ANALYTICAL

BIOCHtMISTR\r

153, 536-541

(1986)

A Sensitive Spectrophotometric Superoxide Dismutase

FRANCEXO PAOLETTI, DONATELLA AND ANNA

Method for the Determination Activity in Tissue Extracts

ALDINUCCI, CAPARRINI ALESSANDRA MOCALI,

of

Received October

3. 1985

Superoxide dismutase (EC I. IS. I. I) has been assayed by a spectrophotometric method based
on the inhibition of a superoxide-driven N.ADH oxidation. The assay consists ofa purely chemical reaction sequence which involves EDTA. Mn(II). mercaptoethanol. and molecular oxygen. re-

quiring neither auxiliary enzymes nor sophisticated equipment. The method is very flexible and
rapid and is applicable with high sensitivity to the determination of both pure and crude superoxide dismutase preparations. The decrease of the rate of NADH oxidation is a function of enzyme concentration. and saturation levels are attainable. Fifty percent inhibition. corresponding to one unit 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. common cellular components do not interfere with the measurement. except for hemoglobin when present at relatively high concentrations. The assay is performed at physiological pH and is unaffected by catalase. 15 1986 Academtc Press. Inc KEY WORDS: superoxide dismutase; spectrophotometric determination: chemical assay: superoxide: NADH oxidation: metal complex.

Superoxide dismutase (SOD) (EC 1.15.1.1) is a family of metalloenzymes which is known to accelerate spontaneous dismutation of the superoxide radical to hydrogen peroxide and molecular oxygen (1). SOD is widely distributed among aerobically living organisms and has been inferred to play an important role in controlling superoxide levels in cellular compartments (2,3). The direct measurement of SOD activity (47) is possible but its application is hampered by the requirement of special apparatus not commonly available in the typical laboratory. The other methods employed for enzyme determination are indirect and rely on the ability of SOD to inhibit a superoxide-driven reaction. The extent to which SOD reduces the rate of that reaction is taken as a measure of enzymic activity. Either enzymatic or nonenzymatic systems are used for the generation
Abbreviations used: SOD. superoxide Dea. triethanolamine-diethanolamine; tetrazolium. 0003-2697/86 $3.00 dismutdse: TeaNBT. nitro blue

of superoxide (see Refs. (8-9) for review): detection is then accomplished by luminometric ( 10). polarographic (1 l), or calorimetric ( 1,12,13) techniques, depending on the different approaches and experimental requirements. Notwithstanding the large number of methods available, the need still exists to increase the specificity. accuracy, and simplicity of the assay. In this paper we describe a spectrophotometric method for SOD determination based on a purely chemical reaction sequence which eventually leads to NADH oxidation. This procedure, involving stable and inexpensive reagents. allows a rapid and highly sensitive measurement of SOD activity in pure and crude enzyme preparations, with negligible interference by cellular components.
MATERIALS AND METHODS

Chemicals. Reduced adenine dinucleotide

(P-NADH,
536

disodium salt) was obtained from

Copyright 2 1986 by Academic Press. Inc All rights of reproductmn in any form reserved.

