Francesco Regoli1, Raffaella Bocchetti1,and Danilo Wilhelm Filho21Dipartimento di Biochimica, Biologia e Genetica, Universita Politecnica delle Marche,Ancona, Italy2Departamento de Ecologia e Zoologia, Centro de Ciencias Biologicas, UniversidadeFederal de Santa Catarina, Florianopolis, Brazil
T his chapter provides technical protocols formeasuring the most common antioxidantsin tissues of marine organisms by means
of simple spectrophotometric assays. After a briefdescription of sample preparation procedures, methodsfor the determination of superoxide dismutase (SOD),catalase (CAT), glutathione peroxidase (GPx; bothSe-dependent and the sum of Se-dependent andSe-independent forms), glutathione S-transferase(GST), glutathione reductase (GR), glyoxalase I (GI),and glyoxalase II (GII) activities, as well as glutathione(GSH) content, will be presented. Dilutions andvolumes of samples in assay conditions are oftensuggested, based on our experience with temperateinvertebrates and fish. However, it should always beconsidered that marine organisms might present hugevariations in the levels or activities of antioxidantdefenses. Thus, although the principle and reagents ofvarious assays are of general applicability, appropriatedilutions of samples should always be checkedbefore starting a working session with new speciesand/or tissues.
It is highly recommended that tissue samples for anal-ysis of antioxidants are frozen in liquid N2 immediatelyafter animal dissection, and stored at −80◦C untilhomogenate preparation. A suitable homogenizationbuffer is 100 mM K-phosphate buffer (KH2PO4), pH 7.5,with 2.5% and 1.8% NaCl for marine invertebrates andmarine vertebrates, respectively, and 0.1 mM phenyl-methylsulphonyl fluoride (PMSF). The addition of pro-tease inhibitors is required when homogenizing tissueswith elevated levels of these enzymes (typically diges-tive glands). An appropriate mix of inhibitors is 0.008trypsin inhibitor unit (TIU) mL−1 aprotinin, 1 ng mL−1
leupeptin, 0.5 ng mL−1 pepstatin, and 0.1 mg mL−1
bacitracin.Samples are homogenized on ice, preferably using a
hand-held potter with Teflon or glass pestle. A commonhomogenization ratio of 1:5 weight : volume (w : v)is recommended. Higher volumes of buffer facilitatetissue disintegration, while diluting the proteins.Conversely, lower buffer volumes make samples
367
368 F. Regoli, R. Bocchetti, and D. Wilhelm Filho
more concentrated but may reduce the efficiencyof homogenization. Homogenization of each sampleshould take approximately 30–60 s. To obtain thepost-mitochondrial S9 fraction, containing micro-somes and cytosol, homogenates are centrifuged at12,000 g for 15 min at 4◦C. Supernatants are collectedwithout the lipid phase, immediately assayed, orsubdivided into small aliquots and stored at −80◦C.For most antioxidants, however, it is recommendedto use a more purified, cytosolic fraction due to thepresence of potentially interfering molecules in thepost-mitochondrial S9 fraction.
If microsomes should also be collected, the super-natants obtained after the 12,000 g centrifugation arefurther centrifuged at 100,000 g for 1 h 10 min at 4◦C,to obtain the cytosolic fraction whereas the pelletsrepresent the microsomes. Supernatants are removed,immediately assayed, or subdivided in small aliquots(100–200 μL), and stored at −80◦C. Before freezing,add 1 μL of dithiothreitol (DTT; from a 100 mM stocksolution in methanol stored at 4◦C) to 100 μL of thecytosolic fractions designated for GPx analyses.
HOMOGENIZATION REAGENTS ANDSOLUTIONS
• 100 mM potassium phosphate (KPi) buffer, pH7.5 (homogenization buffer). Dissolve 0.680 g ofKH2PO4 in 50 mL of distilled water; adjust pH to7.5 with concentrated KOH.
• 1.8% or 2.5% NaCl. Dissolve 1.8 g or 2.5 g of NaClin 100 mL of homogenization buffer.
• 100 mM PMSF. Dissolve 0.174 g of PMSF in 10 mLof methanol. This stock solution is stable for 6months at 4◦C. Add 100 μL of stock solutionto 100 mL of homogenization buffer to obtain a0.1 mM concentration.
• Aprotinin (0.008 TIU mL−1). This trypsin inhibitorcan be purchased as a powder or solution. Itsactivity is expressed as TIU and reported in theproduct specification as total activity (for pow-ders) or activity mL−1 (for solutions). Aprotininconcentration in homogenization buffer is 0.008TIU mL−1. Depending on the aprotinin stock solu-tion, calculate the volume containing 0.8 TIU andadd to 100 mL of homogenization buffer.
• Leupeptin (1 ng mL−1). Dissolve 1 mg in 1 mL ofdistilled water to obtain a stock solution. Add100 μL to 100 mL of homogenization buffer.
• Pepstatin (5 ng mL−1). Dissolve 1 mg in 1 mL ofmethanol 100% to obtain a stock solution. Add50 μL to 100 mL of homogenization buffer.
• Bacitracin (0.1 mg mL−1 solution). Dissolve100 mg in 1 mL of distilled water to obtaina stock solution. Add 100 μL to 100 mL ofhomogenization buffer.
• 100 mM DTT. Dissolve 0.01542 g of DTT in 1 mLof methanol to obtain a stock solution.
