been explained by the production of short-livedhighly reactive intermediates such as O
2SCNminus and O
3SCNminus
by the enzyme or by the oxidation of OSCNminus in conditionsof excess H
2O2[65 73 81] The activity of hypothiocyanite
has been described against bacteria such as Actinomyces sppBacillus cereus Lactobacillus spp Staphylococcus albus Saureus Streptococcus spp Escherichia coli Legionella pneu-mophila Salmonella typhimurium Pseudomonas fluorescensP aeruginosa Campylobacter jejuni C coli and Listeriamonocytogenes [14 32] Reversible inhibition is observed
when cells recover after OSCNminus is depleted [14 59] Irre-versible inhibition is obtained with long-term incubation andhigh level of OSCNminus [59] Higher concentration of SCNminuscompared to Iminus is necessary to obtain inhibition against E coliand accumulation of OSCNminus is observed as it is not reactiveagainst all thiols [59]Therefore the activity of the SCNminus-LPOsystem appears to be more bacteriostatic than bactericidal
32 Activity of LPO Related to Oxidized Iodide
321 Chemistry of Oxidized Iodide Iodide is oxidized byCompound I through a single two-electron transfer thatyields oxidized Iminus in the form of I
2or HOI [14 24 82ndash85]
The active agent is composed of a mixture of species that arenot yet formally detailed due to the very complex behaviorand stability of I
2and HOI in aqueous environments that
strongly depend on pH values and iodide concentrations[66 82 83 86]
8 Enzyme Research
Based on the inorganic chemistry of iodine in waterand literature on enzymatic oxidation of iodide the activemolecules have been described as follows (Figure 8)
(i) Under pH 6 and in the presence of iodide only I2
Iminus and I3
minus are present and the only active moleculeis I2 I2concentrations decrease with increasing
concentrations of Iminus At an initial 1mM I2 with Iminus
concentrations ranging from 1mM to 100mM I2
concentrations fall from almost 1mM to 001mM asdescribed by the following association reaction [2482 83 86]
I2+ Iminus 999447999472 I
3
minus (8)
(ii) In solution within a 6ndash9 pH range and with a max-imum 1mM iodide a mixture of HOII
2OHI2I3
minus
is formed in which I3
minus is not active and I2OH is
probably less reactive than HOI or I2[86 87] If
Iminus concentrations are above 10mM I3
minus representsthe main species formed and the concentration ofactive molecules relatively drops The mechanism issummarized in the following net equations
HOI + Iminus +H+ 999447999472 I2OHminus +H+
999447999472 I2+H2O 999447999472 I
2+ Iminus 999447999472 I
3
minus
(9)
(iii) In iodine solution without iodide or when availableiodide has been oxidized the number of I
2-derived
molecules decreases with decreasing I2concentra-
tions At 1000 120583M I2 with pH-related ratios five
relevant species are observed (I2 HOI I
3
minus HI2Ominus
and OIminus) At 10 120583M I2 the main species are only I
2
HOI and OIminus and HOI could represent up to 90 ofthe active oxidant molecules at pH 8-9 [86] Below apH of 106 the following reactions are involved
I2+H2O 999447999472 HOI + Iminus +H+ (hydrolysis of I
2)
I2+ Iminus 999447999472 I
3
minus(triiodide formation independent of pH)
(10)
(iv) At high Iminus and 1 I2concentrations as in Lugol
solution I5
minus and I6
minus are formed and represent 82of the active oxidative agents [86] after the followingreaction
I3
minus+ I2999447999472 I5
minus(pentaiodide formation)
2I3
minus999447999472 I6
2minus(dimerization of I
3
minus)
(11)
The stability of HOI and I2is linked to their dispro-
portionation in iodate which has no oxidative activity inneutral and basic pH conditions [86]The disproportionationreactions read as follows
3HOI 999447999472 IO3
minus+ 2Iminus + 3H+ (disproportionation of HOI)
3I2999447999472 IO
3
minus+ 5Iminus + 6H+ (disproportionation of I
2)
(12)
I2stability increases at higher pHvalues andhigher iodide
concentrations [86] In drinking water HOI disproportion-ation is slow and varies substantially HOI has a half-life of4 days to 35 years depending on (i) the initial level of HOIthat speeds its decomposition and (ii) the presence of boratephosphate or carbonate that catalyzes its decomposition [8889]
322 Mode of Action of Oxidized Iodide The oxidativestrength of I
2is between that of the corresponding hypo-
halous acid HOI and the hypoiodite ion OIminus and ranks asfollows 0485V (OIminus) lt 0536V (I
2) lt 0987V (HOI) [66]
HOI reacts through very rapid oxidation of thiolgroups oxidation of NAD(P)H oxidation of 120573-nicotinamidemononucleotide direct reaction with thioether groupsthrough sulfoxidation and slow oxidation of the aminemoiety (Figure 5) [87 90 91] At low Iminus concentrationsiodination of tyrosine residues is catalyzed by the enzyme[14] In a cellular environment HOI seems to be more selec-tively directed against the degradation of reduced pyridinenucleotides thanHOCL andHOBr because even the presenceof excess glutathione methionine or oxidized glutathionedoes not thoroughly inhibit their oxidation [87]
In some conditions that is (i) enough iodide H2O2 and
peroxidase (ii) no accumulation of oxidized iodide and (iii)no incorporation of iodide into stable byproducts such astyrosine residues iodide acts as a cofactor (Figure 6) andthe proportion of oxidized sulfhydryls is proportional to theamount of H
2O2as described below [85 92]
2Iminus +H2O2+ LPO (native enzyme)
997888rarr I2+ 2H2O + LPO (native enzyme)
R-SH + I2997888rarr R-S-I + Iminus +H+
R-S-I+H2O 997888rarr R-S-OH + Iminus +H+
(13)
In the case of high concentrations of Iminus andor H2O2
inhibition of tyrosine iodation has been observed [83] andrelated to the pseudocatalytic redox degradation of H
2O2
with formation of O2when excessive H
2O2is present (reac-
tion 1) and production of I3
minus when excessive amounts of Iminusare present (reaction 2)
I2+H2O 997888rarr O
2+ 2Iminus + 2H+ (reaction 1)
I2+ Iminus 999447999472 I
3
minus(reaction 2)
(14)
Both reactions deplete the amount of the active oxidizingagent I
2 In the absence of tyrosine oxidized iodide reacts
with nucleophilic molecules such as Iminus Clminus or OHminus to formI2 I3
minus ICl ICl2 IOH and I
2OH [82] Some anions such as
Clminus HPO4
minus or OHminus reduce the amount of I2I3
minus but thiseffect is inversely proportional to the concentration of Iminusabove pH 9 I
2is hydrolyzed and IO
3
minus is formed [82]HOI can be produced chemically through oxidation of Iminus
by Cl2or O3 with a short half-life due to overoxidation of
HOI byCl2andO
3[89] and through oxidation of Iminus byHOCl
HOBr or NH2Cl with a longer half-life [87 89]
Enzyme Research 9
Bacteria
Inhibition of glycolysis Inhibition of respirationInhibition of glucose transportInhibition of the pentose phosphate pathway
- Peptide protein with- SH moiety- thioether moiety- NAD(P)H
HOIOIminus
I2
Figure 9 Biological activity of hypoiodite or iodine on bacteria Irreversible inhibition is observed and could be linked to (i) oxidation ofthiol groups NAD(P)H and thioether groups (ii) high reactivity of HOII
2against thiol and reduced nicotinamide nucleotides and (iii) the
incorporation of iodide in tyrosine residue of protein (iodination of protein) HOIOIminus acid or basic form of hypoiodite and I2 iodine
323 Biological Action of Oxidized Iodide The biologicalaction of oxidized iodide (Figure 9) is similar to that ofhypothiocyanite but differs in that (i) the reactivity of oxi-dized iodide is complete against thiol group and (ii) cells didnot recover after removing of oxidized iodide [59]
Due to the cofactor role of Iminus inhibition of respirationin Escherichia coli in the presence of LPO H
2O2 and Iminus is
complete with only 10120583M NaI whereas 100 120583M of solely I2
is necessary to obtain complete inhibition This is directlyrelated to the oxidation of sulfhydryls not to the percentageof iodine incorporation [92 93]
E coli seems to be more sensitive if the bacteria areincubated together with the entire system (enzyme H
2O2
and iodide) rather than adding several minutes after mixingthe enzyme with its substrates This could be linked to theformation of an unstable reactive intermediate [52]
The activity of the Iminus peroxidase system is more effectiveagainst E coli than the SCNminus system in that lower Iminusconcentrations are necessary all sulfhydryls are oxidized andcells do not recover even if the amount of I
2is not sufficient
to oxidize all SH groups [59 80] Against L acidophilushigh non physiological amounts of Iminus are necessary to obtaininhibitionwhereas small concentrations of SCNminus are effective[70]
CNminus azide EDTA and SCNminus inhibit the formation ofoxidized iodide [50 52] Increased pH values and increasedamounts of thiol and NAD(P)H compounds reduce theactivity of the iodide peroxidase system [52]
LPO-H2O2-Iminus in presence of Streptococcus mitis is active
against Staphylococcus aureus and E coli [94] LPO-H2O2-
Iminus is active against Micrococcus S aureus Listeria monocy-togenes Bacillus cereus E coli and Candida albicans [12 1980] In the presence of other peroxidases the Iminus peroxidasesystem is active against Schistosoma mansoni Fusariumnucleatum andActinobacillus actinomycetemcomitans [31 9596] Compared to SCNminus Iminus-LPO shows bactericidal activities[14 19 80]
33 Activity of LPO Related to Hypoiodite and Hypothiocyan-ite The combination of SCNminus with Iminus in the lactoperoxidasesystem has been poorly studied Tackling the enzymaticmechanism is tricky and contradictory results have been
found about microbial activity in the concomitant presenceof SCNminus and Iminus
In the presence of SCNminus and Iminus there is competitionbetween the two substrates for oxidation by lactoperoxidase[14 36] Iminus alone exhibits bactericidal activity but an SCNminusIminusratio of 01 inhibits that bactericidal effect and an SCNminusIminusratio of 1 antagonizes it due to competition for oxidation andfaster decomposition of HOSCN in the presence of Iminus [14]Against A actinomycetemcomitans the peroxidase systemwith Iminus Clminus or a combination of Iminus and Clminus is effective butaddition of SCNminus cancels the antibacterial effect [96] On theother hand a synergistic or unaffected effect of iodide in theSCNminus-H
2O2-LPO system has been shown against Candida
albicans E coli S aureusAspergillus niger and Pseudomonasaeruginosa [19 97]
4 Conclusion
Themolecular evolution of heme peroxidases and the preser-vation of their catalytic domain [6] show that the productionof strong oxidants is a powerful part of the nonimmunedefense mechanisms against pathogenic bacteria fungi orparasite which made the use of those enzymes in practicalapplications worthwhile
The enzymatic reactions involving mammalian peroxi-dases are complex and various molecules can promote orreduce dramatically the antibacterial activity of the per-oxidase system In order to favor the halogenation cyclerequired in in vitro and in vivo antimicrobial applicationsseveral points have to be taken into account (i) to avoid thepresence of competitors to iodide or thiocyanate for bindingto Compound I and to avoid the presence of inhibitorsof the enzyme or of active molecules (ii) to avoid excessH2O2concentration which is able to destruct the enzyme
and to react with iodine or hypoiodite with loosing of activemolecules (iii) to favor the presence of hypoiodite instead ofiodine due to the association reaction of iodine with iodide(iv) to avoid excess concentration of thiocyanate which caninhibit formation of Compound I (v) to use the entiresystem (enzyme + substrates) instead of active moleculesalone (vi) to favor moderate acid pH when hypothiocyaniteis the active molecule (vii) for bactericidal fungicidal or
10 Enzyme Research
parasitical applications the use of iodide has to be preferred(viii) the use of combined presence of iodide and thiocyanatehas to be checked carefully for efficacy and (ix) to favor thecofactor role of iodide or thiocyanate
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] P J OrsquoBrien ldquoPeroxidasesrdquoChemico-Biological Interactions vol129 no 1-2 pp 113ndash139 2000
[2] W Jantschko P G Furtmuller M Allegra et al ldquoRedoxintermediates of plant and mammalian peroxidases a compar-ative transient-kinetic study of their reactivity toward indolederivativesrdquo Archives of Biochemistry and Biophysics vol 398no 1 pp 12ndash22 2002
[3] S Kimura and M Ikeda-Saito ldquoHuman myeloperoxidase andthyroid peroxidase two enzymes with separate and distinctphysiological functions are evolutionarily related membersof the same gene familyrdquo Proteins Structure Function andGenetics vol 3 no 2 pp 113ndash120 1988
[4] G Battistuzzi M Bellei C A Bortolotti and M Sola ldquoRedoxproperties of heme peroxidasesrdquo Archives of Biochemistry andBiophysics vol 500 no 1 pp 21ndash36 2010
[5] M Zamocky C Jakopitsch P G Furtmuller C Dunand and CObinger ldquoThe peroxidase-cyclooxygenase superfamily recon-structed evolution of critical enzymes of the innate immunesystemrdquo Proteins Structure Function and Genetics vol 72 no2 pp 589ndash605 2008
[6] H Daiyasu and H Toh ldquoMolecular evolution of the myeloper-oxidase familyrdquo Journal of Molecular Evolution vol 51 no 5 pp433ndash445 2000
[7] D Serteyn S Grulke T Franck A Mouithys-Mickalad andG Deby-Dupont ldquoNeutrophile myeloperoxidase protectiveenzyme with strong oxidative activitiesrdquo Annales de MedecineVeterinaire vol 147 no 2 pp 79ndash93 2003
[8] S C Whitman S L Hazen D B Miller R A HegeleJ W Heinecke and M W Huff ldquoModification of type IIIVLDL their remnants and VLDL from apoE- knockout miceby p-hydroxyphenylacetaldehyde a product of myeloperox-idase activity causes marked cholesteryl ester accumulationin macrophagesrdquo Arteriosclerosis Thrombosis and VascularBiology vol 19 no 5 pp 1238ndash1249 1999
[9] T J Barrett and C L Hawkins ldquoHypothiocyanous acid benignor deadlyrdquo Chemical Research in Toxicology vol 25 no 2 pp263ndash273 2012
[10] M M Lloyd D M van Reyk M J Davies and C L HawkinsldquoHypothiocyanous acid is a more potent inducer of apoptosisand protein thiol depletion in murine macrophage cells thanhypochlorous acid or hypobromous acidrdquo Biochemical Journalvol 414 no 2 pp 271ndash280 2008
[11] J Wang and A Slungaard ldquoRole of eosinophil peroxidase inhost defense and disease pathologyrdquo Archives of Biochemistryand Biophysics vol 445 no 2 pp 256ndash260 2006
[12] M Ahariz and P Courtois ldquoCandida albicans susceptibility tolactoperoxidase-generated hypoioditerdquo Clinical Cosmetic andInvestigational Dentistry vol 2 pp 69ndash78 2010
[13] A Welk C Meller R Schubert C Schwahn A Kramerand H Below ldquoEffect of lactoperoxidase on the antimicrobialeffectiveness of the thiocyanate hydrogen peroxide combinationin a quantitative suspension testrdquo BMC Microbiology vol 9article 134 2009
[14] K M Pruitt and J O Tenovuo Eds The Lactoperoxidase Sys-tem Chemistry and Biological Significance vol 27 of Immunol-ogy Series Marcel Dekker New York NY USA 1985
[15] P G Furtmuller W Jantschko G Regelsberger C JakopitschJ Arnhold and C Obinger ldquoReaction of lactoperoxidasecompound I with halides and thiocyanaterdquo Biochemistry vol41 no 39 pp 11895ndash11900 2002
[16] P G Furtmuller U Burner and C Obinger ldquoReaction ofmyeloperoxidase compound I with chloride bromide iodideand thiocyanaterdquo Biochemistry vol 37 no 51 pp 17923ndash179301998
[17] J Arnhold E Monzani P G Furtmuller M Zederbauer LCasella and C Obinger ldquoKinetics and thermodynamics ofhalide and nitrite oxidation by mammalian heme peroxidasesrdquoEuropean Journal of Inorganic Chemistry no 19 pp 3801ndash38112006
[18] M J Davies C L Hawkins D I Pattison and M D ReesldquoMammalian heme peroxidases from molecular mechanismsto health implicationsrdquo Antioxidants and Redox Signaling vol10 no 7 pp 1199ndash1234 2008
[19] J N de Wit and A C M van Hooydonk ldquoStructure functionsand applications of lactoperoxidase in natural antimicrobialsystemsrdquo Nederlands melk en Zuiveltijdschrift vol 50 no 2 pp227ndash244 1996
[20] P G Furtmuller M Zederbauer W Jantschko et al ldquoActivesite structure and catalytic mechanisms of human peroxidasesrdquoArchives of Biochemistry and Biophysics vol 445 no 2 pp 199ndash213 2006
[21] M Zederbauer P G Furtmuller S Brogioni C JakopitschG Smulevich and C Obinger ldquoHeme to protein linkages inmammalian peroxidases impact on spectroscopic redox andcatalytic propertiesrdquo Natural Product Reports vol 24 no 3 pp571ndash584 2007
[22] G Battistuzzi M Bellei J Vlasits et al ldquoRedox thermodynam-ics of lactoperoxidase and eosinophil peroxidaserdquo Archives ofBiochemistry and Biophysics vol 494 no 1 pp 72ndash77 2010
[23] I A Sheikh A Singh N Singh et al ldquoStructural evidence ofsubstrate specificity inmammalian peroxidases structure of thethiocyanate complex with lactoperoxidase and its interactionsat 24 a 24 A resolutionrdquo The Journal of Biological Chemistryvol 284 no 22 pp 14849ndash14856 2009
[24] H Kohler and H Jenzer ldquoInteraction of lactoperoxidase withhydrogen peroxide Formation of enzyme intermediates andgeneration of free radicalsrdquo Free Radical Biology and Medicinevol 6 no 3 pp 323ndash339 1989
[25] P G Furtmuller U Burner W Jantschko G Regelsberger andC Obinger ldquoTwo-electron reduction and one-electron oxida-tion of organic hydroperoxides by human myeloperoxidaserdquoFEBS Letters vol 484 no 2 pp 139ndash143 2000
[26] A Taurog M L Dorris and D R Doerge ldquoMechanism ofsimultaneous iodination and coupling catalyzed by thyroidperoxidaserdquo Archives of Biochemistry and Biophysics vol 330no 1 pp 24ndash32 1996
[27] J E Erman L B Vitello J Matthew Mauro and J KrautldquoDetection of an oxyferryl porphyrin 120587-cation-radical interme-diate in the reaction between hydrogen peroxide and a mutant
Enzyme Research 11
yeast cytochrome c peroxidase Evidence for tryptophan-191involvement in the radical site of compound Irdquo Biochemistryvol 28 no 20 pp 7992ndash7995 1989
[28] M T Ashby ldquoInorganic chemistry of defensive peroxidases inthe human oral cavityrdquo Journal of Dental Research vol 87 no10 pp 900ndash914 2008
[29] J D Chandler and B J Day ldquoThiocyanate a potentially usefultherapeutic agent with host defense and antioxidant propertiesrdquoBiochemical Pharmacology vol 84 no 11 pp 1381ndash1387 2012
[30] E C Jong W R Henderson and S J Klebanoff ldquoBactericidalactivity of eosinophil peroxidaserdquo Journal of Immunology vol124 no 3 pp 1378ndash1382 1980
[31] E C Jong A A F Mahmoud and S J Kelbanoff ldquoPeroxidase-mediated toxicity to schistosomula of Schistosoma mansonirdquoJournal of Immunology vol 126 no 2 pp 468ndash471 1981
[32] L M Wolfson and S S Sumner ldquoAntibacterial activity of thelactoperoxidase system a reviewrdquo Journal of Food Protectionvol 56 no 10 pp 887ndash892 1993
[33] J Arnhold P G Furtmuller G Regelsberger and C ObingerldquoRedox properties of the couple compound Inative enzyme ofmyeloperoxidase and eosinophil peroxidaserdquo European Journalof Biochemistry vol 268 no 19 pp 5142ndash5148 2001
[34] P G Furtmuller J Arnhold W Jantschko M Zederbauer CJakopitsch and C Obinger ldquoStandard reduction potentials ofall couples of the peroxidase cycle of lactoperoxidaserdquo Journalof Inorganic Biochemistry vol 99 no 5 pp 1220ndash1229 2005
[35] C J van Dalen M W Whitehouse C C Winterbourn and AJ Kettle ldquoThiocyanate and chloride as competing substrates formyeloperoxidaserdquo Biochemical Journal vol 327 no 2 pp 487ndash492 1997
[36] A Slungaard and J R Mahoney Jr ldquoThiocyanate is the majorsubstrate for eosinophil peroxidase in physiologic fluids impli-cations for cytotoxicityrdquoThe Journal of Biological Chemistry vol266 no 8 pp 4903ndash4910 1991
[37] J Tenovuo ldquoAntimicrobial function of human salivamdashhowimportant is it for oral healthrdquoActaOdontologica Scandinavicavol 56 no 5 pp 250ndash256 1998
[38] R Ihalin V Loimaranta and J Tenovuo ldquoOrigin structure andbiological activities of peroxidases in human salivardquo Archives ofBiochemistry and Biophysics vol 445 no 2 pp 261ndash268 2006
[39] J A Rooke J F Flockhart and N H Sparks ldquoThe potentialfor increasing the concentrations of micro-nutrients relevant tohuman nutrition inmeat milk and eggsrdquo Journal of AgriculturalScience vol 148 no 5 pp 603ndash614 2010
[40] H Kohler A Taurog and H B Dunford ldquoSpectral studieswith lactoperoxidase and thyroid peroxidase interconversionsbetween native enzyme compound II and compound IIIrdquoArchives of Biochemistry and Biophysics vol 264 no 2 pp 438ndash449 1988
[41] I Yamazaki H S Mason and L Piette ldquoIdentification byelectron paramagnetic resonance spectroscopy of free radicalsgenerated from substrates by peroxidaserdquoThe Journal of Biolog-ical Chemistry vol 235 pp 2444ndash2449 1960
[42] B Chance ldquoThe kinetics and stoichiometry of the transitionfrom the primary to the secondary peroxidase peroxide com-plexesrdquo Archives of Biochemistry and Biophysics vol 41 no 2pp 416ndash424 1952
[43] K M Pruitt B Mansson-Rahemtulla D C Baldone andF Rahemtulla ldquoSteady-state kinetics of thiocyanate oxidationcatalyzed by human salivary peroxidaserdquo Biochemistry vol 27no 1 pp 240ndash245 1988
[44] B G J M Bolscher and R Wever ldquoA kinetic study of thereaction between humanmyeloperoxidase hydroperoxides andcyanide inhibition by chloride and thiocyanaterdquo Biochimica etBiophysica Acta Protein Structure and Molecular Enzymologyvol 788 no 1 pp 1ndash10 1984
[45] L A Marquez J T Huang and H Brian Dunford ldquoSpectraland kinetic studies on the formation of myeloperoxidase com-pounds I and II roles of hydrogen peroxide and superoxiderdquoBiochemistry vol 33 no 6 pp 1447ndash1454 1994
[46] HMAbu-Soud and S LHazen ldquoNitric oxide is a physiologicalsubstrate for mammalian peroxidasesrdquoThe Journal of BiologicalChemistry vol 275 no 48 pp 37524ndash37532 2000
[47] Y R Tahboub S Galijasevic M P Diamond and H MAbu-Soud ldquoThiocyanate modulates the catalytic activity ofmammalian peroxidasesrdquo Journal of Biological Chemistry vol280 no 28 pp 26129ndash26136 2005
[48] H Jenzer W Jones and H Kohler ldquoOn the molecularmechanismof lactoperoxidase-catalyzedH
2O2metabolism and
irreversible enzyme inactivationrdquo The Journal of BiologicalChemistry vol 261 no 33 pp 15550ndash15556 1986
[49] R P Magnusson A Taurog and M L Dorris ldquoMechanism ofiodide-dependent catalatic activity of thyroid peroxidase andlactoperoxidaserdquo The Journal of Biological Chemistry vol 259no 1 pp 197ndash205 1984
[50] D K Bhattacharyya U Bandyopadhyay and R K BanerjeeldquoEDTA inhibits lactoperoxidase-catalyzed iodide oxidation byacting as an electron-donor and interacting near the iodidebinding siterdquoMolecular and Cellular Biochemistry vol 162 no2 pp 105ndash111 1996
[51] C L Hawkins ldquoThe role of hypothiocyanous acid (HOSCN) inbiological systems HOSCN in biological systemsrdquo Free RadicalResearch vol 43 no 12 pp 1147ndash1158 2009
[52] S J Klebanoff ldquoIodination of bacteria a bactericidal mecha-nismrdquo Journal of Experimental Medicine vol 126 no 6 pp1063ndash1078 1967
[53] S J Klebanoff ldquoMyeloperoxidase-halide-hydrogen peroxideantibacterial systemrdquo Journal of Bacteriology vol 95 no 6 pp2131ndash2138 1968
[54] R K Banerjee and A G Datta ldquoSalivary peroxidasesrdquoMolecu-lar and Cellular Biochemistry vol 70 no 1 pp 21ndash29 1986
[55] MHuwiler H Jenzer andHKohler ldquoThe role of compound IIIin reversible and irreversible inactivation of lactoperoxidaserdquoEuropean Journal of Biochemistry vol 158 no 3 pp 609ndash6141986
[56] R Wever W M Kast J H Kasinoedin and R Boelens ldquoTheperoxidation of thiocyanate catalysed by myeloperoxidase andlactoperoxidaserdquo Biochimica et Biophysica Acta (BBA)ProteinStructure and Molecular vol 709 no 2 pp 212ndash219 1982
[57] C E A Souza D Maitra G M Saed et al ldquoHypochlorousacid-induced heme degradation from lactoperoxidase as anovel mechanism of free iron release and tissue injury ininflammatory diseasesrdquo PLoS ONE vol 6 no 11 Article IDe27641 2011
[58] J Carlsson ldquoBactericidal effect of hydrogen peroxide is pre-vented by the lactoperoxidase-thiocyanate system under anaer-obic conditionsrdquo Infection and Immunity vol 29 no 3 pp 1190ndash1192 1980
[59] E L Thomas and T M Aune ldquoLactoperoxidase peroxidethiocyanate antimicrobial system correlation of sulfhydryloxidation with antimicrobial actionrdquo Infection and Immunityvol 20 no 2 pp 456ndash463 1978
12 Enzyme Research
[60] J Carlsson Y Iwami and T Yamada ldquoHydrogen peroxideexcretion by oral streptococci and effect of lactoperoxidase-thiocyanate-hydrogen peroxiderdquo Infection and Immunity vol40 no 1 pp 70ndash80 1983
[61] K D Kussendrager and A C M van Hooijdonk ldquoLactoperox-idase physico-chemical properties occurrence mechanism ofaction and applicationsrdquoTheBritish Journal of Nutrition vol 84supplement 1 pp S19ndashS25 2000
[62] J P Perraudin ldquoProteines a activites biologiques lactoferrineet lactoperoxydase Connaissances recemment acquises et tech-nologies drsquoobtentionrdquo Lait vol 71 no 2 pp 191ndash211 1991
[63] J-W Boots and R Floris ldquoLactoperoxidase From catalyticmechanism to practical applicationsrdquo International Dairy Jour-nal vol 16 no 11 pp 1272ndash1276 2006
[64] A C M van Hooijdonk K D Kussendrager and J M SteijnsldquoIn vivo antimicrobial and antiviral activity of components inbovine milk and colostrum involved in non-specific defencerdquoBritish Journal of Nutrition vol 84 supplement 1 pp S127ndashS1342000
[65] D M Hogg and G R Jago ldquoThe antibacterial action of lac-toperoxidaseThe nature of the bacterial inhibitorrdquo BiochemicalJournal vol 117 no 4 pp 779ndash790 1970
[66] M T Ashby ldquoHypothiocyaniterdquo in Advances in InorganicChemistry R van Eldik and I-B Ivana Eds chapter 8 pp 263ndash303 Academic Press New York NY USA 2012
[67] E L Thomas ldquoLactoperoxidase-catalyzed oxidation of thio-cyanate equilibria between oxidized forms of thiocyanaterdquoBiochemistry vol 20 no 11 pp 3273ndash3280 1981
[68] T M Aune and E LThomas ldquoOxidation of protein sulfhydrylsby products of peroxidase-catalyzed oxidation of thiocyanateionrdquo Biochemistry vol 17 no 6 pp 1005ndash1010 1978
[69] T M Aune and E L Thomas ldquoAccumulation of hypothiocyan-ite ion during peroxidase-catalyzed oxidation of thiocyanateionrdquo European Journal of Biochemistry vol 80 no 1 pp 209ndash214 1977
[70] J D Oram and B Reiter ldquoThe inhibition of streptococci bylactoperoxidase thiocyanate and hydrogen peroxideThe effectof the inhibitory system on susceptible and resistant strains ofgroup N streptococcirdquo Biochemical Journal vol 100 no 2 pp373ndash381 1966
[71] J Kalmar K L Woldegiorgis B Biri and M T AshbyldquoMechanism of decomposition of the human defense factorhypothiocyanite near physiological pHrdquo Journal of the Ameri-can Chemical Society vol 133 no 49 pp 19911ndash19921 2011
[72] H Hoogendoorn J P PiessensW Scholtes and L A StoddardldquoHypothiocyanite ion the inhibitor formed by the system lac-toperoxidase thiocyanate hydrogen peroxide I Identification ofthe inhibiting compoundrdquoCaries Research vol 11 no 2 pp 77ndash84 1977
[73] L Bjorck and O Claesson ldquoCorrelation between concentrationof hypothiocyanate and antibacterial effect of the lactoperoxi-dase system against Escherichia colirdquo Journal of Dairy Sciencevol 63 no 6 pp 919ndash922 1980
[74] P Nagy S S Alguindigue and M T Ashby ldquoLactoperoxidase-catalyzed oxidation of thiocyanate by hydrogen peroxide areinvestigation of hypothiocyanite by nuclear magnetic reso-nance and optical spectroscopyrdquo Biochemistry vol 45 no 41pp 12610ndash12616 2006
[75] Y Adolphe M Jacquot M Linder A-M Revol-Junelles andJ-B Milliere ldquoOptimization of the components concentrationsof the lactoperoxidase system by RSMrdquo Journal of AppliedMicrobiology vol 100 no 5 pp 1034ndash1042 2006
[76] M Adamson and K M Pruitt ldquoLactoperoxidase-catalyzedinactivation of hexokinaserdquo Biochimica et Biophysica Acta vol658 no 2 pp 238ndash247 1981
[77] M N Mickelson ldquoGlucose transport in Streptococcus agalac-tiae and its inhibition by lactoperoxidase-thiocyanate-hydrogenperoxiderdquo Journal of Bacteriology vol 132 no 2 pp 541ndash5481977
[78] E L Thomas K A Pera K W Smith and A K ChwangldquoInhibition of Streptococcus mutans by the lactoperoxidaseantimicrobial systemrdquo Infection and Immunity vol 39 no 2 pp767ndash778 1983
[79] J Sermon K Vanoirbeek P De Spiegeleer R Van Houdt AAertsen and C W Michiels ldquoUnique stress response to thelactoperoxidase-thiocyanate enzyme system in Escherichia colirdquoResearch in Microbiology vol 156 no 2 pp 225ndash232 2005
[80] E L Thomas and T M Aune ldquoSusceptibility of Escherichia colito bactericidal action of lactoperoxidase peroxide and iodideor thiocyanaterdquoAntimicrobial Agents andChemotherapy vol 13no 2 pp 261ndash265 1978
[81] K M Pruitt J Tenovuo R W Andrews and T McKaneldquoLactoperoxidase-catalyzed oxidation of thiocyanate polaro-graphic study of the oxidation productsrdquo Biochemistry vol 21no 3 pp 562ndash567 1982
[82] M Huwiler and H Kohler ldquoPseudo-catalytic degradation ofhydrogen peroxide in the lactoperoxidaseH
2O2iodide sys-
temrdquo European Journal of Biochemistry vol 141 no 1 pp 69ndash741984
[83] M Huwiler U Burgi and H Kohler ldquoMechanism of enzymaticand non-enzymatic tyrosine iodination Inhibition by excesshydrogen peroxide andor iodiderdquo European Journal of Bio-chemistry vol 147 no 3 pp 469ndash476 1985
[84] M Morrison G S Bayse and A W Michaels ldquoDeterminationof spectral properties of aqueous I2 and I3- and the equilibriumconstantrdquo Analytical Biochemistry vol 42 no 1 pp 195ndash2011971
[85] E L Thomas and T M Aune ldquoPeroxidase catalyzed oxidationof protein sulfhydrylsmediated by iodinerdquoBiochemistry vol 16no 16 pp 3581ndash3586 1977
[86] W Gottardi ldquoIodine and disinfection theoretical study onmode of action efficiency stability and analytical aspects in theaqueous systemrdquo Archiv der Pharmazie vol 332 no 5 pp 151ndash157 1999
[87] W A Prutz R Kissner W H Koppenol and H RueggerldquoOn the irreversible destruction of reduced nicotinamidenucleotides by hypohalous acidsrdquo Archives of Biochemistry andBiophysics vol 380 no 1 pp 181ndash191 2000
[88] Y Bichsel and U Von Gunten ldquoHypoiodous acid kinetics ofthe buffer-catalyzed disproportionationrdquo Water Research vol34 no 12 pp 3197ndash3203 2000
[89] Y Bichsel and U von Gunten ldquoOxidation of iodide andhypoiodous acid in the disinfection of natural watersrdquo Environ-mental Science and Technology vol 33 no 22 pp 4040ndash40451999
[90] W A Prutz R Kissner T Nauser and W H Koppenol ldquoOnthe oxidation of cytochrome c by hypohalous acidsrdquo Archives ofBiochemistry and Biophysics vol 389 no 1 pp 110ndash122 2001
[91] A Virion J L Michot D Deme and J Pommier ldquoNADPHoxidation catalyzed by the peroxidaseH
2O2system Iodide-
mediated oxidation of NADPH to iodinated NADPrdquo EuropeanJournal of Biochemistry vol 148 no 2 pp 239ndash243 1985
Enzyme Research 13
[92] E L Thomas and T M Aune ldquoCofactor role of iodide in per-oxidase antimicrobial action against Escherichia colirdquo Antimic-robial Agents and Chemotherapy vol 13 no 6 pp 1000ndash10051978
[93] E L Thomas and T M Aune ldquoOxidation of Escherichiacoli sulfhydryl components by the peroxidase-hydrogenperoxide-iodide antimicrobial systemrdquo Antimicrobial Agentsand Chemotherapy vol 13 no 6 pp 1006ndash1010 1978
[94] C B Hamon and S J Klebanoff ldquoA peroxidase-mediatedstreptococcus mitis-dependent antimicrobial system in salivardquoJournal of Experimental Medicine vol 137 no 2 pp 438ndash4501973
[95] R Ihalin J Nuutila V Loimaranta M Lenander J Tenovuoand E-M Lilius ldquoSusceptibility of Fusobacterium nucleatum tokilling by peroxidase-iodide-hydrogen peroxide combinationin buffer solution and in human whole salivardquo Anaerobe vol9 no 1 pp 23ndash30 2003
[96] R Ihalin V Loimaranta M Lenander-Lumikari and J Ten-ovuo ldquoThe effects of different (pseudo)halide substrates onperoxidase-mediated killing of Actinobacillus actinomycetem-comitansrdquo Journal of Periodontal Research vol 33 no 7 pp 421ndash427 1998
[97] E H Bosch H van doorne and S de Vries ldquoThe lactoper-oxidase system the influence of iodide and the chemical andantimicrobial stability over the period of about 18 monthsrdquoJournal of AppliedMicrobiology vol 89 no 2 pp 215ndash224 2000
MPO Compound I apparent second-order rate constant at
LPO Compound I apparent second-order rate constant at
Clminus chloride Brminus bromide Iminus iodide and SCNminus thiocyanate
pH 7 from [16]
pH 7 from [15]
Figure 2 Apparent second-order rate constant at pH 7 (times104Mminus1 sminus1) of the reaction between myeloperoxidase Compound I or lactoper-oxidase Compound I with (pseudo)halides [15 16]
is the standard reduction potential of the enzyme whichdiffers among peroxidases and plays a role in their capacityto oxidize specific (pseudo)halides The redox reaction canoccur only if the reduction potential of the enzyme is equalor superior to the reduction potential of the substrate Thestandard reduction potential at pH 7 of Compound I perox-idases and the couple of two-electron reduction HOXXminus isranking in the following ascending rank LPOCompound I ltEPO Compound I lt MPO Compound I HOSCNSCNminus ltHOIIminus ≪ HOBrBrminus lt HOClminusClminus [17 18 33 34] Thisinvolves that only the MPO Compound I is able to oxidizeClminus with appropriate rates LPO being able to oxidize withhigh rates Iminus and SCNminus and slowly Brminus [16 17 20 33 34]Interestingly although LPO Compound I has the lowestreduction potential compared to EPOCompound I andMPOCompound I it catalyzes the oxidation of Iminus and SCNminus withthe highest rates (Figure 2) [15ndash17 20] This suggests thatother factors play a role such as anion size anion access andanion binding as well as structural and amino acid differencesin the active and binding site between enzymes [17 18 20]
The reduction potential of the Compound Inativeenzyme and HOXXminus redox couples depends on reactantconcentrations and pH values At a specific reactant concen-tration it decreases with increasing pH values but slopesdiffer (Figure 3) [17] This means that there exists a thresholdpH value above which the oxidation of halides becomesthermodynamically unfavorable especially for halides withhigh reduction potential such as Clminus and Brminus [17]
Concentrations of (pseudo)halogens also affect theiraffinity to Compound I (Figure 4)
Although plasma Clminus concentrations are 1000-foldhigher than Brminus and SCNminus MPO Compound I oxidizessimilar amounts of SCNminus and Clminus [35] EPO Compound I
Compound Inative enzyme
Threshold pH value
Redu
ctio
n po
tent
ial (
V)
pH
HOXOXminus H2O
Figure 3 Illustration (according to [17]) of the pH thresholdvalue above which the oxidation of the halogen by mammalianheme peroxidase will be thermodically unfavorable The reductionpotential of the couple Compound Inative enzyme and the couplehalogen (X = chloride bromide) HOXOXminus is expressed with anillustrative function of the pH at a specific concentration of enzymeand substrates
preferentially oxidizes SCNminus in the presence of physiologicalconcentrations of SCNminus Brminus and Iminus [36] In the salivaof healthy adults where Clminus concentrations are only about25-fold higher than SCNminus SPO Compound I and MPOCompound I primarily generate hypothiocyanite [37 38]The levels of Iminus in human milk saliva blood and tissuesexcept the thyroid gland are below 1120583M and its in vivooxidation by Compound I is negligible [17 38] In humanmilk peroxidase activity is only derived from leucocytes AsMPO is able to oxidize Clminus and Clminus milk concentration is
Iodide 2120583MThiocyanate 120120583MChloride 13 mM
Figure 4 Illustration of the interaction between the biodisponibility of a peroxidase the (pseudo)halogen concentration in plasma in salivaand in milk and the production of oxidant molecules MPO myeloperoxidase SPO salivary peroxidase LPO bovine lactoperoxidaseOClminus hypochlorite and OSCNminus hypothiocyanite Although chloride is the most available substrate compared to thiocyanate bromide andiodide thiocyanate is the most effective substrate for the Compound I and hypothiocyanite could be produced at equal or superior levelscompared to hypohalides
high oxidation of Clminus is possible although it has never beenreported [14] In bovine milk lactoperoxidase is an abundantenzyme and with mean concentrations of Iminus and SCNminus of310 120583gkg and 02ndash15mgkg respectively oxidation is possi-ble [19 39] Nevertheless the relative abundance of SCNminus inall secretions blood and tissues and its better capacity as anelectron donor make it one of the main in vivo substratesof Compound I lactoperoxidase and myeloperoxidase for2-electron oxidation compared to halides [15] In in vitroapplications the ratio between (pseudo)halides regulates theratio of hypohalides generated by the reaction However asSCNminus is the most effective substrate for Compound I itspresence even in small quantities enhances its oxidation[14 35 36]
23 The Peroxidase Cycle Alternatively Compound I canshift to the peroxidase cycle which consists of two sequen-tial one-electron transfers back to the enzyme that yield(i) Compound II and (ii) the native enzyme while thesubstrate is oxidized into a radical (Figure 1) [40ndash42] Theperoxidase cycle is summarized in the following equations
Compound I + AH 997888rarr Compound II + A∙
Compound II + AH 997888rarr Native enzyme + A∙(3)
Compound I is not specific regarding the one-electrondonor it can be exogenous or endogenous and a lot of candi-dates have been described [18 20 43]Hydrogen peroxide canundergo a one-electron oxidation onlywithMPOCompoundI with the formation of superoxide [16 20 44 45]
During the first step of the peroxidase cycle the cationlocated in the porphyrin ring undergoes a one-electronreduction with formation of Compound II and concomitantoxidizing of one one-electron substrate [4 24] Compound II
maintains one oxidizing equivalent in the oxyferryl center[4 24] Finally this latter is reduced back to the native enzymewith the oxidation of a second one-electron donor
The standard reduction potential of the couple Com-pound ICompound II is high and allowed the one-electronoxidation by Compound I of a wide range of substrates[18 20] In contrast the standard reduction potential of thecouple Compound IInative enzyme is low and restrains thenumbers of possible substrates for Compound II [18 20]With the result that (i) the Compound IInative enzymestandard reduction potential is too low to react with halogensand (ii) the nature of substrates strongly influenced theirability to be oxidized by mammalian peroxidase compoundII [2 14 20] therefore when the enzyme is in this state it hasto be first reduced to the ground state before possibly partici-pating to the halogenation cycle and producing antimicrobialmolecules [14] Moreover the reduction of Compound II tothe ground state is the rate-limiting step [45 46] that is theperoxidase cycle interferes with the halogenation cycle andslows down antimicrobial activity [47]
The peroxidase cycle has been described as a possiblecatalytic sink for nitric oxide (NO) [46] but also for hydrogenperoxide in the case of a moderate excess of H
2O2relative
to LPO [24] Increase of NO removal from media evenin presence of Clminus after addition of MPO EPO or LPOand accelerated rates of Compound I and Compound IIreduction in presence of NO show that peroxidases mayregulate the bioavailability of NO [46] In conditions ofhigh excess of hydrogen peroxide relative to LPO and inthe absence of an exogenous electron donor CompoundII is transformed into Compound III which is 3 oxidativeequivalents above the native enzyme In moderate excessconditions Compound III can be partially reconverted intoCompound II and can reenter the peroxidase cycle [24 40]
Enzyme Research 5
Otherwise the enzyme is irreversibly inactivated the hemefraction is cleaved and iron is released [48] In the presenceof an exogenous two-electron donor the enzyme is largelyprotected from hydrogen peroxide because the halogenationcycle is favored Furthermore protection is higher withiodide because oxidized iodide consumes H
2O2to produce
oxygen and iodide in a reaction called the pseudocatalyticactivity of peroxidase [24 40 49]
However thiocyanate can act as a one-electron donor andbe part of the peroxidase cycle with the sequential formationof two thiocyanate radicals [47] With 200120583M SCNminus LPOis predominantly in its native form this indicates that thehalogenation cycle prevails [47]
In the presence of both one- and two-electron donorscompetition for oxidation can occur and favor the halogena-tion or the peroxidase cycle The presence of EDTA inhibitsthe oxidation of iodide due to competition for binding toCompound I [50] The standard reduction potential betweenthe donors favors the molecule with the lowest reductionpotential Thereby the respective reduction potentials of theone- and two-electron oxidation of thiocyanate at very lowpH are 165V and 082V and promote the halogenationcycle [51] In the case of low concentrations of halides orthiocyanate below 10 120583M Iminus or 3 120583M SCNminus CompoundI reacts with any suitable exogenous or endogenous one-electron donor with the subsequent formation of CompoundII and a negligible oxidation rate of halides and thiocyanate[14]
24 Inhibition of the Function of Mammalian Heme Peroxi-dase The function of heme peroxidases can be inhibited inseveral ways that could be classified into three categoriesThe first one could represent an inhibition of the enzymeby (i) molecules or proteins and (ii) external conditionssuch as pH and temperature For example cyanide azidenitrite mercaptomethylimidazole thiourea superoxide highlevels of nitric oxide and high levels of thiocyanate bindto the native enzyme and alter Compound I formation [2046 47 52ndash54] With thiocyanate inhibition is linked to therestriction of the binding site to hydrogen peroxide andthe interaction of SCNminus with a water molecule [23] Highconcentration of H
2O2or Iminus will inactivate irreversibly LPO
with liberation of free iron [48 55] Temperature between73∘C and 83∘C depending on the heating time results inunfolding and inactivation of LPO [19] Extreme pH isinactivating enzymes and at low pH an amino acid groupprobably histidine is protonated which prevents the bindingof H2O2[56] Some proteases such as pepsin and pronase are
able to inactivate LPO by proteolysis but chymotrypsin did itvery slowly and trypsin and thermolysin are not active againstLPO [19]
The second group of inhibitors could concern substancesor proteins which are able to interfere with the catalyticmechanism For example catalase consumes H
2O2and will
stop the formation of Compound I [30 52] Competitionbetween substrates can also interfere with the reaction cyclesuch as SCNminus which competes very effectively with Clminus Brminusand Iminus [52 53] HOCl has the capacity to bind to LPO native
enzyme and convert it into Compound I Above 100 120583MHOCl mediates the destruction of the LPO heme center[57]
The third class could be related to substances or pro-teins which are buffering active molecules produced duringthe catalytic reaction For example presence of thiosulfatethioglycolate glutathione dithiothreitol cysteineNAD(P)Hand tyrosine will reduce the antimicrobial activity throughreacting with OClminus OBrminus OIminus or OSCNminus [52 53 58 59]The enzyme NADH-OSCN oxidoreductase is able to reduceOSCNminus in SCNminus [60]
3 Activity of Lactoperoxidase withThiocyanate andor Iodide
LPO concentrations in cowrsquos milk are around 30mg Lminus1depending on season diet and calving and breeding season[61] LPO extraction from whey or milk is based on awell-developed industrial process [62] Compared to MPOand EPO LPO is easily isolated and manufactured in largequantities As a result cowrsquos milk peroxidase is the favoritemolecule for in vitro or in vivo applications such as con-servation of raw and pasteurized milk storage of emulsionsand cosmetics moisturizing gel and toothpaste in human drymouth veterinary products and preservation of foodstuffs[19 61 63 64]
31 Activity of LPO Related to Hypothiocyanite