SUPEROXIDE

DISMUTASE

DETERMINATION

537

Boehringer-Mannheim (West Germany), while MnClz . 2H20, ethylenedinitrilotetraacetic acid (EDTA), and 2-mercaptoethanol were supplied by Merck. Darmstad (West Germany). Pure SOD preparations were obtained from Diagnostic Data Inc. (beef erythrocytes, 3300 U/mg protein) and from Sigma Chemical Company (human erythrocytes, ca. 2500 U/ mg protein). Catalase (beef liver. 350 mg/ml ammonium sulfate suspension) was provided by Boehringer-Mannheim. All other chemicals were analytical grade and used without further purification. Eqrr;pmenr. Assays were performed with a Gilford spectrophotometer (Model 350) connected to a recorder. Rcqynts l~inrlsolutions. All solutions were made up with deionized or well-aerated distilled water. according to the following scheme. ( 1) Triethanolamine-diethanolamine (Tea-Dea) buffer. 100 mM each, pH 7.4. Dilute 14.9 g Tea. 10.5 g Dea. and ca. 13.8 ml of 37%, HCI up to 1 liter with water, taking care to maintain the pH around 7.4-7.5. (3) NADH, 7.5 mM. For 100 assays, dissolve 20 mg of reduced nucleotide (disodium salt) in 4 ml of water. (3) EDTA/MnCI,, 100 mM/50 mM. Make a stock solution of EDTA. 0.3 M (i.e., dissolve 5.85 g of EDTA-acid in a final volume of 100 ml. adjusting the pH to around 7 with molar NaOH solution) and of MnCL2. 0.1 M (by dissolving 1.62 g of MnC12. 7Hz0 in 100 ml water). The mixture of equal volumes of these two stock solutions yields our third reagent (EDTA/MnCI:). (4) Mercaptoethanol, 10 mrvr. Dilute 0.05 ml of concentrated thiol. 14.2 M, up to 7 I ml with water. The NADH solution is stable for at least 3 days 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. Reagent 3, once made up. can be used over a 2week period (see further comments in l.he Results section).

Preparation qf rat liver c~msoi. For the measurement of SOD in rat liver, the tissue extract is first prepared by homogenizing the liver in 3 vol of Tea-Dea buffer 25 mM, pH 7.4, and then cleared by centrifugation at 30,000 rpm for 60 min at 4C. The supernatant is dialyzed against cold homogenization buffer and then employed for enzymatic assays. Protein determination was carried out by the L.owry method ( 14) on samples dialyzed against 0.9% NaCl buffered at pH 7.5-8 with molar NaHCOI solution.

RESULTS

Description oJt/re method. The principle of this method is based on the oxidation of NADH, mediated by superoxide radical, in a purely chemical system recently developed in our laboratory. Coenzyme oxidation occurs in the presence of suitable concentrations of EDTA. Mr? and mercaptoethanol (see below) through a free-radical chain of reactions involving thiol oxidation and univalent 02 reduction. A detailed explanation of the reaction mechanism is beyond the scope of the present paper and will be reported elsewhere (manuscript submitted). The addition of SOD to the reaction mixture causes a proportionate inhibition, of the rate of NADH oxidation, thus confirming the involvement of superoxide in the process and providing the basis for SOD activity determination. To perform the assay sequentially add the following (see Materials and Methods) to a cuvette: Tea-Dea buffer. 0.800 ml: NADH solution, 0.040 ml; EDTA/MnC12 solution, 0.035 ml; sample (or water or buffer) 0.100 ml: mix thoroughly and read against air at 340 nm for a stable baseline: then add mercaptoethanol solution. 0.100 ml. Mix and monitor the decrease in absorbance using 0.5-I full scale deflection. The final volume in the cuvette is 1.065 ml and the light path is IO mm. A typical analysis of SOD activity is shown in Fig. 1 where the kinetics of NADH oxidation in the absence (control) and in the pres-

538
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PAOLETTI

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--_ --__

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--__ -SOD 2

z 0 z

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24

FIG. I. Effect of superoxide dismutase on the rate of NADH oxidation. Four assays are carried out simultaneously in the absence (control) and in the presence of SOD (sample 1. IO ng; sample 2, 80 ng: sample 3, 380 ng of 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 for about 15 min after mercaptoethanol addition. The rate of NADH oxidation in the control is ca. 5 nmol per min. at room temperature.