Stock solutions of PMSF, leupeptin, pepstatin, baci-tracin, and DTT are stable for 6 months at 4◦C.
DETERMINATION OF SUPEROXIDEDISMUTASES ACTIVITY (EC1.15.1.1)
SOD catalyzes the dismutation of O •−2 into O2 and H2O2
(Fridovich 1986):
2O •−2 + 2H+ → H2O2 + O2
SOD activity is determined in an indirect spec-trophotometric assay by monitoring the reduction ofcytochrome c by O •−
2 at 550 nm. O •−2 is generated by
a HX/XO system (McCord and Fridovich 1976). Oneunit of SOD is defined as the amount of enzyme neededto inhibit the reduction of cytochrome c by 50%.Different volumes of sample are used to determine 50%inhibition of the reaction rate.
Reagents and Solutions
• 100 mM KPi buffer, pH 7.8. Dissolve 0.680 g ofKH2PO4 in 50 mL of distilled water; adjust pH to7.8 with concentrated KOH.
• 100 mM EDTA stock solution. Dissolve 3.7224 gof EDTA (disodium salt, dehydrated) in 100 mL ofdistilled water. This solution is stable for 3 monthsat 4◦C.
• 300 mU mL−1 xanthine oxidase (XO, prepareimmediately before the use). Depending on theproduct, dilute the XO to 300 mU mL−1 with colddistilled water.
• Working buffer: 100 mM KPi buffer, pH 7.8;0.2 mM EDTA; 100 μM hypoxanthine (HX);20 μM cytochrome c. Prepare immediately beforeuse:• 50 mL of 100 mM KPi buffer• + 100 μL of 100 mM EDTA• + 0.68 mg of HX• + 12.3 mg of cytochrome c.
Spectrophotometric Assays of Antioxidants 369
Procedure
Spectrophotometric analyses are carried out atλ = 550 nm (light path 1 cm) at constant temperature(18 ± 1◦C for temperate species). A reference (unin-hibited) reaction is carried out to calculate the changein absorbance per minute (�Abs), which reflects thereduction of cytochrome c by O •−
2 in the absence ofSOD. For each sample at least three different samplevolumes should be tested in the reaction, to calculatethe amount needed to inhibit the reference reactionby 50%.
Reference Reaction
Add to a plastic cuvette:
• 500 μL working buffer• 480 μL of 100 mM KPi buffer• + 20 μL of XO.
Mix vigorously after the addition of XO. Read theincrease in absorbance for at least 1 min. The reactionshould be linear. �Abs should be approximately 0.1.
Sample Reactions
Three readings (R-I to R-III) with three different vol-umes for each sample are performed. Note that the finalvolume in the assay is always 1000 μL.
Start reaction by adding XO and mix vigorously.Read the increase in absorbance for at least 1 min.Reactions should also be linear. Increasing the amountof sample in the assay should decrease the �Abscompared to the reference reaction (reduction ofcytochrome c is lowered by a higher content of SOD inthe samples). The three volumes of sample should givean inhibition of between 20 and 70% compared to the
Abs Ref
R-I
R-II
R-III
Time
Fig. 27.1 The reaction starts by adding XO. Absorbanceincrease must be linear for 1 min.
reference reaction range (Fig. 27.1). It is important tonote that the three volumes reported in the exampleare suggestive and should be adjusted according to theobserved inhibition of the reference reaction for eachtype of sample. However, in a final volume of 1 mL,different aliquots of samples will be compensated bydifferent volumes of KPi buffer. In this way, the volumeof working buffer does not change in different readings,so that the applied concentrations of HX, cytochromec, and XO in the assay remain constant.
Calculation of the Volume Necessary toReduce the Reference Reaction by 50%
The relationship between added volume of sample andobserved �Abs is often described as linear in a semilog-arithmic scale (Fig. 27.2).
Plotting the relationship between added volume ofsample (μL) and observed �Abs (as shown in Fig. 27.2)it is possible in the majority of conventional worksheets
Add
ed v
olum
e of
sam
ple
ΔAbs min−1
Fig. 27.2 Relationship between added volume of sampleand observed �Abs.
370 F. Regoli, R. Bocchetti, and D. Wilhelm Filho
to interpolate these points choosing among differentequations (i.e linear, polynomial, exponential). Byselecting a second-order regression on a linear scale,the worksheets also provide the coefficients for thisequation, which relates absorbance to added volume ofsample (μL):
y = ax2 + bx + c
Using the coefficients a, b, c from the equation and set-ting x to the value corresponding to 50% of the �Absmeasured in the reference reaction, y will yield thevolume (μL) of sample needed to reduce the referencereaction by 50%, e.g. the volume of sample (Vol) thatcontains 1 Unit of SOD. According to the present proto-col, the total volume in the assay is 1000 μL (see tablefor RI–RIII above), in which the number of containedSOD Units will thus correspond to 1000/Vol.
Calculations
SOD units are typically normalized to protein content,tissue weight, or mL (for blood, plasma or hemolymph).Normalization to tissue weight often precludes compar-isons between different species or organs with differentrelative water content. When normalizing the activityto mg protein, comparability of the values from differ-ent tissues may be influenced by the basal content oftotal proteins, which can be highly variable betweenorgans (Vlahogianni et al. 2007; Fernandez et al.2009).