311 Mode of Action of Hypothiocyanite Thiocyanate is oxi-dized in a two-electron reaction that yields hypothiocyaniteHypothiocyanite has a pKa of 53 [65] It is more acidic thanhypohalides that have pKas of 75 (HOCl) 86 (HOBr) and106 (HOI) [14 66] All hypo(pseudo)halides (OXminus) are in anacid-base equilibrium association with their correspondingacid hypo(pseudo)halide (HOX) For example in the case ofhypothiocyanite
HOSCN 999447999472 OSCNminus +H+ (4)
The acid form has a higher oxidation potential and ismore soluble in nonpolar media so that it passes throughhydrophobic barriers such as cell membranes more easilybut it is less stable than the basic form (OXminus) [14 66]Hypohalide acids are predominant in acidic to neutral mediaand even in basic conditions for HOBr and HOI whereashypothiocyanite needs a pH below 53 to be predominant inthe acid form [66 67]
SCNminus is the two-electron donorwith the lowest reductionpotential and therefore forms the hypothiocyanite acid withthe lowest oxidative power compared to hypohalous acidsHypohalous acids rank as follows with increasing oxidativestrength OSCNminus lt OIminus lt OBrminus lt OClminus [28 66]These characteristicsmake hypothiocyanite relatively specificregarding its molecular target (Figure 5) that is a thiolmoiety [28 59 68]
6 Enzyme Research
- SH group- NAD(P)H
- SH group- NAD(P)H- Reduced pyridine nucleotide- R-S-R (thioether group)
HOSCNOSCNminus
HOIOIminusI2
- NH2 group
Figure 5 Target group of hypothiocyanite hypoiodite and iodineDue to its low oxidation power hypothiocyanite is relatively specificand is not reactive against all thiols In vivo hypoiodite seems tobe selectively directed against reduced pyridine nucleotide becauseeven the presence of excess glutathione and methionine doesnot thoroughly inhibit their oxidation HOSCNOSCNminus acidic orbasic form of hypothiocyanite HOIOIminus acidic or basic form ofhypoiodite and I
2 iodine
R-S-SCN or R-S-I
LPO R-SH
+ R-S-OHOSCNminusSCNminus SCNminus
or Iminus or Iminusor minusOI
H2O
H2O
2
Figure 6 Illustration of the cofactor role of SCNminus or Iminus Whenthe necessary conditions are fulfilled that is (i) no substratecompetitor for SCNminus or Iminus for binding to lactoperoxidase (ii)enough peroxidase H
2O2and SCNminus or Iminus (iii) enough R-SH
and (iv) no incorporation of SCNminus or Iminus in stable byproducts thequantity of OSCNminus or OIminus produced depends only on the amountof H2O2 SCNminus thiocyanate Iminus iodide H
2O2 hydrogen peroxide
LPO lactoperoxidase R-SH peptide or protein with a thiol moietyR-S-SCN or R-S-I sulfenyl thiocyanate or iodide R-SOH sulfenicacid OSCNminus hypothiocyanite and OIminus hypoiodite
Sulfhydryl oxidation by OSCNminus generates sulfenyl thio-cyanate in equilibrium with sulfenic acid [68]
SCNminus +H2O2+ LPO 997888rarr OSCNminus + LPO
R-SH +OSCNminus 997888rarr R-S-SCN +OHminus
R-S-SCN +H2O 997888rarr R-S-OH + SCNminus +H+
(5)
The cycle of reactions shows that thiocyanate acts likea cofactor for LPO (Figure 6) so that the total number ofoxidized sulfhydryls is independent of SCNminus as long as(i) thiocyanate is not exhausted (ii) thiocyanate is not incompetition with other substrates for the binding to Com-pound I (iii) thiocyanate is not incorporated into an aromaticamino acid (iv) enoughH
2O2is present and (v) thiol moiety
is still available [68 69]Although the target of OSCNminus is a thiol moiety not
all sulfhydryls are equally sensitive to OSCNminus albumincysteine mercaptoethanol dithiothreitol glutathione and 5-thio-2-nitrobenzoic acid are all oxidized but 120573-lactoglobulinis poorly oxidized probably due to a limited accessibility ofsulfhydryls to OSCNminus [68] In some conditions that is thejoint presence of LPO enough H
2O2and SCNminus and after the
oxidation of available sulfhydryls modification of tyrosinetryptophan and histidine protein residues can occur and that
could be linked to the formation of a labile powerful oxidantsuch as sulfur dicyanide [68]
Some authors suggest that (SCN)2is formed during the
enzymatic reaction and then chemically hydrolyzed intohypothiocyanite [14 69 70] However a recent publicationdemonstrates that (SCN)
2cannot be a precursor during the
enzymatic oxidation of SCNminus at neutral pH inmammals [71]Hypothiocyanite is less stable in acid conditions with
high concentrations of SCNminus and in the presence of (SCN)2
and it is thought to break down via the following net reaction[14]
4HOSCN +H2O 997888rarr 3SCNminus + CNOminus + SO
4
2minus+ 6H+ (6)
A recent study based notably on spectroscopic and chro-matographic methods proposes the following net equationwithin the 4ndash7 pH range
3HOSCN +H2O 997888rarr XSO
4
2minus+ XHCN
+ (1 minus X) SO3
2minus+ (1 minus X)CNOminus
+ 2SCNminus + (5 minus X)H+
(7)
The proportions of end anions were different at pH 4 andpH 7 at pH 7 the proportion of CNOminus was higher SCNminusformation was slower and no CNminus was detected [71]
It might seem easier to produce hypothiocyanite chemi-cally in in vitro applications but producing hypothiocyanitechemically from the oxidation of SCNminus by a halogen (Cl
2or
Br2) or by a hypohalous acid (HOCl or HOBr) in basic media
is tricky due to overoxidation of SCNminus [66] The referencemethod in the literature to produce 1- to 2-day stable OSCNminusis by hydrolyzing (SCN)
2in basic conditions [72ndash74]
Hypothiocyanite inhibitors have been described Forexample CNminus a weak acid buffer dissolved carbonate excesshydrogen peroxide hydrofluoric acid metallic ions glyc-erol or ammonium sulfate accelerates the decomposition ofOSCNminus whereas sulfonamide stabilizes it [67 72]
Appropriate concentrations of substrates induce en-hanced activity [75]
312 Biological Activity of Hypothiocyanite The biologicalactivity of hypothiocyanite is summarized in Figure 7
The sulfhydryl moiety is essential for the activityof numerous enzymes and proteins Inhibition of bacte-rial glycolysis through the oxidation of hexokinase gly-ceraldehyde-3-phosphate dehydrogenase (GAPDH) aldo-lase and glucose-6-phosphate dehydrogenase has beenobserved [14 51 65 70 76] Inhibition of respiration andglucose transport is associated with the alteration of cellmembranes or transporters [14 51 65 77] Irreversible inhi-bition is linked to long periods of incubation and bacterialsensitivity depends on the bacterial species and on hypothio-cyanite concentrations [14 51 59] Increased concentrationsof reducing agents such as glutathione and cysteine canreverse the inhibition through buffering hypothiocyaniteand converting the reduced thiol back into sulfhydryl [1478] This defense mechanism is used by Escherichia coli itinduces the CysJ promoter during the stress response to the
Enzyme Research 7
Bacteria
Inhibition of glycolysis Inhibition of respirationInhibition of glucose transport
Possible defense mechanism of bacteria- Increased GSH and cysteine content
Peptide proteinwith SH moiety
HOSCNOSCNminus
- NAD(P)H-dependent reduction of OSCNminus
Figure 7 Biological activity of hypothiocyanite on bacteria and possible defensemechanism of the bacteria Reversible inhibition is observedin that (i) hypothiocyanite is not reactive against all thiols and (ii) if hypothiocyanite is removed or diluted the pathogen recovers Irreversibleinhibition is linked to (i) long period of incubation (ii) the bacterial species and (iii) hypothiocyanite concentration HOSCNOSCNminus acidicor basic form of hypothiocyanite and GSH glutathione
pH lt 6
Influence of iodide concentration
Influence of the pH
1 I2 and high Iminus
I5minusI6
minus
I2I3minus
6 lt pH lt 9
HOII2OHI2I3minus
Iminus + H2O2 + LPO rarr active molecules
I2 (without Iminus)HOIOIminusI2I3
minusHI2Ominus
Figure 8 Illustration of the molecules that can be present after oxidation of iodide by lactoperoxidase in presence of H2O2The active species
depend mainly on the concentration of iodide (upper part) and the pH (lower part) The species with an oxidant power are represented inbold
lactoperoxidase system [79] Another resistance mechanismcould be the NAD(P)H-dependent reduction of OSCNminuswithout any loss of the sulfhydryl compound [14 72 78]Alteration of the bacterial membrane increases the efficacyof hypothiocyanite [80]
Furthermore the activity of the entire system (enzyme +substrates) is known to be more effective than hypothiocyan-ite alone whether enzymatically or chemically producedThis has been explained by the production of short-livedhighly reactive intermediates such as O
2SCNminus and O
3SCNminus
by the enzyme or by the oxidation of OSCNminus in conditionsof excess H
2O2[65 73 81] The activity of hypothiocyanite
has been described against bacteria such as Actinomyces sppBacillus cereus Lactobacillus spp Staphylococcus albus Saureus Streptococcus spp Escherichia coli Legionella pneu-mophila Salmonella typhimurium Pseudomonas fluorescensP aeruginosa Campylobacter jejuni C coli and Listeriamonocytogenes [14 32] Reversible inhibition is observed
when cells recover after OSCNminus is depleted [14 59] Irre-versible inhibition is obtained with long-term incubation andhigh level of OSCNminus [59] Higher concentration of SCNminuscompared to Iminus is necessary to obtain inhibition against E coliand accumulation of OSCNminus is observed as it is not reactiveagainst all thiols [59]Therefore the activity of the SCNminus-LPOsystem appears to be more bacteriostatic than bactericidal
32 Activity of LPO Related to Oxidized Iodide
321 Chemistry of Oxidized Iodide Iodide is oxidized byCompound I through a single two-electron transfer thatyields oxidized Iminus in the form of I
2or HOI [14 24 82ndash85]
The active agent is composed of a mixture of species that arenot yet formally detailed due to the very complex behaviorand stability of I
2and HOI in aqueous environments that
strongly depend on pH values and iodide concentrations[66 82 83 86]
8 Enzyme Research
Based on the inorganic chemistry of iodine in waterand literature on enzymatic oxidation of iodide the activemolecules have been described as follows (Figure 8)
(i) Under pH 6 and in the presence of iodide only I2
Iminus and I3
minus are present and the only active moleculeis I2 I2concentrations decrease with increasing
concentrations of Iminus At an initial 1mM I2 with Iminus
concentrations ranging from 1mM to 100mM I2
concentrations fall from almost 1mM to 001mM asdescribed by the following association reaction [2482 83 86]
I2+ Iminus 999447999472 I
3
minus (8)
(ii) In solution within a 6ndash9 pH range and with a max-imum 1mM iodide a mixture of HOII
2OHI2I3
minus
is formed in which I3
minus is not active and I2OH is
probably less reactive than HOI or I2[86 87] If
Iminus concentrations are above 10mM I3
minus representsthe main species formed and the concentration ofactive molecules relatively drops The mechanism issummarized in the following net equations
HOI + Iminus +H+ 999447999472 I2OHminus +H+
999447999472 I2+H2O 999447999472 I
2+ Iminus 999447999472 I
3
minus
(9)
(iii) In iodine solution without iodide or when availableiodide has been oxidized the number of I
2-derived
molecules decreases with decreasing I2concentra-
tions At 1000 120583M I2 with pH-related ratios five
relevant species are observed (I2 HOI I
3
minus HI2Ominus
and OIminus) At 10 120583M I2 the main species are only I
2
HOI and OIminus and HOI could represent up to 90 ofthe active oxidant molecules at pH 8-9 [86] Below apH of 106 the following reactions are involved
I2+H2O 999447999472 HOI + Iminus +H+ (hydrolysis of I
2)
I2+ Iminus 999447999472 I
3
minus(triiodide formation independent of pH)
(10)
(iv) At high Iminus and 1 I2concentrations as in Lugol
solution I5
minus and I6
minus are formed and represent 82of the active oxidative agents [86] after the followingreaction
I3
minus+ I2999447999472 I5
minus(pentaiodide formation)
2I3
minus999447999472 I6
2minus(dimerization of I
3
minus)
(11)
The stability of HOI and I2is linked to their dispro-
portionation in iodate which has no oxidative activity inneutral and basic pH conditions [86]The disproportionationreactions read as follows
3HOI 999447999472 IO3
minus+ 2Iminus + 3H+ (disproportionation of HOI)
3I2999447999472 IO
3
minus+ 5Iminus + 6H+ (disproportionation of I
2)
(12)
I2stability increases at higher pHvalues andhigher iodide
concentrations [86] In drinking water HOI disproportion-ation is slow and varies substantially HOI has a half-life of4 days to 35 years depending on (i) the initial level of HOIthat speeds its decomposition and (ii) the presence of boratephosphate or carbonate that catalyzes its decomposition [8889]
322 Mode of Action of Oxidized Iodide The oxidativestrength of I
2is between that of the corresponding hypo-
halous acid HOI and the hypoiodite ion OIminus and ranks asfollows 0485V (OIminus) lt 0536V (I
2) lt 0987V (HOI) [66]
HOI reacts through very rapid oxidation of thiolgroups oxidation of NAD(P)H oxidation of 120573-nicotinamidemononucleotide direct reaction with thioether groupsthrough sulfoxidation and slow oxidation of the aminemoiety (Figure 5) [87 90 91] At low Iminus concentrationsiodination of tyrosine residues is catalyzed by the enzyme[14] In a cellular environment HOI seems to be more selec-tively directed against the degradation of reduced pyridinenucleotides thanHOCL andHOBr because even the presenceof excess glutathione methionine or oxidized glutathionedoes not thoroughly inhibit their oxidation [87]
In some conditions that is (i) enough iodide H2O2 and
peroxidase (ii) no accumulation of oxidized iodide and (iii)no incorporation of iodide into stable byproducts such astyrosine residues iodide acts as a cofactor (Figure 6) andthe proportion of oxidized sulfhydryls is proportional to theamount of H
2O2as described below [85 92]
2Iminus +H2O2+ LPO (native enzyme)
997888rarr I2+ 2H2O + LPO (native enzyme)
R-SH + I2997888rarr R-S-I + Iminus +H+
R-S-I+H2O 997888rarr R-S-OH + Iminus +H+
(13)
In the case of high concentrations of Iminus andor H2O2
inhibition of tyrosine iodation has been observed [83] andrelated to the pseudocatalytic redox degradation of H
2O2
with formation of O2when excessive H
2O2is present (reac-
tion 1) and production of I3
minus when excessive amounts of Iminusare present (reaction 2)
I2+H2O 997888rarr O
2+ 2Iminus + 2H+ (reaction 1)
I2+ Iminus 999447999472 I
3
minus(reaction 2)
(14)
Both reactions deplete the amount of the active oxidizingagent I
2 In the absence of tyrosine oxidized iodide reacts
with nucleophilic molecules such as Iminus Clminus or OHminus to formI2 I3
minus ICl ICl2 IOH and I
2OH [82] Some anions such as
Clminus HPO4
minus or OHminus reduce the amount of I2I3
minus but thiseffect is inversely proportional to the concentration of Iminusabove pH 9 I
2is hydrolyzed and IO
3
minus is formed [82]HOI can be produced chemically through oxidation of Iminus
by Cl2or O3 with a short half-life due to overoxidation of
HOI byCl2andO
3[89] and through oxidation of Iminus byHOCl
HOBr or NH2Cl with a longer half-life [87 89]
Enzyme Research 9
Bacteria
Inhibition of glycolysis Inhibition of respirationInhibition of glucose transportInhibition of the pentose phosphate pathway
- Peptide protein with- SH moiety- thioether moiety- NAD(P)H
HOIOIminus
I2
Figure 9 Biological activity of hypoiodite or iodine on bacteria Irreversible inhibition is observed and could be linked to (i) oxidation ofthiol groups NAD(P)H and thioether groups (ii) high reactivity of HOII
2against thiol and reduced nicotinamide nucleotides and (iii) the
incorporation of iodide in tyrosine residue of protein (iodination of protein) HOIOIminus acid or basic form of hypoiodite and I2 iodine
323 Biological Action of Oxidized Iodide The biologicalaction of oxidized iodide (Figure 9) is similar to that ofhypothiocyanite but differs in that (i) the reactivity of oxi-dized iodide is complete against thiol group and (ii) cells didnot recover after removing of oxidized iodide [59]
Due to the cofactor role of Iminus inhibition of respirationin Escherichia coli in the presence of LPO H
2O2 and Iminus is
complete with only 10120583M NaI whereas 100 120583M of solely I2
is necessary to obtain complete inhibition This is directlyrelated to the oxidation of sulfhydryls not to the percentageof iodine incorporation [92 93]
E coli seems to be more sensitive if the bacteria areincubated together with the entire system (enzyme H
2O2
and iodide) rather than adding several minutes after mixingthe enzyme with its substrates This could be linked to theformation of an unstable reactive intermediate [52]
The activity of the Iminus peroxidase system is more effectiveagainst E coli than the SCNminus system in that lower Iminusconcentrations are necessary all sulfhydryls are oxidized andcells do not recover even if the amount of I
2is not sufficient
to oxidize all SH groups [59 80] Against L acidophilushigh non physiological amounts of Iminus are necessary to obtaininhibitionwhereas small concentrations of SCNminus are effective[70]
CNminus azide EDTA and SCNminus inhibit the formation ofoxidized iodide [50 52] Increased pH values and increasedamounts of thiol and NAD(P)H compounds reduce theactivity of the iodide peroxidase system [52]
LPO-H2O2-Iminus in presence of Streptococcus mitis is active
against Staphylococcus aureus and E coli [94] LPO-H2O2-
Iminus is active against Micrococcus S aureus Listeria monocy-togenes Bacillus cereus E coli and Candida albicans [12 1980] In the presence of other peroxidases the Iminus peroxidasesystem is active against Schistosoma mansoni Fusariumnucleatum andActinobacillus actinomycetemcomitans [31 9596] Compared to SCNminus Iminus-LPO shows bactericidal activities[14 19 80]
33 Activity of LPO Related to Hypoiodite and Hypothiocyan-ite The combination of SCNminus with Iminus in the lactoperoxidasesystem has been poorly studied Tackling the enzymaticmechanism is tricky and contradictory results have been
found about microbial activity in the concomitant presenceof SCNminus and Iminus
In the presence of SCNminus and Iminus there is competitionbetween the two substrates for oxidation by lactoperoxidase[14 36] Iminus alone exhibits bactericidal activity but an SCNminusIminusratio of 01 inhibits that bactericidal effect and an SCNminusIminusratio of 1 antagonizes it due to competition for oxidation andfaster decomposition of HOSCN in the presence of Iminus [14]Against A actinomycetemcomitans the peroxidase systemwith Iminus Clminus or a combination of Iminus and Clminus is effective butaddition of SCNminus cancels the antibacterial effect [96] On theother hand a synergistic or unaffected effect of iodide in theSCNminus-H
2O2-LPO system has been shown against Candida
albicans E coli S aureusAspergillus niger and Pseudomonasaeruginosa [19 97]
4 Conclusion
Themolecular evolution of heme peroxidases and the preser-vation of their catalytic domain [6] show that the productionof strong oxidants is a powerful part of the nonimmunedefense mechanisms against pathogenic bacteria fungi orparasite which made the use of those enzymes in practicalapplications worthwhile
The enzymatic reactions involving mammalian peroxi-dases are complex and various molecules can promote orreduce dramatically the antibacterial activity of the per-oxidase system In order to favor the halogenation cyclerequired in in vitro and in vivo antimicrobial applicationsseveral points have to be taken into account (i) to avoid thepresence of competitors to iodide or thiocyanate for bindingto Compound I and to avoid the presence of inhibitorsof the enzyme or of active molecules (ii) to avoid excessH2O2concentration which is able to destruct the enzyme
and to react with iodine or hypoiodite with loosing of activemolecules (iii) to favor the presence of hypoiodite instead ofiodine due to the association reaction of iodine with iodide(iv) to avoid excess concentration of thiocyanate which caninhibit formation of Compound I (v) to use the entiresystem (enzyme + substrates) instead of active moleculesalone (vi) to favor moderate acid pH when hypothiocyaniteis the active molecule (vii) for bactericidal fungicidal or
10 Enzyme Research
parasitical applications the use of iodide has to be preferred(viii) the use of combined presence of iodide and thiocyanatehas to be checked carefully for efficacy and (ix) to favor thecofactor role of iodide or thiocyanate
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] P J OrsquoBrien ldquoPeroxidasesrdquoChemico-Biological Interactions vol129 no 1-2 pp 113ndash139 2000
[2] W Jantschko P G Furtmuller M Allegra et al ldquoRedoxintermediates of plant and mammalian peroxidases a compar-ative transient-kinetic study of their reactivity toward indolederivativesrdquo Archives of Biochemistry and Biophysics vol 398no 1 pp 12ndash22 2002
[3] S Kimura and M Ikeda-Saito ldquoHuman myeloperoxidase andthyroid peroxidase two enzymes with separate and distinctphysiological functions are evolutionarily related membersof the same gene familyrdquo Proteins Structure Function andGenetics vol 3 no 2 pp 113ndash120 1988
[4] G Battistuzzi M Bellei C A Bortolotti and M Sola ldquoRedoxproperties of heme peroxidasesrdquo Archives of Biochemistry andBiophysics vol 500 no 1 pp 21ndash36 2010
[5] M Zamocky C Jakopitsch P G Furtmuller C Dunand and CObinger ldquoThe peroxidase-cyclooxygenase superfamily recon-structed evolution of critical enzymes of the innate immunesystemrdquo Proteins Structure Function and Genetics vol 72 no2 pp 589ndash605 2008
[6] H Daiyasu and H Toh ldquoMolecular evolution of the myeloper-oxidase familyrdquo Journal of Molecular Evolution vol 51 no 5 pp433ndash445 2000
[7] D Serteyn S Grulke T Franck A Mouithys-Mickalad andG Deby-Dupont ldquoNeutrophile myeloperoxidase protectiveenzyme with strong oxidative activitiesrdquo Annales de MedecineVeterinaire vol 147 no 2 pp 79ndash93 2003
[8] S C Whitman S L Hazen D B Miller R A HegeleJ W Heinecke and M W Huff ldquoModification of type IIIVLDL their remnants and VLDL from apoE- knockout miceby p-hydroxyphenylacetaldehyde a product of myeloperox-idase activity causes marked cholesteryl ester accumulationin macrophagesrdquo Arteriosclerosis Thrombosis and VascularBiology vol 19 no 5 pp 1238ndash1249 1999
[9] T J Barrett and C L Hawkins ldquoHypothiocyanous acid benignor deadlyrdquo Chemical Research in Toxicology vol 25 no 2 pp263ndash273 2012
[10] M M Lloyd D M van Reyk M J Davies and C L HawkinsldquoHypothiocyanous acid is a more potent inducer of apoptosisand protein thiol depletion in murine macrophage cells thanhypochlorous acid or hypobromous acidrdquo Biochemical Journalvol 414 no 2 pp 271ndash280 2008
[11] J Wang and A Slungaard ldquoRole of eosinophil peroxidase inhost defense and disease pathologyrdquo Archives of Biochemistryand Biophysics vol 445 no 2 pp 256ndash260 2006
[12] M Ahariz and P Courtois ldquoCandida albicans susceptibility tolactoperoxidase-generated hypoioditerdquo Clinical Cosmetic andInvestigational Dentistry vol 2 pp 69ndash78 2010
[13] A Welk C Meller R Schubert C Schwahn A Kramerand H Below ldquoEffect of lactoperoxidase on the antimicrobialeffectiveness of the thiocyanate hydrogen peroxide combinationin a quantitative suspension testrdquo BMC Microbiology vol 9article 134 2009
[14] K M Pruitt and J O Tenovuo Eds The Lactoperoxidase Sys-tem Chemistry and Biological Significance vol 27 of Immunol-ogy Series Marcel Dekker New York NY USA 1985
[15] P G Furtmuller W Jantschko G Regelsberger C JakopitschJ Arnhold and C Obinger ldquoReaction of lactoperoxidasecompound I with halides and thiocyanaterdquo Biochemistry vol41 no 39 pp 11895ndash11900 2002
[16] P G Furtmuller U Burner and C Obinger ldquoReaction ofmyeloperoxidase compound I with chloride bromide iodideand thiocyanaterdquo Biochemistry vol 37 no 51 pp 17923ndash179301998
[17] J Arnhold E Monzani P G Furtmuller M Zederbauer LCasella and C Obinger ldquoKinetics and thermodynamics ofhalide and nitrite oxidation by mammalian heme peroxidasesrdquoEuropean Journal of Inorganic Chemistry no 19 pp 3801ndash38112006
[18] M J Davies C L Hawkins D I Pattison and M D ReesldquoMammalian heme peroxidases from molecular mechanismsto health implicationsrdquo Antioxidants and Redox Signaling vol10 no 7 pp 1199ndash1234 2008
[19] J N de Wit and A C M van Hooydonk ldquoStructure functionsand applications of lactoperoxidase in natural antimicrobialsystemsrdquo Nederlands melk en Zuiveltijdschrift vol 50 no 2 pp227ndash244 1996
[20] P G Furtmuller M Zederbauer W Jantschko et al ldquoActivesite structure and catalytic mechanisms of human peroxidasesrdquoArchives of Biochemistry and Biophysics vol 445 no 2 pp 199ndash213 2006
[21] M Zederbauer P G Furtmuller S Brogioni C JakopitschG Smulevich and C Obinger ldquoHeme to protein linkages inmammalian peroxidases impact on spectroscopic redox andcatalytic propertiesrdquo Natural Product Reports vol 24 no 3 pp571ndash584 2007
[22] G Battistuzzi M Bellei J Vlasits et al ldquoRedox thermodynam-ics of lactoperoxidase and eosinophil peroxidaserdquo Archives ofBiochemistry and Biophysics vol 494 no 1 pp 72ndash77 2010
[23] I A Sheikh A Singh N Singh et al ldquoStructural evidence ofsubstrate specificity inmammalian peroxidases structure of thethiocyanate complex with lactoperoxidase and its interactionsat 24 a 24 A resolutionrdquo The Journal of Biological Chemistryvol 284 no 22 pp 14849ndash14856 2009
[24] H Kohler and H Jenzer ldquoInteraction of lactoperoxidase withhydrogen peroxide Formation of enzyme intermediates andgeneration of free radicalsrdquo Free Radical Biology and Medicinevol 6 no 3 pp 323ndash339 1989
[25] P G Furtmuller U Burner W Jantschko G Regelsberger andC Obinger ldquoTwo-electron reduction and one-electron oxida-tion of organic hydroperoxides by human myeloperoxidaserdquoFEBS Letters vol 484 no 2 pp 139ndash143 2000
[26] A Taurog M L Dorris and D R Doerge ldquoMechanism ofsimultaneous iodination and coupling catalyzed by thyroidperoxidaserdquo Archives of Biochemistry and Biophysics vol 330no 1 pp 24ndash32 1996
[27] J E Erman L B Vitello J Matthew Mauro and J KrautldquoDetection of an oxyferryl porphyrin 120587-cation-radical interme-diate in the reaction between hydrogen peroxide and a mutant
Enzyme Research 11
yeast cytochrome c peroxidase Evidence for tryptophan-191involvement in the radical site of compound Irdquo Biochemistryvol 28 no 20 pp 7992ndash7995 1989
[28] M T Ashby ldquoInorganic chemistry of defensive peroxidases inthe human oral cavityrdquo Journal of Dental Research vol 87 no10 pp 900ndash914 2008
[29] J D Chandler and B J Day ldquoThiocyanate a potentially usefultherapeutic agent with host defense and antioxidant propertiesrdquoBiochemical Pharmacology vol 84 no 11 pp 1381ndash1387 2012
[30] E C Jong W R Henderson and S J Klebanoff ldquoBactericidalactivity of eosinophil peroxidaserdquo Journal of Immunology vol124 no 3 pp 1378ndash1382 1980
[31] E C Jong A A F Mahmoud and S J Kelbanoff ldquoPeroxidase-mediated toxicity to schistosomula of Schistosoma mansonirdquoJournal of Immunology vol 126 no 2 pp 468ndash471 1981
[32] L M Wolfson and S S Sumner ldquoAntibacterial activity of thelactoperoxidase system a reviewrdquo Journal of Food Protectionvol 56 no 10 pp 887ndash892 1993
[33] J Arnhold P G Furtmuller G Regelsberger and C ObingerldquoRedox properties of the couple compound Inative enzyme ofmyeloperoxidase and eosinophil peroxidaserdquo European Journalof Biochemistry vol 268 no 19 pp 5142ndash5148 2001
[34] P G Furtmuller J Arnhold W Jantschko M Zederbauer CJakopitsch and C Obinger ldquoStandard reduction potentials ofall couples of the peroxidase cycle of lactoperoxidaserdquo Journalof Inorganic Biochemistry vol 99 no 5 pp 1220ndash1229 2005
[35] C J van Dalen M W Whitehouse C C Winterbourn and AJ Kettle ldquoThiocyanate and chloride as competing substrates formyeloperoxidaserdquo Biochemical Journal vol 327 no 2 pp 487ndash492 1997
[36] A Slungaard and J R Mahoney Jr ldquoThiocyanate is the majorsubstrate for eosinophil peroxidase in physiologic fluids impli-cations for cytotoxicityrdquoThe Journal of Biological Chemistry vol266 no 8 pp 4903ndash4910 1991
[37] J Tenovuo ldquoAntimicrobial function of human salivamdashhowimportant is it for oral healthrdquoActaOdontologica Scandinavicavol 56 no 5 pp 250ndash256 1998
[38] R Ihalin V Loimaranta and J Tenovuo ldquoOrigin structure andbiological activities of peroxidases in human salivardquo Archives ofBiochemistry and Biophysics vol 445 no 2 pp 261ndash268 2006
[39] J A Rooke J F Flockhart and N H Sparks ldquoThe potentialfor increasing the concentrations of micro-nutrients relevant tohuman nutrition inmeat milk and eggsrdquo Journal of AgriculturalScience vol 148 no 5 pp 603ndash614 2010
[40] H Kohler A Taurog and H B Dunford ldquoSpectral studieswith lactoperoxidase and thyroid peroxidase interconversionsbetween native enzyme compound II and compound IIIrdquoArchives of Biochemistry and Biophysics vol 264 no 2 pp 438ndash449 1988
[41] I Yamazaki H S Mason and L Piette ldquoIdentification byelectron paramagnetic resonance spectroscopy of free radicalsgenerated from substrates by peroxidaserdquoThe Journal of Biolog-ical Chemistry vol 235 pp 2444ndash2449 1960
[42] B Chance ldquoThe kinetics and stoichiometry of the transitionfrom the primary to the secondary peroxidase peroxide com-plexesrdquo Archives of Biochemistry and Biophysics vol 41 no 2pp 416ndash424 1952
[43] K M Pruitt B Mansson-Rahemtulla D C Baldone andF Rahemtulla ldquoSteady-state kinetics of thiocyanate oxidationcatalyzed by human salivary peroxidaserdquo Biochemistry vol 27no 1 pp 240ndash245 1988
[44] B G J M Bolscher and R Wever ldquoA kinetic study of thereaction between humanmyeloperoxidase hydroperoxides andcyanide inhibition by chloride and thiocyanaterdquo Biochimica etBiophysica Acta Protein Structure and Molecular Enzymologyvol 788 no 1 pp 1ndash10 1984
[45] L A Marquez J T Huang and H Brian Dunford ldquoSpectraland kinetic studies on the formation of myeloperoxidase com-pounds I and II roles of hydrogen peroxide and superoxiderdquoBiochemistry vol 33 no 6 pp 1447ndash1454 1994
[46] HMAbu-Soud and S LHazen ldquoNitric oxide is a physiologicalsubstrate for mammalian peroxidasesrdquoThe Journal of BiologicalChemistry vol 275 no 48 pp 37524ndash37532 2000
[47] Y R Tahboub S Galijasevic M P Diamond and H MAbu-Soud ldquoThiocyanate modulates the catalytic activity ofmammalian peroxidasesrdquo Journal of Biological Chemistry vol280 no 28 pp 26129ndash26136 2005
[48] H Jenzer W Jones and H Kohler ldquoOn the molecularmechanismof lactoperoxidase-catalyzedH
2O2metabolism and
irreversible enzyme inactivationrdquo The Journal of BiologicalChemistry vol 261 no 33 pp 15550ndash15556 1986
[49] R P Magnusson A Taurog and M L Dorris ldquoMechanism ofiodide-dependent catalatic activity of thyroid peroxidase andlactoperoxidaserdquo The Journal of Biological Chemistry vol 259no 1 pp 197ndash205 1984
[50] D K Bhattacharyya U Bandyopadhyay and R K BanerjeeldquoEDTA inhibits lactoperoxidase-catalyzed iodide oxidation byacting as an electron-donor and interacting near the iodidebinding siterdquoMolecular and Cellular Biochemistry vol 162 no2 pp 105ndash111 1996
[51] C L Hawkins ldquoThe role of hypothiocyanous acid (HOSCN) inbiological systems HOSCN in biological systemsrdquo Free RadicalResearch vol 43 no 12 pp 1147ndash1158 2009
[52] S J Klebanoff ldquoIodination of bacteria a bactericidal mecha-nismrdquo Journal of Experimental Medicine vol 126 no 6 pp1063ndash1078 1967
[53] S J Klebanoff ldquoMyeloperoxidase-halide-hydrogen peroxideantibacterial systemrdquo Journal of Bacteriology vol 95 no 6 pp2131ndash2138 1968
[54] R K Banerjee and A G Datta ldquoSalivary peroxidasesrdquoMolecu-lar and Cellular Biochemistry vol 70 no 1 pp 21ndash29 1986
[55] MHuwiler H Jenzer andHKohler ldquoThe role of compound IIIin reversible and irreversible inactivation of lactoperoxidaserdquoEuropean Journal of Biochemistry vol 158 no 3 pp 609ndash6141986
[56] R Wever W M Kast J H Kasinoedin and R Boelens ldquoTheperoxidation of thiocyanate catalysed by myeloperoxidase andlactoperoxidaserdquo Biochimica et Biophysica Acta (BBA)ProteinStructure and Molecular vol 709 no 2 pp 212ndash219 1982
[57] C E A Souza D Maitra G M Saed et al ldquoHypochlorousacid-induced heme degradation from lactoperoxidase as anovel mechanism of free iron release and tissue injury ininflammatory diseasesrdquo PLoS ONE vol 6 no 11 Article IDe27641 2011
[58] J Carlsson ldquoBactericidal effect of hydrogen peroxide is pre-vented by the lactoperoxidase-thiocyanate system under anaer-obic conditionsrdquo Infection and Immunity vol 29 no 3 pp 1190ndash1192 1980
[59] E L Thomas and T M Aune ldquoLactoperoxidase peroxidethiocyanate antimicrobial system correlation of sulfhydryloxidation with antimicrobial actionrdquo Infection and Immunityvol 20 no 2 pp 456ndash463 1978
12 Enzyme Research
[60] J Carlsson Y Iwami and T Yamada ldquoHydrogen peroxideexcretion by oral streptococci and effect of lactoperoxidase-thiocyanate-hydrogen peroxiderdquo Infection and Immunity vol40 no 1 pp 70ndash80 1983
[61] K D Kussendrager and A C M van Hooijdonk ldquoLactoperox-idase physico-chemical properties occurrence mechanism ofaction and applicationsrdquoTheBritish Journal of Nutrition vol 84supplement 1 pp S19ndashS25 2000
[62] J P Perraudin ldquoProteines a activites biologiques lactoferrineet lactoperoxydase Connaissances recemment acquises et tech-nologies drsquoobtentionrdquo Lait vol 71 no 2 pp 191ndash211 1991
[63] J-W Boots and R Floris ldquoLactoperoxidase From catalyticmechanism to practical applicationsrdquo International Dairy Jour-nal vol 16 no 11 pp 1272ndash1276 2006
[64] A C M van Hooijdonk K D Kussendrager and J M SteijnsldquoIn vivo antimicrobial and antiviral activity of components inbovine milk and colostrum involved in non-specific defencerdquoBritish Journal of Nutrition vol 84 supplement 1 pp S127ndashS1342000
[65] D M Hogg and G R Jago ldquoThe antibacterial action of lac-toperoxidaseThe nature of the bacterial inhibitorrdquo BiochemicalJournal vol 117 no 4 pp 779ndash790 1970
[66] M T Ashby ldquoHypothiocyaniterdquo in Advances in InorganicChemistry R van Eldik and I-B Ivana Eds chapter 8 pp 263ndash303 Academic Press New York NY USA 2012
[67] E L Thomas ldquoLactoperoxidase-catalyzed oxidation of thio-cyanate equilibria between oxidized forms of thiocyanaterdquoBiochemistry vol 20 no 11 pp 3273ndash3280 1981
[68] T M Aune and E LThomas ldquoOxidation of protein sulfhydrylsby products of peroxidase-catalyzed oxidation of thiocyanateionrdquo Biochemistry vol 17 no 6 pp 1005ndash1010 1978
[69] T M Aune and E L Thomas ldquoAccumulation of hypothiocyan-ite ion during peroxidase-catalyzed oxidation of thiocyanateionrdquo European Journal of Biochemistry vol 80 no 1 pp 209ndash214 1977
[70] J D Oram and B Reiter ldquoThe inhibition of streptococci bylactoperoxidase thiocyanate and hydrogen peroxideThe effectof the inhibitory system on susceptible and resistant strains ofgroup N streptococcirdquo Biochemical Journal vol 100 no 2 pp373ndash381 1966
[71] J Kalmar K L Woldegiorgis B Biri and M T AshbyldquoMechanism of decomposition of the human defense factorhypothiocyanite near physiological pHrdquo Journal of the Ameri-can Chemical Society vol 133 no 49 pp 19911ndash19921 2011
[72] H Hoogendoorn J P PiessensW Scholtes and L A StoddardldquoHypothiocyanite ion the inhibitor formed by the system lac-toperoxidase thiocyanate hydrogen peroxide I Identification ofthe inhibiting compoundrdquoCaries Research vol 11 no 2 pp 77ndash84 1977
[73] L Bjorck and O Claesson ldquoCorrelation between concentrationof hypothiocyanate and antibacterial effect of the lactoperoxi-dase system against Escherichia colirdquo Journal of Dairy Sciencevol 63 no 6 pp 919ndash922 1980
[74] P Nagy S S Alguindigue and M T Ashby ldquoLactoperoxidase-catalyzed oxidation of thiocyanate by hydrogen peroxide areinvestigation of hypothiocyanite by nuclear magnetic reso-nance and optical spectroscopyrdquo Biochemistry vol 45 no 41pp 12610ndash12616 2006
[75] Y Adolphe M Jacquot M Linder A-M Revol-Junelles andJ-B Milliere ldquoOptimization of the components concentrationsof the lactoperoxidase system by RSMrdquo Journal of AppliedMicrobiology vol 100 no 5 pp 1034ndash1042 2006
[76] M Adamson and K M Pruitt ldquoLactoperoxidase-catalyzedinactivation of hexokinaserdquo Biochimica et Biophysica Acta vol658 no 2 pp 238ndash247 1981
[77] M N Mickelson ldquoGlucose transport in Streptococcus agalac-tiae and its inhibition by lactoperoxidase-thiocyanate-hydrogenperoxiderdquo Journal of Bacteriology vol 132 no 2 pp 541ndash5481977
[78] E L Thomas K A Pera K W Smith and A K ChwangldquoInhibition of Streptococcus mutans by the lactoperoxidaseantimicrobial systemrdquo Infection and Immunity vol 39 no 2 pp767ndash778 1983
[79] J Sermon K Vanoirbeek P De Spiegeleer R Van Houdt AAertsen and C W Michiels ldquoUnique stress response to thelactoperoxidase-thiocyanate enzyme system in Escherichia colirdquoResearch in Microbiology vol 156 no 2 pp 225ndash232 2005
[80] E L Thomas and T M Aune ldquoSusceptibility of Escherichia colito bactericidal action of lactoperoxidase peroxide and iodideor thiocyanaterdquoAntimicrobial Agents andChemotherapy vol 13no 2 pp 261ndash265 1978
[81] K M Pruitt J Tenovuo R W Andrews and T McKaneldquoLactoperoxidase-catalyzed oxidation of thiocyanate polaro-graphic study of the oxidation productsrdquo Biochemistry vol 21no 3 pp 562ndash567 1982
[82] M Huwiler and H Kohler ldquoPseudo-catalytic degradation ofhydrogen peroxide in the lactoperoxidaseH
2O2iodide sys-
temrdquo European Journal of Biochemistry vol 141 no 1 pp 69ndash741984
[83] M Huwiler U Burgi and H Kohler ldquoMechanism of enzymaticand non-enzymatic tyrosine iodination Inhibition by excesshydrogen peroxide andor iodiderdquo European Journal of Bio-chemistry vol 147 no 3 pp 469ndash476 1985
[84] M Morrison G S Bayse and A W Michaels ldquoDeterminationof spectral properties of aqueous I2 and I3- and the equilibriumconstantrdquo Analytical Biochemistry vol 42 no 1 pp 195ndash2011971
[85] E L Thomas and T M Aune ldquoPeroxidase catalyzed oxidationof protein sulfhydrylsmediated by iodinerdquoBiochemistry vol 16no 16 pp 3581ndash3586 1977
[86] W Gottardi ldquoIodine and disinfection theoretical study onmode of action efficiency stability and analytical aspects in theaqueous systemrdquo Archiv der Pharmazie vol 332 no 5 pp 151ndash157 1999
[87] W A Prutz R Kissner W H Koppenol and H RueggerldquoOn the irreversible destruction of reduced nicotinamidenucleotides by hypohalous acidsrdquo Archives of Biochemistry andBiophysics vol 380 no 1 pp 181ndash191 2000
[88] Y Bichsel and U Von Gunten ldquoHypoiodous acid kinetics ofthe buffer-catalyzed disproportionationrdquo Water Research vol34 no 12 pp 3197ndash3203 2000
[89] Y Bichsel and U von Gunten ldquoOxidation of iodide andhypoiodous acid in the disinfection of natural watersrdquo Environ-mental Science and Technology vol 33 no 22 pp 4040ndash40451999
[90] W A Prutz R Kissner T Nauser and W H Koppenol ldquoOnthe oxidation of cytochrome c by hypohalous acidsrdquo Archives ofBiochemistry and Biophysics vol 389 no 1 pp 110ndash122 2001
[91] A Virion J L Michot D Deme and J Pommier ldquoNADPHoxidation catalyzed by the peroxidaseH
2O2system Iodide-
mediated oxidation of NADPH to iodinated NADPrdquo EuropeanJournal of Biochemistry vol 148 no 2 pp 239ndash243 1985
Enzyme Research 13
[92] E L Thomas and T M Aune ldquoCofactor role of iodide in per-oxidase antimicrobial action against Escherichia colirdquo Antimic-robial Agents and Chemotherapy vol 13 no 6 pp 1000ndash10051978
[93] E L Thomas and T M Aune ldquoOxidation of Escherichiacoli sulfhydryl components by the peroxidase-hydrogenperoxide-iodide antimicrobial systemrdquo Antimicrobial Agentsand Chemotherapy vol 13 no 6 pp 1006ndash1010 1978
[94] C B Hamon and S J Klebanoff ldquoA peroxidase-mediatedstreptococcus mitis-dependent antimicrobial system in salivardquoJournal of Experimental Medicine vol 137 no 2 pp 438ndash4501973
[95] R Ihalin J Nuutila V Loimaranta M Lenander J Tenovuoand E-M Lilius ldquoSusceptibility of Fusobacterium nucleatum tokilling by peroxidase-iodide-hydrogen peroxide combinationin buffer solution and in human whole salivardquo Anaerobe vol9 no 1 pp 23ndash30 2003
[96] R Ihalin V Loimaranta M Lenander-Lumikari and J Ten-ovuo ldquoThe effects of different (pseudo)halide substrates onperoxidase-mediated killing of Actinobacillus actinomycetem-comitansrdquo Journal of Periodontal Research vol 33 no 7 pp 421ndash427 1998
[97] E H Bosch H van doorne and S de Vries ldquoThe lactoper-oxidase system the influence of iodide and the chemical andantimicrobial stability over the period of about 18 monthsrdquoJournal of AppliedMicrobiology vol 89 no 2 pp 215ndash224 2000
Iodide 2120583MThiocyanate 120120583MChloride 13 mM
Figure 4 Illustration of the interaction between the biodisponibility of a peroxidase the (pseudo)halogen concentration in plasma in salivaand in milk and the production of oxidant molecules MPO myeloperoxidase SPO salivary peroxidase LPO bovine lactoperoxidaseOClminus hypochlorite and OSCNminus hypothiocyanite Although chloride is the most available substrate compared to thiocyanate bromide andiodide thiocyanate is the most effective substrate for the Compound I and hypothiocyanite could be produced at equal or superior levelscompared to hypohalides
high oxidation of Clminus is possible although it has never beenreported [14] In bovine milk lactoperoxidase is an abundantenzyme and with mean concentrations of Iminus and SCNminus of310 120583gkg and 02ndash15mgkg respectively oxidation is possi-ble [19 39] Nevertheless the relative abundance of SCNminus inall secretions blood and tissues and its better capacity as anelectron donor make it one of the main in vivo substratesof Compound I lactoperoxidase and myeloperoxidase for2-electron oxidation compared to halides [15] In in vitroapplications the ratio between (pseudo)halides regulates theratio of hypohalides generated by the reaction However asSCNminus is the most effective substrate for Compound I itspresence even in small quantities enhances its oxidation[14 35 36]
23 The Peroxidase Cycle Alternatively Compound I canshift to the peroxidase cycle which consists of two sequen-tial one-electron transfers back to the enzyme that yield(i) Compound II and (ii) the native enzyme while thesubstrate is oxidized into a radical (Figure 1) [40ndash42] Theperoxidase cycle is summarized in the following equations
Compound I + AH 997888rarr Compound II + A∙
Compound II + AH 997888rarr Native enzyme + A∙(3)
Compound I is not specific regarding the one-electrondonor it can be exogenous or endogenous and a lot of candi-dates have been described [18 20 43]Hydrogen peroxide canundergo a one-electron oxidation onlywithMPOCompoundI with the formation of superoxide [16 20 44 45]
During the first step of the peroxidase cycle the cationlocated in the porphyrin ring undergoes a one-electronreduction with formation of Compound II and concomitantoxidizing of one one-electron substrate [4 24] Compound II
maintains one oxidizing equivalent in the oxyferryl center[4 24] Finally this latter is reduced back to the native enzymewith the oxidation of a second one-electron donor
The standard reduction potential of the couple Com-pound ICompound II is high and allowed the one-electronoxidation by Compound I of a wide range of substrates[18 20] In contrast the standard reduction potential of thecouple Compound IInative enzyme is low and restrains thenumbers of possible substrates for Compound II [18 20]With the result that (i) the Compound IInative enzymestandard reduction potential is too low to react with halogensand (ii) the nature of substrates strongly influenced theirability to be oxidized by mammalian peroxidase compoundII [2 14 20] therefore when the enzyme is in this state it hasto be first reduced to the ground state before possibly partici-pating to the halogenation cycle and producing antimicrobialmolecules [14] Moreover the reduction of Compound II tothe ground state is the rate-limiting step [45 46] that is theperoxidase cycle interferes with the halogenation cycle andslows down antimicrobial activity [47]
