SOD Assay Paoletti Et Al

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ANALYTICAL BIOCHtMISTR\r 153, 536-541 (1986)

A Sensitive Spectrophotometric Method for the Determination ofSuperoxide Dismutase Activity in Tissue Extracts

FRANCEXO PAOLETTI, DONATELLA ALDINUC CI, ALESSANDRA MOCALI,AND ANNA CAPARRINI

ReceivedOctober 3. 1985

Superoxide dismutase (EC I. IS. I. I) has been assayed by a spectrophotometric method basedon the inhi bitio n of a superoxide-driven N.ADH oxidation. The assay consists ofa purely chemical

reaction sequence which involves EDTA . Mn(II). mercaptoe thanol. and molecular oxygen. re-quiring neither auxiliary enz ym es nor soph isticated equipm ent. The method is very flexible andrapid and is applic able with high sensitivity to the determ ination of both pure and crude superoxidedismutase preparations. The decrease of the rate of NADH oxidation is a function of enzyme

concentration. and saturation levels are attainab le. Fifty percent inhib ition . corresponding to oneunit ofthe enzyme, is produced by approximately I5 ng of pure superoxide dismutase. Experiments

on rat liver cytosol have shown the specificity of the method for superoxide dismutase. Moreover.comm on cellular com ponents do not interfere with the measuremen t. except for hem oglob in

when present at relatively high concentrations. The assay is performed at physiologica l pH andis unaffected by catalase. 15 1986 Academtc Press. Inc

KE Y WORDS: superoxide dismutase; spectrophotometric determ ination: chemical assay: su-peroxide: NADH oxidation: metal complex.

Superoxide dismutase (SOD) (EC 1.15.1.1)is a family of metalloenzymes which is knownto accelerate spontaneous dismutation of thesuperoxide radical to hydrogen peroxide andmolecular oxygen (1). SOD is widely distrib-uted among aerobically living organisms andhas been inferred to play an important role incontrolling superoxide levels in cellular com-

partments (2,3).The direct measurement of SOD activity (4-

7) is possible but its application is hamperedby the requirement of special apparatus notcommonly available in the typical laboratory.The other methods employed for enzyme de-termination are indirect and rely on the abilityof SOD to inhibit a superoxide-driven reac-tion. The extent to which SOD reduces therate of that reaction is taken as a measure ofenzymic activity. Either enzymatic or non-enzymatic systems are used for the generation

Abbreviations used: SOD. superoxide dismutdse: Tea-Dea. triethanolamine-diethanolamine; NBT. nitro bluetetrazolium.

of superoxide (see Refs. (8-9) for review): de-tection is then accomplished by luminometric( 10). polarographic (1 l), or calorimetric( 1,12,13) techniques, depending on the differ-ent approaches and experimental require-ments.

Notwithstanding the large number ofmethods available, the need sti ll exists to in-

crease the specificity. accuracy, and simplicityof the assay. In this paper we describe a spec-trophotometric method for SOD determina-tion based on a purely chemical reaction se-quence which eventually leads to NADH ox-idation. This procedure, involving stable andinexpensive reagents. allows a rapid and highlysensitive measurement of SOD activity in pureand crude enzyme preparations, with negli-gible interference by cellular components.

MATERIALS AND METHODS

Chemicals. Reduced adenine dinucleotide(P-NADH, disodium salt) was obtained from

0003-2697/86 3.00 536Copyrigh t 2 198 6 by Acade mic Press. IncAll rights of reproductmn in any form reserved.


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SUPEROXIDE DISMUTASE DETERMINATION 537

Boehringer-Mannheim (West Germany),while MnClz . 2H20, ethylenedinitrilotetraac-etic acid (EDTA), and 2-mercaptoethanol weresupplied by Merck. Darmstad (West Ger-many). Pure SOD preparations were obtainedfrom Diagnostic Data Inc. (beef erythrocytes,3300 U/mg protein) and from Sigma ChemicalCompany (human erythrocytes, ca. 2500 U/mg protein). Catalase (beef liver. 350 mg/mlammonium sulfate suspension) was providedby Boehringer-Mannheim. Al l other chemicalswere analytical grade and used without further

purification.Eqrr;pmenr. Assays were performed with a

Gilford spectrophotometer (Model 350) con-nected to a recorder.