When normalizing to protein content:
SOD (U mg protein−1)
= (1000/Vol) × (sample dilution)/proteins
where ‘‘1000/Vol’’ corresponds to the number ofSOD units contained in 1 mL (final assay volume, seeabove), ‘‘sample dilution’’ is the dilution factor applied(if any) to the cytosolic fractions before the assay,and ‘‘proteins’’ is the protein content in the cytoso-lic fractions (e.g. undiluted sample extract expressedas mg mL−1).
DETERMINATION OF CATALASEACTIVITY (EC1.11.1.6)
CAT is a heme containing enzyme found in peroxisomesof all aerobic organisms (Mueller et al. 1997). This
enzyme catalyzes the decomposition of H2O2 to H2Oand O2.
2H2O2 −→ 2H2O + O2
The physiological role of CAT is fundamental in coun-teracting the production of H2O2, which can reactwith transition metals to form HO •. By removing oneof the main precursors of these highly toxic reactiveoxygen species (ROS), CAT has been indicated as one ofthe main antioxidant defenses towards HO • in marineorganisms (Halliwell and Aruoma 1991; Halliwell andGutteridge 2001; Regoli et al. 2002). The spectropho-tometric assay described by Aebi (1984) is one ofthe most used methods to measure CAT activity invertebrate and invertebrate tissues. Other methods,such as the polarographic determination, are less com-mon. The spectrophotometric assay quantifies the lossof absorbance at 240 nm due to the decomposition of5–12 mM H2O2. The rate of H2O2 decay is proportionalto the amount of CAT contained in the sample, andis calculated using an extinction coefficient ε = 0.04mM−1 cm−1.
Since H2O2 is not stable, the stock solution needs to betitrated before performing the assay to test its effectiveconcentration. The titration consists in reading theabsorbance of different dilutions of the H2O2 stock,and calculating the concentrations with the extinctioncoefficient:
H2O2 concentration in the stock solution is calcu-lated by multiplying the concentrations obtained indiluted solutions with their respective dilution factors:
c1 ∗ X1 = C1
c2 ∗ X2 = C2
c3 ∗ X3 = C3
The mean of these values is the effective H2O2 concen-tration in the stock solution.
Importantly: to increase lifetime of the originallypurchased stock solution, do not dip the pipette-tipinto the stock bottle. Instead pour an aliquot into asmall, clean beaker, use it to make your solutions, anddischarge the rest of the aliquot.
Spectrophotometric Assays of Antioxidants 371
Reagents and Solutions
• 100 mM KPi buffer, pH 7.0 (adjust pH with con-centrated KOH).
• 1.2 M H2O2. Prepare fresh by adding 100 μL of thealiquot taken from the 12 M H2O2 stock to 900 μLof distilled water.
Procedure
Spectrophotometric analyses are carried out at λ =240 nm (light path 1 cm) and at constant temperature(18± 1◦C for temperate species).
Add to a quartz cuvette (final volume 1 mL):
• 980 μL of KPi buffer
INSTRUMENT AUTOZERO
• 10 μL of 1.2 M H2O2
Read the absorbance and add:
• 10 μL of sample.
Mix vigorously after the addition of sample. The instru-ment autozero before the addition of H2O2 allows acheck of the proper assay concentration of this sub-strate (12 mM), which should give an absorbance valueof 0.48. Other protocols ‘‘autozero’’ with buffer andsample, and start the reaction by adding a known vol-ume of a previously prepared 12 mM H2O2 dilution(35 μL of commercial 30% H2O2 solution is added to10 mL buffer, absorbance ≈ 1.400–1.500 AU).
Register the decrease in the absorbance for at least1 min. Reactions should be linear for at least 30 s;
otherwise a different dilution or sample volume shouldbe used. Each sample is measured twice. Maintain afinal volume of 1 mL even if different sample dilutionsare needed. Compensate by changing the volume ofKPi buffer. Appropriate dilutions for measuring CAT incytosolic fractions of marine organisms can be highlyvariable ranging for samples homogenized at (w : v) 1:5to 1:10 to 1:100 in digestive gland of invertebrates, andbetween 1:100 and 1:500 in fish liver. Proper dilutionsto obtain reliable readings need to be predetermined foreach batch of samples.
Calculations
Results are usually expressed in μmol or nmol H2O2
min−1 mg protein−1, g of wet tissues, or mL of blood,plasma or hemolymph (see Box 27.1). When evaluatingblood, hemolysates must be diluted at least 500 times inthe cuvette, to avoid interference of peroxidase activityfrom hemoglobin (Aebi 1984). Normalization to tissueweight often precludes comparisons between differentspecies or organs with different relative water content(see Box 27.2). When normalizing to mg of protein,the values might also be distorted by different proteincontent of specific tissues.
When the enzymatic activity is normalized to proteincontent:
CAT activity (μmol min−1 mg−1 proteins)
= (�Abs/−0.04) × (sample dilution)/proteins
where ‘‘�Abs’’ is the change in absorbance per minute,‘‘−0.04’’ is the extinction coefficient (mM−1 cm−1),‘‘sample dilution’’ is the dilution factor (if any)applied to the cytosolic fractions before the assay,
Box 27.1 Less common units for expressing CAT activity
Other units are less common for expressing CAT activity and might confound or make comparisons moredifficult. Among these, the Bergmeyer Unit (UB) corresponds to the amount of CAT that converts half of theO2 from a H2O2 solution of any concentration in 100 s at 25◦C (Bergmeyer 1965). International Units (UI)correspond to μmoles of substrate converted in 1 min at 25◦C per mg of protein (Bergmeyer 1965). Finally, oneSigma Unit (SU) corresponds to the amount of CAT that converts 1 mol of H2O2 per minute at 25◦C, pH 7.0,while the decay in H2O2 concentration falls in the interval of 10.3–9.2 mM. Therefore, the interconversionsare: (i) for pure enzyme concentration (considering the MW of CAT as 30,000; Chance at al. 1973) – 1 pmolof CAT = 0.033 UB = 0.2 UI; or 1 UB = 13 UI; (ii) for CAT activity, multiply the value of CAT concentration inpmoles for 28.2 to obtain the amount of H2O2 min−1 ( μmoles min−1 g−1), or for 0.47 if the activity is recordedper second (μmoles s−1 g−1 or mg protein−1).