The peroxidase cycle has been described as a possiblecatalytic sink for nitric oxide (NO) [46] but also for hydrogenperoxide in the case of a moderate excess of H
2O2relative
to LPO [24] Increase of NO removal from media evenin presence of Clminus after addition of MPO EPO or LPOand accelerated rates of Compound I and Compound IIreduction in presence of NO show that peroxidases mayregulate the bioavailability of NO [46] In conditions ofhigh excess of hydrogen peroxide relative to LPO and inthe absence of an exogenous electron donor CompoundII is transformed into Compound III which is 3 oxidativeequivalents above the native enzyme In moderate excessconditions Compound III can be partially reconverted intoCompound II and can reenter the peroxidase cycle [24 40]
Enzyme Research 5
Otherwise the enzyme is irreversibly inactivated the hemefraction is cleaved and iron is released [48] In the presenceof an exogenous two-electron donor the enzyme is largelyprotected from hydrogen peroxide because the halogenationcycle is favored Furthermore protection is higher withiodide because oxidized iodide consumes H
2O2to produce
oxygen and iodide in a reaction called the pseudocatalyticactivity of peroxidase [24 40 49]
However thiocyanate can act as a one-electron donor andbe part of the peroxidase cycle with the sequential formationof two thiocyanate radicals [47] With 200120583M SCNminus LPOis predominantly in its native form this indicates that thehalogenation cycle prevails [47]
In the presence of both one- and two-electron donorscompetition for oxidation can occur and favor the halogena-tion or the peroxidase cycle The presence of EDTA inhibitsthe oxidation of iodide due to competition for binding toCompound I [50] The standard reduction potential betweenthe donors favors the molecule with the lowest reductionpotential Thereby the respective reduction potentials of theone- and two-electron oxidation of thiocyanate at very lowpH are 165V and 082V and promote the halogenationcycle [51] In the case of low concentrations of halides orthiocyanate below 10 120583M Iminus or 3 120583M SCNminus CompoundI reacts with any suitable exogenous or endogenous one-electron donor with the subsequent formation of CompoundII and a negligible oxidation rate of halides and thiocyanate[14]
24 Inhibition of the Function of Mammalian Heme Peroxi-dase The function of heme peroxidases can be inhibited inseveral ways that could be classified into three categoriesThe first one could represent an inhibition of the enzymeby (i) molecules or proteins and (ii) external conditionssuch as pH and temperature For example cyanide azidenitrite mercaptomethylimidazole thiourea superoxide highlevels of nitric oxide and high levels of thiocyanate bindto the native enzyme and alter Compound I formation [2046 47 52ndash54] With thiocyanate inhibition is linked to therestriction of the binding site to hydrogen peroxide andthe interaction of SCNminus with a water molecule [23] Highconcentration of H
2O2or Iminus will inactivate irreversibly LPO
with liberation of free iron [48 55] Temperature between73∘C and 83∘C depending on the heating time results inunfolding and inactivation of LPO [19] Extreme pH isinactivating enzymes and at low pH an amino acid groupprobably histidine is protonated which prevents the bindingof H2O2[56] Some proteases such as pepsin and pronase are
able to inactivate LPO by proteolysis but chymotrypsin did itvery slowly and trypsin and thermolysin are not active againstLPO [19]
The second group of inhibitors could concern substancesor proteins which are able to interfere with the catalyticmechanism For example catalase consumes H
2O2and will
stop the formation of Compound I [30 52] Competitionbetween substrates can also interfere with the reaction cyclesuch as SCNminus which competes very effectively with Clminus Brminusand Iminus [52 53] HOCl has the capacity to bind to LPO native
enzyme and convert it into Compound I Above 100 120583MHOCl mediates the destruction of the LPO heme center[57]
The third class could be related to substances or pro-teins which are buffering active molecules produced duringthe catalytic reaction For example presence of thiosulfatethioglycolate glutathione dithiothreitol cysteineNAD(P)Hand tyrosine will reduce the antimicrobial activity throughreacting with OClminus OBrminus OIminus or OSCNminus [52 53 58 59]The enzyme NADH-OSCN oxidoreductase is able to reduceOSCNminus in SCNminus [60]
3 Activity of Lactoperoxidase withThiocyanate andor Iodide
LPO concentrations in cowrsquos milk are around 30mg Lminus1depending on season diet and calving and breeding season[61] LPO extraction from whey or milk is based on awell-developed industrial process [62] Compared to MPOand EPO LPO is easily isolated and manufactured in largequantities As a result cowrsquos milk peroxidase is the favoritemolecule for in vitro or in vivo applications such as con-servation of raw and pasteurized milk storage of emulsionsand cosmetics moisturizing gel and toothpaste in human drymouth veterinary products and preservation of foodstuffs[19 61 63 64]
31 Activity of LPO Related to Hypothiocyanite
311 Mode of Action of Hypothiocyanite Thiocyanate is oxi-dized in a two-electron reaction that yields hypothiocyaniteHypothiocyanite has a pKa of 53 [65] It is more acidic thanhypohalides that have pKas of 75 (HOCl) 86 (HOBr) and106 (HOI) [14 66] All hypo(pseudo)halides (OXminus) are in anacid-base equilibrium association with their correspondingacid hypo(pseudo)halide (HOX) For example in the case ofhypothiocyanite
HOSCN 999447999472 OSCNminus +H+ (4)
The acid form has a higher oxidation potential and ismore soluble in nonpolar media so that it passes throughhydrophobic barriers such as cell membranes more easilybut it is less stable than the basic form (OXminus) [14 66]Hypohalide acids are predominant in acidic to neutral mediaand even in basic conditions for HOBr and HOI whereashypothiocyanite needs a pH below 53 to be predominant inthe acid form [66 67]
SCNminus is the two-electron donorwith the lowest reductionpotential and therefore forms the hypothiocyanite acid withthe lowest oxidative power compared to hypohalous acidsHypohalous acids rank as follows with increasing oxidativestrength OSCNminus lt OIminus lt OBrminus lt OClminus [28 66]These characteristicsmake hypothiocyanite relatively specificregarding its molecular target (Figure 5) that is a thiolmoiety [28 59 68]
6 Enzyme Research
- SH group- NAD(P)H
- SH group- NAD(P)H- Reduced pyridine nucleotide- R-S-R (thioether group)
HOSCNOSCNminus
HOIOIminusI2
- NH2 group
Figure 5 Target group of hypothiocyanite hypoiodite and iodineDue to its low oxidation power hypothiocyanite is relatively specificand is not reactive against all thiols In vivo hypoiodite seems tobe selectively directed against reduced pyridine nucleotide becauseeven the presence of excess glutathione and methionine doesnot thoroughly inhibit their oxidation HOSCNOSCNminus acidic orbasic form of hypothiocyanite HOIOIminus acidic or basic form ofhypoiodite and I
2 iodine
R-S-SCN or R-S-I
LPO R-SH
+ R-S-OHOSCNminusSCNminus SCNminus
or Iminus or Iminusor minusOI
H2O
H2O
2
Figure 6 Illustration of the cofactor role of SCNminus or Iminus Whenthe necessary conditions are fulfilled that is (i) no substratecompetitor for SCNminus or Iminus for binding to lactoperoxidase (ii)enough peroxidase H
2O2and SCNminus or Iminus (iii) enough R-SH
and (iv) no incorporation of SCNminus or Iminus in stable byproducts thequantity of OSCNminus or OIminus produced depends only on the amountof H2O2 SCNminus thiocyanate Iminus iodide H
2O2 hydrogen peroxide
LPO lactoperoxidase R-SH peptide or protein with a thiol moietyR-S-SCN or R-S-I sulfenyl thiocyanate or iodide R-SOH sulfenicacid OSCNminus hypothiocyanite and OIminus hypoiodite
Sulfhydryl oxidation by OSCNminus generates sulfenyl thio-cyanate in equilibrium with sulfenic acid [68]
SCNminus +H2O2+ LPO 997888rarr OSCNminus + LPO
R-SH +OSCNminus 997888rarr R-S-SCN +OHminus
R-S-SCN +H2O 997888rarr R-S-OH + SCNminus +H+
(5)
The cycle of reactions shows that thiocyanate acts likea cofactor for LPO (Figure 6) so that the total number ofoxidized sulfhydryls is independent of SCNminus as long as(i) thiocyanate is not exhausted (ii) thiocyanate is not incompetition with other substrates for the binding to Com-pound I (iii) thiocyanate is not incorporated into an aromaticamino acid (iv) enoughH
2O2is present and (v) thiol moiety
is still available [68 69]Although the target of OSCNminus is a thiol moiety not
all sulfhydryls are equally sensitive to OSCNminus albumincysteine mercaptoethanol dithiothreitol glutathione and 5-thio-2-nitrobenzoic acid are all oxidized but 120573-lactoglobulinis poorly oxidized probably due to a limited accessibility ofsulfhydryls to OSCNminus [68] In some conditions that is thejoint presence of LPO enough H
2O2and SCNminus and after the
oxidation of available sulfhydryls modification of tyrosinetryptophan and histidine protein residues can occur and that
could be linked to the formation of a labile powerful oxidantsuch as sulfur dicyanide [68]
Some authors suggest that (SCN)2is formed during the
enzymatic reaction and then chemically hydrolyzed intohypothiocyanite [14 69 70] However a recent publicationdemonstrates that (SCN)
2cannot be a precursor during the
enzymatic oxidation of SCNminus at neutral pH inmammals [71]Hypothiocyanite is less stable in acid conditions with
high concentrations of SCNminus and in the presence of (SCN)2
and it is thought to break down via the following net reaction[14]
4HOSCN +H2O 997888rarr 3SCNminus + CNOminus + SO
4
2minus+ 6H+ (6)
A recent study based notably on spectroscopic and chro-matographic methods proposes the following net equationwithin the 4ndash7 pH range
3HOSCN +H2O 997888rarr XSO
4
2minus+ XHCN
+ (1 minus X) SO3
2minus+ (1 minus X)CNOminus
+ 2SCNminus + (5 minus X)H+
(7)
The proportions of end anions were different at pH 4 andpH 7 at pH 7 the proportion of CNOminus was higher SCNminusformation was slower and no CNminus was detected [71]
It might seem easier to produce hypothiocyanite chemi-cally in in vitro applications but producing hypothiocyanitechemically from the oxidation of SCNminus by a halogen (Cl
2or
Br2) or by a hypohalous acid (HOCl or HOBr) in basic media
is tricky due to overoxidation of SCNminus [66] The referencemethod in the literature to produce 1- to 2-day stable OSCNminusis by hydrolyzing (SCN)
2in basic conditions [72ndash74]
Hypothiocyanite inhibitors have been described Forexample CNminus a weak acid buffer dissolved carbonate excesshydrogen peroxide hydrofluoric acid metallic ions glyc-erol or ammonium sulfate accelerates the decomposition ofOSCNminus whereas sulfonamide stabilizes it [67 72]
Appropriate concentrations of substrates induce en-hanced activity [75]
312 Biological Activity of Hypothiocyanite The biologicalactivity of hypothiocyanite is summarized in Figure 7
The sulfhydryl moiety is essential for the activityof numerous enzymes and proteins Inhibition of bacte-rial glycolysis through the oxidation of hexokinase gly-ceraldehyde-3-phosphate dehydrogenase (GAPDH) aldo-lase and glucose-6-phosphate dehydrogenase has beenobserved [14 51 65 70 76] Inhibition of respiration andglucose transport is associated with the alteration of cellmembranes or transporters [14 51 65 77] Irreversible inhi-bition is linked to long periods of incubation and bacterialsensitivity depends on the bacterial species and on hypothio-cyanite concentrations [14 51 59] Increased concentrationsof reducing agents such as glutathione and cysteine canreverse the inhibition through buffering hypothiocyaniteand converting the reduced thiol back into sulfhydryl [1478] This defense mechanism is used by Escherichia coli itinduces the CysJ promoter during the stress response to the
Enzyme Research 7
Bacteria
Inhibition of glycolysis Inhibition of respirationInhibition of glucose transport
Possible defense mechanism of bacteria- Increased GSH and cysteine content
Peptide proteinwith SH moiety
HOSCNOSCNminus
- NAD(P)H-dependent reduction of OSCNminus
Figure 7 Biological activity of hypothiocyanite on bacteria and possible defensemechanism of the bacteria Reversible inhibition is observedin that (i) hypothiocyanite is not reactive against all thiols and (ii) if hypothiocyanite is removed or diluted the pathogen recovers Irreversibleinhibition is linked to (i) long period of incubation (ii) the bacterial species and (iii) hypothiocyanite concentration HOSCNOSCNminus acidicor basic form of hypothiocyanite and GSH glutathione
pH lt 6
Influence of iodide concentration
Influence of the pH
1 I2 and high Iminus
I5minusI6
minus
I2I3minus
6 lt pH lt 9
HOII2OHI2I3minus
Iminus + H2O2 + LPO rarr active molecules
I2 (without Iminus)HOIOIminusI2I3
minusHI2Ominus
Figure 8 Illustration of the molecules that can be present after oxidation of iodide by lactoperoxidase in presence of H2O2The active species
depend mainly on the concentration of iodide (upper part) and the pH (lower part) The species with an oxidant power are represented inbold
lactoperoxidase system [79] Another resistance mechanismcould be the NAD(P)H-dependent reduction of OSCNminuswithout any loss of the sulfhydryl compound [14 72 78]Alteration of the bacterial membrane increases the efficacyof hypothiocyanite [80]
Furthermore the activity of the entire system (enzyme +substrates) is known to be more effective than hypothiocyan-ite alone whether enzymatically or chemically producedThis has been explained by the production of short-livedhighly reactive intermediates such as O
2SCNminus and O
3SCNminus
by the enzyme or by the oxidation of OSCNminus in conditionsof excess H
2O2[65 73 81] The activity of hypothiocyanite
has been described against bacteria such as Actinomyces sppBacillus cereus Lactobacillus spp Staphylococcus albus Saureus Streptococcus spp Escherichia coli Legionella pneu-mophila Salmonella typhimurium Pseudomonas fluorescensP aeruginosa Campylobacter jejuni C coli and Listeriamonocytogenes [14 32] Reversible inhibition is observed
when cells recover after OSCNminus is depleted [14 59] Irre-versible inhibition is obtained with long-term incubation andhigh level of OSCNminus [59] Higher concentration of SCNminuscompared to Iminus is necessary to obtain inhibition against E coliand accumulation of OSCNminus is observed as it is not reactiveagainst all thiols [59]Therefore the activity of the SCNminus-LPOsystem appears to be more bacteriostatic than bactericidal
32 Activity of LPO Related to Oxidized Iodide
321 Chemistry of Oxidized Iodide Iodide is oxidized byCompound I through a single two-electron transfer thatyields oxidized Iminus in the form of I
2or HOI [14 24 82ndash85]
The active agent is composed of a mixture of species that arenot yet formally detailed due to the very complex behaviorand stability of I
2and HOI in aqueous environments that
strongly depend on pH values and iodide concentrations[66 82 83 86]
8 Enzyme Research
Based on the inorganic chemistry of iodine in waterand literature on enzymatic oxidation of iodide the activemolecules have been described as follows (Figure 8)
(i) Under pH 6 and in the presence of iodide only I2
Iminus and I3
minus are present and the only active moleculeis I2 I2concentrations decrease with increasing
concentrations of Iminus At an initial 1mM I2 with Iminus
concentrations ranging from 1mM to 100mM I2
concentrations fall from almost 1mM to 001mM asdescribed by the following association reaction [2482 83 86]
I2+ Iminus 999447999472 I
3
minus (8)
(ii) In solution within a 6ndash9 pH range and with a max-imum 1mM iodide a mixture of HOII
2OHI2I3
minus
is formed in which I3
minus is not active and I2OH is
probably less reactive than HOI or I2[86 87] If
Iminus concentrations are above 10mM I3
minus representsthe main species formed and the concentration ofactive molecules relatively drops The mechanism issummarized in the following net equations
HOI + Iminus +H+ 999447999472 I2OHminus +H+
999447999472 I2+H2O 999447999472 I
2+ Iminus 999447999472 I
3
minus
(9)
(iii) In iodine solution without iodide or when availableiodide has been oxidized the number of I
2-derived
molecules decreases with decreasing I2concentra-
tions At 1000 120583M I2 with pH-related ratios five
relevant species are observed (I2 HOI I
3
minus HI2Ominus
and OIminus) At 10 120583M I2 the main species are only I
2
HOI and OIminus and HOI could represent up to 90 ofthe active oxidant molecules at pH 8-9 [86] Below apH of 106 the following reactions are involved
I2+H2O 999447999472 HOI + Iminus +H+ (hydrolysis of I
2)
I2+ Iminus 999447999472 I
3
minus(triiodide formation independent of pH)
(10)
(iv) At high Iminus and 1 I2concentrations as in Lugol
solution I5
minus and I6
minus are formed and represent 82of the active oxidative agents [86] after the followingreaction
I3
minus+ I2999447999472 I5
minus(pentaiodide formation)
2I3
minus999447999472 I6
2minus(dimerization of I
3
minus)
(11)
The stability of HOI and I2is linked to their dispro-
portionation in iodate which has no oxidative activity inneutral and basic pH conditions [86]The disproportionationreactions read as follows
3HOI 999447999472 IO3
minus+ 2Iminus + 3H+ (disproportionation of HOI)
3I2999447999472 IO
3
minus+ 5Iminus + 6H+ (disproportionation of I
2)
(12)
I2stability increases at higher pHvalues andhigher iodide
concentrations [86] In drinking water HOI disproportion-ation is slow and varies substantially HOI has a half-life of4 days to 35 years depending on (i) the initial level of HOIthat speeds its decomposition and (ii) the presence of boratephosphate or carbonate that catalyzes its decomposition [8889]
322 Mode of Action of Oxidized Iodide The oxidativestrength of I
2is between that of the corresponding hypo-
halous acid HOI and the hypoiodite ion OIminus and ranks asfollows 0485V (OIminus) lt 0536V (I
2) lt 0987V (HOI) [66]
HOI reacts through very rapid oxidation of thiolgroups oxidation of NAD(P)H oxidation of 120573-nicotinamidemononucleotide direct reaction with thioether groupsthrough sulfoxidation and slow oxidation of the aminemoiety (Figure 5) [87 90 91] At low Iminus concentrationsiodination of tyrosine residues is catalyzed by the enzyme[14] In a cellular environment HOI seems to be more selec-tively directed against the degradation of reduced pyridinenucleotides thanHOCL andHOBr because even the presenceof excess glutathione methionine or oxidized glutathionedoes not thoroughly inhibit their oxidation [87]
In some conditions that is (i) enough iodide H2O2 and
peroxidase (ii) no accumulation of oxidized iodide and (iii)no incorporation of iodide into stable byproducts such astyrosine residues iodide acts as a cofactor (Figure 6) andthe proportion of oxidized sulfhydryls is proportional to theamount of H
2O2as described below [85 92]
2Iminus +H2O2+ LPO (native enzyme)
997888rarr I2+ 2H2O + LPO (native enzyme)
R-SH + I2997888rarr R-S-I + Iminus +H+
R-S-I+H2O 997888rarr R-S-OH + Iminus +H+
(13)
In the case of high concentrations of Iminus andor H2O2
inhibition of tyrosine iodation has been observed [83] andrelated to the pseudocatalytic redox degradation of H
2O2
with formation of O2when excessive H
2O2is present (reac-
tion 1) and production of I3
minus when excessive amounts of Iminusare present (reaction 2)
I2+H2O 997888rarr O
2+ 2Iminus + 2H+ (reaction 1)
I2+ Iminus 999447999472 I
3
minus(reaction 2)
(14)
Both reactions deplete the amount of the active oxidizingagent I
2 In the absence of tyrosine oxidized iodide reacts
with nucleophilic molecules such as Iminus Clminus or OHminus to formI2 I3
minus ICl ICl2 IOH and I
2OH [82] Some anions such as
Clminus HPO4
minus or OHminus reduce the amount of I2I3
minus but thiseffect is inversely proportional to the concentration of Iminusabove pH 9 I
2is hydrolyzed and IO
3
minus is formed [82]HOI can be produced chemically through oxidation of Iminus
by Cl2or O3 with a short half-life due to overoxidation of
HOI byCl2andO
3[89] and through oxidation of Iminus byHOCl
HOBr or NH2Cl with a longer half-life [87 89]
Enzyme Research 9
Bacteria
Inhibition of glycolysis Inhibition of respirationInhibition of glucose transportInhibition of the pentose phosphate pathway
- Peptide protein with- SH moiety- thioether moiety- NAD(P)H
HOIOIminus
I2
Figure 9 Biological activity of hypoiodite or iodine on bacteria Irreversible inhibition is observed and could be linked to (i) oxidation ofthiol groups NAD(P)H and thioether groups (ii) high reactivity of HOII
2against thiol and reduced nicotinamide nucleotides and (iii) the
incorporation of iodide in tyrosine residue of protein (iodination of protein) HOIOIminus acid or basic form of hypoiodite and I2 iodine
323 Biological Action of Oxidized Iodide The biologicalaction of oxidized iodide (Figure 9) is similar to that ofhypothiocyanite but differs in that (i) the reactivity of oxi-dized iodide is complete against thiol group and (ii) cells didnot recover after removing of oxidized iodide [59]
Due to the cofactor role of Iminus inhibition of respirationin Escherichia coli in the presence of LPO H
2O2 and Iminus is
complete with only 10120583M NaI whereas 100 120583M of solely I2
is necessary to obtain complete inhibition This is directlyrelated to the oxidation of sulfhydryls not to the percentageof iodine incorporation [92 93]
E coli seems to be more sensitive if the bacteria areincubated together with the entire system (enzyme H
2O2
and iodide) rather than adding several minutes after mixingthe enzyme with its substrates This could be linked to theformation of an unstable reactive intermediate [52]
The activity of the Iminus peroxidase system is more effectiveagainst E coli than the SCNminus system in that lower Iminusconcentrations are necessary all sulfhydryls are oxidized andcells do not recover even if the amount of I
2is not sufficient
to oxidize all SH groups [59 80] Against L acidophilushigh non physiological amounts of Iminus are necessary to obtaininhibitionwhereas small concentrations of SCNminus are effective[70]
CNminus azide EDTA and SCNminus inhibit the formation ofoxidized iodide [50 52] Increased pH values and increasedamounts of thiol and NAD(P)H compounds reduce theactivity of the iodide peroxidase system [52]
LPO-H2O2-Iminus in presence of Streptococcus mitis is active
against Staphylococcus aureus and E coli [94] LPO-H2O2-
Iminus is active against Micrococcus S aureus Listeria monocy-togenes Bacillus cereus E coli and Candida albicans [12 1980] In the presence of other peroxidases the Iminus peroxidasesystem is active against Schistosoma mansoni Fusariumnucleatum andActinobacillus actinomycetemcomitans [31 9596] Compared to SCNminus Iminus-LPO shows bactericidal activities[14 19 80]
33 Activity of LPO Related to Hypoiodite and Hypothiocyan-ite The combination of SCNminus with Iminus in the lactoperoxidasesystem has been poorly studied Tackling the enzymaticmechanism is tricky and contradictory results have been
found about microbial activity in the concomitant presenceof SCNminus and Iminus
In the presence of SCNminus and Iminus there is competitionbetween the two substrates for oxidation by lactoperoxidase[14 36] Iminus alone exhibits bactericidal activity but an SCNminusIminusratio of 01 inhibits that bactericidal effect and an SCNminusIminusratio of 1 antagonizes it due to competition for oxidation andfaster decomposition of HOSCN in the presence of Iminus [14]Against A actinomycetemcomitans the peroxidase systemwith Iminus Clminus or a combination of Iminus and Clminus is effective butaddition of SCNminus cancels the antibacterial effect [96] On theother hand a synergistic or unaffected effect of iodide in theSCNminus-H
2O2-LPO system has been shown against Candida
albicans E coli S aureusAspergillus niger and Pseudomonasaeruginosa [19 97]
4 Conclusion
Themolecular evolution of heme peroxidases and the preser-vation of their catalytic domain [6] show that the productionof strong oxidants is a powerful part of the nonimmunedefense mechanisms against pathogenic bacteria fungi orparasite which made the use of those enzymes in practicalapplications worthwhile
The enzymatic reactions involving mammalian peroxi-dases are complex and various molecules can promote orreduce dramatically the antibacterial activity of the per-oxidase system In order to favor the halogenation cyclerequired in in vitro and in vivo antimicrobial applicationsseveral points have to be taken into account (i) to avoid thepresence of competitors to iodide or thiocyanate for bindingto Compound I and to avoid the presence of inhibitorsof the enzyme or of active molecules (ii) to avoid excessH2O2concentration which is able to destruct the enzyme
and to react with iodine or hypoiodite with loosing of activemolecules (iii) to favor the presence of hypoiodite instead ofiodine due to the association reaction of iodine with iodide(iv) to avoid excess concentration of thiocyanate which caninhibit formation of Compound I (v) to use the entiresystem (enzyme + substrates) instead of active moleculesalone (vi) to favor moderate acid pH when hypothiocyaniteis the active molecule (vii) for bactericidal fungicidal or
10 Enzyme Research
parasitical applications the use of iodide has to be preferred(viii) the use of combined presence of iodide and thiocyanatehas to be checked carefully for efficacy and (ix) to favor thecofactor role of iodide or thiocyanate
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] P J OrsquoBrien ldquoPeroxidasesrdquoChemico-Biological Interactions vol129 no 1-2 pp 113ndash139 2000
[2] W Jantschko P G Furtmuller M Allegra et al ldquoRedoxintermediates of plant and mammalian peroxidases a compar-ative transient-kinetic study of their reactivity toward indolederivativesrdquo Archives of Biochemistry and Biophysics vol 398no 1 pp 12ndash22 2002
[3] S Kimura and M Ikeda-Saito ldquoHuman myeloperoxidase andthyroid peroxidase two enzymes with separate and distinctphysiological functions are evolutionarily related membersof the same gene familyrdquo Proteins Structure Function andGenetics vol 3 no 2 pp 113ndash120 1988
[4] G Battistuzzi M Bellei C A Bortolotti and M Sola ldquoRedoxproperties of heme peroxidasesrdquo Archives of Biochemistry andBiophysics vol 500 no 1 pp 21ndash36 2010
[5] M Zamocky C Jakopitsch P G Furtmuller C Dunand and CObinger ldquoThe peroxidase-cyclooxygenase superfamily recon-structed evolution of critical enzymes of the innate immunesystemrdquo Proteins Structure Function and Genetics vol 72 no2 pp 589ndash605 2008
[6] H Daiyasu and H Toh ldquoMolecular evolution of the myeloper-oxidase familyrdquo Journal of Molecular Evolution vol 51 no 5 pp433ndash445 2000
[7] D Serteyn S Grulke T Franck A Mouithys-Mickalad andG Deby-Dupont ldquoNeutrophile myeloperoxidase protectiveenzyme with strong oxidative activitiesrdquo Annales de MedecineVeterinaire vol 147 no 2 pp 79ndash93 2003
[8] S C Whitman S L Hazen D B Miller R A HegeleJ W Heinecke and M W Huff ldquoModification of type IIIVLDL their remnants and VLDL from apoE- knockout miceby p-hydroxyphenylacetaldehyde a product of myeloperox-idase activity causes marked cholesteryl ester accumulationin macrophagesrdquo Arteriosclerosis Thrombosis and VascularBiology vol 19 no 5 pp 1238ndash1249 1999
[9] T J Barrett and C L Hawkins ldquoHypothiocyanous acid benignor deadlyrdquo Chemical Research in Toxicology vol 25 no 2 pp263ndash273 2012
[10] M M Lloyd D M van Reyk M J Davies and C L HawkinsldquoHypothiocyanous acid is a more potent inducer of apoptosisand protein thiol depletion in murine macrophage cells thanhypochlorous acid or hypobromous acidrdquo Biochemical Journalvol 414 no 2 pp 271ndash280 2008
[11] J Wang and A Slungaard ldquoRole of eosinophil peroxidase inhost defense and disease pathologyrdquo Archives of Biochemistryand Biophysics vol 445 no 2 pp 256ndash260 2006
[12] M Ahariz and P Courtois ldquoCandida albicans susceptibility tolactoperoxidase-generated hypoioditerdquo Clinical Cosmetic andInvestigational Dentistry vol 2 pp 69ndash78 2010
[13] A Welk C Meller R Schubert C Schwahn A Kramerand H Below ldquoEffect of lactoperoxidase on the antimicrobialeffectiveness of the thiocyanate hydrogen peroxide combinationin a quantitative suspension testrdquo BMC Microbiology vol 9article 134 2009
[14] K M Pruitt and J O Tenovuo Eds The Lactoperoxidase Sys-tem Chemistry and Biological Significance vol 27 of Immunol-ogy Series Marcel Dekker New York NY USA 1985
[15] P G Furtmuller W Jantschko G Regelsberger C JakopitschJ Arnhold and C Obinger ldquoReaction of lactoperoxidasecompound I with halides and thiocyanaterdquo Biochemistry vol41 no 39 pp 11895ndash11900 2002
[16] P G Furtmuller U Burner and C Obinger ldquoReaction ofmyeloperoxidase compound I with chloride bromide iodideand thiocyanaterdquo Biochemistry vol 37 no 51 pp 17923ndash179301998
[17] J Arnhold E Monzani P G Furtmuller M Zederbauer LCasella and C Obinger ldquoKinetics and thermodynamics ofhalide and nitrite oxidation by mammalian heme peroxidasesrdquoEuropean Journal of Inorganic Chemistry no 19 pp 3801ndash38112006
[18] M J Davies C L Hawkins D I Pattison and M D ReesldquoMammalian heme peroxidases from molecular mechanismsto health implicationsrdquo Antioxidants and Redox Signaling vol10 no 7 pp 1199ndash1234 2008
[19] J N de Wit and A C M van Hooydonk ldquoStructure functionsand applications of lactoperoxidase in natural antimicrobialsystemsrdquo Nederlands melk en Zuiveltijdschrift vol 50 no 2 pp227ndash244 1996
[20] P G Furtmuller M Zederbauer W Jantschko et al ldquoActivesite structure and catalytic mechanisms of human peroxidasesrdquoArchives of Biochemistry and Biophysics vol 445 no 2 pp 199ndash213 2006
[21] M Zederbauer P G Furtmuller S Brogioni C JakopitschG Smulevich and C Obinger ldquoHeme to protein linkages inmammalian peroxidases impact on spectroscopic redox andcatalytic propertiesrdquo Natural Product Reports vol 24 no 3 pp571ndash584 2007
[22] G Battistuzzi M Bellei J Vlasits et al ldquoRedox thermodynam-ics of lactoperoxidase and eosinophil peroxidaserdquo Archives ofBiochemistry and Biophysics vol 494 no 1 pp 72ndash77 2010
[23] I A Sheikh A Singh N Singh et al ldquoStructural evidence ofsubstrate specificity inmammalian peroxidases structure of thethiocyanate complex with lactoperoxidase and its interactionsat 24 a 24 A resolutionrdquo The Journal of Biological Chemistryvol 284 no 22 pp 14849ndash14856 2009
[24] H Kohler and H Jenzer ldquoInteraction of lactoperoxidase withhydrogen peroxide Formation of enzyme intermediates andgeneration of free radicalsrdquo Free Radical Biology and Medicinevol 6 no 3 pp 323ndash339 1989
[25] P G Furtmuller U Burner W Jantschko G Regelsberger andC Obinger ldquoTwo-electron reduction and one-electron oxida-tion of organic hydroperoxides by human myeloperoxidaserdquoFEBS Letters vol 484 no 2 pp 139ndash143 2000
[26] A Taurog M L Dorris and D R Doerge ldquoMechanism ofsimultaneous iodination and coupling catalyzed by thyroidperoxidaserdquo Archives of Biochemistry and Biophysics vol 330no 1 pp 24ndash32 1996
[27] J E Erman L B Vitello J Matthew Mauro and J KrautldquoDetection of an oxyferryl porphyrin 120587-cation-radical interme-diate in the reaction between hydrogen peroxide and a mutant
Enzyme Research 11
yeast cytochrome c peroxidase Evidence for tryptophan-191involvement in the radical site of compound Irdquo Biochemistryvol 28 no 20 pp 7992ndash7995 1989
[28] M T Ashby ldquoInorganic chemistry of defensive peroxidases inthe human oral cavityrdquo Journal of Dental Research vol 87 no10 pp 900ndash914 2008
[29] J D Chandler and B J Day ldquoThiocyanate a potentially usefultherapeutic agent with host defense and antioxidant propertiesrdquoBiochemical Pharmacology vol 84 no 11 pp 1381ndash1387 2012
[30] E C Jong W R Henderson and S J Klebanoff ldquoBactericidalactivity of eosinophil peroxidaserdquo Journal of Immunology vol124 no 3 pp 1378ndash1382 1980
[31] E C Jong A A F Mahmoud and S J Kelbanoff ldquoPeroxidase-mediated toxicity to schistosomula of Schistosoma mansonirdquoJournal of Immunology vol 126 no 2 pp 468ndash471 1981
[32] L M Wolfson and S S Sumner ldquoAntibacterial activity of thelactoperoxidase system a reviewrdquo Journal of Food Protectionvol 56 no 10 pp 887ndash892 1993
[33] J Arnhold P G Furtmuller G Regelsberger and C ObingerldquoRedox properties of the couple compound Inative enzyme ofmyeloperoxidase and eosinophil peroxidaserdquo European Journalof Biochemistry vol 268 no 19 pp 5142ndash5148 2001
[34] P G Furtmuller J Arnhold W Jantschko M Zederbauer CJakopitsch and C Obinger ldquoStandard reduction potentials ofall couples of the peroxidase cycle of lactoperoxidaserdquo Journalof Inorganic Biochemistry vol 99 no 5 pp 1220ndash1229 2005
[35] C J van Dalen M W Whitehouse C C Winterbourn and AJ Kettle ldquoThiocyanate and chloride as competing substrates formyeloperoxidaserdquo Biochemical Journal vol 327 no 2 pp 487ndash492 1997
[36] A Slungaard and J R Mahoney Jr ldquoThiocyanate is the majorsubstrate for eosinophil peroxidase in physiologic fluids impli-cations for cytotoxicityrdquoThe Journal of Biological Chemistry vol266 no 8 pp 4903ndash4910 1991
[37] J Tenovuo ldquoAntimicrobial function of human salivamdashhowimportant is it for oral healthrdquoActaOdontologica Scandinavicavol 56 no 5 pp 250ndash256 1998
[38] R Ihalin V Loimaranta and J Tenovuo ldquoOrigin structure andbiological activities of peroxidases in human salivardquo Archives ofBiochemistry and Biophysics vol 445 no 2 pp 261ndash268 2006
[39] J A Rooke J F Flockhart and N H Sparks ldquoThe potentialfor increasing the concentrations of micro-nutrients relevant tohuman nutrition inmeat milk and eggsrdquo Journal of AgriculturalScience vol 148 no 5 pp 603ndash614 2010
[40] H Kohler A Taurog and H B Dunford ldquoSpectral studieswith lactoperoxidase and thyroid peroxidase interconversionsbetween native enzyme compound II and compound IIIrdquoArchives of Biochemistry and Biophysics vol 264 no 2 pp 438ndash449 1988
[41] I Yamazaki H S Mason and L Piette ldquoIdentification byelectron paramagnetic resonance spectroscopy of free radicalsgenerated from substrates by peroxidaserdquoThe Journal of Biolog-ical Chemistry vol 235 pp 2444ndash2449 1960
[42] B Chance ldquoThe kinetics and stoichiometry of the transitionfrom the primary to the secondary peroxidase peroxide com-plexesrdquo Archives of Biochemistry and Biophysics vol 41 no 2pp 416ndash424 1952
[43] K M Pruitt B Mansson-Rahemtulla D C Baldone andF Rahemtulla ldquoSteady-state kinetics of thiocyanate oxidationcatalyzed by human salivary peroxidaserdquo Biochemistry vol 27no 1 pp 240ndash245 1988
[44] B G J M Bolscher and R Wever ldquoA kinetic study of thereaction between humanmyeloperoxidase hydroperoxides andcyanide inhibition by chloride and thiocyanaterdquo Biochimica etBiophysica Acta Protein Structure and Molecular Enzymologyvol 788 no 1 pp 1ndash10 1984
[45] L A Marquez J T Huang and H Brian Dunford ldquoSpectraland kinetic studies on the formation of myeloperoxidase com-pounds I and II roles of hydrogen peroxide and superoxiderdquoBiochemistry vol 33 no 6 pp 1447ndash1454 1994
[46] HMAbu-Soud and S LHazen ldquoNitric oxide is a physiologicalsubstrate for mammalian peroxidasesrdquoThe Journal of BiologicalChemistry vol 275 no 48 pp 37524ndash37532 2000
[47] Y R Tahboub S Galijasevic M P Diamond and H MAbu-Soud ldquoThiocyanate modulates the catalytic activity ofmammalian peroxidasesrdquo Journal of Biological Chemistry vol280 no 28 pp 26129ndash26136 2005
[48] H Jenzer W Jones and H Kohler ldquoOn the molecularmechanismof lactoperoxidase-catalyzedH
2O2metabolism and
irreversible enzyme inactivationrdquo The Journal of BiologicalChemistry vol 261 no 33 pp 15550ndash15556 1986
[49] R P Magnusson A Taurog and M L Dorris ldquoMechanism ofiodide-dependent catalatic activity of thyroid peroxidase andlactoperoxidaserdquo The Journal of Biological Chemistry vol 259no 1 pp 197ndash205 1984
[50] D K Bhattacharyya U Bandyopadhyay and R K BanerjeeldquoEDTA inhibits lactoperoxidase-catalyzed iodide oxidation byacting as an electron-donor and interacting near the iodidebinding siterdquoMolecular and Cellular Biochemistry vol 162 no2 pp 105ndash111 1996
[51] C L Hawkins ldquoThe role of hypothiocyanous acid (HOSCN) inbiological systems HOSCN in biological systemsrdquo Free RadicalResearch vol 43 no 12 pp 1147ndash1158 2009
[52] S J Klebanoff ldquoIodination of bacteria a bactericidal mecha-nismrdquo Journal of Experimental Medicine vol 126 no 6 pp1063ndash1078 1967
[53] S J Klebanoff ldquoMyeloperoxidase-halide-hydrogen peroxideantibacterial systemrdquo Journal of Bacteriology vol 95 no 6 pp2131ndash2138 1968
[54] R K Banerjee and A G Datta ldquoSalivary peroxidasesrdquoMolecu-lar and Cellular Biochemistry vol 70 no 1 pp 21ndash29 1986
[55] MHuwiler H Jenzer andHKohler ldquoThe role of compound IIIin reversible and irreversible inactivation of lactoperoxidaserdquoEuropean Journal of Biochemistry vol 158 no 3 pp 609ndash6141986
[56] R Wever W M Kast J H Kasinoedin and R Boelens ldquoTheperoxidation of thiocyanate catalysed by myeloperoxidase andlactoperoxidaserdquo Biochimica et Biophysica Acta (BBA)ProteinStructure and Molecular vol 709 no 2 pp 212ndash219 1982
[57] C E A Souza D Maitra G M Saed et al ldquoHypochlorousacid-induced heme degradation from lactoperoxidase as anovel mechanism of free iron release and tissue injury ininflammatory diseasesrdquo PLoS ONE vol 6 no 11 Article IDe27641 2011
[58] J Carlsson ldquoBactericidal effect of hydrogen peroxide is pre-vented by the lactoperoxidase-thiocyanate system under anaer-obic conditionsrdquo Infection and Immunity vol 29 no 3 pp 1190ndash1192 1980
[59] E L Thomas and T M Aune ldquoLactoperoxidase peroxidethiocyanate antimicrobial system correlation of sulfhydryloxidation with antimicrobial actionrdquo Infection and Immunityvol 20 no 2 pp 456ndash463 1978
12 Enzyme Research
[60] J Carlsson Y Iwami and T Yamada ldquoHydrogen peroxideexcretion by oral streptococci and effect of lactoperoxidase-thiocyanate-hydrogen peroxiderdquo Infection and Immunity vol40 no 1 pp 70ndash80 1983
[61] K D Kussendrager and A C M van Hooijdonk ldquoLactoperox-idase physico-chemical properties occurrence mechanism ofaction and applicationsrdquoTheBritish Journal of Nutrition vol 84supplement 1 pp S19ndashS25 2000
[62] J P Perraudin ldquoProteines a activites biologiques lactoferrineet lactoperoxydase Connaissances recemment acquises et tech-nologies drsquoobtentionrdquo Lait vol 71 no 2 pp 191ndash211 1991
[63] J-W Boots and R Floris ldquoLactoperoxidase From catalyticmechanism to practical applicationsrdquo International Dairy Jour-nal vol 16 no 11 pp 1272ndash1276 2006
[64] A C M van Hooijdonk K D Kussendrager and J M SteijnsldquoIn vivo antimicrobial and antiviral activity of components inbovine milk and colostrum involved in non-specific defencerdquoBritish Journal of Nutrition vol 84 supplement 1 pp S127ndashS1342000
[65] D M Hogg and G R Jago ldquoThe antibacterial action of lac-toperoxidaseThe nature of the bacterial inhibitorrdquo BiochemicalJournal vol 117 no 4 pp 779ndash790 1970
[66] M T Ashby ldquoHypothiocyaniterdquo in Advances in InorganicChemistry R van Eldik and I-B Ivana Eds chapter 8 pp 263ndash303 Academic Press New York NY USA 2012
[67] E L Thomas ldquoLactoperoxidase-catalyzed oxidation of thio-cyanate equilibria between oxidized forms of thiocyanaterdquoBiochemistry vol 20 no 11 pp 3273ndash3280 1981
[68] T M Aune and E LThomas ldquoOxidation of protein sulfhydrylsby products of peroxidase-catalyzed oxidation of thiocyanateionrdquo Biochemistry vol 17 no 6 pp 1005ndash1010 1978
[69] T M Aune and E L Thomas ldquoAccumulation of hypothiocyan-ite ion during peroxidase-catalyzed oxidation of thiocyanateionrdquo European Journal of Biochemistry vol 80 no 1 pp 209ndash214 1977
[70] J D Oram and B Reiter ldquoThe inhibition of streptococci bylactoperoxidase thiocyanate and hydrogen peroxideThe effectof the inhibitory system on susceptible and resistant strains ofgroup N streptococcirdquo Biochemical Journal vol 100 no 2 pp373ndash381 1966
[71] J Kalmar K L Woldegiorgis B Biri and M T AshbyldquoMechanism of decomposition of the human defense factorhypothiocyanite near physiological pHrdquo Journal of the Ameri-can Chemical Society vol 133 no 49 pp 19911ndash19921 2011
[72] H Hoogendoorn J P PiessensW Scholtes and L A StoddardldquoHypothiocyanite ion the inhibitor formed by the system lac-toperoxidase thiocyanate hydrogen peroxide I Identification ofthe inhibiting compoundrdquoCaries Research vol 11 no 2 pp 77ndash84 1977
[73] L Bjorck and O Claesson ldquoCorrelation between concentrationof hypothiocyanate and antibacterial effect of the lactoperoxi-dase system against Escherichia colirdquo Journal of Dairy Sciencevol 63 no 6 pp 919ndash922 1980
[74] P Nagy S S Alguindigue and M T Ashby ldquoLactoperoxidase-catalyzed oxidation of thiocyanate by hydrogen peroxide areinvestigation of hypothiocyanite by nuclear magnetic reso-nance and optical spectroscopyrdquo Biochemistry vol 45 no 41pp 12610ndash12616 2006
[75] Y Adolphe M Jacquot M Linder A-M Revol-Junelles andJ-B Milliere ldquoOptimization of the components concentrationsof the lactoperoxidase system by RSMrdquo Journal of AppliedMicrobiology vol 100 no 5 pp 1034ndash1042 2006
[76] M Adamson and K M Pruitt ldquoLactoperoxidase-catalyzedinactivation of hexokinaserdquo Biochimica et Biophysica Acta vol658 no 2 pp 238ndash247 1981
[77] M N Mickelson ldquoGlucose transport in Streptococcus agalac-tiae and its inhibition by lactoperoxidase-thiocyanate-hydrogenperoxiderdquo Journal of Bacteriology vol 132 no 2 pp 541ndash5481977
[78] E L Thomas K A Pera K W Smith and A K ChwangldquoInhibition of Streptococcus mutans by the lactoperoxidaseantimicrobial systemrdquo Infection and Immunity vol 39 no 2 pp767ndash778 1983
[79] J Sermon K Vanoirbeek P De Spiegeleer R Van Houdt AAertsen and C W Michiels ldquoUnique stress response to thelactoperoxidase-thiocyanate enzyme system in Escherichia colirdquoResearch in Microbiology vol 156 no 2 pp 225ndash232 2005
[80] E L Thomas and T M Aune ldquoSusceptibility of Escherichia colito bactericidal action of lactoperoxidase peroxide and iodideor thiocyanaterdquoAntimicrobial Agents andChemotherapy vol 13no 2 pp 261ndash265 1978
[81] K M Pruitt J Tenovuo R W Andrews and T McKaneldquoLactoperoxidase-catalyzed oxidation of thiocyanate polaro-graphic study of the oxidation productsrdquo Biochemistry vol 21no 3 pp 562ndash567 1982
[82] M Huwiler and H Kohler ldquoPseudo-catalytic degradation ofhydrogen peroxide in the lactoperoxidaseH
2O2iodide sys-
temrdquo European Journal of Biochemistry vol 141 no 1 pp 69ndash741984
[83] M Huwiler U Burgi and H Kohler ldquoMechanism of enzymaticand non-enzymatic tyrosine iodination Inhibition by excesshydrogen peroxide andor iodiderdquo European Journal of Bio-chemistry vol 147 no 3 pp 469ndash476 1985
[84] M Morrison G S Bayse and A W Michaels ldquoDeterminationof spectral properties of aqueous I2 and I3- and the equilibriumconstantrdquo Analytical Biochemistry vol 42 no 1 pp 195ndash2011971
[85] E L Thomas and T M Aune ldquoPeroxidase catalyzed oxidationof protein sulfhydrylsmediated by iodinerdquoBiochemistry vol 16no 16 pp 3581ndash3586 1977
[86] W Gottardi ldquoIodine and disinfection theoretical study onmode of action efficiency stability and analytical aspects in theaqueous systemrdquo Archiv der Pharmazie vol 332 no 5 pp 151ndash157 1999
[87] W A Prutz R Kissner W H Koppenol and H RueggerldquoOn the irreversible destruction of reduced nicotinamidenucleotides by hypohalous acidsrdquo Archives of Biochemistry andBiophysics vol 380 no 1 pp 181ndash191 2000
[88] Y Bichsel and U Von Gunten ldquoHypoiodous acid kinetics ofthe buffer-catalyzed disproportionationrdquo Water Research vol34 no 12 pp 3197ndash3203 2000
[89] Y Bichsel and U von Gunten ldquoOxidation of iodide andhypoiodous acid in the disinfection of natural watersrdquo Environ-mental Science and Technology vol 33 no 22 pp 4040ndash40451999
[90] W A Prutz R Kissner T Nauser and W H Koppenol ldquoOnthe oxidation of cytochrome c by hypohalous acidsrdquo Archives ofBiochemistry and Biophysics vol 389 no 1 pp 110ndash122 2001
[91] A Virion J L Michot D Deme and J Pommier ldquoNADPHoxidation catalyzed by the peroxidaseH
