\’(>I. 266, No. 26, Issue olSepternher 1.5. pp. 17201-17211, 1991 I’rinlrd in I f. S . A

Fenton Chemistry AMINO ACID OXIDATION*

(Received for publication, March 28, 1991)

Earl R. StadtmanS and Barbara S . Berlett From the Labf1rator.y o/ Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health. Bethesda, Maryland 20892

The oxidation of amino acids by Fenton reagent (H,O, + Fe(I1)) leads mainly to the formation of NH:, a-ketoacids, CO,, oximes, and aldehydes or carboxylic acids containing one less carbon atom. Oxidation is almost completely dependent on the presence of bicar- bonate ion and is stimulated by iron chelators at levels which are substoichiometric with respect to the iron concentration but is inhibited at higher concentrations. The stimulatory effect of chelators is not due merely to solubilization of catalytically inactive polymeric forms of Fe(OH)3 nor to the conversion of Fe(I1) to complexes incapable of scavenging hydroxyl radicals. The results suggest that an iron chelate and another as yet unidentified form of iron are both required for maximal rates of amino acid oxidation. The metal ion- catalyzed oxidation of amino acids is likely a “caged” process, since the oxidation is not inhibited by hy- droxyl radical scavengers, and the relative rates of oxidation of various amino acids by the Fenton system as well as the distribution of products formed (espe- cially products of aromatic amino acids) are signifi- cantly different from those reported for amino acid oxidation by ionizing radiation. Several iron-binding proteins, peptides, and hemoglobin degradation prod- ucts can replace Fe(1I) or Fe(II1) in the bicarbonate- dependent oxidation of amino acids. In view of their ability to sequester metal ions and their susceptibility to oxidation by H202 in the presence of physiological concentrations of bicarbonate, amino acids may serve an important role in antioxidant defense against tissue damage.

Various enzymic and nonenzymic mixed-function oxidation (MFO)‘ systems’ catalyze the oxidative inactivation of en- zymes (1-4). It has been established that these oxidations

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

.$ To whom correspondence should be addressed Bldg. 3, Rm. 222, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-496-4096.

’ The abbreviations used are: MFO, mixed-function oxidation; desferal, desferrioxamine; PRI, product recovery index; MCO, metal- catalyzed oxidation; G(NH,,), number of NH., molecules produced per 100 eV of radiation energy consumed; y(NH.<), moles of NH,] produced per mol of H,02 consumed; DMPO, 5,5-dimethyl-l-pyrroline-N-ox- ide; HPLC, high pressure liquid chromatography; Hepes, 4-(2-hy- droxyethy1)-1-piperazineethanesulfonic acid; EGTA, [ethylene- his(oxyethylenenitrilo)]tetraacetic acid.

‘I In order to avoid confusion between these mixed-function oxida- tion systems and mixed-function oxidases, the MFO systems are now referred to as metal-catalyzed oxidation (MCO) systems.

involve the conversion of histidine residues to asparagine residues (5), of proline residues to glutamic semialdehyde and to pyroglutamic or glutamic acid residues (6), of arginine residues to glutamic semialdehyde residues (6), and of all these and other amino acid (especially lysine) residues to carbonyl derivatives (6, 7 ) . Such oxidations are marking steps in protein turnover (1, 8-13); many are implicated in the accumulation of altered forms of enzymes during aging (14- 18), during oxidative stress (16), in neutrophil function (19), in various pathological conditions (ZO), and in ischemia-re- perfusion injury (21). Results of mechanistic studies are con- sistent with the view that Fe(I1) and H,Oe which are produced by the MFO systems undergo site-specific Fenton reactions at metal-binding sites on the proteins to generate an active oxygen species (OH’, ferry1 ion, singlet oxygen), which at- tacks side chains of amino acid residues at the metal-binding site (4).

In contrast to the site-specific reactions catalyzed by metal ions, the exposure of proteins to ionizing radiation leads to modification of virtually all amino acid residues, albeit cys- teine, histidine, tyrosine, methionine, and tryptophan resi- dues are preferential targets (23-25). Curiously, the latter three amino acid residues are not common targets for the metal ion-catalyzed reaction, presumably because they are not usually present at the protein metal-binding sites. Neverthe- less, results of studies on the oxidation of peptides (26, 23) and proteins (23-30) by ionizing radiation have contributed significantly t,o our understanding of how these molecules interact with oxygen radicals. Moreover, detailed studies of the oxidation of amino acids by ionizing radiation have estab- lished that NH:, a-ketoacids, aldehydes, and hydrogen per- oxide are among the major products (24,31,32), and plausible mechanisms for the generation of these products have been proposed (23,24,31). In view of the fact that the modification of proteins by ionizing radiation is at least qualitatively dif- ferent from those catalyzed by metal ions, it was of interest to investigate further the oxidation of amino acids by the Fenton system.

The present study was prompted by the report of Zs-Nagy and Floyd (22) showing that the reaction of H,O, with Fe(I1) complexes of ADP or ATP leads to the generation of OH’ radicals which could be trapped by the spin trap 5,5-dimethyl- 1-pyrroline-N-oxide (DMPO) and quantitated by means of electron spin resonance spectroscopy. Formation of the DMPO-OH’ spin adduct was quenched by the addition of amino acids, suggesting that at high concentrations amino acids compete effectively with DMPO for reaction with OH ’ . We presumed therefore that a detailed analysis of the reaction of amino acids with the Fenton reagent under the conditions used by Nagy and Floyd might contribute to a better under- standing of the chemistry of enzyme oxidation by MFO sys- tems.

17201

17202 Fenton Chemistry: Amino Acid Oxidation

Results summarized here show that the Fenton reagent catalyzes the oxidative deamination-decarboxylation of all amino acids to mixtures of the corresponding a-ketoacid and of aldehydes and carboxylic acids containing one less carbon atom. Surprisingly, these oxidative reactions are greatly stim- ulated by bicarbonate ion and are enhanced by substoichio- metric amounts but are inhibited by greater than stoichio- metric amounts of various chelators, including nucleoside di- and triphosphates, EDTA, desferal, etc.

EXPERIMENTAL PROCEDURES

Amino Acid Oxidation-The oxidation of amino acids was contin- uously monitored by manometric measurements of the increase in pressure which occurs when the reactions are carried out in a bicar- bonate/COe buffer (pH 7.6) in a Warburg apparatus, as described below. After termination of the manometric measurements, suitable aliquots of the reaction mixtures were used to determine the concen- trations of carbonyl compounds, NH:, amino acid, and H,O, (ferri- thiocyanate method) as previously reported (33). In some cases, the concentrations of oximes and ether extractable acids were also deter- mined and were identified as described below.