372 F. Regoli, R. Bocchetti, and D. Wilhelm Filho
Box 27.2 Cautionary notes
Some organisms, especially invertebrates, might have some peculiarities which complicate determination ofCAT activity. For instance, fresh homogenates from two mollusks cultivated in South Brazil, Perna perna andCrassostea gigas, revealed a sort of lag phase, contrasting with the ‘‘normal’’ rapid and sharp profile from differenttissues of many aquatic species so far studied (De Almeida et al. 2007). In short, there is a relatively long delayof approximately 1 min in P. perna and of about 3 min in C. gigas before a decay in H2O2 can be detected. If theresearcher is not aware about this delay and intends to record only the first seconds or minute, no apparentCAT activity is detected.
If the researcher is interested in the calculation of the Vmax of CAT activity, then a cautious procedure shouldbe considered. With long monitoring periods, e.g. 1–3 min, the profile would not reflect the Vmax value ofCAT activity (better measured in the first seconds of reaction), and a relatively low or underestimated valueis obtained. CAT activity is characterized as a pseudo-first-order category, which makes this enzyme uniquecompared to the other antioxidant enzymes (Chance et al. 1973). As a consequence, the comparison of resultson CAT activities coming from different laboratories can be more difficult.
and ‘‘proteins’’ is the protein content in the cytosolicfractions expressed as mg mL−1.
DETERMINATION OF GLUTATHIONEPEROXIDASES ACTIVITY (EC1.11.1.9AND EC2.5.1.18)
GPx are a large family of selenium-dependent andselenium-independent enzymes with peroxidase activ-ities. The main biological role of GPx is to protectcells from oxidative damage by reducing both organichydroperoxides to their corresponding alcohols andfree H2O2 to water. The Se-dependent GPx react witha wide variety of hydroperoxides, including both H2O2
and organic peroxides, whereas the Se-independentforms reduce only organic hydroperoxides. GPx havevarious cellular localizations including cytosol, mito-chondrial matrix, and membranes, thus combiningwith CAT in removing H2O2 from different cellularcompartments (Halliwell and Gutteridge 2007).
2GSH + H2O2 → GSSG + H2O
2GSH + ROOH → GSSG + ROH + H2O
GPx activities are measured by a coupled spectropho-tometric assay (Lawrence and Burk 1976). GPx reduceH2O2 or organic hydroperoxides using GSH as cofac-tor. Variation in absorbance is thus measured due toconsumption of reduced nicotinamide adenine dinu-cleotide phosphate (NADPH), used by GR to reconvertglutathione disulphide (GSSG) to GSH. The decrease
in NADPH concentration is proportional to the GPxactivity in the sample and it is followed at 340 nm(ε = 6.22 mM−1 cm−1).
2GSH + H2O2 → GSSG + H2O
(Se-dependent forms)
2GSH + ROOH → GSSG + ROH + H2O
(both Se-dependent and Se-independent forms)
GSSG + NADPH + H+ → 2GSH + NADP+ (GR)
Reagents and Solutions
• 100 mM KPi buffer (adjust pH with concentratedKOH).
• 100 mM EDTA.• 100 mM GSH working solution (freshly prepared).
Dissolve 0.0307 g of GSH (MW 307.32) in 1 mL ofdistilled water.
• NADPH (20 mg mL−1) working solution (freshlyprepared). Dissolve 2 mg of NADPH in 100 μL ofdistilled water.
• GR (100 U mL−1) working solution (prepare imme-diately before use). Depending on the product,dilute GR to 100 U mL−1 in cold distilled water.
• 100 mM sodium azide (NaN3) working solution(freshly prepared). Dissolve 6.5 mg in 1 mL of dis-tilled water. NaN3 is an inhibitor of CAT and isadded only when measuring GPx activity towardsH2O2.
Spectrophotometric Assays of Antioxidants 373
• 200 mM cumene hydroperoxide (CHP) workingsolution (freshly prepared). Add 38 μL of a 5.2 MCHP stock solution to 962 μL of methanol.
• 100 mM H2O2 working solution (freshly pre-pared). Add 83 μL of the 12 M H2O2 stock solutionto 9917 μL of distilled water. Since H2O2 is notstable, the stock solution needs to be titratedbefore performing the assay to test the effectiveconcentration (see above).
Again: take care not to contaminate the original H2O2
stock solution!
Procedure
Spectrophotometric analyses are carried out at λ =340 nm (light pass 1 cm) at constant temperature(18 ± 1◦C for temperate species). Before measuringGPx activities, the rate of the blank reaction has tobe carefully determined. At least 10 readings withoutsample should be performed at the beginning of thesession to evaluate the decrease in absorbance due tothe oxidation of NADPH by H2O2 or CHP. This valuewill be subtracted from the total rate of sample reac-tion (�Abssample − �Absblank = �Absfinal sample). Runa new blank reaction following every 20 samples.