Rcqynts l~inrlsolutions. All solutions weremade up with deionized or well-aerated dis-tilled water. according to the following scheme.

( 1) Triethanolamine-diethanolamine(Tea-Dea) buffer. 100 mM each, pH 7.4. Di-lute 14.9 g Tea. 10.5 g Dea. and ca. 13.8 mlof 37 , HCI up to 1 liter with water, takingcare to maintain the pH around 7.4-7.5.

(3) NADH, 7.5 mM. For 100 assays, dis-solve 20 mg of reduced nucleotide (disodiumsalt) in 4 ml of water.

(3) EDTA/MnCI,, 100 mM/50 mM. Makea stock solution of EDTA. 0.3 M (i.e., dissolve5.85 g of EDTA-acid in a final volume of 100

ml. adjusting the pH to around 7 with molarNaOH solution) and of MnCL2. 0.1 M (by dis-solving 1.62 g of MnC12. 7Hz0 in 100 ml wa-ter). The mixture of equal volumes of thesetwo stock solutions yields our third reagent(EDTA/MnCI:).

(4) Mercaptoethanol, 10 mrvr. Dilute 0.05ml of concentrated thiol. 14.2 M, up to 7 I mlwith water.

The NADH solution is stable for at least 3days in the refrigerator. For longer storage.keep it at ~20C. The solutions of EDTA.MnCl?, and mercaptoethanol are quite stable,even at room temperature. for months. Re-agent 3, once made up. can be used over a 2-week period (see further comments in l.he Re-sults section).

Preparation qf rat liver c~msoi. For themeasurement of SOD in rat liver, the tissueextract is first prepared by homogenizing theliver in 3 vol of Tea-Dea buffer 25 mM, pH7.4, and then cleared by centrifugation at30,000 rpm for 60 min at 4C. The superna-tant is dialyzed against cold homogenizationbuffer and then employed for enzymatic as-says. Protein determination was carried outby the L.owry method ( 14) on samples dialyzedagainst 0.9 NaCl buffered at pH 7.5-8 withmolar NaHCOI solution.

RESULTS

Description oJt/re method. The principle ofthis method is based on the oxidation ofNADH, mediated by superoxide radical, in apurely chemical system recently developed inour laboratory. Coenzyme oxidation occurs inthe presence of suitable concentrations ofEDTA. Mr? and mercaptoethanol (see be-low) through a free-radical chain of reactionsinvolving thiol oxidation and univalent 02 re-duction. A detailed explanation of the reactionmechanism is beyond the scope of the presentpaper and wil l be reported elsewhere (manu-script submitted). The addition of SOD to thereaction mixture causes a proportionate in-hibition, of the rate of NADH oxidation, thus

confirming the involvement of superoxide inthe process and providing the basis for SODactivity determination.

To perform the assay sequentially add thefollowing (see Materials and Methods) to acuvette: Tea-Dea buffer. 0.800 ml: NADHsolution, 0.040 ml; EDTA/MnC12 solution,0.035 ml; sample (or water or buffer) 0.100ml: mix thoroughly and read against air at 340nm for a stable baseline: then add mercapto-ethanol solution. 0.100 ml.

Mix and monitor the decrease in absorbanceusing 0.5-I full scale deflection. The final vol-ume in the cuvette is 1.065 ml and the lightpath is IO mm.

A typical analysis of SOD activity is shownin Fig. 1 where the kinet ics of NADH oxida-tion in the absence (control) and in the pres-


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538 PAOLETTI ET AL

, SgAq

1.4 -.-*--

i

-----__ ---_- - -SOD 3

- -.. _

\

11__

1

__

E\ S O D 2

c

z 1.2

I

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' \0 \ \

z\ \

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L \CONIROI

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0 8 16 24

INCUBATION (mtnl

FIG. I. Effect of superoxide dismutase on the rate ofNADH oxidation. Four assays are carried out simulta-

neously in the absence (control) and in the presence ofSOD (sample 1. IO ng; sample 2, 80 ng: sample 3, 380 ngof pure enzyme from bovine erythrocytes. Diagnostic Data

Inc.). Assay mixtures are prepared as described in the text

and decreases in absorbance. at 340 nm. are recorded forabout 15 min after mercaptoethanol addition. The rate of

NADH oxidation in the control is ca. 5 nmol per min. atroom temperature.