2O2system Iodide-
mediated oxidation of NADPH to iodinated NADPrdquo EuropeanJournal of Biochemistry vol 148 no 2 pp 239ndash243 1985
Enzyme Research 13
[92] E L Thomas and T M Aune ldquoCofactor role of iodide in per-oxidase antimicrobial action against Escherichia colirdquo Antimic-robial Agents and Chemotherapy vol 13 no 6 pp 1000ndash10051978
[93] E L Thomas and T M Aune ldquoOxidation of Escherichiacoli sulfhydryl components by the peroxidase-hydrogenperoxide-iodide antimicrobial systemrdquo Antimicrobial Agentsand Chemotherapy vol 13 no 6 pp 1006ndash1010 1978
[94] C B Hamon and S J Klebanoff ldquoA peroxidase-mediatedstreptococcus mitis-dependent antimicrobial system in salivardquoJournal of Experimental Medicine vol 137 no 2 pp 438ndash4501973
[95] R Ihalin J Nuutila V Loimaranta M Lenander J Tenovuoand E-M Lilius ldquoSusceptibility of Fusobacterium nucleatum tokilling by peroxidase-iodide-hydrogen peroxide combinationin buffer solution and in human whole salivardquo Anaerobe vol9 no 1 pp 23ndash30 2003
[96] R Ihalin V Loimaranta M Lenander-Lumikari and J Ten-ovuo ldquoThe effects of different (pseudo)halide substrates onperoxidase-mediated killing of Actinobacillus actinomycetem-comitansrdquo Journal of Periodontal Research vol 33 no 7 pp 421ndash427 1998
[97] E H Bosch H van doorne and S de Vries ldquoThe lactoper-oxidase system the influence of iodide and the chemical andantimicrobial stability over the period of about 18 monthsrdquoJournal of AppliedMicrobiology vol 89 no 2 pp 215ndash224 2000
Otherwise the enzyme is irreversibly inactivated the hemefraction is cleaved and iron is released [48] In the presenceof an exogenous two-electron donor the enzyme is largelyprotected from hydrogen peroxide because the halogenationcycle is favored Furthermore protection is higher withiodide because oxidized iodide consumes H
2O2to produce
oxygen and iodide in a reaction called the pseudocatalyticactivity of peroxidase [24 40 49]
However thiocyanate can act as a one-electron donor andbe part of the peroxidase cycle with the sequential formationof two thiocyanate radicals [47] With 200120583M SCNminus LPOis predominantly in its native form this indicates that thehalogenation cycle prevails [47]
In the presence of both one- and two-electron donorscompetition for oxidation can occur and favor the halogena-tion or the peroxidase cycle The presence of EDTA inhibitsthe oxidation of iodide due to competition for binding toCompound I [50] The standard reduction potential betweenthe donors favors the molecule with the lowest reductionpotential Thereby the respective reduction potentials of theone- and two-electron oxidation of thiocyanate at very lowpH are 165V and 082V and promote the halogenationcycle [51] In the case of low concentrations of halides orthiocyanate below 10 120583M Iminus or 3 120583M SCNminus CompoundI reacts with any suitable exogenous or endogenous one-electron donor with the subsequent formation of CompoundII and a negligible oxidation rate of halides and thiocyanate[14]
24 Inhibition of the Function of Mammalian Heme Peroxi-dase The function of heme peroxidases can be inhibited inseveral ways that could be classified into three categoriesThe first one could represent an inhibition of the enzymeby (i) molecules or proteins and (ii) external conditionssuch as pH and temperature For example cyanide azidenitrite mercaptomethylimidazole thiourea superoxide highlevels of nitric oxide and high levels of thiocyanate bindto the native enzyme and alter Compound I formation [2046 47 52ndash54] With thiocyanate inhibition is linked to therestriction of the binding site to hydrogen peroxide andthe interaction of SCNminus with a water molecule [23] Highconcentration of H
2O2or Iminus will inactivate irreversibly LPO
with liberation of free iron [48 55] Temperature between73∘C and 83∘C depending on the heating time results inunfolding and inactivation of LPO [19] Extreme pH isinactivating enzymes and at low pH an amino acid groupprobably histidine is protonated which prevents the bindingof H2O2[56] Some proteases such as pepsin and pronase are
able to inactivate LPO by proteolysis but chymotrypsin did itvery slowly and trypsin and thermolysin are not active againstLPO [19]
The second group of inhibitors could concern substancesor proteins which are able to interfere with the catalyticmechanism For example catalase consumes H
2O2and will
stop the formation of Compound I [30 52] Competitionbetween substrates can also interfere with the reaction cyclesuch as SCNminus which competes very effectively with Clminus Brminusand Iminus [52 53] HOCl has the capacity to bind to LPO native
enzyme and convert it into Compound I Above 100 120583MHOCl mediates the destruction of the LPO heme center[57]
The third class could be related to substances or pro-teins which are buffering active molecules produced duringthe catalytic reaction For example presence of thiosulfatethioglycolate glutathione dithiothreitol cysteineNAD(P)Hand tyrosine will reduce the antimicrobial activity throughreacting with OClminus OBrminus OIminus or OSCNminus [52 53 58 59]The enzyme NADH-OSCN oxidoreductase is able to reduceOSCNminus in SCNminus [60]
3 Activity of Lactoperoxidase withThiocyanate andor Iodide
LPO concentrations in cowrsquos milk are around 30mg Lminus1depending on season diet and calving and breeding season[61] LPO extraction from whey or milk is based on awell-developed industrial process [62] Compared to MPOand EPO LPO is easily isolated and manufactured in largequantities As a result cowrsquos milk peroxidase is the favoritemolecule for in vitro or in vivo applications such as con-servation of raw and pasteurized milk storage of emulsionsand cosmetics moisturizing gel and toothpaste in human drymouth veterinary products and preservation of foodstuffs[19 61 63 64]
31 Activity of LPO Related to Hypothiocyanite
311 Mode of Action of Hypothiocyanite Thiocyanate is oxi-dized in a two-electron reaction that yields hypothiocyaniteHypothiocyanite has a pKa of 53 [65] It is more acidic thanhypohalides that have pKas of 75 (HOCl) 86 (HOBr) and106 (HOI) [14 66] All hypo(pseudo)halides (OXminus) are in anacid-base equilibrium association with their correspondingacid hypo(pseudo)halide (HOX) For example in the case ofhypothiocyanite
HOSCN 999447999472 OSCNminus +H+ (4)
The acid form has a higher oxidation potential and ismore soluble in nonpolar media so that it passes throughhydrophobic barriers such as cell membranes more easilybut it is less stable than the basic form (OXminus) [14 66]Hypohalide acids are predominant in acidic to neutral mediaand even in basic conditions for HOBr and HOI whereashypothiocyanite needs a pH below 53 to be predominant inthe acid form [66 67]
SCNminus is the two-electron donorwith the lowest reductionpotential and therefore forms the hypothiocyanite acid withthe lowest oxidative power compared to hypohalous acidsHypohalous acids rank as follows with increasing oxidativestrength OSCNminus lt OIminus lt OBrminus lt OClminus [28 66]These characteristicsmake hypothiocyanite relatively specificregarding its molecular target (Figure 5) that is a thiolmoiety [28 59 68]
6 Enzyme Research
- SH group- NAD(P)H
- SH group- NAD(P)H- Reduced pyridine nucleotide- R-S-R (thioether group)
HOSCNOSCNminus
HOIOIminusI2
- NH2 group
Figure 5 Target group of hypothiocyanite hypoiodite and iodineDue to its low oxidation power hypothiocyanite is relatively specificand is not reactive against all thiols In vivo hypoiodite seems tobe selectively directed against reduced pyridine nucleotide becauseeven the presence of excess glutathione and methionine doesnot thoroughly inhibit their oxidation HOSCNOSCNminus acidic orbasic form of hypothiocyanite HOIOIminus acidic or basic form ofhypoiodite and I
2 iodine
R-S-SCN or R-S-I
LPO R-SH
+ R-S-OHOSCNminusSCNminus SCNminus
or Iminus or Iminusor minusOI
H2O
H2O
2
Figure 6 Illustration of the cofactor role of SCNminus or Iminus Whenthe necessary conditions are fulfilled that is (i) no substratecompetitor for SCNminus or Iminus for binding to lactoperoxidase (ii)enough peroxidase H
2O2and SCNminus or Iminus (iii) enough R-SH
and (iv) no incorporation of SCNminus or Iminus in stable byproducts thequantity of OSCNminus or OIminus produced depends only on the amountof H2O2 SCNminus thiocyanate Iminus iodide H
2O2 hydrogen peroxide
LPO lactoperoxidase R-SH peptide or protein with a thiol moietyR-S-SCN or R-S-I sulfenyl thiocyanate or iodide R-SOH sulfenicacid OSCNminus hypothiocyanite and OIminus hypoiodite
Sulfhydryl oxidation by OSCNminus generates sulfenyl thio-cyanate in equilibrium with sulfenic acid [68]
SCNminus +H2O2+ LPO 997888rarr OSCNminus + LPO
R-SH +OSCNminus 997888rarr R-S-SCN +OHminus
R-S-SCN +H2O 997888rarr R-S-OH + SCNminus +H+
(5)
The cycle of reactions shows that thiocyanate acts likea cofactor for LPO (Figure 6) so that the total number ofoxidized sulfhydryls is independent of SCNminus as long as(i) thiocyanate is not exhausted (ii) thiocyanate is not incompetition with other substrates for the binding to Com-pound I (iii) thiocyanate is not incorporated into an aromaticamino acid (iv) enoughH
2O2is present and (v) thiol moiety
is still available [68 69]Although the target of OSCNminus is a thiol moiety not
all sulfhydryls are equally sensitive to OSCNminus albumincysteine mercaptoethanol dithiothreitol glutathione and 5-thio-2-nitrobenzoic acid are all oxidized but 120573-lactoglobulinis poorly oxidized probably due to a limited accessibility ofsulfhydryls to OSCNminus [68] In some conditions that is thejoint presence of LPO enough H
2O2and SCNminus and after the
oxidation of available sulfhydryls modification of tyrosinetryptophan and histidine protein residues can occur and that
could be linked to the formation of a labile powerful oxidantsuch as sulfur dicyanide [68]
Some authors suggest that (SCN)2is formed during the
enzymatic reaction and then chemically hydrolyzed intohypothiocyanite [14 69 70] However a recent publicationdemonstrates that (SCN)
2cannot be a precursor during the
enzymatic oxidation of SCNminus at neutral pH inmammals [71]Hypothiocyanite is less stable in acid conditions with
high concentrations of SCNminus and in the presence of (SCN)2
and it is thought to break down via the following net reaction[14]
4HOSCN +H2O 997888rarr 3SCNminus + CNOminus + SO
4
2minus+ 6H+ (6)
A recent study based notably on spectroscopic and chro-matographic methods proposes the following net equationwithin the 4ndash7 pH range
3HOSCN +H2O 997888rarr XSO
4
2minus+ XHCN
+ (1 minus X) SO3
2minus+ (1 minus X)CNOminus
+ 2SCNminus + (5 minus X)H+
(7)
The proportions of end anions were different at pH 4 andpH 7 at pH 7 the proportion of CNOminus was higher SCNminusformation was slower and no CNminus was detected [71]
It might seem easier to produce hypothiocyanite chemi-cally in in vitro applications but producing hypothiocyanitechemically from the oxidation of SCNminus by a halogen (Cl
2or
Br2) or by a hypohalous acid (HOCl or HOBr) in basic media
is tricky due to overoxidation of SCNminus [66] The referencemethod in the literature to produce 1- to 2-day stable OSCNminusis by hydrolyzing (SCN)
2in basic conditions [72ndash74]
Hypothiocyanite inhibitors have been described Forexample CNminus a weak acid buffer dissolved carbonate excesshydrogen peroxide hydrofluoric acid metallic ions glyc-erol or ammonium sulfate accelerates the decomposition ofOSCNminus whereas sulfonamide stabilizes it [67 72]
Appropriate concentrations of substrates induce en-hanced activity [75]
312 Biological Activity of Hypothiocyanite The biologicalactivity of hypothiocyanite is summarized in Figure 7
The sulfhydryl moiety is essential for the activityof numerous enzymes and proteins Inhibition of bacte-rial glycolysis through the oxidation of hexokinase gly-ceraldehyde-3-phosphate dehydrogenase (GAPDH) aldo-lase and glucose-6-phosphate dehydrogenase has beenobserved [14 51 65 70 76] Inhibition of respiration andglucose transport is associated with the alteration of cellmembranes or transporters [14 51 65 77] Irreversible inhi-bition is linked to long periods of incubation and bacterialsensitivity depends on the bacterial species and on hypothio-cyanite concentrations [14 51 59] Increased concentrationsof reducing agents such as glutathione and cysteine canreverse the inhibition through buffering hypothiocyaniteand converting the reduced thiol back into sulfhydryl [1478] This defense mechanism is used by Escherichia coli itinduces the CysJ promoter during the stress response to the
Enzyme Research 7
Bacteria
Inhibition of glycolysis Inhibition of respirationInhibition of glucose transport
Possible defense mechanism of bacteria- Increased GSH and cysteine content
Peptide proteinwith SH moiety
HOSCNOSCNminus
- NAD(P)H-dependent reduction of OSCNminus
Figure 7 Biological activity of hypothiocyanite on bacteria and possible defensemechanism of the bacteria Reversible inhibition is observedin that (i) hypothiocyanite is not reactive against all thiols and (ii) if hypothiocyanite is removed or diluted the pathogen recovers Irreversibleinhibition is linked to (i) long period of incubation (ii) the bacterial species and (iii) hypothiocyanite concentration HOSCNOSCNminus acidicor basic form of hypothiocyanite and GSH glutathione
pH lt 6
Influence of iodide concentration
Influence of the pH
1 I2 and high Iminus
I5minusI6
minus
I2I3minus
6 lt pH lt 9
HOII2OHI2I3minus
Iminus + H2O2 + LPO rarr active molecules
I2 (without Iminus)HOIOIminusI2I3
minusHI2Ominus
Figure 8 Illustration of the molecules that can be present after oxidation of iodide by lactoperoxidase in presence of H2O2The active species
depend mainly on the concentration of iodide (upper part) and the pH (lower part) The species with an oxidant power are represented inbold
lactoperoxidase system [79] Another resistance mechanismcould be the NAD(P)H-dependent reduction of OSCNminuswithout any loss of the sulfhydryl compound [14 72 78]Alteration of the bacterial membrane increases the efficacyof hypothiocyanite [80]
Furthermore the activity of the entire system (enzyme +substrates) is known to be more effective than hypothiocyan-ite alone whether enzymatically or chemically producedThis has been explained by the production of short-livedhighly reactive intermediates such as O
2SCNminus and O
3SCNminus
by the enzyme or by the oxidation of OSCNminus in conditionsof excess H
2O2[65 73 81] The activity of hypothiocyanite
has been described against bacteria such as Actinomyces sppBacillus cereus Lactobacillus spp Staphylococcus albus Saureus Streptococcus spp Escherichia coli Legionella pneu-mophila Salmonella typhimurium Pseudomonas fluorescensP aeruginosa Campylobacter jejuni C coli and Listeriamonocytogenes [14 32] Reversible inhibition is observed
when cells recover after OSCNminus is depleted [14 59] Irre-versible inhibition is obtained with long-term incubation andhigh level of OSCNminus [59] Higher concentration of SCNminuscompared to Iminus is necessary to obtain inhibition against E coliand accumulation of OSCNminus is observed as it is not reactiveagainst all thiols [59]Therefore the activity of the SCNminus-LPOsystem appears to be more bacteriostatic than bactericidal
32 Activity of LPO Related to Oxidized Iodide
321 Chemistry of Oxidized Iodide Iodide is oxidized byCompound I through a single two-electron transfer thatyields oxidized Iminus in the form of I
2or HOI [14 24 82ndash85]
The active agent is composed of a mixture of species that arenot yet formally detailed due to the very complex behaviorand stability of I
2and HOI in aqueous environments that
strongly depend on pH values and iodide concentrations[66 82 83 86]
8 Enzyme Research
Based on the inorganic chemistry of iodine in waterand literature on enzymatic oxidation of iodide the activemolecules have been described as follows (Figure 8)
(i) Under pH 6 and in the presence of iodide only I2
Iminus and I3
minus are present and the only active moleculeis I2 I2concentrations decrease with increasing
concentrations of Iminus At an initial 1mM I2 with Iminus
concentrations ranging from 1mM to 100mM I2
concentrations fall from almost 1mM to 001mM asdescribed by the following association reaction [2482 83 86]
I2+ Iminus 999447999472 I
3
minus (8)
(ii) In solution within a 6ndash9 pH range and with a max-imum 1mM iodide a mixture of HOII
2OHI2I3
minus
is formed in which I3
minus is not active and I2OH is
probably less reactive than HOI or I2[86 87] If
Iminus concentrations are above 10mM I3
minus representsthe main species formed and the concentration ofactive molecules relatively drops The mechanism issummarized in the following net equations
HOI + Iminus +H+ 999447999472 I2OHminus +H+
999447999472 I2+H2O 999447999472 I
2+ Iminus 999447999472 I
3
minus
(9)
(iii) In iodine solution without iodide or when availableiodide has been oxidized the number of I
2-derived
molecules decreases with decreasing I2concentra-
tions At 1000 120583M I2 with pH-related ratios five
relevant species are observed (I2 HOI I
3
minus HI2Ominus
and OIminus) At 10 120583M I2 the main species are only I
2
HOI and OIminus and HOI could represent up to 90 ofthe active oxidant molecules at pH 8-9 [86] Below apH of 106 the following reactions are involved
I2+H2O 999447999472 HOI + Iminus +H+ (hydrolysis of I
2)
I2+ Iminus 999447999472 I
3
minus(triiodide formation independent of pH)
(10)
(iv) At high Iminus and 1 I2concentrations as in Lugol
solution I5
minus and I6
minus are formed and represent 82of the active oxidative agents [86] after the followingreaction
I3
minus+ I2999447999472 I5
minus(pentaiodide formation)
2I3
minus999447999472 I6
2minus(dimerization of I
3
minus)
(11)
The stability of HOI and I2is linked to their dispro-
portionation in iodate which has no oxidative activity inneutral and basic pH conditions [86]The disproportionationreactions read as follows
3HOI 999447999472 IO3
minus+ 2Iminus + 3H+ (disproportionation of HOI)
3I2999447999472 IO
3
minus+ 5Iminus + 6H+ (disproportionation of I
2)
(12)
I2stability increases at higher pHvalues andhigher iodide
concentrations [86] In drinking water HOI disproportion-ation is slow and varies substantially HOI has a half-life of4 days to 35 years depending on (i) the initial level of HOIthat speeds its decomposition and (ii) the presence of boratephosphate or carbonate that catalyzes its decomposition [8889]
322 Mode of Action of Oxidized Iodide The oxidativestrength of I
2is between that of the corresponding hypo-
halous acid HOI and the hypoiodite ion OIminus and ranks asfollows 0485V (OIminus) lt 0536V (I
2) lt 0987V (HOI) [66]
HOI reacts through very rapid oxidation of thiolgroups oxidation of NAD(P)H oxidation of 120573-nicotinamidemononucleotide direct reaction with thioether groupsthrough sulfoxidation and slow oxidation of the aminemoiety (Figure 5) [87 90 91] At low Iminus concentrationsiodination of tyrosine residues is catalyzed by the enzyme[14] In a cellular environment HOI seems to be more selec-tively directed against the degradation of reduced pyridinenucleotides thanHOCL andHOBr because even the presenceof excess glutathione methionine or oxidized glutathionedoes not thoroughly inhibit their oxidation [87]
In some conditions that is (i) enough iodide H2O2 and
peroxidase (ii) no accumulation of oxidized iodide and (iii)no incorporation of iodide into stable byproducts such astyrosine residues iodide acts as a cofactor (Figure 6) andthe proportion of oxidized sulfhydryls is proportional to theamount of H
2O2as described below [85 92]
2Iminus +H2O2+ LPO (native enzyme)
997888rarr I2+ 2H2O + LPO (native enzyme)
R-SH + I2997888rarr R-S-I + Iminus +H+
R-S-I+H2O 997888rarr R-S-OH + Iminus +H+
(13)
In the case of high concentrations of Iminus andor H2O2
inhibition of tyrosine iodation has been observed [83] andrelated to the pseudocatalytic redox degradation of H
2O2
with formation of O2when excessive H
2O2is present (reac-
tion 1) and production of I3
minus when excessive amounts of Iminusare present (reaction 2)
I2+H2O 997888rarr O
2+ 2Iminus + 2H+ (reaction 1)
I2+ Iminus 999447999472 I
3
minus(reaction 2)
(14)
Both reactions deplete the amount of the active oxidizingagent I
2 In the absence of tyrosine oxidized iodide reacts
with nucleophilic molecules such as Iminus Clminus or OHminus to formI2 I3
minus ICl ICl2 IOH and I
2OH [82] Some anions such as
Clminus HPO4
minus or OHminus reduce the amount of I2I3
minus but thiseffect is inversely proportional to the concentration of Iminusabove pH 9 I
2is hydrolyzed and IO
3
minus is formed [82]HOI can be produced chemically through oxidation of Iminus
by Cl2or O3 with a short half-life due to overoxidation of
HOI byCl2andO
3[89] and through oxidation of Iminus byHOCl
HOBr or NH2Cl with a longer half-life [87 89]
Enzyme Research 9
Bacteria
Inhibition of glycolysis Inhibition of respirationInhibition of glucose transportInhibition of the pentose phosphate pathway
- Peptide protein with- SH moiety- thioether moiety- NAD(P)H
HOIOIminus
I2
Figure 9 Biological activity of hypoiodite or iodine on bacteria Irreversible inhibition is observed and could be linked to (i) oxidation ofthiol groups NAD(P)H and thioether groups (ii) high reactivity of HOII
2against thiol and reduced nicotinamide nucleotides and (iii) the
incorporation of iodide in tyrosine residue of protein (iodination of protein) HOIOIminus acid or basic form of hypoiodite and I2 iodine
323 Biological Action of Oxidized Iodide The biologicalaction of oxidized iodide (Figure 9) is similar to that ofhypothiocyanite but differs in that (i) the reactivity of oxi-dized iodide is complete against thiol group and (ii) cells didnot recover after removing of oxidized iodide [59]
Due to the cofactor role of Iminus inhibition of respirationin Escherichia coli in the presence of LPO H
2O2 and Iminus is
complete with only 10120583M NaI whereas 100 120583M of solely I2
is necessary to obtain complete inhibition This is directlyrelated to the oxidation of sulfhydryls not to the percentageof iodine incorporation [92 93]
E coli seems to be more sensitive if the bacteria areincubated together with the entire system (enzyme H
2O2
and iodide) rather than adding several minutes after mixingthe enzyme with its substrates This could be linked to theformation of an unstable reactive intermediate [52]
The activity of the Iminus peroxidase system is more effectiveagainst E coli than the SCNminus system in that lower Iminusconcentrations are necessary all sulfhydryls are oxidized andcells do not recover even if the amount of I
2is not sufficient
to oxidize all SH groups [59 80] Against L acidophilushigh non physiological amounts of Iminus are necessary to obtaininhibitionwhereas small concentrations of SCNminus are effective[70]
CNminus azide EDTA and SCNminus inhibit the formation ofoxidized iodide [50 52] Increased pH values and increasedamounts of thiol and NAD(P)H compounds reduce theactivity of the iodide peroxidase system [52]
LPO-H2O2-Iminus in presence of Streptococcus mitis is active
against Staphylococcus aureus and E coli [94] LPO-H2O2-
Iminus is active against Micrococcus S aureus Listeria monocy-togenes Bacillus cereus E coli and Candida albicans [12 1980] In the presence of other peroxidases the Iminus peroxidasesystem is active against Schistosoma mansoni Fusariumnucleatum andActinobacillus actinomycetemcomitans [31 9596] Compared to SCNminus Iminus-LPO shows bactericidal activities[14 19 80]
33 Activity of LPO Related to Hypoiodite and Hypothiocyan-ite The combination of SCNminus with Iminus in the lactoperoxidasesystem has been poorly studied Tackling the enzymaticmechanism is tricky and contradictory results have been
found about microbial activity in the concomitant presenceof SCNminus and Iminus
In the presence of SCNminus and Iminus there is competitionbetween the two substrates for oxidation by lactoperoxidase[14 36] Iminus alone exhibits bactericidal activity but an SCNminusIminusratio of 01 inhibits that bactericidal effect and an SCNminusIminusratio of 1 antagonizes it due to competition for oxidation andfaster decomposition of HOSCN in the presence of Iminus [14]Against A actinomycetemcomitans the peroxidase systemwith Iminus Clminus or a combination of Iminus and Clminus is effective butaddition of SCNminus cancels the antibacterial effect [96] On theother hand a synergistic or unaffected effect of iodide in theSCNminus-H
2O2-LPO system has been shown against Candida
albicans E coli S aureusAspergillus niger and Pseudomonasaeruginosa [19 97]
4 Conclusion
Themolecular evolution of heme peroxidases and the preser-vation of their catalytic domain [6] show that the productionof strong oxidants is a powerful part of the nonimmunedefense mechanisms against pathogenic bacteria fungi orparasite which made the use of those enzymes in practicalapplications worthwhile
The enzymatic reactions involving mammalian peroxi-dases are complex and various molecules can promote orreduce dramatically the antibacterial activity of the per-oxidase system In order to favor the halogenation cyclerequired in in vitro and in vivo antimicrobial applicationsseveral points have to be taken into account (i) to avoid thepresence of competitors to iodide or thiocyanate for bindingto Compound I and to avoid the presence of inhibitorsof the enzyme or of active molecules (ii) to avoid excessH2O2concentration which is able to destruct the enzyme
and to react with iodine or hypoiodite with loosing of activemolecules (iii) to favor the presence of hypoiodite instead ofiodine due to the association reaction of iodine with iodide(iv) to avoid excess concentration of thiocyanate which caninhibit formation of Compound I (v) to use the entiresystem (enzyme + substrates) instead of active moleculesalone (vi) to favor moderate acid pH when hypothiocyaniteis the active molecule (vii) for bactericidal fungicidal or
10 Enzyme Research
parasitical applications the use of iodide has to be preferred(viii) the use of combined presence of iodide and thiocyanatehas to be checked carefully for efficacy and (ix) to favor thecofactor role of iodide or thiocyanate
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] P J OrsquoBrien ldquoPeroxidasesrdquoChemico-Biological Interactions vol129 no 1-2 pp 113ndash139 2000
[2] W Jantschko P G Furtmuller M Allegra et al ldquoRedoxintermediates of plant and mammalian peroxidases a compar-ative transient-kinetic study of their reactivity toward indolederivativesrdquo Archives of Biochemistry and Biophysics vol 398no 1 pp 12ndash22 2002
[3] S Kimura and M Ikeda-Saito ldquoHuman myeloperoxidase andthyroid peroxidase two enzymes with separate and distinctphysiological functions are evolutionarily related membersof the same gene familyrdquo Proteins Structure Function andGenetics vol 3 no 2 pp 113ndash120 1988
[4] G Battistuzzi M Bellei C A Bortolotti and M Sola ldquoRedoxproperties of heme peroxidasesrdquo Archives of Biochemistry andBiophysics vol 500 no 1 pp 21ndash36 2010
[5] M Zamocky C Jakopitsch P G Furtmuller C Dunand and CObinger ldquoThe peroxidase-cyclooxygenase superfamily recon-structed evolution of critical enzymes of the innate immunesystemrdquo Proteins Structure Function and Genetics vol 72 no2 pp 589ndash605 2008
[6] H Daiyasu and H Toh ldquoMolecular evolution of the myeloper-oxidase familyrdquo Journal of Molecular Evolution vol 51 no 5 pp433ndash445 2000
[7] D Serteyn S Grulke T Franck A Mouithys-Mickalad andG Deby-Dupont ldquoNeutrophile myeloperoxidase protectiveenzyme with strong oxidative activitiesrdquo Annales de MedecineVeterinaire vol 147 no 2 pp 79ndash93 2003
[8] S C Whitman S L Hazen D B Miller R A HegeleJ W Heinecke and M W Huff ldquoModification of type IIIVLDL their remnants and VLDL from apoE- knockout miceby p-hydroxyphenylacetaldehyde a product of myeloperox-idase activity causes marked cholesteryl ester accumulationin macrophagesrdquo Arteriosclerosis Thrombosis and VascularBiology vol 19 no 5 pp 1238ndash1249 1999
[9] T J Barrett and C L Hawkins ldquoHypothiocyanous acid benignor deadlyrdquo Chemical Research in Toxicology vol 25 no 2 pp263ndash273 2012
[10] M M Lloyd D M van Reyk M J Davies and C L HawkinsldquoHypothiocyanous acid is a more potent inducer of apoptosisand protein thiol depletion in murine macrophage cells thanhypochlorous acid or hypobromous acidrdquo Biochemical Journalvol 414 no 2 pp 271ndash280 2008
[11] J Wang and A Slungaard ldquoRole of eosinophil peroxidase inhost defense and disease pathologyrdquo Archives of Biochemistryand Biophysics vol 445 no 2 pp 256ndash260 2006
[12] M Ahariz and P Courtois ldquoCandida albicans susceptibility tolactoperoxidase-generated hypoioditerdquo Clinical Cosmetic andInvestigational Dentistry vol 2 pp 69ndash78 2010
[13] A Welk C Meller R Schubert C Schwahn A Kramerand H Below ldquoEffect of lactoperoxidase on the antimicrobialeffectiveness of the thiocyanate hydrogen peroxide combinationin a quantitative suspension testrdquo BMC Microbiology vol 9article 134 2009
[14] K M Pruitt and J O Tenovuo Eds The Lactoperoxidase Sys-tem Chemistry and Biological Significance vol 27 of Immunol-ogy Series Marcel Dekker New York NY USA 1985
[15] P G Furtmuller W Jantschko G Regelsberger C JakopitschJ Arnhold and C Obinger ldquoReaction of lactoperoxidasecompound I with halides and thiocyanaterdquo Biochemistry vol41 no 39 pp 11895ndash11900 2002
[16] P G Furtmuller U Burner and C Obinger ldquoReaction ofmyeloperoxidase compound I with chloride bromide iodideand thiocyanaterdquo Biochemistry vol 37 no 51 pp 17923ndash179301998
[17] J Arnhold E Monzani P G Furtmuller M Zederbauer LCasella and C Obinger ldquoKinetics and thermodynamics ofhalide and nitrite oxidation by mammalian heme peroxidasesrdquoEuropean Journal of Inorganic Chemistry no 19 pp 3801ndash38112006
[18] M J Davies C L Hawkins D I Pattison and M D ReesldquoMammalian heme peroxidases from molecular mechanismsto health implicationsrdquo Antioxidants and Redox Signaling vol10 no 7 pp 1199ndash1234 2008
[19] J N de Wit and A C M van Hooydonk ldquoStructure functionsand applications of lactoperoxidase in natural antimicrobialsystemsrdquo Nederlands melk en Zuiveltijdschrift vol 50 no 2 pp227ndash244 1996
[20] P G Furtmuller M Zederbauer W Jantschko et al ldquoActivesite structure and catalytic mechanisms of human peroxidasesrdquoArchives of Biochemistry and Biophysics vol 445 no 2 pp 199ndash213 2006
[21] M Zederbauer P G Furtmuller S Brogioni C JakopitschG Smulevich and C Obinger ldquoHeme to protein linkages inmammalian peroxidases impact on spectroscopic redox andcatalytic propertiesrdquo Natural Product Reports vol 24 no 3 pp571ndash584 2007
[22] G Battistuzzi M Bellei J Vlasits et al ldquoRedox thermodynam-ics of lactoperoxidase and eosinophil peroxidaserdquo Archives ofBiochemistry and Biophysics vol 494 no 1 pp 72ndash77 2010
[23] I A Sheikh A Singh N Singh et al ldquoStructural evidence ofsubstrate specificity inmammalian peroxidases structure of thethiocyanate complex with lactoperoxidase and its interactionsat 24 a 24 A resolutionrdquo The Journal of Biological Chemistryvol 284 no 22 pp 14849ndash14856 2009
[24] H Kohler and H Jenzer ldquoInteraction of lactoperoxidase withhydrogen peroxide Formation of enzyme intermediates andgeneration of free radicalsrdquo Free Radical Biology and Medicinevol 6 no 3 pp 323ndash339 1989
[25] P G Furtmuller U Burner W Jantschko G Regelsberger andC Obinger ldquoTwo-electron reduction and one-electron oxida-tion of organic hydroperoxides by human myeloperoxidaserdquoFEBS Letters vol 484 no 2 pp 139ndash143 2000
[26] A Taurog M L Dorris and D R Doerge ldquoMechanism ofsimultaneous iodination and coupling catalyzed by thyroidperoxidaserdquo Archives of Biochemistry and Biophysics vol 330no 1 pp 24ndash32 1996
[27] J E Erman L B Vitello J Matthew Mauro and J KrautldquoDetection of an oxyferryl porphyrin 120587-cation-radical interme-diate in the reaction between hydrogen peroxide and a mutant
Enzyme Research 11
yeast cytochrome c peroxidase Evidence for tryptophan-191involvement in the radical site of compound Irdquo Biochemistryvol 28 no 20 pp 7992ndash7995 1989
[28] M T Ashby ldquoInorganic chemistry of defensive peroxidases inthe human oral cavityrdquo Journal of Dental Research vol 87 no10 pp 900ndash914 2008
[29] J D Chandler and B J Day ldquoThiocyanate a potentially usefultherapeutic agent with host defense and antioxidant propertiesrdquoBiochemical Pharmacology vol 84 no 11 pp 1381ndash1387 2012
[30] E C Jong W R Henderson and S J Klebanoff ldquoBactericidalactivity of eosinophil peroxidaserdquo Journal of Immunology vol124 no 3 pp 1378ndash1382 1980
[31] E C Jong A A F Mahmoud and S J Kelbanoff ldquoPeroxidase-mediated toxicity to schistosomula of Schistosoma mansonirdquoJournal of Immunology vol 126 no 2 pp 468ndash471 1981
[32] L M Wolfson and S S Sumner ldquoAntibacterial activity of thelactoperoxidase system a reviewrdquo Journal of Food Protectionvol 56 no 10 pp 887ndash892 1993
[33] J Arnhold P G Furtmuller G Regelsberger and C ObingerldquoRedox properties of the couple compound Inative enzyme ofmyeloperoxidase and eosinophil peroxidaserdquo European Journalof Biochemistry vol 268 no 19 pp 5142ndash5148 2001
[34] P G Furtmuller J Arnhold W Jantschko M Zederbauer CJakopitsch and C Obinger ldquoStandard reduction potentials ofall couples of the peroxidase cycle of lactoperoxidaserdquo Journalof Inorganic Biochemistry vol 99 no 5 pp 1220ndash1229 2005
[35] C J van Dalen M W Whitehouse C C Winterbourn and AJ Kettle ldquoThiocyanate and chloride as competing substrates formyeloperoxidaserdquo Biochemical Journal vol 327 no 2 pp 487ndash492 1997
[36] A Slungaard and J R Mahoney Jr ldquoThiocyanate is the majorsubstrate for eosinophil peroxidase in physiologic fluids impli-cations for cytotoxicityrdquoThe Journal of Biological Chemistry vol266 no 8 pp 4903ndash4910 1991
[37] J Tenovuo ldquoAntimicrobial function of human salivamdashhowimportant is it for oral healthrdquoActaOdontologica Scandinavicavol 56 no 5 pp 250ndash256 1998
[38] R Ihalin V Loimaranta and J Tenovuo ldquoOrigin structure andbiological activities of peroxidases in human salivardquo Archives ofBiochemistry and Biophysics vol 445 no 2 pp 261ndash268 2006
[39] J A Rooke J F Flockhart and N H Sparks ldquoThe potentialfor increasing the concentrations of micro-nutrients relevant tohuman nutrition inmeat milk and eggsrdquo Journal of AgriculturalScience vol 148 no 5 pp 603ndash614 2010
[40] H Kohler A Taurog and H B Dunford ldquoSpectral studieswith lactoperoxidase and thyroid peroxidase interconversionsbetween native enzyme compound II and compound IIIrdquoArchives of Biochemistry and Biophysics vol 264 no 2 pp 438ndash449 1988
[41] I Yamazaki H S Mason and L Piette ldquoIdentification byelectron paramagnetic resonance spectroscopy of free radicalsgenerated from substrates by peroxidaserdquoThe Journal of Biolog-ical Chemistry vol 235 pp 2444ndash2449 1960
[42] B Chance ldquoThe kinetics and stoichiometry of the transitionfrom the primary to the secondary peroxidase peroxide com-plexesrdquo Archives of Biochemistry and Biophysics vol 41 no 2pp 416ndash424 1952
[43] K M Pruitt B Mansson-Rahemtulla D C Baldone andF Rahemtulla ldquoSteady-state kinetics of thiocyanate oxidationcatalyzed by human salivary peroxidaserdquo Biochemistry vol 27no 1 pp 240ndash245 1988
[44] B G J M Bolscher and R Wever ldquoA kinetic study of thereaction between humanmyeloperoxidase hydroperoxides andcyanide inhibition by chloride and thiocyanaterdquo Biochimica etBiophysica Acta Protein Structure and Molecular Enzymologyvol 788 no 1 pp 1ndash10 1984
[45] L A Marquez J T Huang and H Brian Dunford ldquoSpectraland kinetic studies on the formation of myeloperoxidase com-pounds I and II roles of hydrogen peroxide and superoxiderdquoBiochemistry vol 33 no 6 pp 1447ndash1454 1994
[46] HMAbu-Soud and S LHazen ldquoNitric oxide is a physiologicalsubstrate for mammalian peroxidasesrdquoThe Journal of BiologicalChemistry vol 275 no 48 pp 37524ndash37532 2000
[47] Y R Tahboub S Galijasevic M P Diamond and H MAbu-Soud ldquoThiocyanate modulates the catalytic activity ofmammalian peroxidasesrdquo Journal of Biological Chemistry vol280 no 28 pp 26129ndash26136 2005
[48] H Jenzer W Jones and H Kohler ldquoOn the molecularmechanismof lactoperoxidase-catalyzedH
2O2metabolism and
irreversible enzyme inactivationrdquo The Journal of BiologicalChemistry vol 261 no 33 pp 15550ndash15556 1986
[49] R P Magnusson A Taurog and M L Dorris ldquoMechanism ofiodide-dependent catalatic activity of thyroid peroxidase andlactoperoxidaserdquo The Journal of Biological Chemistry vol 259no 1 pp 197ndash205 1984
[50] D K Bhattacharyya U Bandyopadhyay and R K BanerjeeldquoEDTA inhibits lactoperoxidase-catalyzed iodide oxidation byacting as an electron-donor and interacting near the iodidebinding siterdquoMolecular and Cellular Biochemistry vol 162 no2 pp 105ndash111 1996
[51] C L Hawkins ldquoThe role of hypothiocyanous acid (HOSCN) inbiological systems HOSCN in biological systemsrdquo Free RadicalResearch vol 43 no 12 pp 1147ndash1158 2009
[52] S J Klebanoff ldquoIodination of bacteria a bactericidal mecha-nismrdquo Journal of Experimental Medicine vol 126 no 6 pp1063ndash1078 1967
[53] S J Klebanoff ldquoMyeloperoxidase-halide-hydrogen peroxideantibacterial systemrdquo Journal of Bacteriology vol 95 no 6 pp2131ndash2138 1968
[54] R K Banerjee and A G Datta ldquoSalivary peroxidasesrdquoMolecu-lar and Cellular Biochemistry vol 70 no 1 pp 21ndash29 1986
[55] MHuwiler H Jenzer andHKohler ldquoThe role of compound IIIin reversible and irreversible inactivation of lactoperoxidaserdquoEuropean Journal of Biochemistry vol 158 no 3 pp 609ndash6141986
[56] R Wever W M Kast J H Kasinoedin and R Boelens ldquoTheperoxidation of thiocyanate catalysed by myeloperoxidase andlactoperoxidaserdquo Biochimica et Biophysica Acta (BBA)ProteinStructure and Molecular vol 709 no 2 pp 212ndash219 1982
[57] C E A Souza D Maitra G M Saed et al ldquoHypochlorousacid-induced heme degradation from lactoperoxidase as anovel mechanism of free iron release and tissue injury ininflammatory diseasesrdquo PLoS ONE vol 6 no 11 Article IDe27641 2011
[58] J Carlsson ldquoBactericidal effect of hydrogen peroxide is pre-vented by the lactoperoxidase-thiocyanate system under anaer-obic conditionsrdquo Infection and Immunity vol 29 no 3 pp 1190ndash1192 1980
[59] E L Thomas and T M Aune ldquoLactoperoxidase peroxidethiocyanate antimicrobial system correlation of sulfhydryloxidation with antimicrobial actionrdquo Infection and Immunityvol 20 no 2 pp 456ndash463 1978
12 Enzyme Research
[60] J Carlsson Y Iwami and T Yamada ldquoHydrogen peroxideexcretion by oral streptococci and effect of lactoperoxidase-thiocyanate-hydrogen peroxiderdquo Infection and Immunity vol40 no 1 pp 70ndash80 1983
[61] K D Kussendrager and A C M van Hooijdonk ldquoLactoperox-idase physico-chemical properties occurrence mechanism ofaction and applicationsrdquoTheBritish Journal of Nutrition vol 84supplement 1 pp S19ndashS25 2000
[62] J P Perraudin ldquoProteines a activites biologiques lactoferrineet lactoperoxydase Connaissances recemment acquises et tech-nologies drsquoobtentionrdquo Lait vol 71 no 2 pp 191ndash211 1991
[63] J-W Boots and R Floris ldquoLactoperoxidase From catalyticmechanism to practical applicationsrdquo International Dairy Jour-nal vol 16 no 11 pp 1272ndash1276 2006
[64] A C M van Hooijdonk K D Kussendrager and J M SteijnsldquoIn vivo antimicrobial and antiviral activity of components inbovine milk and colostrum involved in non-specific defencerdquoBritish Journal of Nutrition vol 84 supplement 1 pp S127ndashS1342000
[65] D M Hogg and G R Jago ldquoThe antibacterial action of lac-toperoxidaseThe nature of the bacterial inhibitorrdquo BiochemicalJournal vol 117 no 4 pp 779ndash790 1970
[66] M T Ashby ldquoHypothiocyaniterdquo in Advances in InorganicChemistry R van Eldik and I-B Ivana Eds chapter 8 pp 263ndash303 Academic Press New York NY USA 2012
[67] E L Thomas ldquoLactoperoxidase-catalyzed oxidation of thio-cyanate equilibria between oxidized forms of thiocyanaterdquoBiochemistry vol 20 no 11 pp 3273ndash3280 1981
[68] T M Aune and E LThomas ldquoOxidation of protein sulfhydrylsby products of peroxidase-catalyzed oxidation of thiocyanateionrdquo Biochemistry vol 17 no 6 pp 1005ndash1010 1978
[69] T M Aune and E L Thomas ldquoAccumulation of hypothiocyan-ite ion during peroxidase-catalyzed oxidation of thiocyanateionrdquo European Journal of Biochemistry vol 80 no 1 pp 209ndash214 1977
[70] J D Oram and B Reiter ldquoThe inhibition of streptococci bylactoperoxidase thiocyanate and hydrogen peroxideThe effectof the inhibitory system on susceptible and resistant strains ofgroup N streptococcirdquo Biochemical Journal vol 100 no 2 pp373ndash381 1966
[71] J Kalmar K L Woldegiorgis B Biri and M T AshbyldquoMechanism of decomposition of the human defense factorhypothiocyanite near physiological pHrdquo Journal of the Ameri-can Chemical Society vol 133 no 49 pp 19911ndash19921 2011
[72] H Hoogendoorn J P PiessensW Scholtes and L A StoddardldquoHypothiocyanite ion the inhibitor formed by the system lac-toperoxidase thiocyanate hydrogen peroxide I Identification ofthe inhibiting compoundrdquoCaries Research vol 11 no 2 pp 77ndash84 1977
[73] L Bjorck and O Claesson ldquoCorrelation between concentrationof hypothiocyanate and antibacterial effect of the lactoperoxi-dase system against Escherichia colirdquo Journal of Dairy Sciencevol 63 no 6 pp 919ndash922 1980
[74] P Nagy S S Alguindigue and M T Ashby ldquoLactoperoxidase-catalyzed oxidation of thiocyanate by hydrogen peroxide areinvestigation of hypothiocyanite by nuclear magnetic reso-nance and optical spectroscopyrdquo Biochemistry vol 45 no 41pp 12610ndash12616 2006
[75] Y Adolphe M Jacquot M Linder A-M Revol-Junelles andJ-B Milliere ldquoOptimization of the components concentrationsof the lactoperoxidase system by RSMrdquo Journal of AppliedMicrobiology vol 100 no 5 pp 1034ndash1042 2006
[76] M Adamson and K M Pruitt ldquoLactoperoxidase-catalyzedinactivation of hexokinaserdquo Biochimica et Biophysica Acta vol658 no 2 pp 238ndash247 1981
[77] M N Mickelson ldquoGlucose transport in Streptococcus agalac-tiae and its inhibition by lactoperoxidase-thiocyanate-hydrogenperoxiderdquo Journal of Bacteriology vol 132 no 2 pp 541ndash5481977
[78] E L Thomas K A Pera K W Smith and A K ChwangldquoInhibition of Streptococcus mutans by the lactoperoxidaseantimicrobial systemrdquo Infection and Immunity vol 39 no 2 pp767ndash778 1983
[79] J Sermon K Vanoirbeek P De Spiegeleer R Van Houdt AAertsen and C W Michiels ldquoUnique stress response to thelactoperoxidase-thiocyanate enzyme system in Escherichia colirdquoResearch in Microbiology vol 156 no 2 pp 225ndash232 2005
[80] E L Thomas and T M Aune ldquoSusceptibility of Escherichia colito bactericidal action of lactoperoxidase peroxide and iodideor thiocyanaterdquoAntimicrobial Agents andChemotherapy vol 13no 2 pp 261ndash265 1978
[81] K M Pruitt J Tenovuo R W Andrews and T McKaneldquoLactoperoxidase-catalyzed oxidation of thiocyanate polaro-graphic study of the oxidation productsrdquo Biochemistry vol 21no 3 pp 562ndash567 1982
[82] M Huwiler and H Kohler ldquoPseudo-catalytic degradation ofhydrogen peroxide in the lactoperoxidaseH
2O2iodide sys-
temrdquo European Journal of Biochemistry vol 141 no 1 pp 69ndash741984
[83] M Huwiler U Burgi and H Kohler ldquoMechanism of enzymaticand non-enzymatic tyrosine iodination Inhibition by excesshydrogen peroxide andor iodiderdquo European Journal of Bio-chemistry vol 147 no 3 pp 469ndash476 1985
[84] M Morrison G S Bayse and A W Michaels ldquoDeterminationof spectral properties of aqueous I2 and I3- and the equilibriumconstantrdquo Analytical Biochemistry vol 42 no 1 pp 195ndash2011971
[85] E L Thomas and T M Aune ldquoPeroxidase catalyzed oxidationof protein sulfhydrylsmediated by iodinerdquoBiochemistry vol 16no 16 pp 3581ndash3586 1977
[86] W Gottardi ldquoIodine and disinfection theoretical study onmode of action efficiency stability and analytical aspects in theaqueous systemrdquo Archiv der Pharmazie vol 332 no 5 pp 151ndash157 1999