Since GPx activities can vary greatly in different tis-sues and species, substrate concentration in the assaymight become limiting for the enzymatic reaction.Although assay concentrations are suggested in the fol-lowing protocols for both H2O2 and CHP, these shouldbe tested when analyzing new organisms, verifyingthe linear relationship between the final rate of samplereaction and the amount of sample added in the assay.
In the assay for measuring the activity of Se-dependent forms, add to a plastic or glass cuvette (finalvolume 1 mL):
• 835 μL of KPi buffer• 10 μL of NaN3 working solution• 10 μL of EDTA• 20 μL of GSH working solution• 10 μL of GR working solution• 100 μL of blank (KPi buffer) or sample
INSTRUMENT AUTOZERO
• 10 μL of NADPH working solution
Read the absorbance (should be 0.9–1.2)
• 5 μL of H2O2 working solution.
For measuring the sum activity of Se-dependent andSe-independent forms add to a plastic/glass cuvette(final volume 1 mL):
• 846 μL of KPi buffer• 10 μL of EDTA• 20 μL of GSH working solution• 10 μL of GR working solution• 100 μL of blank (KPi) or sample
INSTRUMENT AUTOZERO
• 10 μL of NADPH working solution
Read the absorbance (should be 0.9–1.2)
• 4 μL of CHP working solution.
In both assays, mix vigorously after the additionof substrates (H2O2 or CHP). Read decrease in theabsorbance for at least 1 min. Reactions should be lin-ear, otherwise a different sample volume should beused. Each sample is measured twice. Maintain a finalvolume of 1 mL, even if different aliquots of samples areneeded. Compensate by changing the volume of addedKPi buffer. Appropriate dilutions for measuring GPxactivities in cytosolic fractions of marine organismscan vary, e.g. for samples homogenized 1:5, rangingbetween 1:10 and 1:50 in digestive gland of inverte-brates and between 1:10 and 1:100 in fish liver. Properdilutions need to be determined.
Calculations
Results are expressed in nmol min−1 mg−1 protein,or g (tissues), or mL (blood, plasma, or hemolymph).Normalization to tissue weight often precludes compar-isons between different species or organs with differentrelative water content. When normalizing to mg ofprotein, comparability of values may be compromisedby different tissue-specific protein content.
When the enzymatic activity is normalized to proteincontent:
GPx activity (nmol min−1mg−1proteins)
= (�Absfinal sample/−6.22) × (sample dilution)
× 1000/proteins
where ‘‘�Absfinal sample’’ is the change in absorbanceper minute subtracted from the rate of blank reaction(�Absfinal sample = �Abssample − �Absblank), ‘‘6.22’’ is
374 F. Regoli, R. Bocchetti, and D. Wilhelm Filho
the extinction coefficient (mM−1 cm−1), ‘‘sampledilution’’ is the dilution factor (if any) applied tocytosolic fractions before the assay and ‘‘proteins’’ isthe protein content in cytosolic fractions expressed asmg mL−1 (e.g. not diluted samples).
DETERMINATION OF GLUTATHIONES-TRANSFERASES ACTIVITY(EC 2.5.1.18)
GST are a family of multifunctional enzymes withcytosolic, mitochondrial, and microsomal localization.They catalyze the conjugation of GSH to electrophiliccenters of a wide variety of endogenous (e.g. peroxidizedlipids) and exogenous substrates (e.g. organic xenobi-otics). The latter reactions, known as phase II of bio-transformations, facilitate the dissolution of lipophilicchemicals in the aqueous cellular and extracellularfluids, and thus their excretion (George 1994).
The spectrophotometric assay for GST activityis based on the GST-catalyzed reaction betweenGSH and a substrate, among which the 1-chloro-2,4-dinitrobenzene (CDNB) has the broadest rangeof isozyme detectability (e.g. alpha-, mu-, pi- andother GST isoforms). The GST-catalyzed formation ofGS-DNB produces a dinitrophenyl thioether that canbe detected at 340 nm, ε = −9.6 mM−1 cm−1 (Habigand Jacoby 1981).
Reagents and Solutions
• 100 mM KPi buffer, pH 6.5 (adjust pH with con-centrated KOH).
• 50 mM CDNB stock solution. Dissolve 50.6 mg of1-chloro-2,4-dinitrobenzene in 5 mL of methanol.The stock solution is stable in the dark and at 4◦Cfor 1 month.
• 100 mM GSH working solution (freshly prepared).Dissolve 0.0307 g of GSH (MW 307.32) in 1 mL ofdistilled water.
• Working buffer (100 mM KPi buffer with 1.5 mMCDNB). Prepare immediately before use by adding1.5 mL of CDNB stock solution to 50 mL of 100 mMKPi buffer, pH 6.5.
Procedure
Spectrophotometric analyses are carried out atλ = 340 nm (light path 1 cm) at constant temperature(18 ± 1◦C for temperate species).
Add to a plastic cuvette (final volume 1 mL):
• 965 μL of working buffer• 15 μL of GSH working solution• 20 μL of sample.