ence of superoxide dismutase (SODrm3) are

compared by simultaneous assay using a mul-ticell holder.The reaction is started by mercaptoethanol

and changes in absorbances are recorded forabout 15 min. Rates of NADH oxidation areinitially low, then increase progressively (usu-ally 2-4 min after mercaptoethanol addition)to yield good linear kinetics (5- 10 min) whichare used for calculations. Under our conditionsAi1340 over an S-min interval is 0.250 for the

control, while the presence of 10, 80. and 380ng of SOD in the assay mixture. yields AAvalues of 0.150.0.038, and 0.008, respectively.For calculations the maximal rates obtainedare expressed as a percentage of the control(ordinate) and plotted against a suitable ref-erence (abscissa). One unit of the enzyme isthe amount of SOD capable of inhibiting by

50 the rate of NADH oxidation observed inthe control.

Culihration cme \tii/l pure SOD. The de-termination of SOD activi ty in pure enzymepreparations from bovine erythrocytes i sshown in Fig. 2. Relative rates of NADH ox-idation are reported as a function of theamount of enzyme in the assay mixture. Thecurve thus obtained shows that inhibition isnot directly proportionate to SOD concentra-tion, but rather follows an exponential-likefunction. Almost complete saturation levels

(99 inhibition) are obtained with 400 ng ofthe enzyme, while the same amount of heatedSOD fails to inhibit the reaction. One unit ofthe enzyme corresponds to ca. 15 ng of puresuperoxide dismutase from beef erythrocytes.

Least-square linear regression analysis wasused to obtain a best-fitting curve over therange 4-40 ng, by transforming SOD valuesinto logarithms. The equation is ?; = 1 16.638- 55.619 s; the correlation coefficient Y= -0.9925; n = 22.

Deterrninution ofSOD in liver euxtructs.Totest the applicability of this method to SODdetermination in tissue extracts, experimentswere carried out using rat liver cytosol as thesample (Fig. 3). The assay conditions are thesame as described for pure SOD and the per-centage of NADH oxidation is reported as afunction of the total proteins in the extract.

The curve obtained is essentially identical tothat shown in Fig. 2 for the purified enzymeand in this case too, the boiled sample fails toinhibit NADH oxidation at any rate. Fiftypercent inhibition is produced by approxi-mately 10 pg of liver extract which means anaverage of 100 units of SOD per mg proteinof cell cytosol. while the saturation level isreached with ca. 180-200 yg proteir.

Curve fitting to experimental data is com-

parable to that described in the comments toFig. 2. The linear regression equation, over arange of 4-40 pg protein, is c = 115.366- 67.687 s; the correlation coefficient t= -0.9869; n = 28.

Precautions and optimal conditiom ,fi~rrnemuwnmt. EDTA or other chelators forMn2+ (not EGTA) may alter the optimum


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SUPEROXIDE DISMUTASE DETERMINATION 539

--t-l--c-i-+-

20 40 400

SUPEROXIDEDISMlJiASE lngl

FIG.2. Titration curve with pure superoxide dismutase. Increasing amounts of SOD (O-400 ng) frombovine erythrocytes (Diagnostic Data Inc.) are added to incubatio n mixtures and assayed for activity aspreviously reported. The rate of NADH oxidation (g-min linear kinetic) is expressed as a percentage of the

control. which is always run in each set of assays. Val ues (0: n = 27) refer to separate determinatio ns carned

out individ ually by three of us using different stock solutions of the enzyme. Samp les c ontaining inactiveSOD (0). heated at 100C for 2 min. are shown for comparison.

EDTA/Mn+ ratio and affect the rate of re-action. Alternatively, excess of Mn ions inthe sample could slow down the rate of NADHoxidation. Other divalent cations of the secondtransition series, at concentrations comparableto that of Mn. do not start the reaction, butmay compete for the chelator. In addition,when the sample contains free thiols, i.e.,mercaptoethanol, cysteine and reduced glu-tathione. faster reaction rates, as compared tothe control. are observed. To avoid all inter-ference by compounds mentioned above.samples must be dialyzed against suitable me-

dia before the analysis . However, owing tohandle a large number of samples. dialysiscould be conveniently replaced by a rapid de-salting through a small Sephadex G-25 (coarse)column.