Manometric Measurements of CO,, Acid, and 0, Production-For each experimental condition, identical reaction mixtures were intro- duced into each of two Warburg vessels having three side arms. Unless otherwise stated, the complete reaction mixtures (2.0-ml vol- umes) contained 23.5 mM NaHCO:3, 50 mM amino acid, 30 mM H,O,, and either 1.5 or 100 pM FeS04 and 3.75 or 250 p~ ADP, respectively; the gas phase was 5% COP, 95% N2. Initially, the NaHC0,3, amino acid, and ADP in a volume of 1.8-1.9 ml were placed in the main compartment of the vessel. One side arm contained the FeS04 (in 0.05 ml of 1.0 mM HCl); another side arm contained the H20, in 0.1 ml. After attachment to the manometer, the vessels were placed in a water bath at 37 "C, and with shaking (15 min) the gas atmosphere was replaced with a 5% CO,, 95% N, mixture to yield a final pH of 7.6. Then, by means of a gas-tight hypodermic syringe fitted with a 2-inch needle, 100-200 p1 of oxsorbant was introduced through the capillary of the vented side into the third side arm of just one of the pair of flasks containing identical reaction mixtures. After further shaking for 5 min, the FeSOl and H,O,, in that order, were mixed with the solution in the main compartment. The amounts of C02 and 0, produced were monitored continuously by manometric measure- ment of the pressure change with time. The gas produced in the presence and absence of oxsorbant is attributable to CO, only and COY plus O,, respectively; the difference between these two measure- ments is therefore a measure of 0, production. The amounts (mi- cromoles) of CO, and O2 produced were calculated from the pressure changes as described by Umbreit et al. (34). In some experiments, only one double side-armed Warburg vessel was used for each reaction mixture, and oxsorbant was not present during the incubation. How- ever, when the reaction was over (due to consumption of all the H202), the amount of 0, which had been produced was then determined by measuring the decrease in pressure which occurred immediately fol- lowing rapid addition of oxsorbant (200 pl) into the vented side arm. The validity of this procedure was verified by measurement of the 0, released from known amounts of H,Oa by the action of catalase.

Test Tube Experiments-In experiments where the conversion of amino acid to carbonyl derivatives and NH: was followed over short time intervals (5-15 min), the reactions were carried out in 12 X 75- mm test tubes. Unless otherwise noted, the reaction mixtures in 0.4 ml final volume contained 23.5 mM NaHCO:3 and variable specified amounts of amino acid, FeS04, ADP, and Hs02 and were incubated a t 37 "C in an atmosphere of 5% COP, 95% N1, pH 7.6. Initially, 0.32 ml containing the NaHCOTj, amino acid, and ADP was added to each test tube, and a stream of 5% CO,, 95% N, was directed through a 22-gauge, 3-inch hypodermic needle onto the surface of the solution for 2 min. Then, while still gassing, 0.04 ml of the FeS04 stock solution was added; this was followed after 20 s by the addition of 0.04 ml of the H,Oa stock solution. The hypodermic needle was then withdrawn and, simultaneously the tube was sealed with a rubber stopper. After various time intervals, the reaction mixtures were assayed for H202, amino acid, carbonyl compounds, and NH;. Whether the reactions are carried out in Warburg vessels or in test tubes, the order of additions is very important. The FeS04 (or FeC1:J must be added to the amino acid, bicarbonate, ADP solution prior to the addition of HnO,. If the H 2 0 P is added before the iron salts, the

oxidation of amino acid is very much slower and sometimes is signif- icantly delayed.

Acid Production-As is discussed later, the increase in pressure observed in the presence of oxsorbant is almost entirely attributable to the release of CO, associated with the production of isovaleric acid. After the reaction was terminated, the amounts of acid produced in the oxidation of leucine and phenylalanine were measured by direct titration of ether extractable acid. After acidification with H,S04 to pH 1.5-2.0, the reaction mixtures were extracted 3 times with ethyl ether, and the amount of extractable acid was determined by titration with 0.05 N KOH. The amount of a-ketoacid was determined by measuring the carbocyl content of the neutralized ether-extractable acid fraction before and again after destruction of the a-ketoacids which was achieved by incubation with 12 mM HeO, for 12 h a t room temperature.

Chelex Treatment-For most experiments, metal ions in buffers, bicarbonate, leucine, and ADP were removed by passing stock solu- tions through a Chelex resin (sodium form).

Oximes-The concentrations of isovaleraldoxime and phenylace- taldoxime were determined by HPLC and by their capacities to inhibit horse liver alcohol dehydrogenase. Details of these methods and their identification as products of leucine and phenylalanine oxidation are topics of another communication.

Escherichia coli glutamine synthetase activity was measured by the y-glutamyl transfer procedure (35). Free acids, oximes, and the 2,4- dinitrophenylhydrazone derivative of carbonyl compounds formed in the oxidation of leucine were separated by HPLC on a 25-cm IBM C18 column and were identified by comparison of the retention times with those of authentic standards. For determination of the free acids and oximes formed, the reaction mixtures were adjusted to pH 2.25 with phosphoric acid. 100-p1 aliquots of the acidified mixture were injected onto the column, and the reaction products were eluted with a 0-50% methanol gradient (15 min) at a flow rate of 2 ml/min. The elution profile was monitored at 210 nm; amounts of various products were determined by the areas under individual peaks in comparison with authentic standards. The retention times for various compounds were: leucine, 2.5 min; a-ketoisocaproic acid, 9.1 min; isovaleric acid, 10.3 min; a-ketoisocaproic acid oxime, 13.8 min; isovaleraldoxime, 10.8 min. The 2,4-dinitrophenylhydrazones of carboxyl compounds formed in the oxidation of leucine were prepared by mixing 0.5 ml of the oxidation mixture with 1.5 ml of 5 mM 2,4-dinitrophenylhydrazine in 1 M HC1. After 15 min, 0.8 ml of absolute methanol was added to dissolve the hydrazones and 50 pl of the alcoholic solution was injected onto the HPLC column. The hydrazones were separated by gradient elution, 80-94% methanol (15 min) a t 40 "C, and with a flow rate of 1 ml/min. The elution profile was monitored at 360 nm. The hydrazone derivative of isovaleraldehyde and the cis and trans isomers of a-ketoisocaproic acid hydrazones eluted at 4.6, 3.0, and 4.3 min, respectively. They were identified and quantitated by comparisons with authentic samples as described above for the free acids.

dissolving FeS04.7H20 (Allied Chemical Co.) in 1.0 mM HC1. A 1.0 Materials-Solutions of FeS04 were prepared just prior to use by

M stock solution of FeCL was prepared by dissolving FeCla.6H20 (Fisher) in 2 M HCl. This was diluted to yield 1.0 mM FeCh just prior to use. Glycine, potassium thiocyanate, and EDTA were from Fisher Scientific Co.; p-alanine, DL-serine, hydroxy-L-proline were from Nutritional Biochemicals; L-alanine, DL-homoserine, L-lysine, L-as- partic acid, and L-glutamine were from Calbiochem; L-tyrosine, L- histidine, L-threonine, L-leucine, t-glutamate, L-asparagine, L-valine, diethylenetriaminopentaacetic acid (DPTA), and nitrilotriacetic acid (disodium salt) were from Sigma; oxsorbant was from Burrell COT; L-phenylalanine was from Mann Research Laboratories; 4-methyl-2- oxopentanoic acid (sodium salt) was from Aldrich; ferrozine-3-(2- pyridyl)-5,6-bis-(4-phenylsulfonic acid)-1,2,4-triazine monosodium monohydrate was from HACH Chemical Co.; desferrioxamine (des- feral mesylate) was from Ben Venue Laboratories; Chelex 100 (sodium form) was from Bio-Rad.