Mix vigorously after the addition of sample. Readincrease in absorbance for at least 1 min. Reactionsshould be linear, otherwise a different dilution or sam-ple volume should be used. Maintain a final volume of1 mL even if different aliquots of samples are needed.Compensate by changing the volume of working buffer.Appropriated dilutions for measuring GST in cytosolicfractions of marine organisms can be variable, e.g. forsamples homogenized 1:5, normally ranging between1:10 and 1:100 in digestive gland of invertebrates andbetween 1:100 and 1:500 in fish liver. Proper dilutionsmust be tested for each new batch of samples.
Calculations
Results are usually expressed in nmol min−1 mg−1
protein or g of tissues. Normalization to tissue weightoften precludes comparisons between different speciesor organs with different water content. When nor-malizing to mg of protein, comparability of valuesmay be compromised by different tissue-specific proteincontents.
When the enzymatic activity is normalized to proteincontent:
GST activity (nmol min−1mg−1proteins)
= (�Abs/9.6) × (sample dilution)
× 1000/proteins
where ‘‘�Abs’’ is the variation of absorbance perminute, ‘‘9.6’’ is the extinction coefficient (mM−1
cm−1), ‘‘sample dilution’’ is the dilution factor (if any)applied to the cytosolic fractions before the assay, and‘‘proteins’’ is the protein content in cytosolic fractionsexpressed as mg mL−1 (e.g. not diluted samples).
DETERMINATION OF GLUTATHIONEREDUCTASE ACTIVITY (EC 1.6.4.2)
GR is the enzyme responsible for the reduc-tion of GSSG to GSH, using NADPH as an elec-tron donor. Although GR does not directly act asan antioxidant, it plays an indirect but neverthelessessential role in protecting cells from oxidative damage
Spectrophotometric Assays of Antioxidants 375
and in maintaining the proper redox status of GSH. Thespectrophotometric assay for measuring GR activityis based on the absorbance decrease caused by theconsumption of NADPH during the conversion ofGSSG to GSH (λ = 340 nm, ε = −9.6 mM−1 cm−1)(Meister 1989).
GSSG + NADPH → 2GSH + NADP+
Reagents and Solutions
• 100 mM KPi buffer, pH 7.0 (adjust pH to 7.0 withconcentrated KOH).
• 100 mM EDTA stock solution.• 10 mM GSSG working solution (freshly prepared).
Dissolve 0.0061 g of GSSG (MW 612.63) in 1 mLof distilled water.
• NADPH (1 mg mL−1) working solution (freshlyprepared). Dissolve 5 mg of NADPH in 5 mL ofdistilled water.
Procedure
Spectrophotometric analyses are carried out at λ =340 nm (light path 1 cm) at constant temperature(18 ± 1◦C for temperate species).
Add to a plastic cuvette (final volume 1 mL):
• 750 μL of KPi buffer• 10 μL of EDTA stock solution• 100 μL of GSSG working solution
INSTRUMENT AUTOZERO
• 100 μL of NADPH working solution
Read the absorbance, which should be approximately0.6
• 40 μL of sample.
Mix vigorously after the addition of sample. Read thedecrease in absorbance for at least 1 min. Reactionsshould be linear, otherwise a different dilution or sam-ple volume should be used. Each sample is measuredtwice. Maintain a final volume of 1 mL even if differentaliquots of samples are needed. Compensate by chang-ing the volume of working buffer. Appropriate dilutionsfor measuring GR in cytosolic fractions of marine organ-isms usually vary, e.g. for samples homogenized 1:5,ranging between 1:10 and 1:100 in digestive gland ofinvertebrates and in fish liver. Proper dilutions shouldalways be tested prior to new batch measurements.
Calculations
Results are usually expressed in nmol min−1 mg−1
protein or g of tissue. Normalization to the tissue weightoften precludes comparisons between different speciesor organs with different relative water content. Whennormalizing to mg of protein, comparability of valuesmay be compromised by different tissue-specific proteincontents.
When the enzymatic activity is normalized to proteincontent:
GR activity (nmol min−1mg−1proteins)
= (�Abs/−6.22) × (sample dilution)
× 1000/proteins
where ‘‘�Abs’’ is the variation of absorbance perminute, ‘‘−6.22’’ is the extinction coefficient (mM−1
cm−1), ‘‘sample dilution’’ is the dilution factor (if any)applied to the cytosolic fractions before the assay, and‘‘proteins’’ is the protein content in cytosolic fractionsexpressed as mg mL−1.
DETERMINATION OF GLYOXALASEI (EC 4.4.1.5) AND II (EC3.1.2.6)ACTIVITIES
The glyoxalase system catalyses the detoxification ofreactive α-ketoaldehydes formed in cellular oxidativeprocesses. Using GSH as cofactor, GI forms an inter-mediate thiolester, which is subsequently hydrolysedby GII to the corresponding D-hydroxy acid, with GSHregeneration.
R–CO–CHO + GSH → R–CHOH–COSG (GI)
R–CHOH–COSG → GSH + R–CHOH–COOH (GII)
Increased activities of glyoxalase enzymes havebeen observed in marine organisms exposed tochemical pollutants, probably reflecting an efficientdetoxification mechanism against enhanced levelsof α-ketoaldehydes, extremely toxic and reactivecompounds formed in cellular oxidative processes(Mannervik et al. 1989; Viarengo 1989; Viarengoet al. 1990; Regoli 1992; Regoli and Principato 1995).