For precise calculations, NADH-oxidaseactivity, if present in the sample, should beevaluated before mercaptoethanol additionand subtracted from the final rate. Moreover,because of the high sensi tivi ty of the method.samples are usually diluted by such a largefactor that NADH-oxidase or any other inter-fering activity is pract ically undetectable. In

0 L+- ++- t~-+--+-~.+-..++ --+ -0 8 I6 24 32 184

LIVER CYTOSOL ,,,g pratml

FIG.3. Titration of SOD i n rat liver cytosol. The liver extract 1see Materials and Methods) is dilut ed with100 mM Tea-Dea buffer. pH 7.4. to concentrations suitable for the assay. Measurements are carried out asdescribed in the text. Protein content of the sample is withi n a range of O-400 pg as determin ed by theLowry method . Closed dots (0) represent the average o f four separate de termination s and bars (=) indica tethe range of experim ental values. Open squares (0) refer to assays with boil ed samples.


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540 PAOLETTI tT AL.

addition it is worth mentioning that NADPHreacts as well as NADH in the system (datanot shown) without serving as a substrate forNADH-oxidase. This fact confirms the flexi-bility of our method and may turn out veryuseful when assaying for SOD in fractionscontaining high levels of NADH-oxidase.

Changes of temperature, pH and oxygentension in the system may influence the ab-solute rate of NADH oxidation, but are with-out effect on the relative degree of inhibitionobserved. Each set of assays must refer to i ts

own control and best measurements are ob-tained when the values of .&4+,,, of the control.over an S-min interval, are within the rangeof 0.150-0.400. Reactivity of reagent 3 is in-creased by storage: therefore aged solutionsimmediately yield maximal rates of NADHoxidation without the initial delay shown byfresh-made preparation of the complex.

Catalase does not interfere with the assay,while hydrogen peroxide inhibits it at a mil-limolar concentration level.

DISCUSSION

Any reaction inhibitable by superoxide dis-mutase could potentially provide the basis foran indirect assay of the enzyme and, accordingto that principle, several methods have beendeveloped over the years. However. as alsopointed out by Oberley (15). only a few pro-

cedures permit the sensitive and reliable de-termination of enzymatic activity in tissue ex-tracts with low SOD levels.

Our chemical assay seems particularly suit-able to that purpose since it allows the mea-surement of minute amounts of SOD, such as2 ng, which are far below the detection limitof most published methods. In addition. wehave found that fifty percent inhibition, i.e.,one unit of the enzyme, is produced by 15 ng

of pure protein, while values of catalyt ic ac-tivity. as determined by the xanthine-oxidase/cytochrome c ( 1 , NADH-diaphorase/hy-droxylamine ( 13) and xanthine-oxidase/nitroblue tetrazolium (NBT) ( 12) systems, are ca.200,626, and 630 ng, respectively. A substan-tial improvement of the NBT assay has beenobtained by Buettner (16), whose procedure

yields exactly the same sensitivity reportedhere. However. with his method, saturationlevels are not attainable and different valuesfor half-maximal inhibition are obtained forpure and crude SOD preparations. These in-conveniences are frequently observed in assaysinvolving the reduction of NBT. cytochrome(. or other suitable detector and ascribed tothe action of aspecific electron donors onchromogenic substrates. Our method. on thecontrary, relies on the oxidation of the detector(NADH in this case) and therefore wil l not be

affected by the presence of reductants whichare known to occur in tissue extracts ( 12). Thislack of interference is clearly shown by thefact that calibration curves for pure SOD andrat liver cytosol are almost identical and sat-uration levels (99 ) are attainable in bothcases. The latter result implies that in our sys-tem the same value for catalytic activity is ob-tained by using either 50 or half-maximalinhibition for calculation. This avoids the ne-cessity of running a full calibration curve eachtime and is of valuable practical importance,especially when dealing with samples havinglow SOD levels. From our data. a specific ac-tivity of ca. 66.000 unit/mg protein and ca.100 unit/mg protein can be calculated for SODin pure beef erythrocytes preparations (Diag-nostic Data Inc.: see Materials and Methods)and rat liver cytosol, respectively.

In addition to the experiments with rat livercytosol, the present method has been appliedsuccessfully to the measurement of SOD in avariety of other ce ll extracts and body f luids(data not shown). So far, the only major in-convenience encountered comes from he-moglobin; thus, for reliable determination ofSOD in hemolysates. this molecule must beremoved from the sample before assaying.