RESULTS~

Stimulation of Leucine Oxidation by Bicarbonate Zon-In preliminary experiments, the oxidation of amino acids in a

,'' Portions of this paper (including part of "Results," Tables IV and V, and Figs. 5, 8, and 9) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

Fenton Chemistry: Amino Acid Oxidation 17203

bicarbonate-KC1 solution (pH 7.6) was investigated as de- scribed by Zs-Nag and Floyd ( 2 2 ) . Because our experiments were of longer duration and involved the generation of con- siderable amounts of acid, the buffer capacity of this system was inadequate. Attempts to replace bicarbonate buffer with other buffers led to the discovery that the oxidation of amino acids to carbonyl derivatives is greatly stimulated by bicar- bonate ion. For example, as shown in Fig. 1, in the presence of 0.1 M Hepes buffer (pH 7.55), 20 mM H202, 100 FM Fe(II), and 250 ~ L M ADP, the rate of leucine oxidation increased more than 10-fold as the concentration of bicarbonate was in- creased from 0 to 50 mM. The data in Fig. 1 show also that there is a synergistic effect between ADP and bicarbonate ion on the rate of amino acid oxidation. In the absence of ADP, there is a sigmoidal response to increasing HCO; concentra- tion, but in the presence of ADP a hyperbolic response is observed. The effect of ADP is nonspecific. Replacement of ADP with any one of the other common nucleoside di- and triphosphates yielded similar rates (data not shown). These results are consistent with the view that in the absence of ADP more than 1 eq of HCO; is involved in formation of the catalytic complex, but in the presence of ADP only 1 eq is needed.

In view of the fact that HCOB is an essential component of the reaction mixture and also that amino acid oxidation leads to considerable acid production, nearly all subsequent exper- iments were carried out in a Warburg apparatus using a buffer system comprised of 23.5 mM HCO, and an atmosphere of 5% COS, 95% N,, pH 7.6. In addition to providing excellent pH control, use of the Warburg apparatus permitted contin- uous monitoring of amino acid oxidation through manometric measurements of pressure changes attributable to acid and/ or CO, production as described under “Experimental Proce- dures.” When desired, pressure changes due to production of 0, from H20, were prevented by addition of oxsorbant to the vented arm of the Warburg vessels as described under “Ex- perimental Procedures.”

The data in Fig. 2A show the amount of gas which is produced during the oxidation of leucine in the presence and absence of oxsorbant. As will be shown below, gas produced in the presence of oxsorbant is almost entirely attributable to C02 which is associated with the production of isovaleric acid, either by decarboxylation of the a-ketoisocaproic acid inter- mediate (Reaction 2 ) or by oxidation of the isovaleraldehyde intermediates (Reactions 5 and 6). The gas produced in the absence of oxsorbant is a mixture of O2 and CO,. The differ-

I I I I I I I

- 0 10 20 30 40 50

NaHC03 , mM

FIG. 1. Stimulation of leucine oxidation by HCO; and ADP. Reaction mixtures contained 100 mM Hepes buffer (pH 7.55), 50 mM leucine, 20 mM H202, 100 p M FeSO,, and the amount of NaHC03 as indicated, in the presence (0) and absence (A) of 250 p~ ADP. After 5 min at 25 “C, the amount of carbonyl compound was measured as described under “Experimental Procedures.”

28 A

24 c -OXSORBANT / [ I s

I +OXSORBANT 1

0 20 40 60 80 100 120

MINUTES

1.6

\5 1.2 rn 0 4

1 .o

0.8 ’ I I I I I I I

0 10 20 30 40 50 60 70 80 TIME

FIG. 2. Manometric measurement of gas produced during the oxidation of leucine in the presence and absence of oxsor- bant. A, prior to gassing, the main compartments of two triple side- armed Warburg flasks contained 23.5 mM NaHCOs, 25 mM leucine, and 3.75 PM ADP. One side arm for each vessel contained 0.1 ml of 600 mM H20,; another side arm contained 0.05 ml of 60 p M FeS04; total volume, 2 ml. After gassing 15 min with 5% CO,, 95% N,, 0.2 ml of oxsorbant was introduced into the vented side arm of one of the two vessels, as described under “Experimental Procedures.” After equilibration, the FeS04 and H20, were mixed, in that order, with the contents of the main compartments, and the changes in gas pressure were monitored at various time intervals. Gas production in the absence (circles) and the presence (triangles) of oxsorbant is a measure of total gas (CO, + 0,) and of CO, only, respectively. The difference between these measurements is a measure of the 0, pro- duction (inset) . B, the open circles describe the log of [l - ((gas)/ (total gas))] where (gas) is the amount of gas (0, + CO,) formed a t the times indicated on the abscissa and the (total gas) is the amount of gas (25.3 pmol) formed during 120 min of incubation, at the end of which time gas production had stopped. The open squares 3refer to a Guggenheim plot in which the log of the increments in the amounts of gas formed with successive constant increments in time is plotted against time.

ence between gas produced in the presence and absence of oxsorbance is therefore a measure of the 0, formed in the iron-catalyzed decomposition of H202 (Fig. 2 A , inset). The apparently sigmoidal relation between O2 production and time is likely an artifact reflecting the inability of oxsorbant to absorb O2 as rapidly as it is formed at very low partial pressures of 0,.

The decrease in rates of gas production with time reflects the decrease in H20z concentration. This is evident from Fig. 2B in which two different kinds of semi-log plots are pre- sented. In one plot (open circles), the logarithm of the function (1 - [gas]/[total gas]) is plotted against time, where [gas] is

17204 Fenton Chemistry: Amino Acid Oxidation

the amount of gas produced at the time indicated on the abscissa and [total gas] is the amount of gas produced in 120 min, at which time gas production had ceased (see Fig. 2 A ) . The other plot (open squares) is a conventional Guggenheim plot in which the logarithm of successive increments in the amount of gas produced with constant increments in time is plotted against time. Both semi-log plots are linear as is characteristic of a first-order process. Since both plots are linear, it follows that those HzOz consuming reactions that do not contribute directly to gas production (uiz. a-ketoisoca- proic and isovaleraldehyde formation) are also first-order processes. This follows from the consideration that when a given reactant, (A), is converted to several different products by pseudo unimolecular processes then the overall consump- tion of (A) will also be f i r~ t -order .~ At first glance, it would appear difficult to reconcile the first-order kinetics of gas production with the fact that 2 mol of Hz02 are consumed for each mol of gas produced. One mol is consumed in the production of a carbonyl intermediate, and a second mol is consumed in the oxidation of the intermediate to form CO,. The fact that gas production is first-order with respect to H202 concentration indicates that both H20z consuming re- actions are first-order and that the concentration of the carbonyl intermediates is maintained at steady state levels, i.e. the rate of carbonyl compound formation and the rate of its decomposition to yield C02 are This is in fact the case. In a parallel study (test tube experiment), it was found that under the above experimental conditions, the rate of carbonyl compound formation increased rapidly during the first 5 min of incubation and then remained constant for the duration of the incubation period (data not shown).

As noted under “Experimental Procedures,” the order of additions is very important. After equilibration of the bicar- bonate/amino acid mixture with 5% C02, 95% N2, the Fe(I1) must be added to the mixture prior to the addition of H202. If H202 is added first, amino acid oxidation is either com- pletely suppressed or is delayed for a time that depends upon the Fe(II)/chelator ratio (Fig. 3; Refs. 36 and 37).