[87] W A Prutz R Kissner W H Koppenol and H RueggerldquoOn the irreversible destruction of reduced nicotinamidenucleotides by hypohalous acidsrdquo Archives of Biochemistry andBiophysics vol 380 no 1 pp 181ndash191 2000
[88] Y Bichsel and U Von Gunten ldquoHypoiodous acid kinetics ofthe buffer-catalyzed disproportionationrdquo Water Research vol34 no 12 pp 3197ndash3203 2000
[89] Y Bichsel and U von Gunten ldquoOxidation of iodide andhypoiodous acid in the disinfection of natural watersrdquo Environ-mental Science and Technology vol 33 no 22 pp 4040ndash40451999
[90] W A Prutz R Kissner T Nauser and W H Koppenol ldquoOnthe oxidation of cytochrome c by hypohalous acidsrdquo Archives ofBiochemistry and Biophysics vol 389 no 1 pp 110ndash122 2001
[91] A Virion J L Michot D Deme and J Pommier ldquoNADPHoxidation catalyzed by the peroxidaseH
2O2system Iodide-
mediated oxidation of NADPH to iodinated NADPrdquo EuropeanJournal of Biochemistry vol 148 no 2 pp 239ndash243 1985
Enzyme Research 13
[92] E L Thomas and T M Aune ldquoCofactor role of iodide in per-oxidase antimicrobial action against Escherichia colirdquo Antimic-robial Agents and Chemotherapy vol 13 no 6 pp 1000ndash10051978
[93] E L Thomas and T M Aune ldquoOxidation of Escherichiacoli sulfhydryl components by the peroxidase-hydrogenperoxide-iodide antimicrobial systemrdquo Antimicrobial Agentsand Chemotherapy vol 13 no 6 pp 1006ndash1010 1978
[94] C B Hamon and S J Klebanoff ldquoA peroxidase-mediatedstreptococcus mitis-dependent antimicrobial system in salivardquoJournal of Experimental Medicine vol 137 no 2 pp 438ndash4501973
[95] R Ihalin J Nuutila V Loimaranta M Lenander J Tenovuoand E-M Lilius ldquoSusceptibility of Fusobacterium nucleatum tokilling by peroxidase-iodide-hydrogen peroxide combinationin buffer solution and in human whole salivardquo Anaerobe vol9 no 1 pp 23ndash30 2003
[96] R Ihalin V Loimaranta M Lenander-Lumikari and J Ten-ovuo ldquoThe effects of different (pseudo)halide substrates onperoxidase-mediated killing of Actinobacillus actinomycetem-comitansrdquo Journal of Periodontal Research vol 33 no 7 pp 421ndash427 1998
[97] E H Bosch H van doorne and S de Vries ldquoThe lactoper-oxidase system the influence of iodide and the chemical andantimicrobial stability over the period of about 18 monthsrdquoJournal of AppliedMicrobiology vol 89 no 2 pp 215ndash224 2000
- SH group- NAD(P)H- Reduced pyridine nucleotide- R-S-R (thioether group)
HOSCNOSCNminus
HOIOIminusI2
- NH2 group
Figure 5 Target group of hypothiocyanite hypoiodite and iodineDue to its low oxidation power hypothiocyanite is relatively specificand is not reactive against all thiols In vivo hypoiodite seems tobe selectively directed against reduced pyridine nucleotide becauseeven the presence of excess glutathione and methionine doesnot thoroughly inhibit their oxidation HOSCNOSCNminus acidic orbasic form of hypothiocyanite HOIOIminus acidic or basic form ofhypoiodite and I
2 iodine
R-S-SCN or R-S-I
LPO R-SH
+ R-S-OHOSCNminusSCNminus SCNminus
or Iminus or Iminusor minusOI
H2O
H2O
2
Figure 6 Illustration of the cofactor role of SCNminus or Iminus Whenthe necessary conditions are fulfilled that is (i) no substratecompetitor for SCNminus or Iminus for binding to lactoperoxidase (ii)enough peroxidase H
2O2and SCNminus or Iminus (iii) enough R-SH
and (iv) no incorporation of SCNminus or Iminus in stable byproducts thequantity of OSCNminus or OIminus produced depends only on the amountof H2O2 SCNminus thiocyanate Iminus iodide H
2O2 hydrogen peroxide
LPO lactoperoxidase R-SH peptide or protein with a thiol moietyR-S-SCN or R-S-I sulfenyl thiocyanate or iodide R-SOH sulfenicacid OSCNminus hypothiocyanite and OIminus hypoiodite
Sulfhydryl oxidation by OSCNminus generates sulfenyl thio-cyanate in equilibrium with sulfenic acid [68]
SCNminus +H2O2+ LPO 997888rarr OSCNminus + LPO
R-SH +OSCNminus 997888rarr R-S-SCN +OHminus
R-S-SCN +H2O 997888rarr R-S-OH + SCNminus +H+
(5)
The cycle of reactions shows that thiocyanate acts likea cofactor for LPO (Figure 6) so that the total number ofoxidized sulfhydryls is independent of SCNminus as long as(i) thiocyanate is not exhausted (ii) thiocyanate is not incompetition with other substrates for the binding to Com-pound I (iii) thiocyanate is not incorporated into an aromaticamino acid (iv) enoughH
2O2is present and (v) thiol moiety
is still available [68 69]Although the target of OSCNminus is a thiol moiety not
all sulfhydryls are equally sensitive to OSCNminus albumincysteine mercaptoethanol dithiothreitol glutathione and 5-thio-2-nitrobenzoic acid are all oxidized but 120573-lactoglobulinis poorly oxidized probably due to a limited accessibility ofsulfhydryls to OSCNminus [68] In some conditions that is thejoint presence of LPO enough H
2O2and SCNminus and after the
oxidation of available sulfhydryls modification of tyrosinetryptophan and histidine protein residues can occur and that
could be linked to the formation of a labile powerful oxidantsuch as sulfur dicyanide [68]
Some authors suggest that (SCN)2is formed during the
enzymatic reaction and then chemically hydrolyzed intohypothiocyanite [14 69 70] However a recent publicationdemonstrates that (SCN)
2cannot be a precursor during the
enzymatic oxidation of SCNminus at neutral pH inmammals [71]Hypothiocyanite is less stable in acid conditions with
high concentrations of SCNminus and in the presence of (SCN)2
and it is thought to break down via the following net reaction[14]
4HOSCN +H2O 997888rarr 3SCNminus + CNOminus + SO
4
2minus+ 6H+ (6)
A recent study based notably on spectroscopic and chro-matographic methods proposes the following net equationwithin the 4ndash7 pH range
3HOSCN +H2O 997888rarr XSO
4
2minus+ XHCN
+ (1 minus X) SO3
2minus+ (1 minus X)CNOminus
+ 2SCNminus + (5 minus X)H+
(7)
The proportions of end anions were different at pH 4 andpH 7 at pH 7 the proportion of CNOminus was higher SCNminusformation was slower and no CNminus was detected [71]
It might seem easier to produce hypothiocyanite chemi-cally in in vitro applications but producing hypothiocyanitechemically from the oxidation of SCNminus by a halogen (Cl
2or
Br2) or by a hypohalous acid (HOCl or HOBr) in basic media
is tricky due to overoxidation of SCNminus [66] The referencemethod in the literature to produce 1- to 2-day stable OSCNminusis by hydrolyzing (SCN)
2in basic conditions [72ndash74]
Hypothiocyanite inhibitors have been described Forexample CNminus a weak acid buffer dissolved carbonate excesshydrogen peroxide hydrofluoric acid metallic ions glyc-erol or ammonium sulfate accelerates the decomposition ofOSCNminus whereas sulfonamide stabilizes it [67 72]
Appropriate concentrations of substrates induce en-hanced activity [75]
312 Biological Activity of Hypothiocyanite The biologicalactivity of hypothiocyanite is summarized in Figure 7
The sulfhydryl moiety is essential for the activityof numerous enzymes and proteins Inhibition of bacte-rial glycolysis through the oxidation of hexokinase gly-ceraldehyde-3-phosphate dehydrogenase (GAPDH) aldo-lase and glucose-6-phosphate dehydrogenase has beenobserved [14 51 65 70 76] Inhibition of respiration andglucose transport is associated with the alteration of cellmembranes or transporters [14 51 65 77] Irreversible inhi-bition is linked to long periods of incubation and bacterialsensitivity depends on the bacterial species and on hypothio-cyanite concentrations [14 51 59] Increased concentrationsof reducing agents such as glutathione and cysteine canreverse the inhibition through buffering hypothiocyaniteand converting the reduced thiol back into sulfhydryl [1478] This defense mechanism is used by Escherichia coli itinduces the CysJ promoter during the stress response to the
Enzyme Research 7
Bacteria
Inhibition of glycolysis Inhibition of respirationInhibition of glucose transport
Possible defense mechanism of bacteria- Increased GSH and cysteine content
Peptide proteinwith SH moiety
HOSCNOSCNminus
- NAD(P)H-dependent reduction of OSCNminus
Figure 7 Biological activity of hypothiocyanite on bacteria and possible defensemechanism of the bacteria Reversible inhibition is observedin that (i) hypothiocyanite is not reactive against all thiols and (ii) if hypothiocyanite is removed or diluted the pathogen recovers Irreversibleinhibition is linked to (i) long period of incubation (ii) the bacterial species and (iii) hypothiocyanite concentration HOSCNOSCNminus acidicor basic form of hypothiocyanite and GSH glutathione
pH lt 6
Influence of iodide concentration
Influence of the pH
1 I2 and high Iminus
I5minusI6
minus
I2I3minus
6 lt pH lt 9
HOII2OHI2I3minus
Iminus + H2O2 + LPO rarr active molecules
I2 (without Iminus)HOIOIminusI2I3
minusHI2Ominus
Figure 8 Illustration of the molecules that can be present after oxidation of iodide by lactoperoxidase in presence of H2O2The active species
depend mainly on the concentration of iodide (upper part) and the pH (lower part) The species with an oxidant power are represented inbold
lactoperoxidase system [79] Another resistance mechanismcould be the NAD(P)H-dependent reduction of OSCNminuswithout any loss of the sulfhydryl compound [14 72 78]Alteration of the bacterial membrane increases the efficacyof hypothiocyanite [80]
Furthermore the activity of the entire system (enzyme +substrates) is known to be more effective than hypothiocyan-ite alone whether enzymatically or chemically producedThis has been explained by the production of short-livedhighly reactive intermediates such as O
2SCNminus and O
3SCNminus
by the enzyme or by the oxidation of OSCNminus in conditionsof excess H
2O2[65 73 81] The activity of hypothiocyanite
has been described against bacteria such as Actinomyces sppBacillus cereus Lactobacillus spp Staphylococcus albus Saureus Streptococcus spp Escherichia coli Legionella pneu-mophila Salmonella typhimurium Pseudomonas fluorescensP aeruginosa Campylobacter jejuni C coli and Listeriamonocytogenes [14 32] Reversible inhibition is observed
when cells recover after OSCNminus is depleted [14 59] Irre-versible inhibition is obtained with long-term incubation andhigh level of OSCNminus [59] Higher concentration of SCNminuscompared to Iminus is necessary to obtain inhibition against E coliand accumulation of OSCNminus is observed as it is not reactiveagainst all thiols [59]Therefore the activity of the SCNminus-LPOsystem appears to be more bacteriostatic than bactericidal
32 Activity of LPO Related to Oxidized Iodide
321 Chemistry of Oxidized Iodide Iodide is oxidized byCompound I through a single two-electron transfer thatyields oxidized Iminus in the form of I
2or HOI [14 24 82ndash85]
The active agent is composed of a mixture of species that arenot yet formally detailed due to the very complex behaviorand stability of I
2and HOI in aqueous environments that
strongly depend on pH values and iodide concentrations[66 82 83 86]
8 Enzyme Research
Based on the inorganic chemistry of iodine in waterand literature on enzymatic oxidation of iodide the activemolecules have been described as follows (Figure 8)
(i) Under pH 6 and in the presence of iodide only I2
Iminus and I3
minus are present and the only active moleculeis I2 I2concentrations decrease with increasing
concentrations of Iminus At an initial 1mM I2 with Iminus
concentrations ranging from 1mM to 100mM I2
concentrations fall from almost 1mM to 001mM asdescribed by the following association reaction [2482 83 86]
I2+ Iminus 999447999472 I
3
minus (8)
(ii) In solution within a 6ndash9 pH range and with a max-imum 1mM iodide a mixture of HOII
2OHI2I3
minus
is formed in which I3
minus is not active and I2OH is
probably less reactive than HOI or I2[86 87] If
Iminus concentrations are above 10mM I3
minus representsthe main species formed and the concentration ofactive molecules relatively drops The mechanism issummarized in the following net equations
HOI + Iminus +H+ 999447999472 I2OHminus +H+
999447999472 I2+H2O 999447999472 I
2+ Iminus 999447999472 I
3
minus
(9)
(iii) In iodine solution without iodide or when availableiodide has been oxidized the number of I
2-derived
molecules decreases with decreasing I2concentra-
tions At 1000 120583M I2 with pH-related ratios five
relevant species are observed (I2 HOI I
3
minus HI2Ominus
and OIminus) At 10 120583M I2 the main species are only I
2
HOI and OIminus and HOI could represent up to 90 ofthe active oxidant molecules at pH 8-9 [86] Below apH of 106 the following reactions are involved
I2+H2O 999447999472 HOI + Iminus +H+ (hydrolysis of I
2)
I2+ Iminus 999447999472 I
3
minus(triiodide formation independent of pH)
(10)
(iv) At high Iminus and 1 I2concentrations as in Lugol
solution I5
minus and I6
minus are formed and represent 82of the active oxidative agents [86] after the followingreaction
I3
minus+ I2999447999472 I5
minus(pentaiodide formation)
2I3
minus999447999472 I6
2minus(dimerization of I
3
minus)
(11)
The stability of HOI and I2is linked to their dispro-
portionation in iodate which has no oxidative activity inneutral and basic pH conditions [86]The disproportionationreactions read as follows
3HOI 999447999472 IO3
minus+ 2Iminus + 3H+ (disproportionation of HOI)
3I2999447999472 IO
3
minus+ 5Iminus + 6H+ (disproportionation of I
2)
(12)
I2stability increases at higher pHvalues andhigher iodide
concentrations [86] In drinking water HOI disproportion-ation is slow and varies substantially HOI has a half-life of4 days to 35 years depending on (i) the initial level of HOIthat speeds its decomposition and (ii) the presence of boratephosphate or carbonate that catalyzes its decomposition [8889]
322 Mode of Action of Oxidized Iodide The oxidativestrength of I
2is between that of the corresponding hypo-
halous acid HOI and the hypoiodite ion OIminus and ranks asfollows 0485V (OIminus) lt 0536V (I
2) lt 0987V (HOI) [66]
HOI reacts through very rapid oxidation of thiolgroups oxidation of NAD(P)H oxidation of 120573-nicotinamidemononucleotide direct reaction with thioether groupsthrough sulfoxidation and slow oxidation of the aminemoiety (Figure 5) [87 90 91] At low Iminus concentrationsiodination of tyrosine residues is catalyzed by the enzyme[14] In a cellular environment HOI seems to be more selec-tively directed against the degradation of reduced pyridinenucleotides thanHOCL andHOBr because even the presenceof excess glutathione methionine or oxidized glutathionedoes not thoroughly inhibit their oxidation [87]
In some conditions that is (i) enough iodide H2O2 and
peroxidase (ii) no accumulation of oxidized iodide and (iii)no incorporation of iodide into stable byproducts such astyrosine residues iodide acts as a cofactor (Figure 6) andthe proportion of oxidized sulfhydryls is proportional to theamount of H
2O2as described below [85 92]
2Iminus +H2O2+ LPO (native enzyme)
997888rarr I2+ 2H2O + LPO (native enzyme)
R-SH + I2997888rarr R-S-I + Iminus +H+
R-S-I+H2O 997888rarr R-S-OH + Iminus +H+
(13)
In the case of high concentrations of Iminus andor H2O2
inhibition of tyrosine iodation has been observed [83] andrelated to the pseudocatalytic redox degradation of H
2O2
with formation of O2when excessive H
2O2is present (reac-
tion 1) and production of I3
minus when excessive amounts of Iminusare present (reaction 2)
I2+H2O 997888rarr O
2+ 2Iminus + 2H+ (reaction 1)
I2+ Iminus 999447999472 I
3
minus(reaction 2)
(14)
Both reactions deplete the amount of the active oxidizingagent I
2 In the absence of tyrosine oxidized iodide reacts
with nucleophilic molecules such as Iminus Clminus or OHminus to formI2 I3
minus ICl ICl2 IOH and I
2OH [82] Some anions such as
Clminus HPO4
minus or OHminus reduce the amount of I2I3
minus but thiseffect is inversely proportional to the concentration of Iminusabove pH 9 I
2is hydrolyzed and IO
3
minus is formed [82]HOI can be produced chemically through oxidation of Iminus
by Cl2or O3 with a short half-life due to overoxidation of
HOI byCl2andO
3[89] and through oxidation of Iminus byHOCl
HOBr or NH2Cl with a longer half-life [87 89]
Enzyme Research 9
Bacteria
Inhibition of glycolysis Inhibition of respirationInhibition of glucose transportInhibition of the pentose phosphate pathway
- Peptide protein with- SH moiety- thioether moiety- NAD(P)H
HOIOIminus
I2
Figure 9 Biological activity of hypoiodite or iodine on bacteria Irreversible inhibition is observed and could be linked to (i) oxidation ofthiol groups NAD(P)H and thioether groups (ii) high reactivity of HOII
2against thiol and reduced nicotinamide nucleotides and (iii) the
incorporation of iodide in tyrosine residue of protein (iodination of protein) HOIOIminus acid or basic form of hypoiodite and I2 iodine
323 Biological Action of Oxidized Iodide The biologicalaction of oxidized iodide (Figure 9) is similar to that ofhypothiocyanite but differs in that (i) the reactivity of oxi-dized iodide is complete against thiol group and (ii) cells didnot recover after removing of oxidized iodide [59]
Due to the cofactor role of Iminus inhibition of respirationin Escherichia coli in the presence of LPO H
2O2 and Iminus is
complete with only 10120583M NaI whereas 100 120583M of solely I2
is necessary to obtain complete inhibition This is directlyrelated to the oxidation of sulfhydryls not to the percentageof iodine incorporation [92 93]
E coli seems to be more sensitive if the bacteria areincubated together with the entire system (enzyme H
2O2
and iodide) rather than adding several minutes after mixingthe enzyme with its substrates This could be linked to theformation of an unstable reactive intermediate [52]
The activity of the Iminus peroxidase system is more effectiveagainst E coli than the SCNminus system in that lower Iminusconcentrations are necessary all sulfhydryls are oxidized andcells do not recover even if the amount of I
2is not sufficient
to oxidize all SH groups [59 80] Against L acidophilushigh non physiological amounts of Iminus are necessary to obtaininhibitionwhereas small concentrations of SCNminus are effective[70]
CNminus azide EDTA and SCNminus inhibit the formation ofoxidized iodide [50 52] Increased pH values and increasedamounts of thiol and NAD(P)H compounds reduce theactivity of the iodide peroxidase system [52]
LPO-H2O2-Iminus in presence of Streptococcus mitis is active
against Staphylococcus aureus and E coli [94] LPO-H2O2-
Iminus is active against Micrococcus S aureus Listeria monocy-togenes Bacillus cereus E coli and Candida albicans [12 1980] In the presence of other peroxidases the Iminus peroxidasesystem is active against Schistosoma mansoni Fusariumnucleatum andActinobacillus actinomycetemcomitans [31 9596] Compared to SCNminus Iminus-LPO shows bactericidal activities[14 19 80]
33 Activity of LPO Related to Hypoiodite and Hypothiocyan-ite The combination of SCNminus with Iminus in the lactoperoxidasesystem has been poorly studied Tackling the enzymaticmechanism is tricky and contradictory results have been
found about microbial activity in the concomitant presenceof SCNminus and Iminus
In the presence of SCNminus and Iminus there is competitionbetween the two substrates for oxidation by lactoperoxidase[14 36] Iminus alone exhibits bactericidal activity but an SCNminusIminusratio of 01 inhibits that bactericidal effect and an SCNminusIminusratio of 1 antagonizes it due to competition for oxidation andfaster decomposition of HOSCN in the presence of Iminus [14]Against A actinomycetemcomitans the peroxidase systemwith Iminus Clminus or a combination of Iminus and Clminus is effective butaddition of SCNminus cancels the antibacterial effect [96] On theother hand a synergistic or unaffected effect of iodide in theSCNminus-H
2O2-LPO system has been shown against Candida
albicans E coli S aureusAspergillus niger and Pseudomonasaeruginosa [19 97]
4 Conclusion
Themolecular evolution of heme peroxidases and the preser-vation of their catalytic domain [6] show that the productionof strong oxidants is a powerful part of the nonimmunedefense mechanisms against pathogenic bacteria fungi orparasite which made the use of those enzymes in practicalapplications worthwhile
The enzymatic reactions involving mammalian peroxi-dases are complex and various molecules can promote orreduce dramatically the antibacterial activity of the per-oxidase system In order to favor the halogenation cyclerequired in in vitro and in vivo antimicrobial applicationsseveral points have to be taken into account (i) to avoid thepresence of competitors to iodide or thiocyanate for bindingto Compound I and to avoid the presence of inhibitorsof the enzyme or of active molecules (ii) to avoid excessH2O2concentration which is able to destruct the enzyme
and to react with iodine or hypoiodite with loosing of activemolecules (iii) to favor the presence of hypoiodite instead ofiodine due to the association reaction of iodine with iodide(iv) to avoid excess concentration of thiocyanate which caninhibit formation of Compound I (v) to use the entiresystem (enzyme + substrates) instead of active moleculesalone (vi) to favor moderate acid pH when hypothiocyaniteis the active molecule (vii) for bactericidal fungicidal or
10 Enzyme Research
parasitical applications the use of iodide has to be preferred(viii) the use of combined presence of iodide and thiocyanatehas to be checked carefully for efficacy and (ix) to favor thecofactor role of iodide or thiocyanate
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] P J OrsquoBrien ldquoPeroxidasesrdquoChemico-Biological Interactions vol129 no 1-2 pp 113ndash139 2000
[2] W Jantschko P G Furtmuller M Allegra et al ldquoRedoxintermediates of plant and mammalian peroxidases a compar-ative transient-kinetic study of their reactivity toward indolederivativesrdquo Archives of Biochemistry and Biophysics vol 398no 1 pp 12ndash22 2002
[3] S Kimura and M Ikeda-Saito ldquoHuman myeloperoxidase andthyroid peroxidase two enzymes with separate and distinctphysiological functions are evolutionarily related membersof the same gene familyrdquo Proteins Structure Function andGenetics vol 3 no 2 pp 113ndash120 1988
[4] G Battistuzzi M Bellei C A Bortolotti and M Sola ldquoRedoxproperties of heme peroxidasesrdquo Archives of Biochemistry andBiophysics vol 500 no 1 pp 21ndash36 2010
[5] M Zamocky C Jakopitsch P G Furtmuller C Dunand and CObinger ldquoThe peroxidase-cyclooxygenase superfamily recon-structed evolution of critical enzymes of the innate immunesystemrdquo Proteins Structure Function and Genetics vol 72 no2 pp 589ndash605 2008
[6] H Daiyasu and H Toh ldquoMolecular evolution of the myeloper-oxidase familyrdquo Journal of Molecular Evolution vol 51 no 5 pp433ndash445 2000
[7] D Serteyn S Grulke T Franck A Mouithys-Mickalad andG Deby-Dupont ldquoNeutrophile myeloperoxidase protectiveenzyme with strong oxidative activitiesrdquo Annales de MedecineVeterinaire vol 147 no 2 pp 79ndash93 2003
[8] S C Whitman S L Hazen D B Miller R A HegeleJ W Heinecke and M W Huff ldquoModification of type IIIVLDL their remnants and VLDL from apoE- knockout miceby p-hydroxyphenylacetaldehyde a product of myeloperox-idase activity causes marked cholesteryl ester accumulationin macrophagesrdquo Arteriosclerosis Thrombosis and VascularBiology vol 19 no 5 pp 1238ndash1249 1999
[9] T J Barrett and C L Hawkins ldquoHypothiocyanous acid benignor deadlyrdquo Chemical Research in Toxicology vol 25 no 2 pp263ndash273 2012
[10] M M Lloyd D M van Reyk M J Davies and C L HawkinsldquoHypothiocyanous acid is a more potent inducer of apoptosisand protein thiol depletion in murine macrophage cells thanhypochlorous acid or hypobromous acidrdquo Biochemical Journalvol 414 no 2 pp 271ndash280 2008
[11] J Wang and A Slungaard ldquoRole of eosinophil peroxidase inhost defense and disease pathologyrdquo Archives of Biochemistryand Biophysics vol 445 no 2 pp 256ndash260 2006
[12] M Ahariz and P Courtois ldquoCandida albicans susceptibility tolactoperoxidase-generated hypoioditerdquo Clinical Cosmetic andInvestigational Dentistry vol 2 pp 69ndash78 2010
[13] A Welk C Meller R Schubert C Schwahn A Kramerand H Below ldquoEffect of lactoperoxidase on the antimicrobialeffectiveness of the thiocyanate hydrogen peroxide combinationin a quantitative suspension testrdquo BMC Microbiology vol 9article 134 2009
[14] K M Pruitt and J O Tenovuo Eds The Lactoperoxidase Sys-tem Chemistry and Biological Significance vol 27 of Immunol-ogy Series Marcel Dekker New York NY USA 1985
[15] P G Furtmuller W Jantschko G Regelsberger C JakopitschJ Arnhold and C Obinger ldquoReaction of lactoperoxidasecompound I with halides and thiocyanaterdquo Biochemistry vol41 no 39 pp 11895ndash11900 2002
[16] P G Furtmuller U Burner and C Obinger ldquoReaction ofmyeloperoxidase compound I with chloride bromide iodideand thiocyanaterdquo Biochemistry vol 37 no 51 pp 17923ndash179301998
[17] J Arnhold E Monzani P G Furtmuller M Zederbauer LCasella and C Obinger ldquoKinetics and thermodynamics ofhalide and nitrite oxidation by mammalian heme peroxidasesrdquoEuropean Journal of Inorganic Chemistry no 19 pp 3801ndash38112006
[18] M J Davies C L Hawkins D I Pattison and M D ReesldquoMammalian heme peroxidases from molecular mechanismsto health implicationsrdquo Antioxidants and Redox Signaling vol10 no 7 pp 1199ndash1234 2008
[19] J N de Wit and A C M van Hooydonk ldquoStructure functionsand applications of lactoperoxidase in natural antimicrobialsystemsrdquo Nederlands melk en Zuiveltijdschrift vol 50 no 2 pp227ndash244 1996
[20] P G Furtmuller M Zederbauer W Jantschko et al ldquoActivesite structure and catalytic mechanisms of human peroxidasesrdquoArchives of Biochemistry and Biophysics vol 445 no 2 pp 199ndash213 2006
[21] M Zederbauer P G Furtmuller S Brogioni C JakopitschG Smulevich and C Obinger ldquoHeme to protein linkages inmammalian peroxidases impact on spectroscopic redox andcatalytic propertiesrdquo Natural Product Reports vol 24 no 3 pp571ndash584 2007
[22] G Battistuzzi M Bellei J Vlasits et al ldquoRedox thermodynam-ics of lactoperoxidase and eosinophil peroxidaserdquo Archives ofBiochemistry and Biophysics vol 494 no 1 pp 72ndash77 2010
[23] I A Sheikh A Singh N Singh et al ldquoStructural evidence ofsubstrate specificity inmammalian peroxidases structure of thethiocyanate complex with lactoperoxidase and its interactionsat 24 a 24 A resolutionrdquo The Journal of Biological Chemistryvol 284 no 22 pp 14849ndash14856 2009
[24] H Kohler and H Jenzer ldquoInteraction of lactoperoxidase withhydrogen peroxide Formation of enzyme intermediates andgeneration of free radicalsrdquo Free Radical Biology and Medicinevol 6 no 3 pp 323ndash339 1989
[25] P G Furtmuller U Burner W Jantschko G Regelsberger andC Obinger ldquoTwo-electron reduction and one-electron oxida-tion of organic hydroperoxides by human myeloperoxidaserdquoFEBS Letters vol 484 no 2 pp 139ndash143 2000
[26] A Taurog M L Dorris and D R Doerge ldquoMechanism ofsimultaneous iodination and coupling catalyzed by thyroidperoxidaserdquo Archives of Biochemistry and Biophysics vol 330no 1 pp 24ndash32 1996
[27] J E Erman L B Vitello J Matthew Mauro and J KrautldquoDetection of an oxyferryl porphyrin 120587-cation-radical interme-diate in the reaction between hydrogen peroxide and a mutant
Enzyme Research 11
yeast cytochrome c peroxidase Evidence for tryptophan-191involvement in the radical site of compound Irdquo Biochemistryvol 28 no 20 pp 7992ndash7995 1989
[28] M T Ashby ldquoInorganic chemistry of defensive peroxidases inthe human oral cavityrdquo Journal of Dental Research vol 87 no10 pp 900ndash914 2008
[29] J D Chandler and B J Day ldquoThiocyanate a potentially usefultherapeutic agent with host defense and antioxidant propertiesrdquoBiochemical Pharmacology vol 84 no 11 pp 1381ndash1387 2012
[30] E C Jong W R Henderson and S J Klebanoff ldquoBactericidalactivity of eosinophil peroxidaserdquo Journal of Immunology vol124 no 3 pp 1378ndash1382 1980
[31] E C Jong A A F Mahmoud and S J Kelbanoff ldquoPeroxidase-mediated toxicity to schistosomula of Schistosoma mansonirdquoJournal of Immunology vol 126 no 2 pp 468ndash471 1981
[32] L M Wolfson and S S Sumner ldquoAntibacterial activity of thelactoperoxidase system a reviewrdquo Journal of Food Protectionvol 56 no 10 pp 887ndash892 1993
[33] J Arnhold P G Furtmuller G Regelsberger and C ObingerldquoRedox properties of the couple compound Inative enzyme ofmyeloperoxidase and eosinophil peroxidaserdquo European Journalof Biochemistry vol 268 no 19 pp 5142ndash5148 2001
[34] P G Furtmuller J Arnhold W Jantschko M Zederbauer CJakopitsch and C Obinger ldquoStandard reduction potentials ofall couples of the peroxidase cycle of lactoperoxidaserdquo Journalof Inorganic Biochemistry vol 99 no 5 pp 1220ndash1229 2005
[35] C J van Dalen M W Whitehouse C C Winterbourn and AJ Kettle ldquoThiocyanate and chloride as competing substrates formyeloperoxidaserdquo Biochemical Journal vol 327 no 2 pp 487ndash492 1997
[36] A Slungaard and J R Mahoney Jr ldquoThiocyanate is the majorsubstrate for eosinophil peroxidase in physiologic fluids impli-cations for cytotoxicityrdquoThe Journal of Biological Chemistry vol266 no 8 pp 4903ndash4910 1991
[37] J Tenovuo ldquoAntimicrobial function of human salivamdashhowimportant is it for oral healthrdquoActaOdontologica Scandinavicavol 56 no 5 pp 250ndash256 1998
[38] R Ihalin V Loimaranta and J Tenovuo ldquoOrigin structure andbiological activities of peroxidases in human salivardquo Archives ofBiochemistry and Biophysics vol 445 no 2 pp 261ndash268 2006
[39] J A Rooke J F Flockhart and N H Sparks ldquoThe potentialfor increasing the concentrations of micro-nutrients relevant tohuman nutrition inmeat milk and eggsrdquo Journal of AgriculturalScience vol 148 no 5 pp 603ndash614 2010
[40] H Kohler A Taurog and H B Dunford ldquoSpectral studieswith lactoperoxidase and thyroid peroxidase interconversionsbetween native enzyme compound II and compound IIIrdquoArchives of Biochemistry and Biophysics vol 264 no 2 pp 438ndash449 1988
[41] I Yamazaki H S Mason and L Piette ldquoIdentification byelectron paramagnetic resonance spectroscopy of free radicalsgenerated from substrates by peroxidaserdquoThe Journal of Biolog-ical Chemistry vol 235 pp 2444ndash2449 1960
[42] B Chance ldquoThe kinetics and stoichiometry of the transitionfrom the primary to the secondary peroxidase peroxide com-plexesrdquo Archives of Biochemistry and Biophysics vol 41 no 2pp 416ndash424 1952
[43] K M Pruitt B Mansson-Rahemtulla D C Baldone andF Rahemtulla ldquoSteady-state kinetics of thiocyanate oxidationcatalyzed by human salivary peroxidaserdquo Biochemistry vol 27no 1 pp 240ndash245 1988
[44] B G J M Bolscher and R Wever ldquoA kinetic study of thereaction between humanmyeloperoxidase hydroperoxides andcyanide inhibition by chloride and thiocyanaterdquo Biochimica etBiophysica Acta Protein Structure and Molecular Enzymologyvol 788 no 1 pp 1ndash10 1984
[45] L A Marquez J T Huang and H Brian Dunford ldquoSpectraland kinetic studies on the formation of myeloperoxidase com-pounds I and II roles of hydrogen peroxide and superoxiderdquoBiochemistry vol 33 no 6 pp 1447ndash1454 1994
[46] HMAbu-Soud and S LHazen ldquoNitric oxide is a physiologicalsubstrate for mammalian peroxidasesrdquoThe Journal of BiologicalChemistry vol 275 no 48 pp 37524ndash37532 2000
[47] Y R Tahboub S Galijasevic M P Diamond and H MAbu-Soud ldquoThiocyanate modulates the catalytic activity ofmammalian peroxidasesrdquo Journal of Biological Chemistry vol280 no 28 pp 26129ndash26136 2005
[48] H Jenzer W Jones and H Kohler ldquoOn the molecularmechanismof lactoperoxidase-catalyzedH
2O2metabolism and
irreversible enzyme inactivationrdquo The Journal of BiologicalChemistry vol 261 no 33 pp 15550ndash15556 1986
[49] R P Magnusson A Taurog and M L Dorris ldquoMechanism ofiodide-dependent catalatic activity of thyroid peroxidase andlactoperoxidaserdquo The Journal of Biological Chemistry vol 259no 1 pp 197ndash205 1984
[50] D K Bhattacharyya U Bandyopadhyay and R K BanerjeeldquoEDTA inhibits lactoperoxidase-catalyzed iodide oxidation byacting as an electron-donor and interacting near the iodidebinding siterdquoMolecular and Cellular Biochemistry vol 162 no2 pp 105ndash111 1996
[51] C L Hawkins ldquoThe role of hypothiocyanous acid (HOSCN) inbiological systems HOSCN in biological systemsrdquo Free RadicalResearch vol 43 no 12 pp 1147ndash1158 2009
[52] S J Klebanoff ldquoIodination of bacteria a bactericidal mecha-nismrdquo Journal of Experimental Medicine vol 126 no 6 pp1063ndash1078 1967
[53] S J Klebanoff ldquoMyeloperoxidase-halide-hydrogen peroxideantibacterial systemrdquo Journal of Bacteriology vol 95 no 6 pp2131ndash2138 1968
[54] R K Banerjee and A G Datta ldquoSalivary peroxidasesrdquoMolecu-lar and Cellular Biochemistry vol 70 no 1 pp 21ndash29 1986
[55] MHuwiler H Jenzer andHKohler ldquoThe role of compound IIIin reversible and irreversible inactivation of lactoperoxidaserdquoEuropean Journal of Biochemistry vol 158 no 3 pp 609ndash6141986
[56] R Wever W M Kast J H Kasinoedin and R Boelens ldquoTheperoxidation of thiocyanate catalysed by myeloperoxidase andlactoperoxidaserdquo Biochimica et Biophysica Acta (BBA)ProteinStructure and Molecular vol 709 no 2 pp 212ndash219 1982
[57] C E A Souza D Maitra G M Saed et al ldquoHypochlorousacid-induced heme degradation from lactoperoxidase as anovel mechanism of free iron release and tissue injury ininflammatory diseasesrdquo PLoS ONE vol 6 no 11 Article IDe27641 2011
[58] J Carlsson ldquoBactericidal effect of hydrogen peroxide is pre-vented by the lactoperoxidase-thiocyanate system under anaer-obic conditionsrdquo Infection and Immunity vol 29 no 3 pp 1190ndash1192 1980
[59] E L Thomas and T M Aune ldquoLactoperoxidase peroxidethiocyanate antimicrobial system correlation of sulfhydryloxidation with antimicrobial actionrdquo Infection and Immunityvol 20 no 2 pp 456ndash463 1978
12 Enzyme Research
[60] J Carlsson Y Iwami and T Yamada ldquoHydrogen peroxideexcretion by oral streptococci and effect of lactoperoxidase-thiocyanate-hydrogen peroxiderdquo Infection and Immunity vol40 no 1 pp 70ndash80 1983
[61] K D Kussendrager and A C M van Hooijdonk ldquoLactoperox-idase physico-chemical properties occurrence mechanism ofaction and applicationsrdquoTheBritish Journal of Nutrition vol 84supplement 1 pp S19ndashS25 2000
[62] J P Perraudin ldquoProteines a activites biologiques lactoferrineet lactoperoxydase Connaissances recemment acquises et tech-nologies drsquoobtentionrdquo Lait vol 71 no 2 pp 191ndash211 1991
[63] J-W Boots and R Floris ldquoLactoperoxidase From catalyticmechanism to practical applicationsrdquo International Dairy Jour-nal vol 16 no 11 pp 1272ndash1276 2006
[64] A C M van Hooijdonk K D Kussendrager and J M SteijnsldquoIn vivo antimicrobial and antiviral activity of components inbovine milk and colostrum involved in non-specific defencerdquoBritish Journal of Nutrition vol 84 supplement 1 pp S127ndashS1342000
[65] D M Hogg and G R Jago ldquoThe antibacterial action of lac-toperoxidaseThe nature of the bacterial inhibitorrdquo BiochemicalJournal vol 117 no 4 pp 779ndash790 1970
[66] M T Ashby ldquoHypothiocyaniterdquo in Advances in InorganicChemistry R van Eldik and I-B Ivana Eds chapter 8 pp 263ndash303 Academic Press New York NY USA 2012
[67] E L Thomas ldquoLactoperoxidase-catalyzed oxidation of thio-cyanate equilibria between oxidized forms of thiocyanaterdquoBiochemistry vol 20 no 11 pp 3273ndash3280 1981
[68] T M Aune and E LThomas ldquoOxidation of protein sulfhydrylsby products of peroxidase-catalyzed oxidation of thiocyanateionrdquo Biochemistry vol 17 no 6 pp 1005ndash1010 1978
[69] T M Aune and E L Thomas ldquoAccumulation of hypothiocyan-ite ion during peroxidase-catalyzed oxidation of thiocyanateionrdquo European Journal of Biochemistry vol 80 no 1 pp 209ndash214 1977
[70] J D Oram and B Reiter ldquoThe inhibition of streptococci bylactoperoxidase thiocyanate and hydrogen peroxideThe effectof the inhibitory system on susceptible and resistant strains ofgroup N streptococcirdquo Biochemical Journal vol 100 no 2 pp373ndash381 1966
[71] J Kalmar K L Woldegiorgis B Biri and M T AshbyldquoMechanism of decomposition of the human defense factorhypothiocyanite near physiological pHrdquo Journal of the Ameri-can Chemical Society vol 133 no 49 pp 19911ndash19921 2011
[72] H Hoogendoorn J P PiessensW Scholtes and L A StoddardldquoHypothiocyanite ion the inhibitor formed by the system lac-toperoxidase thiocyanate hydrogen peroxide I Identification ofthe inhibiting compoundrdquoCaries Research vol 11 no 2 pp 77ndash84 1977
[73] L Bjorck and O Claesson ldquoCorrelation between concentrationof hypothiocyanate and antibacterial effect of the lactoperoxi-dase system against Escherichia colirdquo Journal of Dairy Sciencevol 63 no 6 pp 919ndash922 1980
[74] P Nagy S S Alguindigue and M T Ashby ldquoLactoperoxidase-catalyzed oxidation of thiocyanate by hydrogen peroxide areinvestigation of hypothiocyanite by nuclear magnetic reso-nance and optical spectroscopyrdquo Biochemistry vol 45 no 41pp 12610ndash12616 2006
[75] Y Adolphe M Jacquot M Linder A-M Revol-Junelles andJ-B Milliere ldquoOptimization of the components concentrationsof the lactoperoxidase system by RSMrdquo Journal of AppliedMicrobiology vol 100 no 5 pp 1034ndash1042 2006
[76] M Adamson and K M Pruitt ldquoLactoperoxidase-catalyzedinactivation of hexokinaserdquo Biochimica et Biophysica Acta vol658 no 2 pp 238ndash247 1981
[77] M N Mickelson ldquoGlucose transport in Streptococcus agalac-tiae and its inhibition by lactoperoxidase-thiocyanate-hydrogenperoxiderdquo Journal of Bacteriology vol 132 no 2 pp 541ndash5481977
[78] E L Thomas K A Pera K W Smith and A K ChwangldquoInhibition of Streptococcus mutans by the lactoperoxidaseantimicrobial systemrdquo Infection and Immunity vol 39 no 2 pp767ndash778 1983
[79] J Sermon K Vanoirbeek P De Spiegeleer R Van Houdt AAertsen and C W Michiels ldquoUnique stress response to thelactoperoxidase-thiocyanate enzyme system in Escherichia colirdquoResearch in Microbiology vol 156 no 2 pp 225ndash232 2005
[80] E L Thomas and T M Aune ldquoSusceptibility of Escherichia colito bactericidal action of lactoperoxidase peroxide and iodideor thiocyanaterdquoAntimicrobial Agents andChemotherapy vol 13no 2 pp 261ndash265 1978
[81] K M Pruitt J Tenovuo R W Andrews and T McKaneldquoLactoperoxidase-catalyzed oxidation of thiocyanate polaro-graphic study of the oxidation productsrdquo Biochemistry vol 21no 3 pp 562ndash567 1982
[82] M Huwiler and H Kohler ldquoPseudo-catalytic degradation ofhydrogen peroxide in the lactoperoxidaseH
2O2iodide sys-
temrdquo European Journal of Biochemistry vol 141 no 1 pp 69ndash741984
[83] M Huwiler U Burgi and H Kohler ldquoMechanism of enzymaticand non-enzymatic tyrosine iodination Inhibition by excesshydrogen peroxide andor iodiderdquo European Journal of Bio-chemistry vol 147 no 3 pp 469ndash476 1985
[84] M Morrison G S Bayse and A W Michaels ldquoDeterminationof spectral properties of aqueous I2 and I3- and the equilibriumconstantrdquo Analytical Biochemistry vol 42 no 1 pp 195ndash2011971
[85] E L Thomas and T M Aune ldquoPeroxidase catalyzed oxidationof protein sulfhydrylsmediated by iodinerdquoBiochemistry vol 16no 16 pp 3581ndash3586 1977
[86] W Gottardi ldquoIodine and disinfection theoretical study onmode of action efficiency stability and analytical aspects in theaqueous systemrdquo Archiv der Pharmazie vol 332 no 5 pp 151ndash157 1999
[87] W A Prutz R Kissner W H Koppenol and H RueggerldquoOn the irreversible destruction of reduced nicotinamidenucleotides by hypohalous acidsrdquo Archives of Biochemistry andBiophysics vol 380 no 1 pp 181ndash191 2000
[88] Y Bichsel and U Von Gunten ldquoHypoiodous acid kinetics ofthe buffer-catalyzed disproportionationrdquo Water Research vol34 no 12 pp 3197ndash3203 2000
[89] Y Bichsel and U von Gunten ldquoOxidation of iodide andhypoiodous acid in the disinfection of natural watersrdquo Environ-mental Science and Technology vol 33 no 22 pp 4040ndash40451999
[90] W A Prutz R Kissner T Nauser and W H Koppenol ldquoOnthe oxidation of cytochrome c by hypohalous acidsrdquo Archives ofBiochemistry and Biophysics vol 389 no 1 pp 110ndash122 2001
[91] A Virion J L Michot D Deme and J Pommier ldquoNADPHoxidation catalyzed by the peroxidaseH
2O2system Iodide-
mediated oxidation of NADPH to iodinated NADPrdquo EuropeanJournal of Biochemistry vol 148 no 2 pp 239ndash243 1985
Enzyme Research 13
[92] E L Thomas and T M Aune ldquoCofactor role of iodide in per-oxidase antimicrobial action against Escherichia colirdquo Antimic-robial Agents and Chemotherapy vol 13 no 6 pp 1000ndash10051978
[93] E L Thomas and T M Aune ldquoOxidation of Escherichiacoli sulfhydryl components by the peroxidase-hydrogenperoxide-iodide antimicrobial systemrdquo Antimicrobial Agentsand Chemotherapy vol 13 no 6 pp 1006ndash1010 1978
[94] C B Hamon and S J Klebanoff ldquoA peroxidase-mediatedstreptococcus mitis-dependent antimicrobial system in salivardquoJournal of Experimental Medicine vol 137 no 2 pp 438ndash4501973
[95] R Ihalin J Nuutila V Loimaranta M Lenander J Tenovuoand E-M Lilius ldquoSusceptibility of Fusobacterium nucleatum tokilling by peroxidase-iodide-hydrogen peroxide combinationin buffer solution and in human whole salivardquo Anaerobe vol9 no 1 pp 23ndash30 2003
[96] R Ihalin V Loimaranta M Lenander-Lumikari and J Ten-ovuo ldquoThe effects of different (pseudo)halide substrates onperoxidase-mediated killing of Actinobacillus actinomycetem-comitansrdquo Journal of Periodontal Research vol 33 no 7 pp 421ndash427 1998
[97] E H Bosch H van doorne and S de Vries ldquoThe lactoper-oxidase system the influence of iodide and the chemical andantimicrobial stability over the period of about 18 monthsrdquoJournal of AppliedMicrobiology vol 89 no 2 pp 215ndash224 2000
Inhibition of glycolysis Inhibition of respirationInhibition of glucose transport
Possible defense mechanism of bacteria- Increased GSH and cysteine content
Peptide proteinwith SH moiety
HOSCNOSCNminus
- NAD(P)H-dependent reduction of OSCNminus
Figure 7 Biological activity of hypothiocyanite on bacteria and possible defensemechanism of the bacteria Reversible inhibition is observedin that (i) hypothiocyanite is not reactive against all thiols and (ii) if hypothiocyanite is removed or diluted the pathogen recovers Irreversibleinhibition is linked to (i) long period of incubation (ii) the bacterial species and (iii) hypothiocyanite concentration HOSCNOSCNminus acidicor basic form of hypothiocyanite and GSH glutathione
pH lt 6
Influence of iodide concentration
Influence of the pH
1 I2 and high Iminus
I5minusI6
minus
I2I3minus
6 lt pH lt 9
HOII2OHI2I3minus
Iminus + H2O2 + LPO rarr active molecules
I2 (without Iminus)HOIOIminusI2I3
minusHI2Ominus
Figure 8 Illustration of the molecules that can be present after oxidation of iodide by lactoperoxidase in presence of H2O2The active species
depend mainly on the concentration of iodide (upper part) and the pH (lower part) The species with an oxidant power are represented inbold
lactoperoxidase system [79] Another resistance mechanismcould be the NAD(P)H-dependent reduction of OSCNminuswithout any loss of the sulfhydryl compound [14 72 78]Alteration of the bacterial membrane increases the efficacyof hypothiocyanite [80]
Furthermore the activity of the entire system (enzyme +substrates) is known to be more effective than hypothiocyan-ite alone whether enzymatically or chemically producedThis has been explained by the production of short-livedhighly reactive intermediates such as O
2SCNminus and O
3SCNminus
by the enzyme or by the oxidation of OSCNminus in conditionsof excess H
2O2[65 73 81] The activity of hypothiocyanite
has been described against bacteria such as Actinomyces sppBacillus cereus Lactobacillus spp Staphylococcus albus Saureus Streptococcus spp Escherichia coli Legionella pneu-mophila Salmonella typhimurium Pseudomonas fluorescensP aeruginosa Campylobacter jejuni C coli and Listeriamonocytogenes [14 32] Reversible inhibition is observed
when cells recover after OSCNminus is depleted [14 59] Irre-versible inhibition is obtained with long-term incubation andhigh level of OSCNminus [59] Higher concentration of SCNminuscompared to Iminus is necessary to obtain inhibition against E coliand accumulation of OSCNminus is observed as it is not reactiveagainst all thiols [59]Therefore the activity of the SCNminus-LPOsystem appears to be more bacteriostatic than bactericidal