The spectrophotometric assay for GI activity is basedon the formation of S-D-lactoyl-glutathione from thehemimercaptal adduct of methylglyoxal (MG) and GSH(Principato et al. 1983). GII activity is followed by thereaction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)
376 F. Regoli, R. Bocchetti, and D. Wilhelm Filho
H
O
OH
O
OH
HO
O
Methylglyoxal
D-Lactate S-Lactoyl-glutathione
Glyoxylase I
Glyoxylase II
O
GS
GS
OH
Hemimercaptal
O
GSH
GSH +
Nonenzymatic
+
Fig. 27.3 Glyoxalase I generatesan intermediate thioester andS-Lactoyl-glutathione from theadduct of methylglyoxal and GSH;glyoxalase II catalyzes theformation of D-lactate with recycleof GSH.
with GSH formed from S-D-lactoylglutathione (Princi-pato et al. 1987) (Fig. 27.3).
Reagents and Solutions
• 100 mM KPi buffer, pH 6.8 (adjust pH with con-centrated KOH).
• 100 mM GSH working solution (freshly prepared).Dissolve 0.0307 g of GSH (MW 307.32) in 1 mL ofdistilled water.
• Methylglyoxal 2% (v/v) working solution. Dilute50 μL of a stock methylglyoxal solution (40%) into950 μL of distilled water.
• 100 mM 3-(N-morpholino)propanesulfonic acid(MOPS), pH 7.2. Dissolve 2.09 g of MOPS in100 mL of distilled water.
• 53 mM S-D- lactoylglutathione. Dissolve 0.02 g in1 mL of distilled water.
• 20 mM DTNB stock solution (freshly prepared).Dissolve 0.07926 g of DTNB in 10 mL of meth-anol. This solution is stable for 3 months in thedark at 4◦C.
Procedure
Spectrophotometric analyses are carried out at λ =240 nm and λ = 412 nm (light path 1 cm), for GI andGII respectively, at constant temperature (18 ± 1◦C).
For GI activity, add to a quartz cuvette (final volume1 mL):
• 840 μL of 100 mM KPi buffer• 100 μL of methylglyoxal working solution• 10 μL of GSH working solution• 50 μL of blank (KPi buffer) or sample.
For GII activity, add to a plastic cuvette (final volume1 mL):
• 930 μL of MOPS buffer• 10 μL of S-D- lactoylglutathione working solution*
• 10 μL of DTNB stock solution• 50 μL of blank (KPi buffer) or sample.
∗After adding S-D-lactoylglutathione a spontaneous hydroly-sis is observed; wait for the stabilization of absorbance beforeadding blank or sample.
In both GI and GII assays, mix vigorously after theaddition of blank/samples. Register the increase inthe absorbance for at least 1 min. Reactions shouldbe linear, otherwise different dilutions or samplevolumes should be used in the assay. Each sample ismeasured twice. Maintain a final volume of 1 mL evenif different aliquots of sample are needed. Compensateby changing the volume of KPi buffer for GI orMOPS for GII. Appropriate dilutions for measuringglyoxalase activities in cytosolic fractions of marineorganisms can vary, e.g. for samples homogenized 1:5,ranging between 1:10 and 1:50 in digestive gland ofinvertebrates and between 1:100 and 1:500 in fishliver (Regoli and Principato 1995; Romero-Ruiz et al.2003). Proper dilutions should always be tested priorto new batch measurements.
Spectrophotometric Assays of Antioxidants 377
Calculations
Results are usually expressed in μmol min−1 mg−1
protein or g of tissue. Normalization to tissue weightoften precludes comparisons between different speciesor organs with different relative water content. Whennormalizing to mg of protein, comparability of valuesmay be compromised by different tissue-specific proteincontents.
When the enzymatic activity is normalized to proteincontent:
GI activity(μmol min−1mg−1proteins)
= (�Abssample/3.37) × (sample dilution)/proteins
GII activity(μmol min−1mg−1proteins)
= (�Abssample/13.6) × (sample dilution)/proteins
where ‘‘�Abssample’’ is the variation of absorbanceper minute, ‘‘3.37’’ and ‘‘13.6’’ are the extinctioncoefficients (mM−1 cm−1) for GI and GII respectively,‘‘sample dilution’’ is the dilution factor (if any) applied tothe cytosolic fractions before the assay, and ‘‘proteins’’is the protein content in cytosolic fractions expressedas mg mL−1 (e.g. not diluted samples).
SPECTROPHOTOMETRICDETERMINATION OF TOTALGLUTATHIONE
GSH is the most abundant cellular thiol involved inmetabolic and transport processes, and in cell protec-tion against the toxic effects of a variety of endogenousand exogenous compounds, including trace metals and
ROS (Meister and Anderson 1983). GSH plays multipleprotective roles against oxidative stress, acting as adirect scavenger of ROS, and also as cofactor of severalantioxidant enzymes. GSSG is reconverted to GSH byGR, but when oxidative processes exceed the reducingcapacity of GR, the excess of GSSG is excreted resultingin a net loss of total GSH from the tissue (Meister 1989).
Total GSH concentration (GSH + 2GSSG) is mea-sured in a colorimetric reaction using DTNB, alsoknown as Ellman’s Reagent, which reacts with thi-olic compounds. 2 GSH react with DTNB to generateGSSG and 2-nitro-5-thiobenzoic acid (Akerboom andSies 1981). GSSG in the sample is reconverted byGR to GSH, which undergoes another reaction, witha continuous formation of 2-nitro-5-thiobenzoic acid,proportional to total GSH content. The formation of2-nitro-5-thiobenzoic acid is monitored at λ = 412 nm(Fig. 27.4).