The low activity observed when Fe(I1) is added to mixtures already containing H202 cannot be due simply to rapid oxi- dation of the Fe(I1) to Fe(III), since very similar results are obtained when the Fe(I1) is replaced by Fe(II1). These results and the curious dependence of the rate of oxidation on the Fe/chelator ratio (discussed below) suggest that in the absence of H202 a multiplicity of complexes between Fe(I1) or Fe(III), amino acids, HCO,, and chelator (ADP, EDTA, etc.) may be formed and that subsequent interaction between these com- plexes in the presence of H20, defines a redox state in which the cyclic oxidation of the Fe(II)/Fe(III) couple is linked to the oxidation of amino acid via hydroxyl radical or ferry1 or perferryl intermediates.

Reaction Products-Under conditions where leucine is pres- ent in excess relative to the amount of H202 added (i.e. when all of the HZO2 is consumed in the course of the reaction), isovaleraldehyde, isovaleric acid, a-ketoisocaproic acid, NH:, and isovaleraldoxime are the major products of leucine oxi- dation (Table I). The corresponding derivatives, phenylacetic acid, phenylacetaldehyde, phenylpyruvic acid, and phenyla- cetaldoxime) are major products of phenylalanine oxidation (data not shown). It is therefore likely that amino acid oxi- dation occurs by three independent pathways described in Reactions 1-8.

‘ We are indebted to Dr. P. B. Chock for the theoretical analysis upon which these conclusions are based.

v) W

0 20 -I

25

5 15

+ 8 1 0 0

5

0 0 10 20 30 0 10 20 30

MINUTES MINUTES

v)

0 Y 3 0”

FIG. 3. The rates of amino acid oxidation and HzO, dispro- portionation are dependent upon the order of addition of HzOz and iron. Initially, the main compartment of the Warburg flasks contained 23.5 mM NaHC03 and 50 mM leucine (panel A j or no leucine (panel B ) . One side arm contained either 0.1 ml of 2 mM FeSO, or 0.1 ml of 2 mM FeSOr plus 0.09 ml of 2 mM desferrioxamine. Another side arm contained 0.1 ml of 0.6 M H,O,. After gassing 15 min with 5% CO,, 95% N,, the contents of the side arms were mixed with the solution in the main compartment. The order of addition was varied as follows: A, the FeSO, was added first and the H202 added second; 0, the FeSO,/desferrioxarnine mixture was added first and the H202 added second W, the H202 was added first and the FeSO,/desferrioxamine was added second; A, the HZOz was added first and the FeS0, added second. Upon mixing with the solution in the main compartment, the final concentrations of FeSO,, desferriox- amine, and H20, were 0.1, 0.09, and 30 mM, respectively.

TABLE I Products formed in the oxidation of leucine

Experimental conditions were as described in the legend to Fig. 2 exceut that the initial concentration of leucine was 50 mM.

Comoound Exot. 1 ExDt. 2 Averaee

H,Oz lost Leucine lost NH; Ether-extractable acids a-Ketoisocaproate Isovalerate Isovaleraldehyde Carbonyl compounds Isovaleraldehyde oxime co:! 0.l

pmollml 27 14.5 12.3 10.0 3.8 4.6 4.8 8.4 0.64 8.3 2.8

pmollml 26 16.1 13.3 9.7 3.7 4.9 5.0 8.6 0.89 7.9 2.8

pmollml 26.5 15.3 12.8 9.9 3.7 4.8 4.9 8.5 0.8 8.1 2.8

Pathway A RCHNH; COO- + H202 + RCOCOO- + NH: + Hz0 (1) RCOCOO- + H,Os + RCOO- + COz + Hz0 (2)

RCHNH: COO- + 2H20, -+ RCOO- + Con + NH; + 2H20 (3)

Pathway B RCHNH: COO- + H202 --f RCHO + NH: + HCOT (4) RCHO + HZ02 + RCOO- + H’ + Hz0 (5) H’ + HCOT -+ Hz0 + COP (6)

RCHNH$ COO- + 2H202 + RCOO- + 2H20 + CO:! + NH: (7)

Pathway C RCHNH: COO- + 2H:!0:! + RCH = NOH + CO:! + 3Hz0 (8) 2H202 + 2H20 + 0 2 (9)

Pathway A involves oxidative deamination of the amino acid to form the corresponding a-ketoacid (Reaction 11, which can undergo further oxidation to form COZ and a carboxylic acid

Fenton Chemistry: Amino Acid Oxidation 17205

with one less carbon atom (Reaction 2). In Pathway B, the amino acid undergoes oxidative deamination-decarboxylation to form HCO: and an aldehyde containing one less carbon atom (Reaction 4). This aldehyde is slowly oxidized further to the corresponding carboxylic acid (Reaction 5). By either pathway, the overall reaction is the same (Reactions 3 and 7). In addition, a small portion of the amino acid undergoes oxidative decarboxylation to form the corresponding aldehy- deoxime (Reaction 8). Finally, O2 is generated via the amino acid-independent iron-catalyzed decomposition of H202 ac- cording to Reaction 9.

It follows from a consideration of Reactions 1-9 that the amount of H202 consumed in the oxidation of leucine would be equal to the sum of the concentrations of any one of various combinations of products as shown in column 2 of Table 11. The respective concentrations of these products from the experiments described in Table I are shown in column 3 of Table 11. The calculated sum for each combination of products (Table 11, column 4) is approximately equal to the actual amount of H20, which was consumed (26.5 Fmol/ml) as determined by direct measurement. I t is therefore evident that all of the major products of leucine oxidation have been accounted for. The susceptibility of other amino acids to oxidation by the Fenton reagent is illustrated in Fig. 4 and Table 111. There is an %fold difference in the rate of oxidation between the most slowly oxidized amino acid, histidine, and the most rapidly oxidized amino acid, valine.

It should be noted that the rates of amino acid oxidation shown in column 2 of Table I11 represent the average rates of CO, production (pmol/min) observed during the first 10 min of incubation. These rates are therefore a measure of Reac- tions 2 and 5 and will reflect the initial rates of primary Reactions 1 and 4 only under conditions where these reactions are rate-limiting. This is the case during the first few minutes of incubation when the level of H,02 is high and the potential rate of a-ketoacid oxidation (Reaction 2) is 10-20 times greater than the observed rates of amino acid oxidation.