32 Activity of LPO Related to Oxidized Iodide
321 Chemistry of Oxidized Iodide Iodide is oxidized byCompound I through a single two-electron transfer thatyields oxidized Iminus in the form of I
2or HOI [14 24 82ndash85]
The active agent is composed of a mixture of species that arenot yet formally detailed due to the very complex behaviorand stability of I
2and HOI in aqueous environments that
strongly depend on pH values and iodide concentrations[66 82 83 86]
8 Enzyme Research
Based on the inorganic chemistry of iodine in waterand literature on enzymatic oxidation of iodide the activemolecules have been described as follows (Figure 8)
(i) Under pH 6 and in the presence of iodide only I2
Iminus and I3
minus are present and the only active moleculeis I2 I2concentrations decrease with increasing
concentrations of Iminus At an initial 1mM I2 with Iminus
concentrations ranging from 1mM to 100mM I2
concentrations fall from almost 1mM to 001mM asdescribed by the following association reaction [2482 83 86]
I2+ Iminus 999447999472 I
3
minus (8)
(ii) In solution within a 6ndash9 pH range and with a max-imum 1mM iodide a mixture of HOII
2OHI2I3
minus
is formed in which I3
minus is not active and I2OH is
probably less reactive than HOI or I2[86 87] If
Iminus concentrations are above 10mM I3
minus representsthe main species formed and the concentration ofactive molecules relatively drops The mechanism issummarized in the following net equations
HOI + Iminus +H+ 999447999472 I2OHminus +H+
999447999472 I2+H2O 999447999472 I
2+ Iminus 999447999472 I
3
minus
(9)
(iii) In iodine solution without iodide or when availableiodide has been oxidized the number of I
2-derived
molecules decreases with decreasing I2concentra-
tions At 1000 120583M I2 with pH-related ratios five
relevant species are observed (I2 HOI I
3
minus HI2Ominus
and OIminus) At 10 120583M I2 the main species are only I
2
HOI and OIminus and HOI could represent up to 90 ofthe active oxidant molecules at pH 8-9 [86] Below apH of 106 the following reactions are involved
I2+H2O 999447999472 HOI + Iminus +H+ (hydrolysis of I
2)
I2+ Iminus 999447999472 I
3
minus(triiodide formation independent of pH)
(10)
(iv) At high Iminus and 1 I2concentrations as in Lugol
solution I5
minus and I6
minus are formed and represent 82of the active oxidative agents [86] after the followingreaction
I3
minus+ I2999447999472 I5
minus(pentaiodide formation)
2I3
minus999447999472 I6
2minus(dimerization of I
3
minus)
(11)
The stability of HOI and I2is linked to their dispro-
portionation in iodate which has no oxidative activity inneutral and basic pH conditions [86]The disproportionationreactions read as follows
3HOI 999447999472 IO3
minus+ 2Iminus + 3H+ (disproportionation of HOI)
3I2999447999472 IO
3
minus+ 5Iminus + 6H+ (disproportionation of I
2)
(12)
I2stability increases at higher pHvalues andhigher iodide
concentrations [86] In drinking water HOI disproportion-ation is slow and varies substantially HOI has a half-life of4 days to 35 years depending on (i) the initial level of HOIthat speeds its decomposition and (ii) the presence of boratephosphate or carbonate that catalyzes its decomposition [8889]
322 Mode of Action of Oxidized Iodide The oxidativestrength of I
2is between that of the corresponding hypo-
halous acid HOI and the hypoiodite ion OIminus and ranks asfollows 0485V (OIminus) lt 0536V (I
2) lt 0987V (HOI) [66]
HOI reacts through very rapid oxidation of thiolgroups oxidation of NAD(P)H oxidation of 120573-nicotinamidemononucleotide direct reaction with thioether groupsthrough sulfoxidation and slow oxidation of the aminemoiety (Figure 5) [87 90 91] At low Iminus concentrationsiodination of tyrosine residues is catalyzed by the enzyme[14] In a cellular environment HOI seems to be more selec-tively directed against the degradation of reduced pyridinenucleotides thanHOCL andHOBr because even the presenceof excess glutathione methionine or oxidized glutathionedoes not thoroughly inhibit their oxidation [87]
In some conditions that is (i) enough iodide H2O2 and
peroxidase (ii) no accumulation of oxidized iodide and (iii)no incorporation of iodide into stable byproducts such astyrosine residues iodide acts as a cofactor (Figure 6) andthe proportion of oxidized sulfhydryls is proportional to theamount of H
2O2as described below [85 92]
2Iminus +H2O2+ LPO (native enzyme)
997888rarr I2+ 2H2O + LPO (native enzyme)
R-SH + I2997888rarr R-S-I + Iminus +H+
R-S-I+H2O 997888rarr R-S-OH + Iminus +H+
(13)
In the case of high concentrations of Iminus andor H2O2
inhibition of tyrosine iodation has been observed [83] andrelated to the pseudocatalytic redox degradation of H
2O2
with formation of O2when excessive H
2O2is present (reac-
tion 1) and production of I3
minus when excessive amounts of Iminusare present (reaction 2)
I2+H2O 997888rarr O
2+ 2Iminus + 2H+ (reaction 1)
I2+ Iminus 999447999472 I
3
minus(reaction 2)
(14)
Both reactions deplete the amount of the active oxidizingagent I
2 In the absence of tyrosine oxidized iodide reacts
with nucleophilic molecules such as Iminus Clminus or OHminus to formI2 I3
minus ICl ICl2 IOH and I
2OH [82] Some anions such as
Clminus HPO4
minus or OHminus reduce the amount of I2I3
minus but thiseffect is inversely proportional to the concentration of Iminusabove pH 9 I
2is hydrolyzed and IO
3
minus is formed [82]HOI can be produced chemically through oxidation of Iminus
by Cl2or O3 with a short half-life due to overoxidation of
HOI byCl2andO
3[89] and through oxidation of Iminus byHOCl
HOBr or NH2Cl with a longer half-life [87 89]
Enzyme Research 9
Bacteria
Inhibition of glycolysis Inhibition of respirationInhibition of glucose transportInhibition of the pentose phosphate pathway
- Peptide protein with- SH moiety- thioether moiety- NAD(P)H
HOIOIminus
I2
Figure 9 Biological activity of hypoiodite or iodine on bacteria Irreversible inhibition is observed and could be linked to (i) oxidation ofthiol groups NAD(P)H and thioether groups (ii) high reactivity of HOII
2against thiol and reduced nicotinamide nucleotides and (iii) the
incorporation of iodide in tyrosine residue of protein (iodination of protein) HOIOIminus acid or basic form of hypoiodite and I2 iodine
323 Biological Action of Oxidized Iodide The biologicalaction of oxidized iodide (Figure 9) is similar to that ofhypothiocyanite but differs in that (i) the reactivity of oxi-dized iodide is complete against thiol group and (ii) cells didnot recover after removing of oxidized iodide [59]
Due to the cofactor role of Iminus inhibition of respirationin Escherichia coli in the presence of LPO H
2O2 and Iminus is
complete with only 10120583M NaI whereas 100 120583M of solely I2
is necessary to obtain complete inhibition This is directlyrelated to the oxidation of sulfhydryls not to the percentageof iodine incorporation [92 93]
E coli seems to be more sensitive if the bacteria areincubated together with the entire system (enzyme H
2O2
and iodide) rather than adding several minutes after mixingthe enzyme with its substrates This could be linked to theformation of an unstable reactive intermediate [52]
The activity of the Iminus peroxidase system is more effectiveagainst E coli than the SCNminus system in that lower Iminusconcentrations are necessary all sulfhydryls are oxidized andcells do not recover even if the amount of I
2is not sufficient
to oxidize all SH groups [59 80] Against L acidophilushigh non physiological amounts of Iminus are necessary to obtaininhibitionwhereas small concentrations of SCNminus are effective[70]
CNminus azide EDTA and SCNminus inhibit the formation ofoxidized iodide [50 52] Increased pH values and increasedamounts of thiol and NAD(P)H compounds reduce theactivity of the iodide peroxidase system [52]
LPO-H2O2-Iminus in presence of Streptococcus mitis is active
against Staphylococcus aureus and E coli [94] LPO-H2O2-
Iminus is active against Micrococcus S aureus Listeria monocy-togenes Bacillus cereus E coli and Candida albicans [12 1980] In the presence of other peroxidases the Iminus peroxidasesystem is active against Schistosoma mansoni Fusariumnucleatum andActinobacillus actinomycetemcomitans [31 9596] Compared to SCNminus Iminus-LPO shows bactericidal activities[14 19 80]
33 Activity of LPO Related to Hypoiodite and Hypothiocyan-ite The combination of SCNminus with Iminus in the lactoperoxidasesystem has been poorly studied Tackling the enzymaticmechanism is tricky and contradictory results have been
found about microbial activity in the concomitant presenceof SCNminus and Iminus
In the presence of SCNminus and Iminus there is competitionbetween the two substrates for oxidation by lactoperoxidase[14 36] Iminus alone exhibits bactericidal activity but an SCNminusIminusratio of 01 inhibits that bactericidal effect and an SCNminusIminusratio of 1 antagonizes it due to competition for oxidation andfaster decomposition of HOSCN in the presence of Iminus [14]Against A actinomycetemcomitans the peroxidase systemwith Iminus Clminus or a combination of Iminus and Clminus is effective butaddition of SCNminus cancels the antibacterial effect [96] On theother hand a synergistic or unaffected effect of iodide in theSCNminus-H
2O2-LPO system has been shown against Candida
albicans E coli S aureusAspergillus niger and Pseudomonasaeruginosa [19 97]
4 Conclusion
Themolecular evolution of heme peroxidases and the preser-vation of their catalytic domain [6] show that the productionof strong oxidants is a powerful part of the nonimmunedefense mechanisms against pathogenic bacteria fungi orparasite which made the use of those enzymes in practicalapplications worthwhile
The enzymatic reactions involving mammalian peroxi-dases are complex and various molecules can promote orreduce dramatically the antibacterial activity of the per-oxidase system In order to favor the halogenation cyclerequired in in vitro and in vivo antimicrobial applicationsseveral points have to be taken into account (i) to avoid thepresence of competitors to iodide or thiocyanate for bindingto Compound I and to avoid the presence of inhibitorsof the enzyme or of active molecules (ii) to avoid excessH2O2concentration which is able to destruct the enzyme
and to react with iodine or hypoiodite with loosing of activemolecules (iii) to favor the presence of hypoiodite instead ofiodine due to the association reaction of iodine with iodide(iv) to avoid excess concentration of thiocyanate which caninhibit formation of Compound I (v) to use the entiresystem (enzyme + substrates) instead of active moleculesalone (vi) to favor moderate acid pH when hypothiocyaniteis the active molecule (vii) for bactericidal fungicidal or
10 Enzyme Research
parasitical applications the use of iodide has to be preferred(viii) the use of combined presence of iodide and thiocyanatehas to be checked carefully for efficacy and (ix) to favor thecofactor role of iodide or thiocyanate
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] P J OrsquoBrien ldquoPeroxidasesrdquoChemico-Biological Interactions vol129 no 1-2 pp 113ndash139 2000
[2] W Jantschko P G Furtmuller M Allegra et al ldquoRedoxintermediates of plant and mammalian peroxidases a compar-ative transient-kinetic study of their reactivity toward indolederivativesrdquo Archives of Biochemistry and Biophysics vol 398no 1 pp 12ndash22 2002
[3] S Kimura and M Ikeda-Saito ldquoHuman myeloperoxidase andthyroid peroxidase two enzymes with separate and distinctphysiological functions are evolutionarily related membersof the same gene familyrdquo Proteins Structure Function andGenetics vol 3 no 2 pp 113ndash120 1988
[4] G Battistuzzi M Bellei C A Bortolotti and M Sola ldquoRedoxproperties of heme peroxidasesrdquo Archives of Biochemistry andBiophysics vol 500 no 1 pp 21ndash36 2010
[5] M Zamocky C Jakopitsch P G Furtmuller C Dunand and CObinger ldquoThe peroxidase-cyclooxygenase superfamily recon-structed evolution of critical enzymes of the innate immunesystemrdquo Proteins Structure Function and Genetics vol 72 no2 pp 589ndash605 2008
[6] H Daiyasu and H Toh ldquoMolecular evolution of the myeloper-oxidase familyrdquo Journal of Molecular Evolution vol 51 no 5 pp433ndash445 2000
[7] D Serteyn S Grulke T Franck A Mouithys-Mickalad andG Deby-Dupont ldquoNeutrophile myeloperoxidase protectiveenzyme with strong oxidative activitiesrdquo Annales de MedecineVeterinaire vol 147 no 2 pp 79ndash93 2003
[8] S C Whitman S L Hazen D B Miller R A HegeleJ W Heinecke and M W Huff ldquoModification of type IIIVLDL their remnants and VLDL from apoE- knockout miceby p-hydroxyphenylacetaldehyde a product of myeloperox-idase activity causes marked cholesteryl ester accumulationin macrophagesrdquo Arteriosclerosis Thrombosis and VascularBiology vol 19 no 5 pp 1238ndash1249 1999
[9] T J Barrett and C L Hawkins ldquoHypothiocyanous acid benignor deadlyrdquo Chemical Research in Toxicology vol 25 no 2 pp263ndash273 2012
[10] M M Lloyd D M van Reyk M J Davies and C L HawkinsldquoHypothiocyanous acid is a more potent inducer of apoptosisand protein thiol depletion in murine macrophage cells thanhypochlorous acid or hypobromous acidrdquo Biochemical Journalvol 414 no 2 pp 271ndash280 2008
[11] J Wang and A Slungaard ldquoRole of eosinophil peroxidase inhost defense and disease pathologyrdquo Archives of Biochemistryand Biophysics vol 445 no 2 pp 256ndash260 2006
[12] M Ahariz and P Courtois ldquoCandida albicans susceptibility tolactoperoxidase-generated hypoioditerdquo Clinical Cosmetic andInvestigational Dentistry vol 2 pp 69ndash78 2010
[13] A Welk C Meller R Schubert C Schwahn A Kramerand H Below ldquoEffect of lactoperoxidase on the antimicrobialeffectiveness of the thiocyanate hydrogen peroxide combinationin a quantitative suspension testrdquo BMC Microbiology vol 9article 134 2009
[14] K M Pruitt and J O Tenovuo Eds The Lactoperoxidase Sys-tem Chemistry and Biological Significance vol 27 of Immunol-ogy Series Marcel Dekker New York NY USA 1985
[15] P G Furtmuller W Jantschko G Regelsberger C JakopitschJ Arnhold and C Obinger ldquoReaction of lactoperoxidasecompound I with halides and thiocyanaterdquo Biochemistry vol41 no 39 pp 11895ndash11900 2002
[16] P G Furtmuller U Burner and C Obinger ldquoReaction ofmyeloperoxidase compound I with chloride bromide iodideand thiocyanaterdquo Biochemistry vol 37 no 51 pp 17923ndash179301998
[17] J Arnhold E Monzani P G Furtmuller M Zederbauer LCasella and C Obinger ldquoKinetics and thermodynamics ofhalide and nitrite oxidation by mammalian heme peroxidasesrdquoEuropean Journal of Inorganic Chemistry no 19 pp 3801ndash38112006
[18] M J Davies C L Hawkins D I Pattison and M D ReesldquoMammalian heme peroxidases from molecular mechanismsto health implicationsrdquo Antioxidants and Redox Signaling vol10 no 7 pp 1199ndash1234 2008
[19] J N de Wit and A C M van Hooydonk ldquoStructure functionsand applications of lactoperoxidase in natural antimicrobialsystemsrdquo Nederlands melk en Zuiveltijdschrift vol 50 no 2 pp227ndash244 1996
[20] P G Furtmuller M Zederbauer W Jantschko et al ldquoActivesite structure and catalytic mechanisms of human peroxidasesrdquoArchives of Biochemistry and Biophysics vol 445 no 2 pp 199ndash213 2006
[21] M Zederbauer P G Furtmuller S Brogioni C JakopitschG Smulevich and C Obinger ldquoHeme to protein linkages inmammalian peroxidases impact on spectroscopic redox andcatalytic propertiesrdquo Natural Product Reports vol 24 no 3 pp571ndash584 2007
[22] G Battistuzzi M Bellei J Vlasits et al ldquoRedox thermodynam-ics of lactoperoxidase and eosinophil peroxidaserdquo Archives ofBiochemistry and Biophysics vol 494 no 1 pp 72ndash77 2010
[23] I A Sheikh A Singh N Singh et al ldquoStructural evidence ofsubstrate specificity inmammalian peroxidases structure of thethiocyanate complex with lactoperoxidase and its interactionsat 24 a 24 A resolutionrdquo The Journal of Biological Chemistryvol 284 no 22 pp 14849ndash14856 2009
[24] H Kohler and H Jenzer ldquoInteraction of lactoperoxidase withhydrogen peroxide Formation of enzyme intermediates andgeneration of free radicalsrdquo Free Radical Biology and Medicinevol 6 no 3 pp 323ndash339 1989
[25] P G Furtmuller U Burner W Jantschko G Regelsberger andC Obinger ldquoTwo-electron reduction and one-electron oxida-tion of organic hydroperoxides by human myeloperoxidaserdquoFEBS Letters vol 484 no 2 pp 139ndash143 2000
[26] A Taurog M L Dorris and D R Doerge ldquoMechanism ofsimultaneous iodination and coupling catalyzed by thyroidperoxidaserdquo Archives of Biochemistry and Biophysics vol 330no 1 pp 24ndash32 1996
[27] J E Erman L B Vitello J Matthew Mauro and J KrautldquoDetection of an oxyferryl porphyrin 120587-cation-radical interme-diate in the reaction between hydrogen peroxide and a mutant
Enzyme Research 11
yeast cytochrome c peroxidase Evidence for tryptophan-191involvement in the radical site of compound Irdquo Biochemistryvol 28 no 20 pp 7992ndash7995 1989
[28] M T Ashby ldquoInorganic chemistry of defensive peroxidases inthe human oral cavityrdquo Journal of Dental Research vol 87 no10 pp 900ndash914 2008
[29] J D Chandler and B J Day ldquoThiocyanate a potentially usefultherapeutic agent with host defense and antioxidant propertiesrdquoBiochemical Pharmacology vol 84 no 11 pp 1381ndash1387 2012
[30] E C Jong W R Henderson and S J Klebanoff ldquoBactericidalactivity of eosinophil peroxidaserdquo Journal of Immunology vol124 no 3 pp 1378ndash1382 1980
[31] E C Jong A A F Mahmoud and S J Kelbanoff ldquoPeroxidase-mediated toxicity to schistosomula of Schistosoma mansonirdquoJournal of Immunology vol 126 no 2 pp 468ndash471 1981
[32] L M Wolfson and S S Sumner ldquoAntibacterial activity of thelactoperoxidase system a reviewrdquo Journal of Food Protectionvol 56 no 10 pp 887ndash892 1993
[33] J Arnhold P G Furtmuller G Regelsberger and C ObingerldquoRedox properties of the couple compound Inative enzyme ofmyeloperoxidase and eosinophil peroxidaserdquo European Journalof Biochemistry vol 268 no 19 pp 5142ndash5148 2001
[34] P G Furtmuller J Arnhold W Jantschko M Zederbauer CJakopitsch and C Obinger ldquoStandard reduction potentials ofall couples of the peroxidase cycle of lactoperoxidaserdquo Journalof Inorganic Biochemistry vol 99 no 5 pp 1220ndash1229 2005
[35] C J van Dalen M W Whitehouse C C Winterbourn and AJ Kettle ldquoThiocyanate and chloride as competing substrates formyeloperoxidaserdquo Biochemical Journal vol 327 no 2 pp 487ndash492 1997
[36] A Slungaard and J R Mahoney Jr ldquoThiocyanate is the majorsubstrate for eosinophil peroxidase in physiologic fluids impli-cations for cytotoxicityrdquoThe Journal of Biological Chemistry vol266 no 8 pp 4903ndash4910 1991
[37] J Tenovuo ldquoAntimicrobial function of human salivamdashhowimportant is it for oral healthrdquoActaOdontologica Scandinavicavol 56 no 5 pp 250ndash256 1998
[38] R Ihalin V Loimaranta and J Tenovuo ldquoOrigin structure andbiological activities of peroxidases in human salivardquo Archives ofBiochemistry and Biophysics vol 445 no 2 pp 261ndash268 2006
[39] J A Rooke J F Flockhart and N H Sparks ldquoThe potentialfor increasing the concentrations of micro-nutrients relevant tohuman nutrition inmeat milk and eggsrdquo Journal of AgriculturalScience vol 148 no 5 pp 603ndash614 2010
[40] H Kohler A Taurog and H B Dunford ldquoSpectral studieswith lactoperoxidase and thyroid peroxidase interconversionsbetween native enzyme compound II and compound IIIrdquoArchives of Biochemistry and Biophysics vol 264 no 2 pp 438ndash449 1988
[41] I Yamazaki H S Mason and L Piette ldquoIdentification byelectron paramagnetic resonance spectroscopy of free radicalsgenerated from substrates by peroxidaserdquoThe Journal of Biolog-ical Chemistry vol 235 pp 2444ndash2449 1960
[42] B Chance ldquoThe kinetics and stoichiometry of the transitionfrom the primary to the secondary peroxidase peroxide com-plexesrdquo Archives of Biochemistry and Biophysics vol 41 no 2pp 416ndash424 1952
[43] K M Pruitt B Mansson-Rahemtulla D C Baldone andF Rahemtulla ldquoSteady-state kinetics of thiocyanate oxidationcatalyzed by human salivary peroxidaserdquo Biochemistry vol 27no 1 pp 240ndash245 1988
[44] B G J M Bolscher and R Wever ldquoA kinetic study of thereaction between humanmyeloperoxidase hydroperoxides andcyanide inhibition by chloride and thiocyanaterdquo Biochimica etBiophysica Acta Protein Structure and Molecular Enzymologyvol 788 no 1 pp 1ndash10 1984
[45] L A Marquez J T Huang and H Brian Dunford ldquoSpectraland kinetic studies on the formation of myeloperoxidase com-pounds I and II roles of hydrogen peroxide and superoxiderdquoBiochemistry vol 33 no 6 pp 1447ndash1454 1994
[46] HMAbu-Soud and S LHazen ldquoNitric oxide is a physiologicalsubstrate for mammalian peroxidasesrdquoThe Journal of BiologicalChemistry vol 275 no 48 pp 37524ndash37532 2000
[47] Y R Tahboub S Galijasevic M P Diamond and H MAbu-Soud ldquoThiocyanate modulates the catalytic activity ofmammalian peroxidasesrdquo Journal of Biological Chemistry vol280 no 28 pp 26129ndash26136 2005
[48] H Jenzer W Jones and H Kohler ldquoOn the molecularmechanismof lactoperoxidase-catalyzedH
2O2metabolism and
irreversible enzyme inactivationrdquo The Journal of BiologicalChemistry vol 261 no 33 pp 15550ndash15556 1986
[49] R P Magnusson A Taurog and M L Dorris ldquoMechanism ofiodide-dependent catalatic activity of thyroid peroxidase andlactoperoxidaserdquo The Journal of Biological Chemistry vol 259no 1 pp 197ndash205 1984
[50] D K Bhattacharyya U Bandyopadhyay and R K BanerjeeldquoEDTA inhibits lactoperoxidase-catalyzed iodide oxidation byacting as an electron-donor and interacting near the iodidebinding siterdquoMolecular and Cellular Biochemistry vol 162 no2 pp 105ndash111 1996
[51] C L Hawkins ldquoThe role of hypothiocyanous acid (HOSCN) inbiological systems HOSCN in biological systemsrdquo Free RadicalResearch vol 43 no 12 pp 1147ndash1158 2009
[52] S J Klebanoff ldquoIodination of bacteria a bactericidal mecha-nismrdquo Journal of Experimental Medicine vol 126 no 6 pp1063ndash1078 1967
[53] S J Klebanoff ldquoMyeloperoxidase-halide-hydrogen peroxideantibacterial systemrdquo Journal of Bacteriology vol 95 no 6 pp2131ndash2138 1968
[54] R K Banerjee and A G Datta ldquoSalivary peroxidasesrdquoMolecu-lar and Cellular Biochemistry vol 70 no 1 pp 21ndash29 1986
[55] MHuwiler H Jenzer andHKohler ldquoThe role of compound IIIin reversible and irreversible inactivation of lactoperoxidaserdquoEuropean Journal of Biochemistry vol 158 no 3 pp 609ndash6141986
[56] R Wever W M Kast J H Kasinoedin and R Boelens ldquoTheperoxidation of thiocyanate catalysed by myeloperoxidase andlactoperoxidaserdquo Biochimica et Biophysica Acta (BBA)ProteinStructure and Molecular vol 709 no 2 pp 212ndash219 1982
[57] C E A Souza D Maitra G M Saed et al ldquoHypochlorousacid-induced heme degradation from lactoperoxidase as anovel mechanism of free iron release and tissue injury ininflammatory diseasesrdquo PLoS ONE vol 6 no 11 Article IDe27641 2011
[58] J Carlsson ldquoBactericidal effect of hydrogen peroxide is pre-vented by the lactoperoxidase-thiocyanate system under anaer-obic conditionsrdquo Infection and Immunity vol 29 no 3 pp 1190ndash1192 1980
[59] E L Thomas and T M Aune ldquoLactoperoxidase peroxidethiocyanate antimicrobial system correlation of sulfhydryloxidation with antimicrobial actionrdquo Infection and Immunityvol 20 no 2 pp 456ndash463 1978
12 Enzyme Research
[60] J Carlsson Y Iwami and T Yamada ldquoHydrogen peroxideexcretion by oral streptococci and effect of lactoperoxidase-thiocyanate-hydrogen peroxiderdquo Infection and Immunity vol40 no 1 pp 70ndash80 1983
[61] K D Kussendrager and A C M van Hooijdonk ldquoLactoperox-idase physico-chemical properties occurrence mechanism ofaction and applicationsrdquoTheBritish Journal of Nutrition vol 84supplement 1 pp S19ndashS25 2000
[62] J P Perraudin ldquoProteines a activites biologiques lactoferrineet lactoperoxydase Connaissances recemment acquises et tech-nologies drsquoobtentionrdquo Lait vol 71 no 2 pp 191ndash211 1991
[63] J-W Boots and R Floris ldquoLactoperoxidase From catalyticmechanism to practical applicationsrdquo International Dairy Jour-nal vol 16 no 11 pp 1272ndash1276 2006
[64] A C M van Hooijdonk K D Kussendrager and J M SteijnsldquoIn vivo antimicrobial and antiviral activity of components inbovine milk and colostrum involved in non-specific defencerdquoBritish Journal of Nutrition vol 84 supplement 1 pp S127ndashS1342000
[65] D M Hogg and G R Jago ldquoThe antibacterial action of lac-toperoxidaseThe nature of the bacterial inhibitorrdquo BiochemicalJournal vol 117 no 4 pp 779ndash790 1970
[66] M T Ashby ldquoHypothiocyaniterdquo in Advances in InorganicChemistry R van Eldik and I-B Ivana Eds chapter 8 pp 263ndash303 Academic Press New York NY USA 2012
[67] E L Thomas ldquoLactoperoxidase-catalyzed oxidation of thio-cyanate equilibria between oxidized forms of thiocyanaterdquoBiochemistry vol 20 no 11 pp 3273ndash3280 1981
[68] T M Aune and E LThomas ldquoOxidation of protein sulfhydrylsby products of peroxidase-catalyzed oxidation of thiocyanateionrdquo Biochemistry vol 17 no 6 pp 1005ndash1010 1978
[69] T M Aune and E L Thomas ldquoAccumulation of hypothiocyan-ite ion during peroxidase-catalyzed oxidation of thiocyanateionrdquo European Journal of Biochemistry vol 80 no 1 pp 209ndash214 1977
[70] J D Oram and B Reiter ldquoThe inhibition of streptococci bylactoperoxidase thiocyanate and hydrogen peroxideThe effectof the inhibitory system on susceptible and resistant strains ofgroup N streptococcirdquo Biochemical Journal vol 100 no 2 pp373ndash381 1966
[71] J Kalmar K L Woldegiorgis B Biri and M T AshbyldquoMechanism of decomposition of the human defense factorhypothiocyanite near physiological pHrdquo Journal of the Ameri-can Chemical Society vol 133 no 49 pp 19911ndash19921 2011
[72] H Hoogendoorn J P PiessensW Scholtes and L A StoddardldquoHypothiocyanite ion the inhibitor formed by the system lac-toperoxidase thiocyanate hydrogen peroxide I Identification ofthe inhibiting compoundrdquoCaries Research vol 11 no 2 pp 77ndash84 1977
[73] L Bjorck and O Claesson ldquoCorrelation between concentrationof hypothiocyanate and antibacterial effect of the lactoperoxi-dase system against Escherichia colirdquo Journal of Dairy Sciencevol 63 no 6 pp 919ndash922 1980
[74] P Nagy S S Alguindigue and M T Ashby ldquoLactoperoxidase-catalyzed oxidation of thiocyanate by hydrogen peroxide areinvestigation of hypothiocyanite by nuclear magnetic reso-nance and optical spectroscopyrdquo Biochemistry vol 45 no 41pp 12610ndash12616 2006
[75] Y Adolphe M Jacquot M Linder A-M Revol-Junelles andJ-B Milliere ldquoOptimization of the components concentrationsof the lactoperoxidase system by RSMrdquo Journal of AppliedMicrobiology vol 100 no 5 pp 1034ndash1042 2006
[76] M Adamson and K M Pruitt ldquoLactoperoxidase-catalyzedinactivation of hexokinaserdquo Biochimica et Biophysica Acta vol658 no 2 pp 238ndash247 1981
[77] M N Mickelson ldquoGlucose transport in Streptococcus agalac-tiae and its inhibition by lactoperoxidase-thiocyanate-hydrogenperoxiderdquo Journal of Bacteriology vol 132 no 2 pp 541ndash5481977
[78] E L Thomas K A Pera K W Smith and A K ChwangldquoInhibition of Streptococcus mutans by the lactoperoxidaseantimicrobial systemrdquo Infection and Immunity vol 39 no 2 pp767ndash778 1983
[79] J Sermon K Vanoirbeek P De Spiegeleer R Van Houdt AAertsen and C W Michiels ldquoUnique stress response to thelactoperoxidase-thiocyanate enzyme system in Escherichia colirdquoResearch in Microbiology vol 156 no 2 pp 225ndash232 2005
[80] E L Thomas and T M Aune ldquoSusceptibility of Escherichia colito bactericidal action of lactoperoxidase peroxide and iodideor thiocyanaterdquoAntimicrobial Agents andChemotherapy vol 13no 2 pp 261ndash265 1978
[81] K M Pruitt J Tenovuo R W Andrews and T McKaneldquoLactoperoxidase-catalyzed oxidation of thiocyanate polaro-graphic study of the oxidation productsrdquo Biochemistry vol 21no 3 pp 562ndash567 1982
[82] M Huwiler and H Kohler ldquoPseudo-catalytic degradation ofhydrogen peroxide in the lactoperoxidaseH
2O2iodide sys-
temrdquo European Journal of Biochemistry vol 141 no 1 pp 69ndash741984
[83] M Huwiler U Burgi and H Kohler ldquoMechanism of enzymaticand non-enzymatic tyrosine iodination Inhibition by excesshydrogen peroxide andor iodiderdquo European Journal of Bio-chemistry vol 147 no 3 pp 469ndash476 1985
[84] M Morrison G S Bayse and A W Michaels ldquoDeterminationof spectral properties of aqueous I2 and I3- and the equilibriumconstantrdquo Analytical Biochemistry vol 42 no 1 pp 195ndash2011971
[85] E L Thomas and T M Aune ldquoPeroxidase catalyzed oxidationof protein sulfhydrylsmediated by iodinerdquoBiochemistry vol 16no 16 pp 3581ndash3586 1977
[86] W Gottardi ldquoIodine and disinfection theoretical study onmode of action efficiency stability and analytical aspects in theaqueous systemrdquo Archiv der Pharmazie vol 332 no 5 pp 151ndash157 1999
[87] W A Prutz R Kissner W H Koppenol and H RueggerldquoOn the irreversible destruction of reduced nicotinamidenucleotides by hypohalous acidsrdquo Archives of Biochemistry andBiophysics vol 380 no 1 pp 181ndash191 2000
[88] Y Bichsel and U Von Gunten ldquoHypoiodous acid kinetics ofthe buffer-catalyzed disproportionationrdquo Water Research vol34 no 12 pp 3197ndash3203 2000
[89] Y Bichsel and U von Gunten ldquoOxidation of iodide andhypoiodous acid in the disinfection of natural watersrdquo Environ-mental Science and Technology vol 33 no 22 pp 4040ndash40451999
[90] W A Prutz R Kissner T Nauser and W H Koppenol ldquoOnthe oxidation of cytochrome c by hypohalous acidsrdquo Archives ofBiochemistry and Biophysics vol 389 no 1 pp 110ndash122 2001
[91] A Virion J L Michot D Deme and J Pommier ldquoNADPHoxidation catalyzed by the peroxidaseH
2O2system Iodide-
mediated oxidation of NADPH to iodinated NADPrdquo EuropeanJournal of Biochemistry vol 148 no 2 pp 239ndash243 1985
Enzyme Research 13
[92] E L Thomas and T M Aune ldquoCofactor role of iodide in per-oxidase antimicrobial action against Escherichia colirdquo Antimic-robial Agents and Chemotherapy vol 13 no 6 pp 1000ndash10051978
[93] E L Thomas and T M Aune ldquoOxidation of Escherichiacoli sulfhydryl components by the peroxidase-hydrogenperoxide-iodide antimicrobial systemrdquo Antimicrobial Agentsand Chemotherapy vol 13 no 6 pp 1006ndash1010 1978
[94] C B Hamon and S J Klebanoff ldquoA peroxidase-mediatedstreptococcus mitis-dependent antimicrobial system in salivardquoJournal of Experimental Medicine vol 137 no 2 pp 438ndash4501973
[95] R Ihalin J Nuutila V Loimaranta M Lenander J Tenovuoand E-M Lilius ldquoSusceptibility of Fusobacterium nucleatum tokilling by peroxidase-iodide-hydrogen peroxide combinationin buffer solution and in human whole salivardquo Anaerobe vol9 no 1 pp 23ndash30 2003
[96] R Ihalin V Loimaranta M Lenander-Lumikari and J Ten-ovuo ldquoThe effects of different (pseudo)halide substrates onperoxidase-mediated killing of Actinobacillus actinomycetem-comitansrdquo Journal of Periodontal Research vol 33 no 7 pp 421ndash427 1998
[97] E H Bosch H van doorne and S de Vries ldquoThe lactoper-oxidase system the influence of iodide and the chemical andantimicrobial stability over the period of about 18 monthsrdquoJournal of AppliedMicrobiology vol 89 no 2 pp 215ndash224 2000
Based on the inorganic chemistry of iodine in waterand literature on enzymatic oxidation of iodide the activemolecules have been described as follows (Figure 8)
(i) Under pH 6 and in the presence of iodide only I2
Iminus and I3
minus are present and the only active moleculeis I2 I2concentrations decrease with increasing
concentrations of Iminus At an initial 1mM I2 with Iminus
concentrations ranging from 1mM to 100mM I2
concentrations fall from almost 1mM to 001mM asdescribed by the following association reaction [2482 83 86]
I2+ Iminus 999447999472 I
3
minus (8)
(ii) In solution within a 6ndash9 pH range and with a max-imum 1mM iodide a mixture of HOII
2OHI2I3
minus
is formed in which I3
minus is not active and I2OH is
probably less reactive than HOI or I2[86 87] If
Iminus concentrations are above 10mM I3
minus representsthe main species formed and the concentration ofactive molecules relatively drops The mechanism issummarized in the following net equations
HOI + Iminus +H+ 999447999472 I2OHminus +H+
999447999472 I2+H2O 999447999472 I
2+ Iminus 999447999472 I
3
minus
(9)
(iii) In iodine solution without iodide or when availableiodide has been oxidized the number of I
2-derived
molecules decreases with decreasing I2concentra-
tions At 1000 120583M I2 with pH-related ratios five
relevant species are observed (I2 HOI I
3
minus HI2Ominus
and OIminus) At 10 120583M I2 the main species are only I
2
HOI and OIminus and HOI could represent up to 90 ofthe active oxidant molecules at pH 8-9 [86] Below apH of 106 the following reactions are involved
I2+H2O 999447999472 HOI + Iminus +H+ (hydrolysis of I
2)
I2+ Iminus 999447999472 I
3
minus(triiodide formation independent of pH)
(10)
(iv) At high Iminus and 1 I2concentrations as in Lugol
solution I5
minus and I6
minus are formed and represent 82of the active oxidative agents [86] after the followingreaction
I3
minus+ I2999447999472 I5
minus(pentaiodide formation)
2I3
minus999447999472 I6
2minus(dimerization of I
3
minus)
(11)
The stability of HOI and I2is linked to their dispro-
portionation in iodate which has no oxidative activity inneutral and basic pH conditions [86]The disproportionationreactions read as follows
3HOI 999447999472 IO3
minus+ 2Iminus + 3H+ (disproportionation of HOI)
3I2999447999472 IO
3
minus+ 5Iminus + 6H+ (disproportionation of I
2)
(12)
I2stability increases at higher pHvalues andhigher iodide
concentrations [86] In drinking water HOI disproportion-ation is slow and varies substantially HOI has a half-life of4 days to 35 years depending on (i) the initial level of HOIthat speeds its decomposition and (ii) the presence of boratephosphate or carbonate that catalyzes its decomposition [8889]
322 Mode of Action of Oxidized Iodide The oxidativestrength of I
2is between that of the corresponding hypo-
halous acid HOI and the hypoiodite ion OIminus and ranks asfollows 0485V (OIminus) lt 0536V (I
2) lt 0987V (HOI) [66]
HOI reacts through very rapid oxidation of thiolgroups oxidation of NAD(P)H oxidation of 120573-nicotinamidemononucleotide direct reaction with thioether groupsthrough sulfoxidation and slow oxidation of the aminemoiety (Figure 5) [87 90 91] At low Iminus concentrationsiodination of tyrosine residues is catalyzed by the enzyme[14] In a cellular environment HOI seems to be more selec-tively directed against the degradation of reduced pyridinenucleotides thanHOCL andHOBr because even the presenceof excess glutathione methionine or oxidized glutathionedoes not thoroughly inhibit their oxidation [87]
In some conditions that is (i) enough iodide H2O2 and
peroxidase (ii) no accumulation of oxidized iodide and (iii)no incorporation of iodide into stable byproducts such astyrosine residues iodide acts as a cofactor (Figure 6) andthe proportion of oxidized sulfhydryls is proportional to theamount of H
2O2as described below [85 92]
2Iminus +H2O2+ LPO (native enzyme)
997888rarr I2+ 2H2O + LPO (native enzyme)
R-SH + I2997888rarr R-S-I + Iminus +H+
R-S-I+H2O 997888rarr R-S-OH + Iminus +H+
(13)
In the case of high concentrations of Iminus andor H2O2
inhibition of tyrosine iodation has been observed [83] andrelated to the pseudocatalytic redox degradation of H
2O2
with formation of O2when excessive H
2O2is present (reac-
tion 1) and production of I3
minus when excessive amounts of Iminusare present (reaction 2)
I2+H2O 997888rarr O
2+ 2Iminus + 2H+ (reaction 1)
I2+ Iminus 999447999472 I
3
minus(reaction 2)
(14)
Both reactions deplete the amount of the active oxidizingagent I
2 In the absence of tyrosine oxidized iodide reacts
with nucleophilic molecules such as Iminus Clminus or OHminus to formI2 I3
minus ICl ICl2 IOH and I
2OH [82] Some anions such as
Clminus HPO4
minus or OHminus reduce the amount of I2I3
minus but thiseffect is inversely proportional to the concentration of Iminusabove pH 9 I
2is hydrolyzed and IO
3
minus is formed [82]HOI can be produced chemically through oxidation of Iminus
by Cl2or O3 with a short half-life due to overoxidation of
HOI byCl2andO
3[89] and through oxidation of Iminus byHOCl
HOBr or NH2Cl with a longer half-life [87 89]
Enzyme Research 9
Bacteria
Inhibition of glycolysis Inhibition of respirationInhibition of glucose transportInhibition of the pentose phosphate pathway
- Peptide protein with- SH moiety- thioether moiety- NAD(P)H
HOIOIminus
I2
Figure 9 Biological activity of hypoiodite or iodine on bacteria Irreversible inhibition is observed and could be linked to (i) oxidation ofthiol groups NAD(P)H and thioether groups (ii) high reactivity of HOII
2against thiol and reduced nicotinamide nucleotides and (iii) the
incorporation of iodide in tyrosine residue of protein (iodination of protein) HOIOIminus acid or basic form of hypoiodite and I2 iodine
323 Biological Action of Oxidized Iodide The biologicalaction of oxidized iodide (Figure 9) is similar to that ofhypothiocyanite but differs in that (i) the reactivity of oxi-dized iodide is complete against thiol group and (ii) cells didnot recover after removing of oxidized iodide [59]
Due to the cofactor role of Iminus inhibition of respirationin Escherichia coli in the presence of LPO H
2O2 and Iminus is
complete with only 10120583M NaI whereas 100 120583M of solely I2
is necessary to obtain complete inhibition This is directlyrelated to the oxidation of sulfhydryls not to the percentageof iodine incorporation [92 93]
E coli seems to be more sensitive if the bacteria areincubated together with the entire system (enzyme H
2O2
and iodide) rather than adding several minutes after mixingthe enzyme with its substrates This could be linked to theformation of an unstable reactive intermediate [52]
The activity of the Iminus peroxidase system is more effectiveagainst E coli than the SCNminus system in that lower Iminusconcentrations are necessary all sulfhydryls are oxidized andcells do not recover even if the amount of I
2is not sufficient
to oxidize all SH groups [59 80] Against L acidophilushigh non physiological amounts of Iminus are necessary to obtaininhibitionwhereas small concentrations of SCNminus are effective[70]
CNminus azide EDTA and SCNminus inhibit the formation ofoxidized iodide [50 52] Increased pH values and increasedamounts of thiol and NAD(P)H compounds reduce theactivity of the iodide peroxidase system [52]
LPO-H2O2-Iminus in presence of Streptococcus mitis is active
against Staphylococcus aureus and E coli [94] LPO-H2O2-
Iminus is active against Micrococcus S aureus Listeria monocy-togenes Bacillus cereus E coli and Candida albicans [12 1980] In the presence of other peroxidases the Iminus peroxidasesystem is active against Schistosoma mansoni Fusariumnucleatum andActinobacillus actinomycetemcomitans [31 9596] Compared to SCNminus Iminus-LPO shows bactericidal activities[14 19 80]
33 Activity of LPO Related to Hypoiodite and Hypothiocyan-ite The combination of SCNminus with Iminus in the lactoperoxidasesystem has been poorly studied Tackling the enzymaticmechanism is tricky and contradictory results have been
found about microbial activity in the concomitant presenceof SCNminus and Iminus
In the presence of SCNminus and Iminus there is competitionbetween the two substrates for oxidation by lactoperoxidase[14 36] Iminus alone exhibits bactericidal activity but an SCNminusIminusratio of 01 inhibits that bactericidal effect and an SCNminusIminusratio of 1 antagonizes it due to competition for oxidation andfaster decomposition of HOSCN in the presence of Iminus [14]Against A actinomycetemcomitans the peroxidase systemwith Iminus Clminus or a combination of Iminus and Clminus is effective butaddition of SCNminus cancels the antibacterial effect [96] On theother hand a synergistic or unaffected effect of iodide in theSCNminus-H
2O2-LPO system has been shown against Candida
albicans E coli S aureusAspergillus niger and Pseudomonasaeruginosa [19 97]
4 Conclusion
Themolecular evolution of heme peroxidases and the preser-vation of their catalytic domain [6] show that the productionof strong oxidants is a powerful part of the nonimmunedefense mechanisms against pathogenic bacteria fungi orparasite which made the use of those enzymes in practicalapplications worthwhile
The enzymatic reactions involving mammalian peroxi-dases are complex and various molecules can promote orreduce dramatically the antibacterial activity of the per-oxidase system In order to favor the halogenation cyclerequired in in vitro and in vivo antimicrobial applicationsseveral points have to be taken into account (i) to avoid thepresence of competitors to iodide or thiocyanate for bindingto Compound I and to avoid the presence of inhibitorsof the enzyme or of active molecules (ii) to avoid excessH2O2concentration which is able to destruct the enzyme
and to react with iodine or hypoiodite with loosing of activemolecules (iii) to favor the presence of hypoiodite instead ofiodine due to the association reaction of iodine with iodide(iv) to avoid excess concentration of thiocyanate which caninhibit formation of Compound I (v) to use the entiresystem (enzyme + substrates) instead of active moleculesalone (vi) to favor moderate acid pH when hypothiocyaniteis the active molecule (vii) for bactericidal fungicidal or
10 Enzyme Research
parasitical applications the use of iodide has to be preferred(viii) the use of combined presence of iodide and thiocyanatehas to be checked carefully for efficacy and (ix) to favor thecofactor role of iodide or thiocyanate
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] P J OrsquoBrien ldquoPeroxidasesrdquoChemico-Biological Interactions vol129 no 1-2 pp 113ndash139 2000
[2] W Jantschko P G Furtmuller M Allegra et al ldquoRedoxintermediates of plant and mammalian peroxidases a compar-ative transient-kinetic study of their reactivity toward indolederivativesrdquo Archives of Biochemistry and Biophysics vol 398no 1 pp 12ndash22 2002
[3] S Kimura and M Ikeda-Saito ldquoHuman myeloperoxidase andthyroid peroxidase two enzymes with separate and distinctphysiological functions are evolutionarily related membersof the same gene familyrdquo Proteins Structure Function andGenetics vol 3 no 2 pp 113ndash120 1988
[4] G Battistuzzi M Bellei C A Bortolotti and M Sola ldquoRedoxproperties of heme peroxidasesrdquo Archives of Biochemistry andBiophysics vol 500 no 1 pp 21ndash36 2010
[5] M Zamocky C Jakopitsch P G Furtmuller C Dunand and CObinger ldquoThe peroxidase-cyclooxygenase superfamily recon-structed evolution of critical enzymes of the innate immunesystemrdquo Proteins Structure Function and Genetics vol 72 no2 pp 589ndash605 2008
[6] H Daiyasu and H Toh ldquoMolecular evolution of the myeloper-oxidase familyrdquo Journal of Molecular Evolution vol 51 no 5 pp433ndash445 2000
[7] D Serteyn S Grulke T Franck A Mouithys-Mickalad andG Deby-Dupont ldquoNeutrophile myeloperoxidase protectiveenzyme with strong oxidative activitiesrdquo Annales de MedecineVeterinaire vol 147 no 2 pp 79ndash93 2003
[8] S C Whitman S L Hazen D B Miller R A HegeleJ W Heinecke and M W Huff ldquoModification of type IIIVLDL their remnants and VLDL from apoE- knockout miceby p-hydroxyphenylacetaldehyde a product of myeloperox-idase activity causes marked cholesteryl ester accumulationin macrophagesrdquo Arteriosclerosis Thrombosis and VascularBiology vol 19 no 5 pp 1238ndash1249 1999
[9] T J Barrett and C L Hawkins ldquoHypothiocyanous acid benignor deadlyrdquo Chemical Research in Toxicology vol 25 no 2 pp263ndash273 2012
[10] M M Lloyd D M van Reyk M J Davies and C L HawkinsldquoHypothiocyanous acid is a more potent inducer of apoptosisand protein thiol depletion in murine macrophage cells thanhypochlorous acid or hypobromous acidrdquo Biochemical Journalvol 414 no 2 pp 271ndash280 2008