Tissue Homogenates
To eliminate the interference of thiolic groups of pro-teins, tissues are homogenized in 5% sulphosalicylicacid with 4 mM EDTA. The homogenization bufferis prepared by dissolving 5 g of sulfosulfosalicylicacid in 100 mL of distilled water and adding 4 mL of100 mM EDTA. This solution is stable for 1 monthat room temperature. Samples are homogenized (1:5w : v) at 4◦C using a hand-held glass potter with ateflon or glass pestle, maintained on ice for 45 minfor de-proteinization and centrifuged at 37,000 g for15 min at 4◦C. Supernatants are collected, assayed orsubdivided into aliquots (100–200 μL) and stored at
Fig. 27.4 Glutathione (GSH)reacts with DTNB to generate2-Nitro-5-thiobenzoic acid andGSSG that is reconverted to GSH byglutathione reductase.
HOOC
HOOC
2
COOH2GSH
GSSG
2-Nitro-5-thiobenzoic acid
Glutathione reductase
NO2
DTNB
SS
S−
O2N
O2N
378 F. Regoli, R. Bocchetti, and D. Wilhelm Filho
−80◦C. Total GSH content can be normalized to tissueweight or protein content (see below). In the lattercase, pellets are re-suspended in 5 mL of 1 M NaOH forprotein measurement.
Reagents and solutions
• 100 mM KPi buffer, pH 7.0 (adjust pH with con-centrated KOH).
• 100 mM EDTA stock solution.• 20 mM DTNB stock solution. Dissolve 0.07927 g
of DTNB in 10 mL of methanol. This solution isstable for 3 months in the dark at 4◦C.
• NADPH (4 mg mL−1) working solution (freshlyprepared). Dissolve 4 mg of NADPH in 1 mL ofdistilled water.
• GR (100 U mL−1) working solution (prepare imme-diately before use). Depending on the product,dilute GR to 100 U mL−1 in cold distilled water.
• Working buffer. Add 1 mL of 100 mM EDTA to100 mL of KPi buffer.
Standards
Both GSH or GSSG can be used as standards.
1. When using GSH standards, prepare a fresh stocksolution of 100 mM GSH by dissolving 0.0307 gof GSH in 1 mL of distilled water. Obtain at leastthree standards by diluting the stock solution:10 μM GSH, 20 μM GSH and 30 μM GSH. Thesestandards are diluted 10-fold in the assay, giving1 μM, 2 μM, 3 μM final GSH concentrations inthe calibration curve.
2. When using GSSG standards, prepare a freshstock solution of 1 mM GSSG by dissolving0.00325 g of GSSG in 5 mL of distilled water.Obtain at least three standards by diluting thestock solution: 5 μM GSSG, 10 μM GSSG and15 μM GSSG. These standards will be diluted10-fold in the assay, giving 0.5 μM, 1 μM,1.5 μM final GSSG concentrations.
Procedure
Spectrophotometric analyses are carried out at λ =412 nm (light path 1 cm) and at constant temperature
(18 ± 1◦C for temperate species). Blank and stan-dard reactions generate a calibration curve to relateabsorbance to glutathione concentration, which willbe used to quantify GSH content in the samples.
For blank and standard reactions, add to a plastic orglass cuvette (final volume 1 mL):
• 835 μL of working buffer• 100 μL of blank (KPi buffer) or standards• 5 μL of DTNB• 50 μL of NADPH• 10 μL of GR.
For sample reactions, add to a plastic cuvette (finalvolume 1 mL):
• 835 μL of working buffer• 100 μL of sample• 5 μL of DTNB• 50 μL of NADPH• 10 μL of GR.
Mix vigorously after the addition of GR. Read increasein absorbance for at least 1 min. Reactions should belinear, otherwise a different dilution or sample volumeshould be used. Maintain a final volume of 1 mL, evenif different aliquots of samples are needed. Compensateby changing the volume of working buffer. Appro-priate dilutions for measuring GSH content in tissuesof marine organisms usually vary, e.g. for sampleshomogenized 1:5, ranging between 1:10 and 1:100 indigestive gland of invertebrates and in fish liver. Properdilutions should always be tested.
Calculations
The �Abs of blank and standards are used to obtainthe linear calibration curve between absorbance andGSH or GSSG concentration (y = ax + b). This equationallows calculation of the GSH concentration in samples(in μM = μmol L−1). Results are expressed in GSHequivalents and normalized to tissue weight (or proteincontent).
To normalize to tissue weight when GSH has beenused as standard:
GSH + 2GSSG (μmol g−1tissue)
= (concentration/1000) × (sample dilution)
× (w/v ratio) × 1
Spectrophotometric Assays of Antioxidants 379
To normalize GSH concentration to tissue weight whenGSSG has been used as standard:
where, ‘‘concentration’’ is glutathione concentration(as GSH or GSSG depending on the standard used)derived from the calibration curve, ‘‘sample dilution’’ isthe dilution factor (if any) applied to homogenizedfractions before the assay, and ‘‘w/v ratio’’ is thehomogenization ratio.
To normalize glutathione concentration to proteincontent:
GSH + 2GSSG(nmol mg−1protein)
= (GSH + 2GSSG)/protein
where ‘‘GSH + 2GSSG’’ is GSH concentration previ-ously calculated and expressed as μmol g−1 of tissue,and ‘‘protein’’ is the protein content measured in thepellet obtained after centrifugation of homogenatesand expressed as mg g−1 of tissue.
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