The overall amounts of NH:, carbonyl compounds, and COz evolved that are associated with the oxidation of the individual amino acids are summarized in Table 111. As noted in Table 11, if the oxidation of an amino acid is described by one or more of Reactions 1-9, then the sum (X,) of the product amounts, (NH:) + (CO,) + 2(02), should be equal to the amount of H202 consumed. The ratio (Z,)/(H202) consumed, referred to here as the product recovery index, PRI, is there- fore an indication of the extent to which amino acid oxidation can be described by Reactions 1-9. As shown in columns 13

and 14 of Table 111, the PRI is approximately 1.0 for about half of the 19 amino acids studied, indicating that their oxidation can be described by Reactions 1-9. However, by another criterion, i.e. (NH:)/(C02 + carbonyl) = 1, it is evident that other mechanisms are involved in the oxidation of some amino acids (Table 111, column 15). The relatively high yield of NH: in the oxidation of the basic amino acids (Lys, Arg) and His is understandable as is also the relatively low yield of NH: in the oxidation of proline. Whether ratios of (NH:)/(CO, + carbonyl) less than 1.0 reflect a greater contribution of Reaction 8 (oxime formation) to the overall reaction or are due to the occurrence of fundamentally differ- ent mechanisms than those described by Reactions 1-8 was not investigated. A more extensive analysis of the products formed in the oxidation of amino acids other than phenylal- anine and leucine was not attempted. Curiously, phenylacetic acid, phenylacetaldehyde, benzoic acid, and phenylacetaldox- ime were the only major products of phenylalanine oxidation. We could obtain no evidence for the formation of phenolic derivatives as judged by spectrophotometric and HPLC anal- yses. It was also surprising that neither methionine sulfone nor methionine sulfoxide could be detected among the prod- ucts of methionine oxidation (data not shown).

Effect of Iron Chelating Agents-The rate of amino acid oxidation varies as a function of the chelator/iron ratio. In general, at a given concentration of Fe(I1) or Fe(II1) the rate of oxidation increases with increasing concentration of che- lator up to the point at which all of the iron is sequestered; but with further additions, the oxidation is inhibited. Data in Fig. 6 summarize the results of a Warburg experiment showing how the desferal/Fe(II) ratio affects the time course of gas production when leucine is oxidized in bicarbonate buffer. The rate of gas production is greatly enhanced by the presence of substoichiometric concentrations of desferal. However, with desferal/Fe(II) ratios greater than 0.4 the time course became sigmoidal, and at ratios greater than 1.0 there is a significant lag before gas is produced; at a ratio of 1.5, no gas was formed during the 30-min incubation period. Similar results were obtained when desferal was replaced with EDTA. It was subsequently demonstrated that under these condi- tions, the iron chelators are slowly oxidized and that the lag in leucine oxidation (gas production) reflects the time required to degrade that amount of chelator that is in excess of the Fe(I1) concentration (36, 37). After the 30-min incubation period, the reaction mixtures were assayed for carbonyl deriv- atives. As shown in Fig. 6, the level of carbonyl compounds increased as the desferal/Fe(II) ratio was increased up to a

TABLE I1 Calculations of the amount of H202 that would be consumed in the conversion of leucine to its products

Abbreviations: a-KIC, a-ketoisocaproic acid IVAL, isovaleraldehyde; IV, isovaleric acid R,C=O, total carbonyl content; EA, ether-extractable acid; [Leuld, leucine decomposed. Product equivalents refer to the moles of H202 required to produce a given product. 2, calculated refers to the total amount of H20, that would be required to oxidize isoleucine to the products indicated in column 2. These calculated fractions should be compared with the value of 26.5 prnol/ml which was observed experimentally. The experimental conditions were as described in the legend to Fig. 2 except that the concentration of leucine was 50 mM.

Product combination

(Theory) product equivalents

(Observed) resuective amounts sum = H,O, consumed

( 2, calculated)

pmollml 1 [a-KIC] + [IVAL] + 2[IV] + 2[oxime] + 2[02] [3.8] + (4.91 + 2[4.8] + 2[0.8] + 2[2.8] 25.5

4 27.9

[NH:] + ([EA] - [a-KIC]) + 2[oxime] + 2[02] [12.8] + ([9.9] - [3.8]) + 2[0.8] + 2[2.81 5

26.1 [NH;] + [CO,] + [oxime] + 2[04 [12.8] + [8.1] + [0.8] + 2[2.8]

6 27.3

[Leuld + [IV] + [oxime] + 2[02] [15.3] + [4.8] + [OB] + 2[2.8] 26.5 26.4"

pmollml

2 [RC,=O] + 2[IV] + 2[oxime] + 2[02] [8.5] + 2[4.8] + 2[0.8] + 2[2.8] 25.3 3 [RC,=O] + 2([EA] - [a-KIC]) + 2[oxime] + 2 [ 0 4 [8.5] + 2([9.9] - [3.81) + 2[0.81 + 2[2.81

-

Average.

Fenton Chemistry: Amino Acid Oxidation

ratio of 1.0 but then decreased precipitously to zero as the ratio was increased to 1.5. The generality of this phenomenon is illustrated by the results of studies using the test tube incubation procedure as described under “Experimental Pro- cedures.” With those chelators that form strong 1:1 complexes with iron (EDTA, desferrioxamine, nitrilotriacetic acid, etc.) even a slight excess of chelator leads to almost complete inhibition of leucine oxidation (Fig. 7). For some chelators (ADP, a,a-dipyridyl, and o-phenanthroline) large excesses are

I I I I I

0 20 40 60 80 Time, minutes

FIG. 4. Manometric measurement of the oxidation of var- ious amino acids. Conditions were as described in Fig. 2, except the concentrations of amino acids, Fe(II), and ADP were 50 mM, 1.5 pM and 3.75 p ~ , respectively. The amount of CO, produced was calculated from pressure changes observed when oxsorbant was present in the side arm.

needed to cause substantial inhibitions (Fig. 7). With ADP. Fe(I1) or ADP. Fe(II1) complexes, optional activity is obtained with molar ratios of ADP/Fe of 2.5-3.0.

DISCUSSION

Of particular significance is the demonstration here that the oxidation of amino acids by Fenton’s reagent (Fe(I1) + H202) is almost completely dependent upon the presence of bicarbonate ion. In this regard it is noteworthy that in a series of articles beginning with the first issue of The Journal of Biological Chemistry, published in 1905, Dakin summarized the results of studies on the oxidation of amino acids by the Fenton reagent (37-39). Although unaware of the requirement for bicarbonate, Dakin noted that the reaction mixtures were “carefully neutralized with sodium carbonate” (37). As in the present study, Dakin demonstrated that NH:, a-ketoacids, and aldehydes containing one less carbon atom were among the products of amino acid oxidation. These are the same products obtained when aliphatic amino acids are exposed to ionizing radiation (23, 24, 40). However, the oxidation of amino acids by MCO systems differs from oxidation by radi- olysis systems in several important respects. (i) The double bonds of aromatic amino acids are primary targets during radiolysis (31). Thus, mono- and dihydroxyphenylalanine de- rivatives are major products of tyrosine and phenylalanine oxidation, and kynurenine is a major product of tryptophan oxidation provoked by ionizing radiation (41, 42). Deamina- tion at the a-carbon atom is a minor process in the radiolysis of the aromatic amino acids (G(NHJ < 0.5) (31). In contrast, as shown here, the oxidation of aromatic amino acids by MCO systems is similar to that of aliphatic amino acids, leading mainly to deamination and decarboxylation via Reactions 1,

TABLE I11 Oxidation of various amino acids by the Fenton reagent i n bicarbonate buffer

The data in columns marked a were from experiments in which the concentrations of Fe(I1) and ADP were 1.5 and 3.75 p ~ , respectively. The data in columns marked b were from experiments in which the concentrations of Fe(I1) and ADP were 100 and 250 p ~ , respectively. Otherwise, the conditions were as described in the legend to Fig. 2, except that the initial concentration of all amino acids, but tyrosine, was 50 mM; the concentration of tyrosine was 5 mM. Reaction mixtures were assayed after 90 min of incubation.