[11] J Wang and A Slungaard ldquoRole of eosinophil peroxidase inhost defense and disease pathologyrdquo Archives of Biochemistryand Biophysics vol 445 no 2 pp 256ndash260 2006
[12] M Ahariz and P Courtois ldquoCandida albicans susceptibility tolactoperoxidase-generated hypoioditerdquo Clinical Cosmetic andInvestigational Dentistry vol 2 pp 69ndash78 2010
[13] A Welk C Meller R Schubert C Schwahn A Kramerand H Below ldquoEffect of lactoperoxidase on the antimicrobialeffectiveness of the thiocyanate hydrogen peroxide combinationin a quantitative suspension testrdquo BMC Microbiology vol 9article 134 2009
[14] K M Pruitt and J O Tenovuo Eds The Lactoperoxidase Sys-tem Chemistry and Biological Significance vol 27 of Immunol-ogy Series Marcel Dekker New York NY USA 1985
[15] P G Furtmuller W Jantschko G Regelsberger C JakopitschJ Arnhold and C Obinger ldquoReaction of lactoperoxidasecompound I with halides and thiocyanaterdquo Biochemistry vol41 no 39 pp 11895ndash11900 2002
[16] P G Furtmuller U Burner and C Obinger ldquoReaction ofmyeloperoxidase compound I with chloride bromide iodideand thiocyanaterdquo Biochemistry vol 37 no 51 pp 17923ndash179301998
[17] J Arnhold E Monzani P G Furtmuller M Zederbauer LCasella and C Obinger ldquoKinetics and thermodynamics ofhalide and nitrite oxidation by mammalian heme peroxidasesrdquoEuropean Journal of Inorganic Chemistry no 19 pp 3801ndash38112006
[18] M J Davies C L Hawkins D I Pattison and M D ReesldquoMammalian heme peroxidases from molecular mechanismsto health implicationsrdquo Antioxidants and Redox Signaling vol10 no 7 pp 1199ndash1234 2008
[19] J N de Wit and A C M van Hooydonk ldquoStructure functionsand applications of lactoperoxidase in natural antimicrobialsystemsrdquo Nederlands melk en Zuiveltijdschrift vol 50 no 2 pp227ndash244 1996
[20] P G Furtmuller M Zederbauer W Jantschko et al ldquoActivesite structure and catalytic mechanisms of human peroxidasesrdquoArchives of Biochemistry and Biophysics vol 445 no 2 pp 199ndash213 2006
[21] M Zederbauer P G Furtmuller S Brogioni C JakopitschG Smulevich and C Obinger ldquoHeme to protein linkages inmammalian peroxidases impact on spectroscopic redox andcatalytic propertiesrdquo Natural Product Reports vol 24 no 3 pp571ndash584 2007
[22] G Battistuzzi M Bellei J Vlasits et al ldquoRedox thermodynam-ics of lactoperoxidase and eosinophil peroxidaserdquo Archives ofBiochemistry and Biophysics vol 494 no 1 pp 72ndash77 2010
[23] I A Sheikh A Singh N Singh et al ldquoStructural evidence ofsubstrate specificity inmammalian peroxidases structure of thethiocyanate complex with lactoperoxidase and its interactionsat 24 a 24 A resolutionrdquo The Journal of Biological Chemistryvol 284 no 22 pp 14849ndash14856 2009
[24] H Kohler and H Jenzer ldquoInteraction of lactoperoxidase withhydrogen peroxide Formation of enzyme intermediates andgeneration of free radicalsrdquo Free Radical Biology and Medicinevol 6 no 3 pp 323ndash339 1989
[25] P G Furtmuller U Burner W Jantschko G Regelsberger andC Obinger ldquoTwo-electron reduction and one-electron oxida-tion of organic hydroperoxides by human myeloperoxidaserdquoFEBS Letters vol 484 no 2 pp 139ndash143 2000
[26] A Taurog M L Dorris and D R Doerge ldquoMechanism ofsimultaneous iodination and coupling catalyzed by thyroidperoxidaserdquo Archives of Biochemistry and Biophysics vol 330no 1 pp 24ndash32 1996
[27] J E Erman L B Vitello J Matthew Mauro and J KrautldquoDetection of an oxyferryl porphyrin 120587-cation-radical interme-diate in the reaction between hydrogen peroxide and a mutant
Enzyme Research 11
yeast cytochrome c peroxidase Evidence for tryptophan-191involvement in the radical site of compound Irdquo Biochemistryvol 28 no 20 pp 7992ndash7995 1989
[28] M T Ashby ldquoInorganic chemistry of defensive peroxidases inthe human oral cavityrdquo Journal of Dental Research vol 87 no10 pp 900ndash914 2008
[29] J D Chandler and B J Day ldquoThiocyanate a potentially usefultherapeutic agent with host defense and antioxidant propertiesrdquoBiochemical Pharmacology vol 84 no 11 pp 1381ndash1387 2012
[30] E C Jong W R Henderson and S J Klebanoff ldquoBactericidalactivity of eosinophil peroxidaserdquo Journal of Immunology vol124 no 3 pp 1378ndash1382 1980
[31] E C Jong A A F Mahmoud and S J Kelbanoff ldquoPeroxidase-mediated toxicity to schistosomula of Schistosoma mansonirdquoJournal of Immunology vol 126 no 2 pp 468ndash471 1981
[32] L M Wolfson and S S Sumner ldquoAntibacterial activity of thelactoperoxidase system a reviewrdquo Journal of Food Protectionvol 56 no 10 pp 887ndash892 1993
[33] J Arnhold P G Furtmuller G Regelsberger and C ObingerldquoRedox properties of the couple compound Inative enzyme ofmyeloperoxidase and eosinophil peroxidaserdquo European Journalof Biochemistry vol 268 no 19 pp 5142ndash5148 2001
[34] P G Furtmuller J Arnhold W Jantschko M Zederbauer CJakopitsch and C Obinger ldquoStandard reduction potentials ofall couples of the peroxidase cycle of lactoperoxidaserdquo Journalof Inorganic Biochemistry vol 99 no 5 pp 1220ndash1229 2005
[35] C J van Dalen M W Whitehouse C C Winterbourn and AJ Kettle ldquoThiocyanate and chloride as competing substrates formyeloperoxidaserdquo Biochemical Journal vol 327 no 2 pp 487ndash492 1997
[36] A Slungaard and J R Mahoney Jr ldquoThiocyanate is the majorsubstrate for eosinophil peroxidase in physiologic fluids impli-cations for cytotoxicityrdquoThe Journal of Biological Chemistry vol266 no 8 pp 4903ndash4910 1991
[37] J Tenovuo ldquoAntimicrobial function of human salivamdashhowimportant is it for oral healthrdquoActaOdontologica Scandinavicavol 56 no 5 pp 250ndash256 1998
[38] R Ihalin V Loimaranta and J Tenovuo ldquoOrigin structure andbiological activities of peroxidases in human salivardquo Archives ofBiochemistry and Biophysics vol 445 no 2 pp 261ndash268 2006
[39] J A Rooke J F Flockhart and N H Sparks ldquoThe potentialfor increasing the concentrations of micro-nutrients relevant tohuman nutrition inmeat milk and eggsrdquo Journal of AgriculturalScience vol 148 no 5 pp 603ndash614 2010
[40] H Kohler A Taurog and H B Dunford ldquoSpectral studieswith lactoperoxidase and thyroid peroxidase interconversionsbetween native enzyme compound II and compound IIIrdquoArchives of Biochemistry and Biophysics vol 264 no 2 pp 438ndash449 1988
[41] I Yamazaki H S Mason and L Piette ldquoIdentification byelectron paramagnetic resonance spectroscopy of free radicalsgenerated from substrates by peroxidaserdquoThe Journal of Biolog-ical Chemistry vol 235 pp 2444ndash2449 1960
[42] B Chance ldquoThe kinetics and stoichiometry of the transitionfrom the primary to the secondary peroxidase peroxide com-plexesrdquo Archives of Biochemistry and Biophysics vol 41 no 2pp 416ndash424 1952
[43] K M Pruitt B Mansson-Rahemtulla D C Baldone andF Rahemtulla ldquoSteady-state kinetics of thiocyanate oxidationcatalyzed by human salivary peroxidaserdquo Biochemistry vol 27no 1 pp 240ndash245 1988
[44] B G J M Bolscher and R Wever ldquoA kinetic study of thereaction between humanmyeloperoxidase hydroperoxides andcyanide inhibition by chloride and thiocyanaterdquo Biochimica etBiophysica Acta Protein Structure and Molecular Enzymologyvol 788 no 1 pp 1ndash10 1984
[45] L A Marquez J T Huang and H Brian Dunford ldquoSpectraland kinetic studies on the formation of myeloperoxidase com-pounds I and II roles of hydrogen peroxide and superoxiderdquoBiochemistry vol 33 no 6 pp 1447ndash1454 1994
[46] HMAbu-Soud and S LHazen ldquoNitric oxide is a physiologicalsubstrate for mammalian peroxidasesrdquoThe Journal of BiologicalChemistry vol 275 no 48 pp 37524ndash37532 2000
[47] Y R Tahboub S Galijasevic M P Diamond and H MAbu-Soud ldquoThiocyanate modulates the catalytic activity ofmammalian peroxidasesrdquo Journal of Biological Chemistry vol280 no 28 pp 26129ndash26136 2005
[48] H Jenzer W Jones and H Kohler ldquoOn the molecularmechanismof lactoperoxidase-catalyzedH
2O2metabolism and
irreversible enzyme inactivationrdquo The Journal of BiologicalChemistry vol 261 no 33 pp 15550ndash15556 1986
[49] R P Magnusson A Taurog and M L Dorris ldquoMechanism ofiodide-dependent catalatic activity of thyroid peroxidase andlactoperoxidaserdquo The Journal of Biological Chemistry vol 259no 1 pp 197ndash205 1984
[50] D K Bhattacharyya U Bandyopadhyay and R K BanerjeeldquoEDTA inhibits lactoperoxidase-catalyzed iodide oxidation byacting as an electron-donor and interacting near the iodidebinding siterdquoMolecular and Cellular Biochemistry vol 162 no2 pp 105ndash111 1996
[51] C L Hawkins ldquoThe role of hypothiocyanous acid (HOSCN) inbiological systems HOSCN in biological systemsrdquo Free RadicalResearch vol 43 no 12 pp 1147ndash1158 2009
[52] S J Klebanoff ldquoIodination of bacteria a bactericidal mecha-nismrdquo Journal of Experimental Medicine vol 126 no 6 pp1063ndash1078 1967
[53] S J Klebanoff ldquoMyeloperoxidase-halide-hydrogen peroxideantibacterial systemrdquo Journal of Bacteriology vol 95 no 6 pp2131ndash2138 1968
[54] R K Banerjee and A G Datta ldquoSalivary peroxidasesrdquoMolecu-lar and Cellular Biochemistry vol 70 no 1 pp 21ndash29 1986
[55] MHuwiler H Jenzer andHKohler ldquoThe role of compound IIIin reversible and irreversible inactivation of lactoperoxidaserdquoEuropean Journal of Biochemistry vol 158 no 3 pp 609ndash6141986
[56] R Wever W M Kast J H Kasinoedin and R Boelens ldquoTheperoxidation of thiocyanate catalysed by myeloperoxidase andlactoperoxidaserdquo Biochimica et Biophysica Acta (BBA)ProteinStructure and Molecular vol 709 no 2 pp 212ndash219 1982
[57] C E A Souza D Maitra G M Saed et al ldquoHypochlorousacid-induced heme degradation from lactoperoxidase as anovel mechanism of free iron release and tissue injury ininflammatory diseasesrdquo PLoS ONE vol 6 no 11 Article IDe27641 2011
[58] J Carlsson ldquoBactericidal effect of hydrogen peroxide is pre-vented by the lactoperoxidase-thiocyanate system under anaer-obic conditionsrdquo Infection and Immunity vol 29 no 3 pp 1190ndash1192 1980
[59] E L Thomas and T M Aune ldquoLactoperoxidase peroxidethiocyanate antimicrobial system correlation of sulfhydryloxidation with antimicrobial actionrdquo Infection and Immunityvol 20 no 2 pp 456ndash463 1978
12 Enzyme Research
[60] J Carlsson Y Iwami and T Yamada ldquoHydrogen peroxideexcretion by oral streptococci and effect of lactoperoxidase-thiocyanate-hydrogen peroxiderdquo Infection and Immunity vol40 no 1 pp 70ndash80 1983
[61] K D Kussendrager and A C M van Hooijdonk ldquoLactoperox-idase physico-chemical properties occurrence mechanism ofaction and applicationsrdquoTheBritish Journal of Nutrition vol 84supplement 1 pp S19ndashS25 2000
[62] J P Perraudin ldquoProteines a activites biologiques lactoferrineet lactoperoxydase Connaissances recemment acquises et tech-nologies drsquoobtentionrdquo Lait vol 71 no 2 pp 191ndash211 1991
[63] J-W Boots and R Floris ldquoLactoperoxidase From catalyticmechanism to practical applicationsrdquo International Dairy Jour-nal vol 16 no 11 pp 1272ndash1276 2006
[64] A C M van Hooijdonk K D Kussendrager and J M SteijnsldquoIn vivo antimicrobial and antiviral activity of components inbovine milk and colostrum involved in non-specific defencerdquoBritish Journal of Nutrition vol 84 supplement 1 pp S127ndashS1342000
[65] D M Hogg and G R Jago ldquoThe antibacterial action of lac-toperoxidaseThe nature of the bacterial inhibitorrdquo BiochemicalJournal vol 117 no 4 pp 779ndash790 1970
[66] M T Ashby ldquoHypothiocyaniterdquo in Advances in InorganicChemistry R van Eldik and I-B Ivana Eds chapter 8 pp 263ndash303 Academic Press New York NY USA 2012
[67] E L Thomas ldquoLactoperoxidase-catalyzed oxidation of thio-cyanate equilibria between oxidized forms of thiocyanaterdquoBiochemistry vol 20 no 11 pp 3273ndash3280 1981
[68] T M Aune and E LThomas ldquoOxidation of protein sulfhydrylsby products of peroxidase-catalyzed oxidation of thiocyanateionrdquo Biochemistry vol 17 no 6 pp 1005ndash1010 1978
[69] T M Aune and E L Thomas ldquoAccumulation of hypothiocyan-ite ion during peroxidase-catalyzed oxidation of thiocyanateionrdquo European Journal of Biochemistry vol 80 no 1 pp 209ndash214 1977
[70] J D Oram and B Reiter ldquoThe inhibition of streptococci bylactoperoxidase thiocyanate and hydrogen peroxideThe effectof the inhibitory system on susceptible and resistant strains ofgroup N streptococcirdquo Biochemical Journal vol 100 no 2 pp373ndash381 1966
[71] J Kalmar K L Woldegiorgis B Biri and M T AshbyldquoMechanism of decomposition of the human defense factorhypothiocyanite near physiological pHrdquo Journal of the Ameri-can Chemical Society vol 133 no 49 pp 19911ndash19921 2011
[72] H Hoogendoorn J P PiessensW Scholtes and L A StoddardldquoHypothiocyanite ion the inhibitor formed by the system lac-toperoxidase thiocyanate hydrogen peroxide I Identification ofthe inhibiting compoundrdquoCaries Research vol 11 no 2 pp 77ndash84 1977
[73] L Bjorck and O Claesson ldquoCorrelation between concentrationof hypothiocyanate and antibacterial effect of the lactoperoxi-dase system against Escherichia colirdquo Journal of Dairy Sciencevol 63 no 6 pp 919ndash922 1980
[74] P Nagy S S Alguindigue and M T Ashby ldquoLactoperoxidase-catalyzed oxidation of thiocyanate by hydrogen peroxide areinvestigation of hypothiocyanite by nuclear magnetic reso-nance and optical spectroscopyrdquo Biochemistry vol 45 no 41pp 12610ndash12616 2006
[75] Y Adolphe M Jacquot M Linder A-M Revol-Junelles andJ-B Milliere ldquoOptimization of the components concentrationsof the lactoperoxidase system by RSMrdquo Journal of AppliedMicrobiology vol 100 no 5 pp 1034ndash1042 2006
[76] M Adamson and K M Pruitt ldquoLactoperoxidase-catalyzedinactivation of hexokinaserdquo Biochimica et Biophysica Acta vol658 no 2 pp 238ndash247 1981
[77] M N Mickelson ldquoGlucose transport in Streptococcus agalac-tiae and its inhibition by lactoperoxidase-thiocyanate-hydrogenperoxiderdquo Journal of Bacteriology vol 132 no 2 pp 541ndash5481977
[78] E L Thomas K A Pera K W Smith and A K ChwangldquoInhibition of Streptococcus mutans by the lactoperoxidaseantimicrobial systemrdquo Infection and Immunity vol 39 no 2 pp767ndash778 1983
[79] J Sermon K Vanoirbeek P De Spiegeleer R Van Houdt AAertsen and C W Michiels ldquoUnique stress response to thelactoperoxidase-thiocyanate enzyme system in Escherichia colirdquoResearch in Microbiology vol 156 no 2 pp 225ndash232 2005
[80] E L Thomas and T M Aune ldquoSusceptibility of Escherichia colito bactericidal action of lactoperoxidase peroxide and iodideor thiocyanaterdquoAntimicrobial Agents andChemotherapy vol 13no 2 pp 261ndash265 1978
[81] K M Pruitt J Tenovuo R W Andrews and T McKaneldquoLactoperoxidase-catalyzed oxidation of thiocyanate polaro-graphic study of the oxidation productsrdquo Biochemistry vol 21no 3 pp 562ndash567 1982
[82] M Huwiler and H Kohler ldquoPseudo-catalytic degradation ofhydrogen peroxide in the lactoperoxidaseH
2O2iodide sys-
temrdquo European Journal of Biochemistry vol 141 no 1 pp 69ndash741984
[83] M Huwiler U Burgi and H Kohler ldquoMechanism of enzymaticand non-enzymatic tyrosine iodination Inhibition by excesshydrogen peroxide andor iodiderdquo European Journal of Bio-chemistry vol 147 no 3 pp 469ndash476 1985
[84] M Morrison G S Bayse and A W Michaels ldquoDeterminationof spectral properties of aqueous I2 and I3- and the equilibriumconstantrdquo Analytical Biochemistry vol 42 no 1 pp 195ndash2011971
[85] E L Thomas and T M Aune ldquoPeroxidase catalyzed oxidationof protein sulfhydrylsmediated by iodinerdquoBiochemistry vol 16no 16 pp 3581ndash3586 1977
[86] W Gottardi ldquoIodine and disinfection theoretical study onmode of action efficiency stability and analytical aspects in theaqueous systemrdquo Archiv der Pharmazie vol 332 no 5 pp 151ndash157 1999
[87] W A Prutz R Kissner W H Koppenol and H RueggerldquoOn the irreversible destruction of reduced nicotinamidenucleotides by hypohalous acidsrdquo Archives of Biochemistry andBiophysics vol 380 no 1 pp 181ndash191 2000
[88] Y Bichsel and U Von Gunten ldquoHypoiodous acid kinetics ofthe buffer-catalyzed disproportionationrdquo Water Research vol34 no 12 pp 3197ndash3203 2000
[89] Y Bichsel and U von Gunten ldquoOxidation of iodide andhypoiodous acid in the disinfection of natural watersrdquo Environ-mental Science and Technology vol 33 no 22 pp 4040ndash40451999
[90] W A Prutz R Kissner T Nauser and W H Koppenol ldquoOnthe oxidation of cytochrome c by hypohalous acidsrdquo Archives ofBiochemistry and Biophysics vol 389 no 1 pp 110ndash122 2001
[91] A Virion J L Michot D Deme and J Pommier ldquoNADPHoxidation catalyzed by the peroxidaseH
2O2system Iodide-
mediated oxidation of NADPH to iodinated NADPrdquo EuropeanJournal of Biochemistry vol 148 no 2 pp 239ndash243 1985
Enzyme Research 13
[92] E L Thomas and T M Aune ldquoCofactor role of iodide in per-oxidase antimicrobial action against Escherichia colirdquo Antimic-robial Agents and Chemotherapy vol 13 no 6 pp 1000ndash10051978
[93] E L Thomas and T M Aune ldquoOxidation of Escherichiacoli sulfhydryl components by the peroxidase-hydrogenperoxide-iodide antimicrobial systemrdquo Antimicrobial Agentsand Chemotherapy vol 13 no 6 pp 1006ndash1010 1978
[94] C B Hamon and S J Klebanoff ldquoA peroxidase-mediatedstreptococcus mitis-dependent antimicrobial system in salivardquoJournal of Experimental Medicine vol 137 no 2 pp 438ndash4501973
[95] R Ihalin J Nuutila V Loimaranta M Lenander J Tenovuoand E-M Lilius ldquoSusceptibility of Fusobacterium nucleatum tokilling by peroxidase-iodide-hydrogen peroxide combinationin buffer solution and in human whole salivardquo Anaerobe vol9 no 1 pp 23ndash30 2003
[96] R Ihalin V Loimaranta M Lenander-Lumikari and J Ten-ovuo ldquoThe effects of different (pseudo)halide substrates onperoxidase-mediated killing of Actinobacillus actinomycetem-comitansrdquo Journal of Periodontal Research vol 33 no 7 pp 421ndash427 1998
[97] E H Bosch H van doorne and S de Vries ldquoThe lactoper-oxidase system the influence of iodide and the chemical andantimicrobial stability over the period of about 18 monthsrdquoJournal of AppliedMicrobiology vol 89 no 2 pp 215ndash224 2000
Inhibition of glycolysis Inhibition of respirationInhibition of glucose transportInhibition of the pentose phosphate pathway
- Peptide protein with- SH moiety- thioether moiety- NAD(P)H
HOIOIminus
I2
Figure 9 Biological activity of hypoiodite or iodine on bacteria Irreversible inhibition is observed and could be linked to (i) oxidation ofthiol groups NAD(P)H and thioether groups (ii) high reactivity of HOII
2against thiol and reduced nicotinamide nucleotides and (iii) the
incorporation of iodide in tyrosine residue of protein (iodination of protein) HOIOIminus acid or basic form of hypoiodite and I2 iodine
323 Biological Action of Oxidized Iodide The biologicalaction of oxidized iodide (Figure 9) is similar to that ofhypothiocyanite but differs in that (i) the reactivity of oxi-dized iodide is complete against thiol group and (ii) cells didnot recover after removing of oxidized iodide [59]
Due to the cofactor role of Iminus inhibition of respirationin Escherichia coli in the presence of LPO H
2O2 and Iminus is
complete with only 10120583M NaI whereas 100 120583M of solely I2
is necessary to obtain complete inhibition This is directlyrelated to the oxidation of sulfhydryls not to the percentageof iodine incorporation [92 93]
E coli seems to be more sensitive if the bacteria areincubated together with the entire system (enzyme H
2O2
and iodide) rather than adding several minutes after mixingthe enzyme with its substrates This could be linked to theformation of an unstable reactive intermediate [52]
The activity of the Iminus peroxidase system is more effectiveagainst E coli than the SCNminus system in that lower Iminusconcentrations are necessary all sulfhydryls are oxidized andcells do not recover even if the amount of I
2is not sufficient
to oxidize all SH groups [59 80] Against L acidophilushigh non physiological amounts of Iminus are necessary to obtaininhibitionwhereas small concentrations of SCNminus are effective[70]
CNminus azide EDTA and SCNminus inhibit the formation ofoxidized iodide [50 52] Increased pH values and increasedamounts of thiol and NAD(P)H compounds reduce theactivity of the iodide peroxidase system [52]
LPO-H2O2-Iminus in presence of Streptococcus mitis is active
against Staphylococcus aureus and E coli [94] LPO-H2O2-
Iminus is active against Micrococcus S aureus Listeria monocy-togenes Bacillus cereus E coli and Candida albicans [12 1980] In the presence of other peroxidases the Iminus peroxidasesystem is active against Schistosoma mansoni Fusariumnucleatum andActinobacillus actinomycetemcomitans [31 9596] Compared to SCNminus Iminus-LPO shows bactericidal activities[14 19 80]
33 Activity of LPO Related to Hypoiodite and Hypothiocyan-ite The combination of SCNminus with Iminus in the lactoperoxidasesystem has been poorly studied Tackling the enzymaticmechanism is tricky and contradictory results have been
found about microbial activity in the concomitant presenceof SCNminus and Iminus
In the presence of SCNminus and Iminus there is competitionbetween the two substrates for oxidation by lactoperoxidase[14 36] Iminus alone exhibits bactericidal activity but an SCNminusIminusratio of 01 inhibits that bactericidal effect and an SCNminusIminusratio of 1 antagonizes it due to competition for oxidation andfaster decomposition of HOSCN in the presence of Iminus [14]Against A actinomycetemcomitans the peroxidase systemwith Iminus Clminus or a combination of Iminus and Clminus is effective butaddition of SCNminus cancels the antibacterial effect [96] On theother hand a synergistic or unaffected effect of iodide in theSCNminus-H
2O2-LPO system has been shown against Candida
albicans E coli S aureusAspergillus niger and Pseudomonasaeruginosa [19 97]
4 Conclusion
Themolecular evolution of heme peroxidases and the preser-vation of their catalytic domain [6] show that the productionof strong oxidants is a powerful part of the nonimmunedefense mechanisms against pathogenic bacteria fungi orparasite which made the use of those enzymes in practicalapplications worthwhile
The enzymatic reactions involving mammalian peroxi-dases are complex and various molecules can promote orreduce dramatically the antibacterial activity of the per-oxidase system In order to favor the halogenation cyclerequired in in vitro and in vivo antimicrobial applicationsseveral points have to be taken into account (i) to avoid thepresence of competitors to iodide or thiocyanate for bindingto Compound I and to avoid the presence of inhibitorsof the enzyme or of active molecules (ii) to avoid excessH2O2concentration which is able to destruct the enzyme
and to react with iodine or hypoiodite with loosing of activemolecules (iii) to favor the presence of hypoiodite instead ofiodine due to the association reaction of iodine with iodide(iv) to avoid excess concentration of thiocyanate which caninhibit formation of Compound I (v) to use the entiresystem (enzyme + substrates) instead of active moleculesalone (vi) to favor moderate acid pH when hypothiocyaniteis the active molecule (vii) for bactericidal fungicidal or
10 Enzyme Research
parasitical applications the use of iodide has to be preferred(viii) the use of combined presence of iodide and thiocyanatehas to be checked carefully for efficacy and (ix) to favor thecofactor role of iodide or thiocyanate
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] P J OrsquoBrien ldquoPeroxidasesrdquoChemico-Biological Interactions vol129 no 1-2 pp 113ndash139 2000
[2] W Jantschko P G Furtmuller M Allegra et al ldquoRedoxintermediates of plant and mammalian peroxidases a compar-ative transient-kinetic study of their reactivity toward indolederivativesrdquo Archives of Biochemistry and Biophysics vol 398no 1 pp 12ndash22 2002
[3] S Kimura and M Ikeda-Saito ldquoHuman myeloperoxidase andthyroid peroxidase two enzymes with separate and distinctphysiological functions are evolutionarily related membersof the same gene familyrdquo Proteins Structure Function andGenetics vol 3 no 2 pp 113ndash120 1988
[4] G Battistuzzi M Bellei C A Bortolotti and M Sola ldquoRedoxproperties of heme peroxidasesrdquo Archives of Biochemistry andBiophysics vol 500 no 1 pp 21ndash36 2010
[5] M Zamocky C Jakopitsch P G Furtmuller C Dunand and CObinger ldquoThe peroxidase-cyclooxygenase superfamily recon-structed evolution of critical enzymes of the innate immunesystemrdquo Proteins Structure Function and Genetics vol 72 no2 pp 589ndash605 2008
[6] H Daiyasu and H Toh ldquoMolecular evolution of the myeloper-oxidase familyrdquo Journal of Molecular Evolution vol 51 no 5 pp433ndash445 2000
[7] D Serteyn S Grulke T Franck A Mouithys-Mickalad andG Deby-Dupont ldquoNeutrophile myeloperoxidase protectiveenzyme with strong oxidative activitiesrdquo Annales de MedecineVeterinaire vol 147 no 2 pp 79ndash93 2003
[8] S C Whitman S L Hazen D B Miller R A HegeleJ W Heinecke and M W Huff ldquoModification of type IIIVLDL their remnants and VLDL from apoE- knockout miceby p-hydroxyphenylacetaldehyde a product of myeloperox-idase activity causes marked cholesteryl ester accumulationin macrophagesrdquo Arteriosclerosis Thrombosis and VascularBiology vol 19 no 5 pp 1238ndash1249 1999
[9] T J Barrett and C L Hawkins ldquoHypothiocyanous acid benignor deadlyrdquo Chemical Research in Toxicology vol 25 no 2 pp263ndash273 2012
[10] M M Lloyd D M van Reyk M J Davies and C L HawkinsldquoHypothiocyanous acid is a more potent inducer of apoptosisand protein thiol depletion in murine macrophage cells thanhypochlorous acid or hypobromous acidrdquo Biochemical Journalvol 414 no 2 pp 271ndash280 2008
[11] J Wang and A Slungaard ldquoRole of eosinophil peroxidase inhost defense and disease pathologyrdquo Archives of Biochemistryand Biophysics vol 445 no 2 pp 256ndash260 2006
[12] M Ahariz and P Courtois ldquoCandida albicans susceptibility tolactoperoxidase-generated hypoioditerdquo Clinical Cosmetic andInvestigational Dentistry vol 2 pp 69ndash78 2010
[13] A Welk C Meller R Schubert C Schwahn A Kramerand H Below ldquoEffect of lactoperoxidase on the antimicrobialeffectiveness of the thiocyanate hydrogen peroxide combinationin a quantitative suspension testrdquo BMC Microbiology vol 9article 134 2009
[14] K M Pruitt and J O Tenovuo Eds The Lactoperoxidase Sys-tem Chemistry and Biological Significance vol 27 of Immunol-ogy Series Marcel Dekker New York NY USA 1985
[15] P G Furtmuller W Jantschko G Regelsberger C JakopitschJ Arnhold and C Obinger ldquoReaction of lactoperoxidasecompound I with halides and thiocyanaterdquo Biochemistry vol41 no 39 pp 11895ndash11900 2002
[16] P G Furtmuller U Burner and C Obinger ldquoReaction ofmyeloperoxidase compound I with chloride bromide iodideand thiocyanaterdquo Biochemistry vol 37 no 51 pp 17923ndash179301998
[17] J Arnhold E Monzani P G Furtmuller M Zederbauer LCasella and C Obinger ldquoKinetics and thermodynamics ofhalide and nitrite oxidation by mammalian heme peroxidasesrdquoEuropean Journal of Inorganic Chemistry no 19 pp 3801ndash38112006
[18] M J Davies C L Hawkins D I Pattison and M D ReesldquoMammalian heme peroxidases from molecular mechanismsto health implicationsrdquo Antioxidants and Redox Signaling vol10 no 7 pp 1199ndash1234 2008
[19] J N de Wit and A C M van Hooydonk ldquoStructure functionsand applications of lactoperoxidase in natural antimicrobialsystemsrdquo Nederlands melk en Zuiveltijdschrift vol 50 no 2 pp227ndash244 1996
[20] P G Furtmuller M Zederbauer W Jantschko et al ldquoActivesite structure and catalytic mechanisms of human peroxidasesrdquoArchives of Biochemistry and Biophysics vol 445 no 2 pp 199ndash213 2006
[21] M Zederbauer P G Furtmuller S Brogioni C JakopitschG Smulevich and C Obinger ldquoHeme to protein linkages inmammalian peroxidases impact on spectroscopic redox andcatalytic propertiesrdquo Natural Product Reports vol 24 no 3 pp571ndash584 2007
[22] G Battistuzzi M Bellei J Vlasits et al ldquoRedox thermodynam-ics of lactoperoxidase and eosinophil peroxidaserdquo Archives ofBiochemistry and Biophysics vol 494 no 1 pp 72ndash77 2010
[23] I A Sheikh A Singh N Singh et al ldquoStructural evidence ofsubstrate specificity inmammalian peroxidases structure of thethiocyanate complex with lactoperoxidase and its interactionsat 24 a 24 A resolutionrdquo The Journal of Biological Chemistryvol 284 no 22 pp 14849ndash14856 2009
[24] H Kohler and H Jenzer ldquoInteraction of lactoperoxidase withhydrogen peroxide Formation of enzyme intermediates andgeneration of free radicalsrdquo Free Radical Biology and Medicinevol 6 no 3 pp 323ndash339 1989
[25] P G Furtmuller U Burner W Jantschko G Regelsberger andC Obinger ldquoTwo-electron reduction and one-electron oxida-tion of organic hydroperoxides by human myeloperoxidaserdquoFEBS Letters vol 484 no 2 pp 139ndash143 2000
[26] A Taurog M L Dorris and D R Doerge ldquoMechanism ofsimultaneous iodination and coupling catalyzed by thyroidperoxidaserdquo Archives of Biochemistry and Biophysics vol 330no 1 pp 24ndash32 1996
[27] J E Erman L B Vitello J Matthew Mauro and J KrautldquoDetection of an oxyferryl porphyrin 120587-cation-radical interme-diate in the reaction between hydrogen peroxide and a mutant
Enzyme Research 11
yeast cytochrome c peroxidase Evidence for tryptophan-191involvement in the radical site of compound Irdquo Biochemistryvol 28 no 20 pp 7992ndash7995 1989
[28] M T Ashby ldquoInorganic chemistry of defensive peroxidases inthe human oral cavityrdquo Journal of Dental Research vol 87 no10 pp 900ndash914 2008
[29] J D Chandler and B J Day ldquoThiocyanate a potentially usefultherapeutic agent with host defense and antioxidant propertiesrdquoBiochemical Pharmacology vol 84 no 11 pp 1381ndash1387 2012
[30] E C Jong W R Henderson and S J Klebanoff ldquoBactericidalactivity of eosinophil peroxidaserdquo Journal of Immunology vol124 no 3 pp 1378ndash1382 1980
[31] E C Jong A A F Mahmoud and S J Kelbanoff ldquoPeroxidase-mediated toxicity to schistosomula of Schistosoma mansonirdquoJournal of Immunology vol 126 no 2 pp 468ndash471 1981
[32] L M Wolfson and S S Sumner ldquoAntibacterial activity of thelactoperoxidase system a reviewrdquo Journal of Food Protectionvol 56 no 10 pp 887ndash892 1993
[33] J Arnhold P G Furtmuller G Regelsberger and C ObingerldquoRedox properties of the couple compound Inative enzyme ofmyeloperoxidase and eosinophil peroxidaserdquo European Journalof Biochemistry vol 268 no 19 pp 5142ndash5148 2001
[34] P G Furtmuller J Arnhold W Jantschko M Zederbauer CJakopitsch and C Obinger ldquoStandard reduction potentials ofall couples of the peroxidase cycle of lactoperoxidaserdquo Journalof Inorganic Biochemistry vol 99 no 5 pp 1220ndash1229 2005
[35] C J van Dalen M W Whitehouse C C Winterbourn and AJ Kettle ldquoThiocyanate and chloride as competing substrates formyeloperoxidaserdquo Biochemical Journal vol 327 no 2 pp 487ndash492 1997
[36] A Slungaard and J R Mahoney Jr ldquoThiocyanate is the majorsubstrate for eosinophil peroxidase in physiologic fluids impli-cations for cytotoxicityrdquoThe Journal of Biological Chemistry vol266 no 8 pp 4903ndash4910 1991
[37] J Tenovuo ldquoAntimicrobial function of human salivamdashhowimportant is it for oral healthrdquoActaOdontologica Scandinavicavol 56 no 5 pp 250ndash256 1998
[38] R Ihalin V Loimaranta and J Tenovuo ldquoOrigin structure andbiological activities of peroxidases in human salivardquo Archives ofBiochemistry and Biophysics vol 445 no 2 pp 261ndash268 2006
[39] J A Rooke J F Flockhart and N H Sparks ldquoThe potentialfor increasing the concentrations of micro-nutrients relevant tohuman nutrition inmeat milk and eggsrdquo Journal of AgriculturalScience vol 148 no 5 pp 603ndash614 2010
[40] H Kohler A Taurog and H B Dunford ldquoSpectral studieswith lactoperoxidase and thyroid peroxidase interconversionsbetween native enzyme compound II and compound IIIrdquoArchives of Biochemistry and Biophysics vol 264 no 2 pp 438ndash449 1988
[41] I Yamazaki H S Mason and L Piette ldquoIdentification byelectron paramagnetic resonance spectroscopy of free radicalsgenerated from substrates by peroxidaserdquoThe Journal of Biolog-ical Chemistry vol 235 pp 2444ndash2449 1960
[42] B Chance ldquoThe kinetics and stoichiometry of the transitionfrom the primary to the secondary peroxidase peroxide com-plexesrdquo Archives of Biochemistry and Biophysics vol 41 no 2pp 416ndash424 1952
[43] K M Pruitt B Mansson-Rahemtulla D C Baldone andF Rahemtulla ldquoSteady-state kinetics of thiocyanate oxidationcatalyzed by human salivary peroxidaserdquo Biochemistry vol 27no 1 pp 240ndash245 1988
[44] B G J M Bolscher and R Wever ldquoA kinetic study of thereaction between humanmyeloperoxidase hydroperoxides andcyanide inhibition by chloride and thiocyanaterdquo Biochimica etBiophysica Acta Protein Structure and Molecular Enzymologyvol 788 no 1 pp 1ndash10 1984
[45] L A Marquez J T Huang and H Brian Dunford ldquoSpectraland kinetic studies on the formation of myeloperoxidase com-pounds I and II roles of hydrogen peroxide and superoxiderdquoBiochemistry vol 33 no 6 pp 1447ndash1454 1994
[46] HMAbu-Soud and S LHazen ldquoNitric oxide is a physiologicalsubstrate for mammalian peroxidasesrdquoThe Journal of BiologicalChemistry vol 275 no 48 pp 37524ndash37532 2000
[47] Y R Tahboub S Galijasevic M P Diamond and H MAbu-Soud ldquoThiocyanate modulates the catalytic activity ofmammalian peroxidasesrdquo Journal of Biological Chemistry vol280 no 28 pp 26129ndash26136 2005
[48] H Jenzer W Jones and H Kohler ldquoOn the molecularmechanismof lactoperoxidase-catalyzedH
2O2metabolism and
irreversible enzyme inactivationrdquo The Journal of BiologicalChemistry vol 261 no 33 pp 15550ndash15556 1986
[49] R P Magnusson A Taurog and M L Dorris ldquoMechanism ofiodide-dependent catalatic activity of thyroid peroxidase andlactoperoxidaserdquo The Journal of Biological Chemistry vol 259no 1 pp 197ndash205 1984
[50] D K Bhattacharyya U Bandyopadhyay and R K BanerjeeldquoEDTA inhibits lactoperoxidase-catalyzed iodide oxidation byacting as an electron-donor and interacting near the iodidebinding siterdquoMolecular and Cellular Biochemistry vol 162 no2 pp 105ndash111 1996
[51] C L Hawkins ldquoThe role of hypothiocyanous acid (HOSCN) inbiological systems HOSCN in biological systemsrdquo Free RadicalResearch vol 43 no 12 pp 1147ndash1158 2009
[52] S J Klebanoff ldquoIodination of bacteria a bactericidal mecha-nismrdquo Journal of Experimental Medicine vol 126 no 6 pp1063ndash1078 1967
[53] S J Klebanoff ldquoMyeloperoxidase-halide-hydrogen peroxideantibacterial systemrdquo Journal of Bacteriology vol 95 no 6 pp2131ndash2138 1968
[54] R K Banerjee and A G Datta ldquoSalivary peroxidasesrdquoMolecu-lar and Cellular Biochemistry vol 70 no 1 pp 21ndash29 1986
[55] MHuwiler H Jenzer andHKohler ldquoThe role of compound IIIin reversible and irreversible inactivation of lactoperoxidaserdquoEuropean Journal of Biochemistry vol 158 no 3 pp 609ndash6141986
[56] R Wever W M Kast J H Kasinoedin and R Boelens ldquoTheperoxidation of thiocyanate catalysed by myeloperoxidase andlactoperoxidaserdquo Biochimica et Biophysica Acta (BBA)ProteinStructure and Molecular vol 709 no 2 pp 212ndash219 1982
[57] C E A Souza D Maitra G M Saed et al ldquoHypochlorousacid-induced heme degradation from lactoperoxidase as anovel mechanism of free iron release and tissue injury ininflammatory diseasesrdquo PLoS ONE vol 6 no 11 Article IDe27641 2011
[58] J Carlsson ldquoBactericidal effect of hydrogen peroxide is pre-vented by the lactoperoxidase-thiocyanate system under anaer-obic conditionsrdquo Infection and Immunity vol 29 no 3 pp 1190ndash1192 1980
[59] E L Thomas and T M Aune ldquoLactoperoxidase peroxidethiocyanate antimicrobial system correlation of sulfhydryloxidation with antimicrobial actionrdquo Infection and Immunityvol 20 no 2 pp 456ndash463 1978
12 Enzyme Research
[60] J Carlsson Y Iwami and T Yamada ldquoHydrogen peroxideexcretion by oral streptococci and effect of lactoperoxidase-thiocyanate-hydrogen peroxiderdquo Infection and Immunity vol40 no 1 pp 70ndash80 1983
[61] K D Kussendrager and A C M van Hooijdonk ldquoLactoperox-idase physico-chemical properties occurrence mechanism ofaction and applicationsrdquoTheBritish Journal of Nutrition vol 84supplement 1 pp S19ndashS25 2000
[62] J P Perraudin ldquoProteines a activites biologiques lactoferrineet lactoperoxydase Connaissances recemment acquises et tech-nologies drsquoobtentionrdquo Lait vol 71 no 2 pp 191ndash211 1991
[63] J-W Boots and R Floris ldquoLactoperoxidase From catalyticmechanism to practical applicationsrdquo International Dairy Jour-nal vol 16 no 11 pp 1272ndash1276 2006
[64] A C M van Hooijdonk K D Kussendrager and J M SteijnsldquoIn vivo antimicrobial and antiviral activity of components inbovine milk and colostrum involved in non-specific defencerdquoBritish Journal of Nutrition vol 84 supplement 1 pp S127ndashS1342000
[65] D M Hogg and G R Jago ldquoThe antibacterial action of lac-toperoxidaseThe nature of the bacterial inhibitorrdquo BiochemicalJournal vol 117 no 4 pp 779ndash790 1970
[66] M T Ashby ldquoHypothiocyaniterdquo in Advances in InorganicChemistry R van Eldik and I-B Ivana Eds chapter 8 pp 263ndash303 Academic Press New York NY USA 2012
[67] E L Thomas ldquoLactoperoxidase-catalyzed oxidation of thio-cyanate equilibria between oxidized forms of thiocyanaterdquoBiochemistry vol 20 no 11 pp 3273ndash3280 1981
[68] T M Aune and E LThomas ldquoOxidation of protein sulfhydrylsby products of peroxidase-catalyzed oxidation of thiocyanateionrdquo Biochemistry vol 17 no 6 pp 1005ndash1010 1978
[69] T M Aune and E L Thomas ldquoAccumulation of hypothiocyan-ite ion during peroxidase-catalyzed oxidation of thiocyanateionrdquo European Journal of Biochemistry vol 80 no 1 pp 209ndash214 1977
[70] J D Oram and B Reiter ldquoThe inhibition of streptococci bylactoperoxidase thiocyanate and hydrogen peroxideThe effectof the inhibitory system on susceptible and resistant strains ofgroup N streptococcirdquo Biochemical Journal vol 100 no 2 pp373ndash381 1966
[71] J Kalmar K L Woldegiorgis B Biri and M T AshbyldquoMechanism of decomposition of the human defense factorhypothiocyanite near physiological pHrdquo Journal of the Ameri-can Chemical Society vol 133 no 49 pp 19911ndash19921 2011
[72] H Hoogendoorn J P PiessensW Scholtes and L A StoddardldquoHypothiocyanite ion the inhibitor formed by the system lac-toperoxidase thiocyanate hydrogen peroxide I Identification ofthe inhibiting compoundrdquoCaries Research vol 11 no 2 pp 77ndash84 1977
[73] L Bjorck and O Claesson ldquoCorrelation between concentrationof hypothiocyanate and antibacterial effect of the lactoperoxi-dase system against Escherichia colirdquo Journal of Dairy Sciencevol 63 no 6 pp 919ndash922 1980
[74] P Nagy S S Alguindigue and M T Ashby ldquoLactoperoxidase-catalyzed oxidation of thiocyanate by hydrogen peroxide areinvestigation of hypothiocyanite by nuclear magnetic reso-nance and optical spectroscopyrdquo Biochemistry vol 45 no 41pp 12610ndash12616 2006
[75] Y Adolphe M Jacquot M Linder A-M Revol-Junelles andJ-B Milliere ldquoOptimization of the components concentrationsof the lactoperoxidase system by RSMrdquo Journal of AppliedMicrobiology vol 100 no 5 pp 1034ndash1042 2006
[76] M Adamson and K M Pruitt ldquoLactoperoxidase-catalyzedinactivation of hexokinaserdquo Biochimica et Biophysica Acta vol658 no 2 pp 238ndash247 1981
[77] M N Mickelson ldquoGlucose transport in Streptococcus agalac-tiae and its inhibition by lactoperoxidase-thiocyanate-hydrogenperoxiderdquo Journal of Bacteriology vol 132 no 2 pp 541ndash5481977
[78] E L Thomas K A Pera K W Smith and A K ChwangldquoInhibition of Streptococcus mutans by the lactoperoxidaseantimicrobial systemrdquo Infection and Immunity vol 39 no 2 pp767ndash778 1983
[79] J Sermon K Vanoirbeek P De Spiegeleer R Van Houdt AAertsen and C W Michiels ldquoUnique stress response to thelactoperoxidase-thiocyanate enzyme system in Escherichia colirdquoResearch in Microbiology vol 156 no 2 pp 225ndash232 2005
[80] E L Thomas and T M Aune ldquoSusceptibility of Escherichia colito bactericidal action of lactoperoxidase peroxide and iodideor thiocyanaterdquoAntimicrobial Agents andChemotherapy vol 13no 2 pp 261ndash265 1978
[81] K M Pruitt J Tenovuo R W Andrews and T McKaneldquoLactoperoxidase-catalyzed oxidation of thiocyanate polaro-graphic study of the oxidation productsrdquo Biochemistry vol 21no 3 pp 562ndash567 1982
[82] M Huwiler and H Kohler ldquoPseudo-catalytic degradation ofhydrogen peroxide in the lactoperoxidaseH
2O2iodide sys-
temrdquo European Journal of Biochemistry vol 141 no 1 pp 69ndash741984
[83] M Huwiler U Burgi and H Kohler ldquoMechanism of enzymaticand non-enzymatic tyrosine iodination Inhibition by excesshydrogen peroxide andor iodiderdquo European Journal of Bio-chemistry vol 147 no 3 pp 469ndash476 1985
[84] M Morrison G S Bayse and A W Michaels ldquoDeterminationof spectral properties of aqueous I2 and I3- and the equilibriumconstantrdquo Analytical Biochemistry vol 42 no 1 pp 195ndash2011971
[85] E L Thomas and T M Aune ldquoPeroxidase catalyzed oxidationof protein sulfhydrylsmediated by iodinerdquoBiochemistry vol 16no 16 pp 3581ndash3586 1977
[86] W Gottardi ldquoIodine and disinfection theoretical study onmode of action efficiency stability and analytical aspects in theaqueous systemrdquo Archiv der Pharmazie vol 332 no 5 pp 151ndash157 1999
[87] W A Prutz R Kissner W H Koppenol and H RueggerldquoOn the irreversible destruction of reduced nicotinamidenucleotides by hypohalous acidsrdquo Archives of Biochemistry andBiophysics vol 380 no 1 pp 181ndash191 2000
[88] Y Bichsel and U Von Gunten ldquoHypoiodous acid kinetics ofthe buffer-catalyzed disproportionationrdquo Water Research vol34 no 12 pp 3197ndash3203 2000
[89] Y Bichsel and U von Gunten ldquoOxidation of iodide andhypoiodous acid in the disinfection of natural watersrdquo Environ-mental Science and Technology vol 33 no 22 pp 4040ndash40451999
[90] W A Prutz R Kissner T Nauser and W H Koppenol ldquoOnthe oxidation of cytochrome c by hypohalous acidsrdquo Archives ofBiochemistry and Biophysics vol 389 no 1 pp 110ndash122 2001
[91] A Virion J L Michot D Deme and J Pommier ldquoNADPHoxidation catalyzed by the peroxidaseH
2O2system Iodide-
mediated oxidation of NADPH to iodinated NADPrdquo EuropeanJournal of Biochemistry vol 148 no 2 pp 239ndash243 1985
Enzyme Research 13
[92] E L Thomas and T M Aune ldquoCofactor role of iodide in per-oxidase antimicrobial action against Escherichia colirdquo Antimic-robial Agents and Chemotherapy vol 13 no 6 pp 1000ndash10051978
[93] E L Thomas and T M Aune ldquoOxidation of Escherichiacoli sulfhydryl components by the peroxidase-hydrogenperoxide-iodide antimicrobial systemrdquo Antimicrobial Agentsand Chemotherapy vol 13 no 6 pp 1006ndash1010 1978
[94] C B Hamon and S J Klebanoff ldquoA peroxidase-mediatedstreptococcus mitis-dependent antimicrobial system in salivardquoJournal of Experimental Medicine vol 137 no 2 pp 438ndash4501973
[95] R Ihalin J Nuutila V Loimaranta M Lenander J Tenovuoand E-M Lilius ldquoSusceptibility of Fusobacterium nucleatum tokilling by peroxidase-iodide-hydrogen peroxide combinationin buffer solution and in human whole salivardquo Anaerobe vol9 no 1 pp 23ndash30 2003
[96] R Ihalin V Loimaranta M Lenander-Lumikari and J Ten-ovuo ldquoThe effects of different (pseudo)halide substrates onperoxidase-mediated killing of Actinobacillus actinomycetem-comitansrdquo Journal of Periodontal Research vol 33 no 7 pp 421ndash427 1998
[97] E H Bosch H van doorne and S de Vries ldquoThe lactoper-oxidase system the influence of iodide and the chemical andantimicrobial stability over the period of about 18 monthsrdquoJournal of AppliedMicrobiology vol 89 no 2 pp 215ndash224 2000
parasitical applications the use of iodide has to be preferred(viii) the use of combined presence of iodide and thiocyanatehas to be checked carefully for efficacy and (ix) to favor thecofactor role of iodide or thiocyanate
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] P J OrsquoBrien ldquoPeroxidasesrdquoChemico-Biological Interactions vol129 no 1-2 pp 113ndash139 2000