An important problem in SOD analysis is

the discrimination between the cuprozinc- andmanganese-form of the enzyme. Cyanide. atconcentrations used for inhibiting the cupro-zinc enzyme, does not interfere with our assayand manganese-SOD (Mn-SOD) can be easilydetermined by differential measurement. Inaddition, this method has proved ofgreat valuein determining traces of Mn-SOD separated


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SUPERO XIDE DISMUT ASE DETERMINATION 541

from rat liver cytosol by means of gel filtration(data not shown). In this regard it is worthrecalling that our assay is carried out at pH7.4 which favors the detection of Mn-SOD. Infact, physiological pH is the most suitable foroptimal activity of Mn-SOD, which is not re-liably assayed at elevated pH values as requiredby other sensitive methods ( 17,18).

On the whole, the present procedure forSOD determination involves stable and in-expensive reagents and consists of a singlespectrophotometric step, easi ly performed on

a time scale of minutes. It appears particularlysuitable for application in the field of bio-chemistry. plant physiology, and clinicalchemistry.

ACKNOWLEDGMENTS

We would like to thank Professor A. Fonnesu. &airm anof the Institute. and Dr. V. Bodd i for his continuous sug-

gestions and statistical analyses. This research was sup-

ported by a grant from the Minister0 dell a Pubbl ica Istru-zione (6Og).

REFERENCES

I. McCord. J. M., and Fridovich, 1. ( 1969) J. Vio l C11crn.

244,6049-6055.

2. McCord. J. M.. Keele. B. B.. Jr.. and Fridovich. I.

(1971) Proi,. Nar. .-I(& SC,; C51 68, 1024-1027.3. Fridovich. I. (1978) Sc~rc~nc~c 201, 875-8 80.

4. Ball ou. D., Palm er. G.. and Massey. V. (1969)Biochrm. Bioqlzm R~.L Cornrnm 36, 898-904.

5. Rotil io. G.. Bray, R. C., and Fielden. E. M. (1972)

Birxhirn. BiuphJ,.r. .1ctu 268. 605-609.6. Klug. D.. Raban i, J.. and Fridovich, 1. (1972) J Bid

C7rrm. 247, 4839-4442.

7. Rigo. A.. Viglino. P.. and Rotilio. G. (1975) A&

Biodxw. 68, 1-X.8. McCord. J. M.. Crapo. J. D., and Fridovich. I. (1977)

in Superoxide and Superoxide Dismutases (Mi-chelson A. M.. McCord. J. M.. and Fridovich, I..eds.), pp. I l- 17. Academic Press. N ew York.

9. Flohe. L.. and Gtting, F. ( 1984) in Methods in En-zymology (Colowick. S. P.. and Kapla n. N. 0..

eds.). Vol. 105, pp. 93-104. Academic Press. NewYork.

IO. Bensinge r. R. E., and Johnson, C. M. ( I98 I ) .I&Biodwnz. 116. I4?- 145.

I I. Tyler. D. D. (1975) B~odw~~~. J.147, 493-504.

12. Beucham p. C.. and Fridovich. I. ( 197 I ) .,lr~ul.B,nchrtn. 44, 276-187.

13. Elstner, E. F.. Young man. R. J.. and OBwal d, W.

( 1383) irz Methods of Enzymatic Analysis (Berg-mmeyer, H. U.. ed.). Vol. III. pp. 293-302, Verlag

Chemie, Weinheim.14. Lowry. 0. H.. Rosebrough. H. J.. Farr. A. L., and

Rand all. R. J. ( 195 I ) J. Bl ol.Ch~7 121, 404-420.15. Oberley. L. W.. and Spitz. D. R. ( 1984) in Methods

in Enzymology (Colowick. S. P.. and Kaplan.

N. 0.. eds.). Vol. 1 05. pp. 457-464. AcademicPress. New York.

16. Buettner. G. R.. Oberley, L. W.. and Leuthauser.

S. W. H. C. ( 1978) Phr~toc~hcr,?. Photo hid 28, 693-695.

17. Marklund, S. (1976) J BirdChn. 251, 7504-7507.

18. Misra. H. P.. and Fridovich, I. (1972) d BidChc177247, 3170-3175.

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SOD Assay Paoletti Et Al