Products formed Amino Initial rate H A N W I

acid of oxidation” consumed NHs co, Carbonyl

xroups

PR1b (C02 + carbonyl) 0,

pmolf min p n o l pmol *mol pmol pmol a a b a b a b a b a b a b a

Valine 0.79 56 59.8 27.5 36.3 20.4 15.9 9.4 23.8 5.4 6.7 1.05 1.10 0.92 Glutamate 0.76 55.5 59.7 24.2 26.6 21.9 15.7 3.9 5.5 5.4 10.1 1.03 1.05 0.94 Isoleucine 0.63 55.8 59.3 31.5 37.8 21.0 13.1 15.4 32.6 3.4 Lysine 0.59 49.8 55.1 31.3 29.2 17.8 19.4 0.5 1.3 1.9 Leucine 0.59 47.8 59.7 23.9 33.7 18.8 16.1 10.8 30.2 5.8 Phenylalanine 0.50 49.3 55.1 20.7 23.8 17.8 17.8 14.8 18.2 4.5 Proline 0.50 46.0 55.1 3.8 14.6 14.5 19.1 1.3 3.0 12.6 Threonine 0.50 42.0 59.8 18.5 19.9 20.2 23.8 9.7 13.0 2.6 Serine 0.49 48.7 59.8 16.3 15.6 17.0 19.6 5.7 6.1 3.3 Tyrosine 0.46 25.6 48.7 3.1 4.4 1.9 4.3 0.9 2.1 10.7 Alanine 0.44 45.7 59.7 23.7 23.0 15.1 19.0 7.8 8.6 9.2 Glycine 0.40 40.0 59.9 7.9 10.9 9.2 15.2 0.8 1.1 18.3 Arginine 0.31 49.6 54.8 22.3 31.8 15.9 16.5 0.9 1.3 3.6 Glutamine 0.39 38.9 59.8 39.6 42.7 10.3 14.4 1.2 1.8 4.1 Tryptophan 0.31 24.0 55.1 4.5 18.6 7.3 9.8 0.7 3.9 0.4 Methionine 0.24 51.4 55.1 5.4 5.6 4.6 2.6 1.6 3.4 1.0 Aspartate 0.16 23.5 59.8 9.4 21.8 10.2 22.8 3.4 8.2 8.4 Asparagine 0.14 41.4 59.8 26.3 29.2 7.0 17.8 5.6 4.1 13.1 Histidine 0.10 23.7 54.2 11.0 32.0 5.6 16.2 1.9 6.8 2.0

1

1

1

6.4 1.06 1.07 4.6 1.06 1.05 5.8 1.14 1.03 4.3 0.96 0.91 4.0 0.95 1.12 3.6 1.05 0.85 7.1 0.82 0.83 8.7 1.03 0.95 8.8 1.25 1.00 3.0 1.34 0.87 5.3 0.92 1.07 5.0 1.49 1.12 7.1 0.53 0.77 3.2 0.23 0.27 3.6 1.55 0.87 2.8 1.44 0.88 3.0 0.87 1.00

0.87 1.71 0.81 0.63 0.24 0.62 0.72 0.62 1.11 0.79 1.33 3.44 0.56 0.87 0.69 2.09 1.47

Rate during the first 10 min. PRI refers to the ratio (&)/(H,O,), where (H202) is the observed amount of H20, consumed and (2 , ) = the

sum; (NH:) + ( C O P ) + 2(02). For this calculation the change in gas pressure which occurs in the presence of oxsorbant is assumed to reflect the sum of the amounts of CO, produced by Reactions 2 and 8.

Fenton Chemistry: Amino Acid Oxidation 17207

-0 10 20 30 0 0.5 1.0 1 . 5 ” --

MINUTES DESFERAL/Fe(ll)

FIG. 6. Dependence of leucine oxidation on the Fe(II)/des- feral ratio. Initially the main compartment of the double side arm Warburg vessels contained 23.5 mM NaHCO:+, 50 mM leucine, and various amounts of desferal as follows: 0, none; A, 40 pM; 0, 80 pM; 0, 100 p ~ ; A, 110 p ~ ; W, 120 pM; X, 150 pM. After equilibration with 5% CO,, 95% N,, 100 p~ FeS04 was added from one side arm, and then 30 mM H202 was added from another side arm. Panel A shows the time course of gases (CO, + 02) which were formed during 30 min. Panel E shows the concentration of carbonyl compounds present in the reaction mixtures after the 30 min of incubation at the indicated chelator/Fe ratios.

00’~” 1

5 -L

10 15 CHELATORIFelllI

CHELATOR/Fe(ll)

FIG. 7. Dependence of leucine oxidation on the chelator/Fe ratio. A , reaction mixtures contained initially, 23.5 mM NaHCO,$,

dipyridyl (DZPYR, 0), ADP (A), o-phenanthroline (0), or EGTA (0) 100 H M FeSO.,, 20 mM H,O,, 50 mM leucine, and amounts of a,a-

to yield chelator/Fe ratios as indicated on the abscissa. The FeS04 and H202 were added after gassing with the 5% CO,, 95% N, mixture. After 30 min at 30 “C, the concentrations of carbonyl compounds were measured as described under “Experimental Procedures.” B, reaction mixture (0.4 ml) contained 23.5 mM NaHCOc3, 100 p~ FeS04, 30 mM H,O,, 50 mM leucine, and concentrations of desferal (0), EDTA (O), or diethylenetriaminopentaacetic acid (A) to yield chela- tor/Fe ratios as indicated on the abscissa. The data are the average of at least three separate experiments. To facilitate comparisons, the data were normalized with respect to the amount of carbonyl com- pounds observed at a cbelator/Fe ratio of 1.0. Otherwise conditions and procedures were as described in A .

2 , and 4. As shown in Table 111, these reactions account for at least 90% of the products formed from phenylalanine and tyrosine; however, the PRI for these amino acids (0.91-0.95) and for tryptophan (0.77) are significantly lower than 1.0, indicating that small amounts of other unidentified products are also formed during the metal ion-catalyzed oxidation of the three amino acids. (ii) The relative rates of oxidation of various amino acids by radiolysis are also different from that observed with MCO systems. Scholes et al. (43) noted that the rates of oxidation of nine different amino acids by OH ‘ radicals generated by radiolysis varied as follows: Trp > Met > His > Arg > Leu > Ser > Ala > Asp > Gly. In the metal- catalyzed oxidation system used here, the relative rates of oxidation of these same amino acids is quite different: Leu > Ser > Ala > Gly > Arg > Trp > Met > Asp > His (Table 111). (iii) The oxidation of amino acid by radiolysis is inhibited by OH’ radical scavengers (31), whereas these scavengers have little or no ability to inhibit the oxidation by MCO systems (Table IV). This suggests that the oxidation of amino acids by MCO systems is a caged process (see below). (iv) The yield of ammonia in the decomposition of simple aliphatic amino acids by radiolysis is inversely proportional to the number of carbon atoms. The G(NH,) values decrease from 2.3 to 0.3 as the number of carbon atoms is increased from 2 to 10 (31). This is attributed to the fact that by increasing the length of the carbon chain, the number of C-H bonds that are suscep- tible to attack by OH’ is increased, and this leads to the generation of more kinds of products. In contrast, the yield of NH, in metal-catalyzed oxidation of aliphatic amino acids is relatively independent of the chain length (Table 111). It follows from Reactions 1-7 that after correcting for the amount of H,O, consumed by disproportionation (i.e. 0, production) and by the secondary reactions (2 and 5 ) (CO, production), the amount of H,02 consumed in the oxidative deamination of amino acids is given by the expression