[2] W Jantschko P G Furtmuller M Allegra et al ldquoRedoxintermediates of plant and mammalian peroxidases a compar-ative transient-kinetic study of their reactivity toward indolederivativesrdquo Archives of Biochemistry and Biophysics vol 398no 1 pp 12ndash22 2002
[3] S Kimura and M Ikeda-Saito ldquoHuman myeloperoxidase andthyroid peroxidase two enzymes with separate and distinctphysiological functions are evolutionarily related membersof the same gene familyrdquo Proteins Structure Function andGenetics vol 3 no 2 pp 113ndash120 1988
[4] G Battistuzzi M Bellei C A Bortolotti and M Sola ldquoRedoxproperties of heme peroxidasesrdquo Archives of Biochemistry andBiophysics vol 500 no 1 pp 21ndash36 2010
[5] M Zamocky C Jakopitsch P G Furtmuller C Dunand and CObinger ldquoThe peroxidase-cyclooxygenase superfamily recon-structed evolution of critical enzymes of the innate immunesystemrdquo Proteins Structure Function and Genetics vol 72 no2 pp 589ndash605 2008
[6] H Daiyasu and H Toh ldquoMolecular evolution of the myeloper-oxidase familyrdquo Journal of Molecular Evolution vol 51 no 5 pp433ndash445 2000
[7] D Serteyn S Grulke T Franck A Mouithys-Mickalad andG Deby-Dupont ldquoNeutrophile myeloperoxidase protectiveenzyme with strong oxidative activitiesrdquo Annales de MedecineVeterinaire vol 147 no 2 pp 79ndash93 2003
[8] S C Whitman S L Hazen D B Miller R A HegeleJ W Heinecke and M W Huff ldquoModification of type IIIVLDL their remnants and VLDL from apoE- knockout miceby p-hydroxyphenylacetaldehyde a product of myeloperox-idase activity causes marked cholesteryl ester accumulationin macrophagesrdquo Arteriosclerosis Thrombosis and VascularBiology vol 19 no 5 pp 1238ndash1249 1999
[9] T J Barrett and C L Hawkins ldquoHypothiocyanous acid benignor deadlyrdquo Chemical Research in Toxicology vol 25 no 2 pp263ndash273 2012
[10] M M Lloyd D M van Reyk M J Davies and C L HawkinsldquoHypothiocyanous acid is a more potent inducer of apoptosisand protein thiol depletion in murine macrophage cells thanhypochlorous acid or hypobromous acidrdquo Biochemical Journalvol 414 no 2 pp 271ndash280 2008
[11] J Wang and A Slungaard ldquoRole of eosinophil peroxidase inhost defense and disease pathologyrdquo Archives of Biochemistryand Biophysics vol 445 no 2 pp 256ndash260 2006
[12] M Ahariz and P Courtois ldquoCandida albicans susceptibility tolactoperoxidase-generated hypoioditerdquo Clinical Cosmetic andInvestigational Dentistry vol 2 pp 69ndash78 2010
[13] A Welk C Meller R Schubert C Schwahn A Kramerand H Below ldquoEffect of lactoperoxidase on the antimicrobialeffectiveness of the thiocyanate hydrogen peroxide combinationin a quantitative suspension testrdquo BMC Microbiology vol 9article 134 2009
[14] K M Pruitt and J O Tenovuo Eds The Lactoperoxidase Sys-tem Chemistry and Biological Significance vol 27 of Immunol-ogy Series Marcel Dekker New York NY USA 1985
[15] P G Furtmuller W Jantschko G Regelsberger C JakopitschJ Arnhold and C Obinger ldquoReaction of lactoperoxidasecompound I with halides and thiocyanaterdquo Biochemistry vol41 no 39 pp 11895ndash11900 2002
[16] P G Furtmuller U Burner and C Obinger ldquoReaction ofmyeloperoxidase compound I with chloride bromide iodideand thiocyanaterdquo Biochemistry vol 37 no 51 pp 17923ndash179301998
[17] J Arnhold E Monzani P G Furtmuller M Zederbauer LCasella and C Obinger ldquoKinetics and thermodynamics ofhalide and nitrite oxidation by mammalian heme peroxidasesrdquoEuropean Journal of Inorganic Chemistry no 19 pp 3801ndash38112006
[18] M J Davies C L Hawkins D I Pattison and M D ReesldquoMammalian heme peroxidases from molecular mechanismsto health implicationsrdquo Antioxidants and Redox Signaling vol10 no 7 pp 1199ndash1234 2008
[19] J N de Wit and A C M van Hooydonk ldquoStructure functionsand applications of lactoperoxidase in natural antimicrobialsystemsrdquo Nederlands melk en Zuiveltijdschrift vol 50 no 2 pp227ndash244 1996
[20] P G Furtmuller M Zederbauer W Jantschko et al ldquoActivesite structure and catalytic mechanisms of human peroxidasesrdquoArchives of Biochemistry and Biophysics vol 445 no 2 pp 199ndash213 2006
[21] M Zederbauer P G Furtmuller S Brogioni C JakopitschG Smulevich and C Obinger ldquoHeme to protein linkages inmammalian peroxidases impact on spectroscopic redox andcatalytic propertiesrdquo Natural Product Reports vol 24 no 3 pp571ndash584 2007
[22] G Battistuzzi M Bellei J Vlasits et al ldquoRedox thermodynam-ics of lactoperoxidase and eosinophil peroxidaserdquo Archives ofBiochemistry and Biophysics vol 494 no 1 pp 72ndash77 2010
[23] I A Sheikh A Singh N Singh et al ldquoStructural evidence ofsubstrate specificity inmammalian peroxidases structure of thethiocyanate complex with lactoperoxidase and its interactionsat 24 a 24 A resolutionrdquo The Journal of Biological Chemistryvol 284 no 22 pp 14849ndash14856 2009
[24] H Kohler and H Jenzer ldquoInteraction of lactoperoxidase withhydrogen peroxide Formation of enzyme intermediates andgeneration of free radicalsrdquo Free Radical Biology and Medicinevol 6 no 3 pp 323ndash339 1989
[25] P G Furtmuller U Burner W Jantschko G Regelsberger andC Obinger ldquoTwo-electron reduction and one-electron oxida-tion of organic hydroperoxides by human myeloperoxidaserdquoFEBS Letters vol 484 no 2 pp 139ndash143 2000
[26] A Taurog M L Dorris and D R Doerge ldquoMechanism ofsimultaneous iodination and coupling catalyzed by thyroidperoxidaserdquo Archives of Biochemistry and Biophysics vol 330no 1 pp 24ndash32 1996
[27] J E Erman L B Vitello J Matthew Mauro and J KrautldquoDetection of an oxyferryl porphyrin 120587-cation-radical interme-diate in the reaction between hydrogen peroxide and a mutant
Enzyme Research 11
yeast cytochrome c peroxidase Evidence for tryptophan-191involvement in the radical site of compound Irdquo Biochemistryvol 28 no 20 pp 7992ndash7995 1989
[28] M T Ashby ldquoInorganic chemistry of defensive peroxidases inthe human oral cavityrdquo Journal of Dental Research vol 87 no10 pp 900ndash914 2008
[29] J D Chandler and B J Day ldquoThiocyanate a potentially usefultherapeutic agent with host defense and antioxidant propertiesrdquoBiochemical Pharmacology vol 84 no 11 pp 1381ndash1387 2012
[30] E C Jong W R Henderson and S J Klebanoff ldquoBactericidalactivity of eosinophil peroxidaserdquo Journal of Immunology vol124 no 3 pp 1378ndash1382 1980
[31] E C Jong A A F Mahmoud and S J Kelbanoff ldquoPeroxidase-mediated toxicity to schistosomula of Schistosoma mansonirdquoJournal of Immunology vol 126 no 2 pp 468ndash471 1981
[32] L M Wolfson and S S Sumner ldquoAntibacterial activity of thelactoperoxidase system a reviewrdquo Journal of Food Protectionvol 56 no 10 pp 887ndash892 1993
[33] J Arnhold P G Furtmuller G Regelsberger and C ObingerldquoRedox properties of the couple compound Inative enzyme ofmyeloperoxidase and eosinophil peroxidaserdquo European Journalof Biochemistry vol 268 no 19 pp 5142ndash5148 2001
[34] P G Furtmuller J Arnhold W Jantschko M Zederbauer CJakopitsch and C Obinger ldquoStandard reduction potentials ofall couples of the peroxidase cycle of lactoperoxidaserdquo Journalof Inorganic Biochemistry vol 99 no 5 pp 1220ndash1229 2005
[35] C J van Dalen M W Whitehouse C C Winterbourn and AJ Kettle ldquoThiocyanate and chloride as competing substrates formyeloperoxidaserdquo Biochemical Journal vol 327 no 2 pp 487ndash492 1997
[36] A Slungaard and J R Mahoney Jr ldquoThiocyanate is the majorsubstrate for eosinophil peroxidase in physiologic fluids impli-cations for cytotoxicityrdquoThe Journal of Biological Chemistry vol266 no 8 pp 4903ndash4910 1991
[37] J Tenovuo ldquoAntimicrobial function of human salivamdashhowimportant is it for oral healthrdquoActaOdontologica Scandinavicavol 56 no 5 pp 250ndash256 1998
[38] R Ihalin V Loimaranta and J Tenovuo ldquoOrigin structure andbiological activities of peroxidases in human salivardquo Archives ofBiochemistry and Biophysics vol 445 no 2 pp 261ndash268 2006
[39] J A Rooke J F Flockhart and N H Sparks ldquoThe potentialfor increasing the concentrations of micro-nutrients relevant tohuman nutrition inmeat milk and eggsrdquo Journal of AgriculturalScience vol 148 no 5 pp 603ndash614 2010
[40] H Kohler A Taurog and H B Dunford ldquoSpectral studieswith lactoperoxidase and thyroid peroxidase interconversionsbetween native enzyme compound II and compound IIIrdquoArchives of Biochemistry and Biophysics vol 264 no 2 pp 438ndash449 1988
[41] I Yamazaki H S Mason and L Piette ldquoIdentification byelectron paramagnetic resonance spectroscopy of free radicalsgenerated from substrates by peroxidaserdquoThe Journal of Biolog-ical Chemistry vol 235 pp 2444ndash2449 1960
[42] B Chance ldquoThe kinetics and stoichiometry of the transitionfrom the primary to the secondary peroxidase peroxide com-plexesrdquo Archives of Biochemistry and Biophysics vol 41 no 2pp 416ndash424 1952
[43] K M Pruitt B Mansson-Rahemtulla D C Baldone andF Rahemtulla ldquoSteady-state kinetics of thiocyanate oxidationcatalyzed by human salivary peroxidaserdquo Biochemistry vol 27no 1 pp 240ndash245 1988
[44] B G J M Bolscher and R Wever ldquoA kinetic study of thereaction between humanmyeloperoxidase hydroperoxides andcyanide inhibition by chloride and thiocyanaterdquo Biochimica etBiophysica Acta Protein Structure and Molecular Enzymologyvol 788 no 1 pp 1ndash10 1984
[45] L A Marquez J T Huang and H Brian Dunford ldquoSpectraland kinetic studies on the formation of myeloperoxidase com-pounds I and II roles of hydrogen peroxide and superoxiderdquoBiochemistry vol 33 no 6 pp 1447ndash1454 1994
[46] HMAbu-Soud and S LHazen ldquoNitric oxide is a physiologicalsubstrate for mammalian peroxidasesrdquoThe Journal of BiologicalChemistry vol 275 no 48 pp 37524ndash37532 2000
[47] Y R Tahboub S Galijasevic M P Diamond and H MAbu-Soud ldquoThiocyanate modulates the catalytic activity ofmammalian peroxidasesrdquo Journal of Biological Chemistry vol280 no 28 pp 26129ndash26136 2005
[48] H Jenzer W Jones and H Kohler ldquoOn the molecularmechanismof lactoperoxidase-catalyzedH
2O2metabolism and
irreversible enzyme inactivationrdquo The Journal of BiologicalChemistry vol 261 no 33 pp 15550ndash15556 1986
[49] R P Magnusson A Taurog and M L Dorris ldquoMechanism ofiodide-dependent catalatic activity of thyroid peroxidase andlactoperoxidaserdquo The Journal of Biological Chemistry vol 259no 1 pp 197ndash205 1984
[50] D K Bhattacharyya U Bandyopadhyay and R K BanerjeeldquoEDTA inhibits lactoperoxidase-catalyzed iodide oxidation byacting as an electron-donor and interacting near the iodidebinding siterdquoMolecular and Cellular Biochemistry vol 162 no2 pp 105ndash111 1996
[51] C L Hawkins ldquoThe role of hypothiocyanous acid (HOSCN) inbiological systems HOSCN in biological systemsrdquo Free RadicalResearch vol 43 no 12 pp 1147ndash1158 2009
[52] S J Klebanoff ldquoIodination of bacteria a bactericidal mecha-nismrdquo Journal of Experimental Medicine vol 126 no 6 pp1063ndash1078 1967
[53] S J Klebanoff ldquoMyeloperoxidase-halide-hydrogen peroxideantibacterial systemrdquo Journal of Bacteriology vol 95 no 6 pp2131ndash2138 1968
[54] R K Banerjee and A G Datta ldquoSalivary peroxidasesrdquoMolecu-lar and Cellular Biochemistry vol 70 no 1 pp 21ndash29 1986
[55] MHuwiler H Jenzer andHKohler ldquoThe role of compound IIIin reversible and irreversible inactivation of lactoperoxidaserdquoEuropean Journal of Biochemistry vol 158 no 3 pp 609ndash6141986
[56] R Wever W M Kast J H Kasinoedin and R Boelens ldquoTheperoxidation of thiocyanate catalysed by myeloperoxidase andlactoperoxidaserdquo Biochimica et Biophysica Acta (BBA)ProteinStructure and Molecular vol 709 no 2 pp 212ndash219 1982
[57] C E A Souza D Maitra G M Saed et al ldquoHypochlorousacid-induced heme degradation from lactoperoxidase as anovel mechanism of free iron release and tissue injury ininflammatory diseasesrdquo PLoS ONE vol 6 no 11 Article IDe27641 2011
[58] J Carlsson ldquoBactericidal effect of hydrogen peroxide is pre-vented by the lactoperoxidase-thiocyanate system under anaer-obic conditionsrdquo Infection and Immunity vol 29 no 3 pp 1190ndash1192 1980
[59] E L Thomas and T M Aune ldquoLactoperoxidase peroxidethiocyanate antimicrobial system correlation of sulfhydryloxidation with antimicrobial actionrdquo Infection and Immunityvol 20 no 2 pp 456ndash463 1978
12 Enzyme Research
[60] J Carlsson Y Iwami and T Yamada ldquoHydrogen peroxideexcretion by oral streptococci and effect of lactoperoxidase-thiocyanate-hydrogen peroxiderdquo Infection and Immunity vol40 no 1 pp 70ndash80 1983
[61] K D Kussendrager and A C M van Hooijdonk ldquoLactoperox-idase physico-chemical properties occurrence mechanism ofaction and applicationsrdquoTheBritish Journal of Nutrition vol 84supplement 1 pp S19ndashS25 2000
[62] J P Perraudin ldquoProteines a activites biologiques lactoferrineet lactoperoxydase Connaissances recemment acquises et tech-nologies drsquoobtentionrdquo Lait vol 71 no 2 pp 191ndash211 1991
[63] J-W Boots and R Floris ldquoLactoperoxidase From catalyticmechanism to practical applicationsrdquo International Dairy Jour-nal vol 16 no 11 pp 1272ndash1276 2006
[64] A C M van Hooijdonk K D Kussendrager and J M SteijnsldquoIn vivo antimicrobial and antiviral activity of components inbovine milk and colostrum involved in non-specific defencerdquoBritish Journal of Nutrition vol 84 supplement 1 pp S127ndashS1342000
[65] D M Hogg and G R Jago ldquoThe antibacterial action of lac-toperoxidaseThe nature of the bacterial inhibitorrdquo BiochemicalJournal vol 117 no 4 pp 779ndash790 1970
[66] M T Ashby ldquoHypothiocyaniterdquo in Advances in InorganicChemistry R van Eldik and I-B Ivana Eds chapter 8 pp 263ndash303 Academic Press New York NY USA 2012
[67] E L Thomas ldquoLactoperoxidase-catalyzed oxidation of thio-cyanate equilibria between oxidized forms of thiocyanaterdquoBiochemistry vol 20 no 11 pp 3273ndash3280 1981
[68] T M Aune and E LThomas ldquoOxidation of protein sulfhydrylsby products of peroxidase-catalyzed oxidation of thiocyanateionrdquo Biochemistry vol 17 no 6 pp 1005ndash1010 1978
[69] T M Aune and E L Thomas ldquoAccumulation of hypothiocyan-ite ion during peroxidase-catalyzed oxidation of thiocyanateionrdquo European Journal of Biochemistry vol 80 no 1 pp 209ndash214 1977
[70] J D Oram and B Reiter ldquoThe inhibition of streptococci bylactoperoxidase thiocyanate and hydrogen peroxideThe effectof the inhibitory system on susceptible and resistant strains ofgroup N streptococcirdquo Biochemical Journal vol 100 no 2 pp373ndash381 1966
[71] J Kalmar K L Woldegiorgis B Biri and M T AshbyldquoMechanism of decomposition of the human defense factorhypothiocyanite near physiological pHrdquo Journal of the Ameri-can Chemical Society vol 133 no 49 pp 19911ndash19921 2011
[72] H Hoogendoorn J P PiessensW Scholtes and L A StoddardldquoHypothiocyanite ion the inhibitor formed by the system lac-toperoxidase thiocyanate hydrogen peroxide I Identification ofthe inhibiting compoundrdquoCaries Research vol 11 no 2 pp 77ndash84 1977
[73] L Bjorck and O Claesson ldquoCorrelation between concentrationof hypothiocyanate and antibacterial effect of the lactoperoxi-dase system against Escherichia colirdquo Journal of Dairy Sciencevol 63 no 6 pp 919ndash922 1980
[74] P Nagy S S Alguindigue and M T Ashby ldquoLactoperoxidase-catalyzed oxidation of thiocyanate by hydrogen peroxide areinvestigation of hypothiocyanite by nuclear magnetic reso-nance and optical spectroscopyrdquo Biochemistry vol 45 no 41pp 12610ndash12616 2006
[75] Y Adolphe M Jacquot M Linder A-M Revol-Junelles andJ-B Milliere ldquoOptimization of the components concentrationsof the lactoperoxidase system by RSMrdquo Journal of AppliedMicrobiology vol 100 no 5 pp 1034ndash1042 2006
[76] M Adamson and K M Pruitt ldquoLactoperoxidase-catalyzedinactivation of hexokinaserdquo Biochimica et Biophysica Acta vol658 no 2 pp 238ndash247 1981
[77] M N Mickelson ldquoGlucose transport in Streptococcus agalac-tiae and its inhibition by lactoperoxidase-thiocyanate-hydrogenperoxiderdquo Journal of Bacteriology vol 132 no 2 pp 541ndash5481977
[78] E L Thomas K A Pera K W Smith and A K ChwangldquoInhibition of Streptococcus mutans by the lactoperoxidaseantimicrobial systemrdquo Infection and Immunity vol 39 no 2 pp767ndash778 1983
[79] J Sermon K Vanoirbeek P De Spiegeleer R Van Houdt AAertsen and C W Michiels ldquoUnique stress response to thelactoperoxidase-thiocyanate enzyme system in Escherichia colirdquoResearch in Microbiology vol 156 no 2 pp 225ndash232 2005
[80] E L Thomas and T M Aune ldquoSusceptibility of Escherichia colito bactericidal action of lactoperoxidase peroxide and iodideor thiocyanaterdquoAntimicrobial Agents andChemotherapy vol 13no 2 pp 261ndash265 1978
[81] K M Pruitt J Tenovuo R W Andrews and T McKaneldquoLactoperoxidase-catalyzed oxidation of thiocyanate polaro-graphic study of the oxidation productsrdquo Biochemistry vol 21no 3 pp 562ndash567 1982
[82] M Huwiler and H Kohler ldquoPseudo-catalytic degradation ofhydrogen peroxide in the lactoperoxidaseH
2O2iodide sys-
temrdquo European Journal of Biochemistry vol 141 no 1 pp 69ndash741984
[83] M Huwiler U Burgi and H Kohler ldquoMechanism of enzymaticand non-enzymatic tyrosine iodination Inhibition by excesshydrogen peroxide andor iodiderdquo European Journal of Bio-chemistry vol 147 no 3 pp 469ndash476 1985
[84] M Morrison G S Bayse and A W Michaels ldquoDeterminationof spectral properties of aqueous I2 and I3- and the equilibriumconstantrdquo Analytical Biochemistry vol 42 no 1 pp 195ndash2011971
[85] E L Thomas and T M Aune ldquoPeroxidase catalyzed oxidationof protein sulfhydrylsmediated by iodinerdquoBiochemistry vol 16no 16 pp 3581ndash3586 1977
[86] W Gottardi ldquoIodine and disinfection theoretical study onmode of action efficiency stability and analytical aspects in theaqueous systemrdquo Archiv der Pharmazie vol 332 no 5 pp 151ndash157 1999
[87] W A Prutz R Kissner W H Koppenol and H RueggerldquoOn the irreversible destruction of reduced nicotinamidenucleotides by hypohalous acidsrdquo Archives of Biochemistry andBiophysics vol 380 no 1 pp 181ndash191 2000
[88] Y Bichsel and U Von Gunten ldquoHypoiodous acid kinetics ofthe buffer-catalyzed disproportionationrdquo Water Research vol34 no 12 pp 3197ndash3203 2000
[89] Y Bichsel and U von Gunten ldquoOxidation of iodide andhypoiodous acid in the disinfection of natural watersrdquo Environ-mental Science and Technology vol 33 no 22 pp 4040ndash40451999
[90] W A Prutz R Kissner T Nauser and W H Koppenol ldquoOnthe oxidation of cytochrome c by hypohalous acidsrdquo Archives ofBiochemistry and Biophysics vol 389 no 1 pp 110ndash122 2001
[91] A Virion J L Michot D Deme and J Pommier ldquoNADPHoxidation catalyzed by the peroxidaseH
2O2system Iodide-
mediated oxidation of NADPH to iodinated NADPrdquo EuropeanJournal of Biochemistry vol 148 no 2 pp 239ndash243 1985
Enzyme Research 13
[92] E L Thomas and T M Aune ldquoCofactor role of iodide in per-oxidase antimicrobial action against Escherichia colirdquo Antimic-robial Agents and Chemotherapy vol 13 no 6 pp 1000ndash10051978
[93] E L Thomas and T M Aune ldquoOxidation of Escherichiacoli sulfhydryl components by the peroxidase-hydrogenperoxide-iodide antimicrobial systemrdquo Antimicrobial Agentsand Chemotherapy vol 13 no 6 pp 1006ndash1010 1978
[94] C B Hamon and S J Klebanoff ldquoA peroxidase-mediatedstreptococcus mitis-dependent antimicrobial system in salivardquoJournal of Experimental Medicine vol 137 no 2 pp 438ndash4501973
[95] R Ihalin J Nuutila V Loimaranta M Lenander J Tenovuoand E-M Lilius ldquoSusceptibility of Fusobacterium nucleatum tokilling by peroxidase-iodide-hydrogen peroxide combinationin buffer solution and in human whole salivardquo Anaerobe vol9 no 1 pp 23ndash30 2003
[96] R Ihalin V Loimaranta M Lenander-Lumikari and J Ten-ovuo ldquoThe effects of different (pseudo)halide substrates onperoxidase-mediated killing of Actinobacillus actinomycetem-comitansrdquo Journal of Periodontal Research vol 33 no 7 pp 421ndash427 1998
[97] E H Bosch H van doorne and S de Vries ldquoThe lactoper-oxidase system the influence of iodide and the chemical andantimicrobial stability over the period of about 18 monthsrdquoJournal of AppliedMicrobiology vol 89 no 2 pp 215ndash224 2000
yeast cytochrome c peroxidase Evidence for tryptophan-191involvement in the radical site of compound Irdquo Biochemistryvol 28 no 20 pp 7992ndash7995 1989
[28] M T Ashby ldquoInorganic chemistry of defensive peroxidases inthe human oral cavityrdquo Journal of Dental Research vol 87 no10 pp 900ndash914 2008
[29] J D Chandler and B J Day ldquoThiocyanate a potentially usefultherapeutic agent with host defense and antioxidant propertiesrdquoBiochemical Pharmacology vol 84 no 11 pp 1381ndash1387 2012
[30] E C Jong W R Henderson and S J Klebanoff ldquoBactericidalactivity of eosinophil peroxidaserdquo Journal of Immunology vol124 no 3 pp 1378ndash1382 1980
[31] E C Jong A A F Mahmoud and S J Kelbanoff ldquoPeroxidase-mediated toxicity to schistosomula of Schistosoma mansonirdquoJournal of Immunology vol 126 no 2 pp 468ndash471 1981
[32] L M Wolfson and S S Sumner ldquoAntibacterial activity of thelactoperoxidase system a reviewrdquo Journal of Food Protectionvol 56 no 10 pp 887ndash892 1993
[33] J Arnhold P G Furtmuller G Regelsberger and C ObingerldquoRedox properties of the couple compound Inative enzyme ofmyeloperoxidase and eosinophil peroxidaserdquo European Journalof Biochemistry vol 268 no 19 pp 5142ndash5148 2001
[34] P G Furtmuller J Arnhold W Jantschko M Zederbauer CJakopitsch and C Obinger ldquoStandard reduction potentials ofall couples of the peroxidase cycle of lactoperoxidaserdquo Journalof Inorganic Biochemistry vol 99 no 5 pp 1220ndash1229 2005
[35] C J van Dalen M W Whitehouse C C Winterbourn and AJ Kettle ldquoThiocyanate and chloride as competing substrates formyeloperoxidaserdquo Biochemical Journal vol 327 no 2 pp 487ndash492 1997
[36] A Slungaard and J R Mahoney Jr ldquoThiocyanate is the majorsubstrate for eosinophil peroxidase in physiologic fluids impli-cations for cytotoxicityrdquoThe Journal of Biological Chemistry vol266 no 8 pp 4903ndash4910 1991
[37] J Tenovuo ldquoAntimicrobial function of human salivamdashhowimportant is it for oral healthrdquoActaOdontologica Scandinavicavol 56 no 5 pp 250ndash256 1998
[38] R Ihalin V Loimaranta and J Tenovuo ldquoOrigin structure andbiological activities of peroxidases in human salivardquo Archives ofBiochemistry and Biophysics vol 445 no 2 pp 261ndash268 2006
[39] J A Rooke J F Flockhart and N H Sparks ldquoThe potentialfor increasing the concentrations of micro-nutrients relevant tohuman nutrition inmeat milk and eggsrdquo Journal of AgriculturalScience vol 148 no 5 pp 603ndash614 2010
[40] H Kohler A Taurog and H B Dunford ldquoSpectral studieswith lactoperoxidase and thyroid peroxidase interconversionsbetween native enzyme compound II and compound IIIrdquoArchives of Biochemistry and Biophysics vol 264 no 2 pp 438ndash449 1988
[41] I Yamazaki H S Mason and L Piette ldquoIdentification byelectron paramagnetic resonance spectroscopy of free radicalsgenerated from substrates by peroxidaserdquoThe Journal of Biolog-ical Chemistry vol 235 pp 2444ndash2449 1960
[42] B Chance ldquoThe kinetics and stoichiometry of the transitionfrom the primary to the secondary peroxidase peroxide com-plexesrdquo Archives of Biochemistry and Biophysics vol 41 no 2pp 416ndash424 1952
[43] K M Pruitt B Mansson-Rahemtulla D C Baldone andF Rahemtulla ldquoSteady-state kinetics of thiocyanate oxidationcatalyzed by human salivary peroxidaserdquo Biochemistry vol 27no 1 pp 240ndash245 1988
[44] B G J M Bolscher and R Wever ldquoA kinetic study of thereaction between humanmyeloperoxidase hydroperoxides andcyanide inhibition by chloride and thiocyanaterdquo Biochimica etBiophysica Acta Protein Structure and Molecular Enzymologyvol 788 no 1 pp 1ndash10 1984
[45] L A Marquez J T Huang and H Brian Dunford ldquoSpectraland kinetic studies on the formation of myeloperoxidase com-pounds I and II roles of hydrogen peroxide and superoxiderdquoBiochemistry vol 33 no 6 pp 1447ndash1454 1994
[46] HMAbu-Soud and S LHazen ldquoNitric oxide is a physiologicalsubstrate for mammalian peroxidasesrdquoThe Journal of BiologicalChemistry vol 275 no 48 pp 37524ndash37532 2000
[47] Y R Tahboub S Galijasevic M P Diamond and H MAbu-Soud ldquoThiocyanate modulates the catalytic activity ofmammalian peroxidasesrdquo Journal of Biological Chemistry vol280 no 28 pp 26129ndash26136 2005
[48] H Jenzer W Jones and H Kohler ldquoOn the molecularmechanismof lactoperoxidase-catalyzedH
2O2metabolism and
irreversible enzyme inactivationrdquo The Journal of BiologicalChemistry vol 261 no 33 pp 15550ndash15556 1986
[49] R P Magnusson A Taurog and M L Dorris ldquoMechanism ofiodide-dependent catalatic activity of thyroid peroxidase andlactoperoxidaserdquo The Journal of Biological Chemistry vol 259no 1 pp 197ndash205 1984
[50] D K Bhattacharyya U Bandyopadhyay and R K BanerjeeldquoEDTA inhibits lactoperoxidase-catalyzed iodide oxidation byacting as an electron-donor and interacting near the iodidebinding siterdquoMolecular and Cellular Biochemistry vol 162 no2 pp 105ndash111 1996
[51] C L Hawkins ldquoThe role of hypothiocyanous acid (HOSCN) inbiological systems HOSCN in biological systemsrdquo Free RadicalResearch vol 43 no 12 pp 1147ndash1158 2009
[52] S J Klebanoff ldquoIodination of bacteria a bactericidal mecha-nismrdquo Journal of Experimental Medicine vol 126 no 6 pp1063ndash1078 1967
[53] S J Klebanoff ldquoMyeloperoxidase-halide-hydrogen peroxideantibacterial systemrdquo Journal of Bacteriology vol 95 no 6 pp2131ndash2138 1968
[54] R K Banerjee and A G Datta ldquoSalivary peroxidasesrdquoMolecu-lar and Cellular Biochemistry vol 70 no 1 pp 21ndash29 1986
[55] MHuwiler H Jenzer andHKohler ldquoThe role of compound IIIin reversible and irreversible inactivation of lactoperoxidaserdquoEuropean Journal of Biochemistry vol 158 no 3 pp 609ndash6141986
[56] R Wever W M Kast J H Kasinoedin and R Boelens ldquoTheperoxidation of thiocyanate catalysed by myeloperoxidase andlactoperoxidaserdquo Biochimica et Biophysica Acta (BBA)ProteinStructure and Molecular vol 709 no 2 pp 212ndash219 1982
[57] C E A Souza D Maitra G M Saed et al ldquoHypochlorousacid-induced heme degradation from lactoperoxidase as anovel mechanism of free iron release and tissue injury ininflammatory diseasesrdquo PLoS ONE vol 6 no 11 Article IDe27641 2011
[58] J Carlsson ldquoBactericidal effect of hydrogen peroxide is pre-vented by the lactoperoxidase-thiocyanate system under anaer-obic conditionsrdquo Infection and Immunity vol 29 no 3 pp 1190ndash1192 1980
[59] E L Thomas and T M Aune ldquoLactoperoxidase peroxidethiocyanate antimicrobial system correlation of sulfhydryloxidation with antimicrobial actionrdquo Infection and Immunityvol 20 no 2 pp 456ndash463 1978
12 Enzyme Research
[60] J Carlsson Y Iwami and T Yamada ldquoHydrogen peroxideexcretion by oral streptococci and effect of lactoperoxidase-thiocyanate-hydrogen peroxiderdquo Infection and Immunity vol40 no 1 pp 70ndash80 1983
[61] K D Kussendrager and A C M van Hooijdonk ldquoLactoperox-idase physico-chemical properties occurrence mechanism ofaction and applicationsrdquoTheBritish Journal of Nutrition vol 84supplement 1 pp S19ndashS25 2000
[62] J P Perraudin ldquoProteines a activites biologiques lactoferrineet lactoperoxydase Connaissances recemment acquises et tech-nologies drsquoobtentionrdquo Lait vol 71 no 2 pp 191ndash211 1991
[63] J-W Boots and R Floris ldquoLactoperoxidase From catalyticmechanism to practical applicationsrdquo International Dairy Jour-nal vol 16 no 11 pp 1272ndash1276 2006
[64] A C M van Hooijdonk K D Kussendrager and J M SteijnsldquoIn vivo antimicrobial and antiviral activity of components inbovine milk and colostrum involved in non-specific defencerdquoBritish Journal of Nutrition vol 84 supplement 1 pp S127ndashS1342000
[65] D M Hogg and G R Jago ldquoThe antibacterial action of lac-toperoxidaseThe nature of the bacterial inhibitorrdquo BiochemicalJournal vol 117 no 4 pp 779ndash790 1970
[66] M T Ashby ldquoHypothiocyaniterdquo in Advances in InorganicChemistry R van Eldik and I-B Ivana Eds chapter 8 pp 263ndash303 Academic Press New York NY USA 2012
[67] E L Thomas ldquoLactoperoxidase-catalyzed oxidation of thio-cyanate equilibria between oxidized forms of thiocyanaterdquoBiochemistry vol 20 no 11 pp 3273ndash3280 1981
[68] T M Aune and E LThomas ldquoOxidation of protein sulfhydrylsby products of peroxidase-catalyzed oxidation of thiocyanateionrdquo Biochemistry vol 17 no 6 pp 1005ndash1010 1978
[69] T M Aune and E L Thomas ldquoAccumulation of hypothiocyan-ite ion during peroxidase-catalyzed oxidation of thiocyanateionrdquo European Journal of Biochemistry vol 80 no 1 pp 209ndash214 1977
[70] J D Oram and B Reiter ldquoThe inhibition of streptococci bylactoperoxidase thiocyanate and hydrogen peroxideThe effectof the inhibitory system on susceptible and resistant strains ofgroup N streptococcirdquo Biochemical Journal vol 100 no 2 pp373ndash381 1966
[71] J Kalmar K L Woldegiorgis B Biri and M T AshbyldquoMechanism of decomposition of the human defense factorhypothiocyanite near physiological pHrdquo Journal of the Ameri-can Chemical Society vol 133 no 49 pp 19911ndash19921 2011
[72] H Hoogendoorn J P PiessensW Scholtes and L A StoddardldquoHypothiocyanite ion the inhibitor formed by the system lac-toperoxidase thiocyanate hydrogen peroxide I Identification ofthe inhibiting compoundrdquoCaries Research vol 11 no 2 pp 77ndash84 1977
[73] L Bjorck and O Claesson ldquoCorrelation between concentrationof hypothiocyanate and antibacterial effect of the lactoperoxi-dase system against Escherichia colirdquo Journal of Dairy Sciencevol 63 no 6 pp 919ndash922 1980
[74] P Nagy S S Alguindigue and M T Ashby ldquoLactoperoxidase-catalyzed oxidation of thiocyanate by hydrogen peroxide areinvestigation of hypothiocyanite by nuclear magnetic reso-nance and optical spectroscopyrdquo Biochemistry vol 45 no 41pp 12610ndash12616 2006
[75] Y Adolphe M Jacquot M Linder A-M Revol-Junelles andJ-B Milliere ldquoOptimization of the components concentrationsof the lactoperoxidase system by RSMrdquo Journal of AppliedMicrobiology vol 100 no 5 pp 1034ndash1042 2006
[76] M Adamson and K M Pruitt ldquoLactoperoxidase-catalyzedinactivation of hexokinaserdquo Biochimica et Biophysica Acta vol658 no 2 pp 238ndash247 1981
[77] M N Mickelson ldquoGlucose transport in Streptococcus agalac-tiae and its inhibition by lactoperoxidase-thiocyanate-hydrogenperoxiderdquo Journal of Bacteriology vol 132 no 2 pp 541ndash5481977
[78] E L Thomas K A Pera K W Smith and A K ChwangldquoInhibition of Streptococcus mutans by the lactoperoxidaseantimicrobial systemrdquo Infection and Immunity vol 39 no 2 pp767ndash778 1983
[79] J Sermon K Vanoirbeek P De Spiegeleer R Van Houdt AAertsen and C W Michiels ldquoUnique stress response to thelactoperoxidase-thiocyanate enzyme system in Escherichia colirdquoResearch in Microbiology vol 156 no 2 pp 225ndash232 2005
[80] E L Thomas and T M Aune ldquoSusceptibility of Escherichia colito bactericidal action of lactoperoxidase peroxide and iodideor thiocyanaterdquoAntimicrobial Agents andChemotherapy vol 13no 2 pp 261ndash265 1978
[81] K M Pruitt J Tenovuo R W Andrews and T McKaneldquoLactoperoxidase-catalyzed oxidation of thiocyanate polaro-graphic study of the oxidation productsrdquo Biochemistry vol 21no 3 pp 562ndash567 1982
[82] M Huwiler and H Kohler ldquoPseudo-catalytic degradation ofhydrogen peroxide in the lactoperoxidaseH
2O2iodide sys-
temrdquo European Journal of Biochemistry vol 141 no 1 pp 69ndash741984
[83] M Huwiler U Burgi and H Kohler ldquoMechanism of enzymaticand non-enzymatic tyrosine iodination Inhibition by excesshydrogen peroxide andor iodiderdquo European Journal of Bio-chemistry vol 147 no 3 pp 469ndash476 1985
[84] M Morrison G S Bayse and A W Michaels ldquoDeterminationof spectral properties of aqueous I2 and I3- and the equilibriumconstantrdquo Analytical Biochemistry vol 42 no 1 pp 195ndash2011971
[85] E L Thomas and T M Aune ldquoPeroxidase catalyzed oxidationof protein sulfhydrylsmediated by iodinerdquoBiochemistry vol 16no 16 pp 3581ndash3586 1977
[86] W Gottardi ldquoIodine and disinfection theoretical study onmode of action efficiency stability and analytical aspects in theaqueous systemrdquo Archiv der Pharmazie vol 332 no 5 pp 151ndash157 1999
[87] W A Prutz R Kissner W H Koppenol and H RueggerldquoOn the irreversible destruction of reduced nicotinamidenucleotides by hypohalous acidsrdquo Archives of Biochemistry andBiophysics vol 380 no 1 pp 181ndash191 2000
[88] Y Bichsel and U Von Gunten ldquoHypoiodous acid kinetics ofthe buffer-catalyzed disproportionationrdquo Water Research vol34 no 12 pp 3197ndash3203 2000
[89] Y Bichsel and U von Gunten ldquoOxidation of iodide andhypoiodous acid in the disinfection of natural watersrdquo Environ-mental Science and Technology vol 33 no 22 pp 4040ndash40451999
[90] W A Prutz R Kissner T Nauser and W H Koppenol ldquoOnthe oxidation of cytochrome c by hypohalous acidsrdquo Archives ofBiochemistry and Biophysics vol 389 no 1 pp 110ndash122 2001
[91] A Virion J L Michot D Deme and J Pommier ldquoNADPHoxidation catalyzed by the peroxidaseH
2O2system Iodide-
mediated oxidation of NADPH to iodinated NADPrdquo EuropeanJournal of Biochemistry vol 148 no 2 pp 239ndash243 1985
Enzyme Research 13
[92] E L Thomas and T M Aune ldquoCofactor role of iodide in per-oxidase antimicrobial action against Escherichia colirdquo Antimic-robial Agents and Chemotherapy vol 13 no 6 pp 1000ndash10051978
[93] E L Thomas and T M Aune ldquoOxidation of Escherichiacoli sulfhydryl components by the peroxidase-hydrogenperoxide-iodide antimicrobial systemrdquo Antimicrobial Agentsand Chemotherapy vol 13 no 6 pp 1006ndash1010 1978
[94] C B Hamon and S J Klebanoff ldquoA peroxidase-mediatedstreptococcus mitis-dependent antimicrobial system in salivardquoJournal of Experimental Medicine vol 137 no 2 pp 438ndash4501973
[95] R Ihalin J Nuutila V Loimaranta M Lenander J Tenovuoand E-M Lilius ldquoSusceptibility of Fusobacterium nucleatum tokilling by peroxidase-iodide-hydrogen peroxide combinationin buffer solution and in human whole salivardquo Anaerobe vol9 no 1 pp 23ndash30 2003
[96] R Ihalin V Loimaranta M Lenander-Lumikari and J Ten-ovuo ldquoThe effects of different (pseudo)halide substrates onperoxidase-mediated killing of Actinobacillus actinomycetem-comitansrdquo Journal of Periodontal Research vol 33 no 7 pp 421ndash427 1998
[97] E H Bosch H van doorne and S de Vries ldquoThe lactoper-oxidase system the influence of iodide and the chemical andantimicrobial stability over the period of about 18 monthsrdquoJournal of AppliedMicrobiology vol 89 no 2 pp 215ndash224 2000
[60] J Carlsson Y Iwami and T Yamada ldquoHydrogen peroxideexcretion by oral streptococci and effect of lactoperoxidase-thiocyanate-hydrogen peroxiderdquo Infection and Immunity vol40 no 1 pp 70ndash80 1983
[61] K D Kussendrager and A C M van Hooijdonk ldquoLactoperox-idase physico-chemical properties occurrence mechanism ofaction and applicationsrdquoTheBritish Journal of Nutrition vol 84supplement 1 pp S19ndashS25 2000
[62] J P Perraudin ldquoProteines a activites biologiques lactoferrineet lactoperoxydase Connaissances recemment acquises et tech-nologies drsquoobtentionrdquo Lait vol 71 no 2 pp 191ndash211 1991
[63] J-W Boots and R Floris ldquoLactoperoxidase From catalyticmechanism to practical applicationsrdquo International Dairy Jour-nal vol 16 no 11 pp 1272ndash1276 2006
[64] A C M van Hooijdonk K D Kussendrager and J M SteijnsldquoIn vivo antimicrobial and antiviral activity of components inbovine milk and colostrum involved in non-specific defencerdquoBritish Journal of Nutrition vol 84 supplement 1 pp S127ndashS1342000
[65] D M Hogg and G R Jago ldquoThe antibacterial action of lac-toperoxidaseThe nature of the bacterial inhibitorrdquo BiochemicalJournal vol 117 no 4 pp 779ndash790 1970
[66] M T Ashby ldquoHypothiocyaniterdquo in Advances in InorganicChemistry R van Eldik and I-B Ivana Eds chapter 8 pp 263ndash303 Academic Press New York NY USA 2012
[67] E L Thomas ldquoLactoperoxidase-catalyzed oxidation of thio-cyanate equilibria between oxidized forms of thiocyanaterdquoBiochemistry vol 20 no 11 pp 3273ndash3280 1981
[68] T M Aune and E LThomas ldquoOxidation of protein sulfhydrylsby products of peroxidase-catalyzed oxidation of thiocyanateionrdquo Biochemistry vol 17 no 6 pp 1005ndash1010 1978
[69] T M Aune and E L Thomas ldquoAccumulation of hypothiocyan-ite ion during peroxidase-catalyzed oxidation of thiocyanateionrdquo European Journal of Biochemistry vol 80 no 1 pp 209ndash214 1977
[70] J D Oram and B Reiter ldquoThe inhibition of streptococci bylactoperoxidase thiocyanate and hydrogen peroxideThe effectof the inhibitory system on susceptible and resistant strains ofgroup N streptococcirdquo Biochemical Journal vol 100 no 2 pp373ndash381 1966
[71] J Kalmar K L Woldegiorgis B Biri and M T AshbyldquoMechanism of decomposition of the human defense factorhypothiocyanite near physiological pHrdquo Journal of the Ameri-can Chemical Society vol 133 no 49 pp 19911ndash19921 2011
[72] H Hoogendoorn J P PiessensW Scholtes and L A StoddardldquoHypothiocyanite ion the inhibitor formed by the system lac-toperoxidase thiocyanate hydrogen peroxide I Identification ofthe inhibiting compoundrdquoCaries Research vol 11 no 2 pp 77ndash84 1977
[73] L Bjorck and O Claesson ldquoCorrelation between concentrationof hypothiocyanate and antibacterial effect of the lactoperoxi-dase system against Escherichia colirdquo Journal of Dairy Sciencevol 63 no 6 pp 919ndash922 1980
[74] P Nagy S S Alguindigue and M T Ashby ldquoLactoperoxidase-catalyzed oxidation of thiocyanate by hydrogen peroxide areinvestigation of hypothiocyanite by nuclear magnetic reso-nance and optical spectroscopyrdquo Biochemistry vol 45 no 41pp 12610ndash12616 2006
[75] Y Adolphe M Jacquot M Linder A-M Revol-Junelles andJ-B Milliere ldquoOptimization of the components concentrationsof the lactoperoxidase system by RSMrdquo Journal of AppliedMicrobiology vol 100 no 5 pp 1034ndash1042 2006
[76] M Adamson and K M Pruitt ldquoLactoperoxidase-catalyzedinactivation of hexokinaserdquo Biochimica et Biophysica Acta vol658 no 2 pp 238ndash247 1981
[77] M N Mickelson ldquoGlucose transport in Streptococcus agalac-tiae and its inhibition by lactoperoxidase-thiocyanate-hydrogenperoxiderdquo Journal of Bacteriology vol 132 no 2 pp 541ndash5481977
[78] E L Thomas K A Pera K W Smith and A K ChwangldquoInhibition of Streptococcus mutans by the lactoperoxidaseantimicrobial systemrdquo Infection and Immunity vol 39 no 2 pp767ndash778 1983
[79] J Sermon K Vanoirbeek P De Spiegeleer R Van Houdt AAertsen and C W Michiels ldquoUnique stress response to thelactoperoxidase-thiocyanate enzyme system in Escherichia colirdquoResearch in Microbiology vol 156 no 2 pp 225ndash232 2005
[80] E L Thomas and T M Aune ldquoSusceptibility of Escherichia colito bactericidal action of lactoperoxidase peroxide and iodideor thiocyanaterdquoAntimicrobial Agents andChemotherapy vol 13no 2 pp 261ndash265 1978
[81] K M Pruitt J Tenovuo R W Andrews and T McKaneldquoLactoperoxidase-catalyzed oxidation of thiocyanate polaro-graphic study of the oxidation productsrdquo Biochemistry vol 21no 3 pp 562ndash567 1982
[82] M Huwiler and H Kohler ldquoPseudo-catalytic degradation ofhydrogen peroxide in the lactoperoxidaseH
2O2iodide sys-
temrdquo European Journal of Biochemistry vol 141 no 1 pp 69ndash741984
[83] M Huwiler U Burgi and H Kohler ldquoMechanism of enzymaticand non-enzymatic tyrosine iodination Inhibition by excesshydrogen peroxide andor iodiderdquo European Journal of Bio-chemistry vol 147 no 3 pp 469ndash476 1985
[84] M Morrison G S Bayse and A W Michaels ldquoDeterminationof spectral properties of aqueous I2 and I3- and the equilibriumconstantrdquo Analytical Biochemistry vol 42 no 1 pp 195ndash2011971
[85] E L Thomas and T M Aune ldquoPeroxidase catalyzed oxidationof protein sulfhydrylsmediated by iodinerdquoBiochemistry vol 16no 16 pp 3581ndash3586 1977
[86] W Gottardi ldquoIodine and disinfection theoretical study onmode of action efficiency stability and analytical aspects in theaqueous systemrdquo Archiv der Pharmazie vol 332 no 5 pp 151ndash157 1999
[87] W A Prutz R Kissner W H Koppenol and H RueggerldquoOn the irreversible destruction of reduced nicotinamidenucleotides by hypohalous acidsrdquo Archives of Biochemistry andBiophysics vol 380 no 1 pp 181ndash191 2000
[88] Y Bichsel and U Von Gunten ldquoHypoiodous acid kinetics ofthe buffer-catalyzed disproportionationrdquo Water Research vol34 no 12 pp 3197ndash3203 2000
[89] Y Bichsel and U von Gunten ldquoOxidation of iodide andhypoiodous acid in the disinfection of natural watersrdquo Environ-mental Science and Technology vol 33 no 22 pp 4040ndash40451999
[90] W A Prutz R Kissner T Nauser and W H Koppenol ldquoOnthe oxidation of cytochrome c by hypohalous acidsrdquo Archives ofBiochemistry and Biophysics vol 389 no 1 pp 110ndash122 2001
[91] A Virion J L Michot D Deme and J Pommier ldquoNADPHoxidation catalyzed by the peroxidaseH
2O2system Iodide-
mediated oxidation of NADPH to iodinated NADPrdquo EuropeanJournal of Biochemistry vol 148 no 2 pp 239ndash243 1985
Enzyme Research 13
[92] E L Thomas and T M Aune ldquoCofactor role of iodide in per-oxidase antimicrobial action against Escherichia colirdquo Antimic-robial Agents and Chemotherapy vol 13 no 6 pp 1000ndash10051978
[93] E L Thomas and T M Aune ldquoOxidation of Escherichiacoli sulfhydryl components by the peroxidase-hydrogenperoxide-iodide antimicrobial systemrdquo Antimicrobial Agentsand Chemotherapy vol 13 no 6 pp 1006ndash1010 1978
[94] C B Hamon and S J Klebanoff ldquoA peroxidase-mediatedstreptococcus mitis-dependent antimicrobial system in salivardquoJournal of Experimental Medicine vol 137 no 2 pp 438ndash4501973
[95] R Ihalin J Nuutila V Loimaranta M Lenander J Tenovuoand E-M Lilius ldquoSusceptibility of Fusobacterium nucleatum tokilling by peroxidase-iodide-hydrogen peroxide combinationin buffer solution and in human whole salivardquo Anaerobe vol9 no 1 pp 23ndash30 2003
[96] R Ihalin V Loimaranta M Lenander-Lumikari and J Ten-ovuo ldquoThe effects of different (pseudo)halide substrates onperoxidase-mediated killing of Actinobacillus actinomycetem-comitansrdquo Journal of Periodontal Research vol 33 no 7 pp 421ndash427 1998
[97] E H Bosch H van doorne and S de Vries ldquoThe lactoper-oxidase system the influence of iodide and the chemical andantimicrobial stability over the period of about 18 monthsrdquoJournal of AppliedMicrobiology vol 89 no 2 pp 215ndash224 2000
[92] E L Thomas and T M Aune ldquoCofactor role of iodide in per-oxidase antimicrobial action against Escherichia colirdquo Antimic-robial Agents and Chemotherapy vol 13 no 6 pp 1000ndash10051978
[93] E L Thomas and T M Aune ldquoOxidation of Escherichiacoli sulfhydryl components by the peroxidase-hydrogenperoxide-iodide antimicrobial systemrdquo Antimicrobial Agentsand Chemotherapy vol 13 no 6 pp 1006ndash1010 1978
[94] C B Hamon and S J Klebanoff ldquoA peroxidase-mediatedstreptococcus mitis-dependent antimicrobial system in salivardquoJournal of Experimental Medicine vol 137 no 2 pp 438ndash4501973
[95] R Ihalin J Nuutila V Loimaranta M Lenander J Tenovuoand E-M Lilius ldquoSusceptibility of Fusobacterium nucleatum tokilling by peroxidase-iodide-hydrogen peroxide combinationin buffer solution and in human whole salivardquo Anaerobe vol9 no 1 pp 23ndash30 2003
[96] R Ihalin V Loimaranta M Lenander-Lumikari and J Ten-ovuo ldquoThe effects of different (pseudo)halide substrates onperoxidase-mediated killing of Actinobacillus actinomycetem-comitansrdquo Journal of Periodontal Research vol 33 no 7 pp 421ndash427 1998
[97] E H Bosch H van doorne and S de Vries ldquoThe lactoper-oxidase system the influence of iodide and the chemical andantimicrobial stability over the period of about 18 monthsrdquoJournal of AppliedMicrobiology vol 89 no 2 pp 215ndash224 2000