(H20,)~ = (HZO,), - (‘20, + 20,)

where (H20Jt = total amount of H202 consumed and (H202)* represents the amount H202 consumed in the primary deam- ination reaction. The ammonia yield, y(NH3), is therefore given by the ratio (NH3)/(H20,) which should be equal to 1.0 if the amino acid oxidation is due only to Reactions 1 and 4. By this criterion, it is evident from the data in Table I11 that the yield of NH, in the oxidation of simple aliphatic amino acids is independent of the number of carbon atoms, i.e. y(NH3) = 1.0 for alanine, leucine, valine, and isoleucine. These results are therefore in contrast to those obtained by the radiolysis mechanism and are consistent with the view that the oxidation by MCO systems is a “cage”-type process in which amino acid-iron chelate complexes are reactive in- termediates (see below).

The multiphasic response of amino acid oxidation to changes in the [chelator]/[iron salt] ratio is not unique; similar effects have been observed for the peroxidation of lipids (44-48) and in the generation of OH’ by xanthine oxidase (49). Among other explanations, it has been suggested that stimulation of the oxidative processes by EDTA and other chelators at [chelator]/[iron salt] ratios of less than 1.0 is due to an increase in the availability of soluble iron com- plexes (44, 47-49). This suggestion is based on the consider- ation that at physiological pH values and the concentrations of iron salts commonly employed (>75 p ~ ) in in vitro studies, Fe(II1) would exist almost exclusively in the form of large insoluble Fe(OH)3 polymeric complexes and would be unavail- able for Fenton-type chemistry. Stimulation of the reaction by substoichiometric amounts of chelators was therefore at-

17208 Fenton Chemistry: Amino Acid Oxidation

tributed to a conversion of Fe(II1) to soluble iron-chelate complexes. However, in our studies, the ability of chelators to stimulate amino acid oxidation cannot be due only to the solubilization of iron, since under our experimental condi- tions, the iron would be present largely in the form of soluble bicarbonate and/or amino acid complexes, even in the absence of other chelators. The solubility argument is discounted also by the results summarized in Fig. 9 showing that the rate of amino acid oxidation is a function of both the unchelated iron (i.e. iron in excess of EDTA) and the EDTA-iron complex. Thus, in the absence of EDTA, the rate of oxidation increases with an increase in the iron concentration over the range 0- 5 p~ but is inhibited by higher concentrations of iron, 10-100 PM (Fig. 9A, solid circles). This result is therefore similar to that observed in the oxidation of other compounds (45, 46). However, as shown in Fig. 9B, when the concentration of unchelated iron is held constant at 50 p ~ , the rate of amino acid oxidation increased progressively as the concentration of EDTA-iron complex was increased over the range of 0-200 FM. Moreover, if the ratio [total iron]/[EDTA] is held con- stant at 1.33 so that there is always an excess of unchelated iron, then the rate of amino acid oxidation increases as the amount of unchelated iron is increased over the range of 5- 20 p~ and is independent of further increments in the unche- lated iron concentration over the range of 20-100 p~ (Fig. 9A, solid triangles). The data in Fig. 9A discount also the possibility that the inhibition of amino acid oxidation by high concentrations of unchelated iron is due to the ability of Fe(I1) to scavenge OH’ by Reaction 10 (50),

Fe(I1) + OH’ + Fe(II1) + OH- (10)

since the inhibition of amino acid oxidation by high concen- trations of iron (Fig. 9A, solid circles) is prevented if some EDTA + iron complex is present in addition to the unchelated iron (Fig. 9A, solid triangles).

Considered together, the results summarized in Figs. 6-9 support the view that two forms of iron, the EDTA.iron complex and another form, possibly an amino acid chelate or a bicarbonate complex, are required for optimal rates of amino acid oxidation. With substoichiometric levels of EDTA or desferal, both chelated and unchelated forms of iron would be present, but where the EDTA is in slight excess, only one form, the EDTA.iron complex, would exist. To account for the multiphasic response of lipid peroxidation to increasing concentrations of EDTA, Tien et al. (46) suggested that the EDTA .Fe(II) chelate complex might be needed for rapid generation of H202 by autooxidation (Reaction 11)

PEDTA.Fe(I1) + 2H+ + 0, + H,O, + BEDTA.Fe(II1) (11)

and that unchelated iron is required for rapid cleavage of the H20, thus formed to produce OH’ (Reaction 12).

Fe(I1) + H,O, + OH’ + OH- + Fe(II1) (12)

Maximal lipid peroxidation would therefore be obtained at an EDTA/Fe(II) ratio where the balance between the two proc- esses is optimized. Such an explanation cannot account for our results because in our experiments, high concentrations of H202 were present from the beginning. Obligatory coupling of Reactions 10 and 11 is therefore not a rate-determining factor. Of particular significance, however, are the results of studies by Aust and co-workers (46, 51, 52) and Braughler et al. (44, 45) showing that the initiation of lipid peroxidation involves the simultaneous presence of both oxidized (Fe(II1)) and reduced (Fe(I1)) forms of iron, optimally in a 1:l ratio. This provides strong evidence for the role of two forms of iron in iron-mediated oxidation reactions, but until now, mecha-

nisms to explain the phenomenon are still lacking. As noted by Gutteridge et al. (47), a complex formed by the binding of two metal ions as might occur a t low [chelator]/[iron] ratios could promote oxygen-catalyzed reactions, whereas 1:l com- plexes or different kinds of complexes at high [chelator]/ [iron] ratios might be inactive or serve as inhibitors of oxi- dation reactions. In view of the fact that iron can form complexes with bicarbonate and amino acid as well as with EDTA and other chelators, the multiphasic response of amino acid oxidation to variations in the [chelator]/[iron] ratio might reflect variations in the composition, concentration, and redox potentials (46, 53, 54) of various iron complexes that are formed under our experimental conditions. We re- ported earlier (55) that the Mn(I1)-catalyzed disproportiona- tion of H202 is a third order function of the bicarbonate ion concentration and is reduced to a second order process by the presence of amino acids. It was proposed that in the absence of ADP 3 eq of HCO, react with Mn(I1) to form the catalyt- ically active complex, but in the presence of amino acid 1 eq of HCO, is replaced by the amino acid to yield an active complex of the composition, amino acid. Mn( 11) . (HCO;),. The formation of a similar complex in which a chelator is also involved is suggested by the data in Fig. 1, showing that in the absence of ADP there is a sigmoidal response of leucine oxidation to increasing concentrations of HCO;, whereas in the presence of ADP a hyperbolic response is observed. This suggests that in the absence of ADP more than 1 eq of HCO; is involved in production of the catalytically active complex, whereas in the presence of ADP only 1 eq is required. Although not shown, a double-reciprocal plot ( l / v versus 1/ HCO;) of the data obtained in the presence of ADP is linear, from which an apparent dissociation constant of 13 mM can be calculated for the binding of HCO; to form the putative catalytic complex (possibly a ADP. Fe(I1). HCO:,. leucine complex). By monitoring spectral changes that occur in var- ious reaction mixtures, we have obtained direct evidence for the formation of a complex between chelator, Fe(II), and amino acid (36).” Thus, when Fe(I1) is added to a mixture containing the iron chelator ferrozine and any amino acid, a blue complex (A,,, = 630 nm) of the composition, (ferro- zine):<.Fe(II). amino acid is formed almost instantly and is slowly decomposed to yield the more stable (ferrozine):<. Fe(I1) complex (A,,, = 590 nm). Whereas the Fe(I1) in the (ferro- zinc):<. Fe(I1) complex is resistant to oxidation of H,02 or 0 2 ,

the Fe(I1) in the ternary complex is almost instantly oxidized by H,O, (36).

We reported earlier that bicarbonate ion is required for the Mn(I1)-dependent oxidation of amino acids by H202 (33, 55- 57) and that in the presence of bicarbonate and H202, OH’ is generated by the CuBn superoxide dismutase (58). Bicarbon- ate has been showa:.hlso to stimulate the iron-catalyzed oxi- dation of deoxyribose by xanthine oxidase (59), the oxidative modification of proteins by radiolysis (25), and the lumines- cence obtained during oxidation of some substances by xan- thine oxidase at pH 10 (60). Because in the latter case, the amount of luminescence was proportional to the square of the COT concentration, it was proposed (60) that OH’ generated in these reactions reacts with COT to form the bicarbonate radical (COS) (61,62) the dimerization of which is responsible for the light emission. As discussed previously (33, 56), it appears more likely that the bicarbonate ion interacts with the amino acid and Fe(II), or Mn(II), to form a ternary complex which in the presence of HzOy facilitates rapid redox cycling of the metal ion, with concomitant generation of an active oxygen species (OH’, ferry1 ion), and oxidation of the

r, B. S. Berlett and E. It. Stadtman, unpublished data.

Fenton Chemistry: Amino Acid Oxidation 17209

amino acid in the complex (see Refs. 33 and 56 for a detailed discussion of this mechanism). An analogous mechanism has been proposed also for the site-specific modification of amino acid residues in proteins (4,20). According to this mechanism, the metal ion-catalyzed oxidation of amino acids would be a “caged” reaction in which the active oxygen species generated within the complex would attack the a-carbon atom of the amino acid before it could escape to the surrounding medium. This would account for the high specificity of the reaction mechanism and for the failure of radical scavengers to inhibit the reaction. Although it appears reasonable in principle, the proposed mechanism must be modified to account for the conclusion that two forms of iron (possibly both oxidized, Fe(III), and reduced, Fe(II), forms) are involved in a rate- determining step (44, 51, 52).

Formation of the DMPO-OH‘ adduct, which occurs when H202 and Fe(I1). ADP chelate are incubated in bicarbonate buffer with the spin trap DMPO, was used by Zs-Nagy and Floyd (22) to verify that OH is generated in this system. The addition of amino acids to the reaction mixtures led to a decrease in the level of DMPO-OH‘ adduct. This was taken as evidence that the amino acids compete with DMPO for reaction with the OH’ formed in solution. This interpretation may not be correct. As noted in the present study, the oxida- tion of amino acids by the H2O2-ADP.Fe(II) system is not inhibited by a number of OH’ radical scavengers including high concentrations of formate, mannitol, dimethyl sulfoxide, and ethanol and urea. This and other considerations sum- marized above suggest that the oxidation of amino acids is a “cage” process in which formation of an amino acid.Fe(I1) chelate complex is a primary step and in which oxygen radicals formed are not released in solution but react directly with the amino acid. In view of this consideration and the fact that amino acids are able to form chelate complexes with Fe(II), we suggest that the quenching of DMPO-OH’ adduct forma- tion in the experiments of Zs-Nagy and Floyd is due to sequestration of the Fe(I1) rather than to competition with DMPO for the hydroxyl radicals in solution.

It is noteworthy that in view of the fact that amino acids can sequester metal ions and the fact that all amino acids undergo rapid metal ion-dependent oxidation by H 2 0 y under physiological conditions ( i e . in the presence of bicarbonate buffer and ADP), the size of the amino acid pool could be an important factor in the protection of cellular constituents (proteins, lipids, nucleic acids) against oxidative damage.

Acknowledgments-We thank Rodney L. Levine for his assistance in the development of the high pressure liquid chromatographic procedures used for the separation and quantitation of the products of leucine oxidation, Lin Tsai for providing authentic samples of N- ketoisocaproic acid oxime, isovaleraldoxime, and the 2,4-dinitrophen- ylhydrazones of wketoisocaproic acid and isovaleraldehyde, and P. R. Chock for useful discussions and helpful suggestions concerning the manuscript presentation.

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Biophys. 246, 501-514

NO"( 20.3 7.8 6 4 1.1 8.8 4.2 0.25 0.86

FcT'lti" 122 PM)

FelTedO*l" 51 0 29 0 22 0 1.8 29.6 25.0 7 5 1.08 (0.3 mg)

Hemi" 51.0 2 9 0 1 6 7 5 2 30.0 29.0 10.0 (50 pM)

I I 1

Hemlli"

52.5 268 180 3.9 25.5 13.0 2.0 0.98

(50 P M )

Tr-femmFeOl) 4.2 -- 0 5 0 0.24 2 . 1 0 0 0.13 ( I LM)

51.0 290 16.7 4.9 30.0 29.0 to o 1.10

Blmmyein.FeU1) 47 5 30 20.4 2.3 27.0 11.4 (SO pM)

9 0 1 0 9

Ferrichmmc A 54 5 30 20.5 3.9 36.0 20 6 iImpM)

2 6 I 18

Nolle 0 6.0 1.6 08 1 3 112 0 1 33 2 0 51 16

DMTU 10 9.2 6.4 2.6 0 3 8.1 0 3 I2 5 I 3 49 50

Manmtul 50 6.2 0.9 .-. 0 9 163 0 2 29 I O 44 5 5

Thloursa SO 10 IO I 4 0 I I 0 7 8 0 7.2 48 48

1l.U SO 5 4 I O I 1 1 3 164 0 33 1.3 51 I ?

Formate 50 5.0 0 8 2 0 I O I5 8 0 10 1.4 SO I6

Elhannl 25 6 0 0 1 09 12 I 7 0 0 2 32 1.5 48 55

o-Tocopherol 0.1 5 9 I 6 0 5 0.8 160 0 2 30 1.7 SO I4

O M S 0 50 54 0 8 I1 I O I 8 0 0 2 33 0 9 51 18

Fenton Chemistry: Amino Acid Oxidation 17211

.*- - -

24 r

20

g 12

N E

-0 2 n

8

I ' I I 1 I L

0 10 20 30 40

Time, minutes

4

-A"- ..A -0.2 I . ~ "

,-. r-. 4 -0

0 I I I

0 10 20 30 40 50 60 Time, minutes

20 p ... .,.... ..,....A 2.5

12

8

4

0 0 30 60 90 120 150 180

Time, minutes

5 I , , , , , I 4 0 0

0 20 40 60 80 1000 50 100 150 200

UNCHELATED Fe(ll), uM EDTA, uM