50% the rate of NADH oxidation observed in the control. Culihration cme \tii/l pure SOD. The determination of SOD activity in pure enzyme preparations from bovine erythrocytes is shown in Fig. 2. Relative rates of NADH oxidation are reported as a function of the amount of enzyme in the assay mixture. The curve thus obtained shows that inhibition is not directly proportionate to SOD concentration, but rather follows an exponential-like function. Almost complete saturation levels (99% inhibition) are obtained with 400 ng of the enzyme, while the same amount of heated SOD fails to inhibit the reaction. One unit of the enzyme corresponds to ca. 15 ng of pure superoxide dismutase from beef erythrocytes. Least-square linear regression analysis was used to obtain a best-fitting curve over the range 4-40 ng, by transforming SOD values into logarithms. The equation is ?; = 1 16.638 - 55.619 s; the correlation coefficient Y

= -0.9925; n = 22. Deterrninution ofSOD in liver euxtructs. To

test the applicability of this method to SOD determination in tissue extracts, experiments were carried out using rat liver cytosol as the sample (Fig. 3). The assay conditions are the same as described for pure SOD and the percentage of NADH oxidation is reported as a function of the total proteins in the extract. The curve obtained is essentially identical to that shown in Fig. 2 for the purified enzyme and in this case too, the boiled sample fails to inhibit NADH oxidation at any rate. Fifty percent inhibition is produced by approximately 10 pg of liver extract which means an average of 100 units of SOD per mg protein of cell cytosol. while the saturation level is reached with ca. 180-200 yg proteir. Curve fitting to experimental data is comparable to that described in the comments to Fig. 2. The linear regression equation, over a range of 4-40 pg protein, is c = 115.366 - 67.687 s; the correlation coefficient t

ence of superoxide dismutase (SODrm3) are compared by simultaneous assay using a multicell holder. The reaction is started by mercaptoethanol and changes in absorbances are recorded for about 15 min. Rates of NADH oxidation are initially low, then increase progressively (usually 2-4 min after mercaptoethanol addition) to yield good linear kinetics (5- 10 min) which are used for calculations. Under our conditions Ai1340 over an S-min interval is 0.250 for the control, while the presence of 10, 80. and 380 ng of SOD in the assay mixture. yields AA values of 0.150.0.038, and 0.008, respectively. For calculations the maximal rates obtained are expressed as a percentage of the control (ordinate) and plotted against a suitable reference (abscissa). One unit of the enzyme is the amount of SOD capable of inhibiting by

= -0.9869; n = 28. Precautions and optimal conditiom ,fi~r rnemuwnmt. EDTA or other chelators for
Mn2+ (not EGTA) may alter the optimum

SUPEROXIDE

DISMUTASE

DETERMINATION

539

--t-l--c-i-+20 SUPEROXIDE DISMlJiASE lngl 40 400

FIG. 2. Titration curve with pure superoxide dismutase. Increasing amounts of SOD (O-400 ng) from bovine erythrocytes (Diagnostic Data Inc.) are added to incubation mixtures and assayed for activity as previously 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. Values (0: n = 27) refer to separate determinations carned out individually by three of us using different stock solutions of the enzyme. Samples containing inactive SOD (0). heated at 100C for 2 min. are shown for comparison.

EDTA/Mn+ ratio and affect the rate of reaction. Alternatively, excess of Mn ions in the sample could slow down the rate of NADH oxidation. Other divalent cations of the second transition series, at concentrations comparable to that of Mn. do not start the reaction, but may compete for the chelator. In addition, when the sample contains free thiols, i.e., mercaptoethanol, cysteine and reduced glutathione. faster reaction rates, as compared to the control. are observed. To avoid all interference by compounds mentioned above. samples must be dialyzed against suitable me-

dia before the analysis. However, owing to handle a large number of samples. dialysis could be conveniently replaced by a rapid desalting through a small Sephadex G-25 (coarse) column. For precise calculations, NADH-oxidase activity, if present in the sample, should be evaluated before mercaptoethanol addition and subtracted from the final rate. Moreover, because of the high sensitivity of the method. samples are usually diluted by such a large factor that NADH-oxidase or any other interfering activity is practically undetectable. In

0 L+0

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t~-+--+-~.+ .-..++
24 CYTOSOL ,,,g pratml

--+
32

184

FIG. 3. Titration of SOD in rat liver cytosol. The liver extract 1see Materials and Methods) is diluted with 100 mM Tea-Dea buffer. pH 7.4. to concentrations suitable for the assay. Measurements are carried out as described in the text. Protein content of the sample is within a range of O-400 pg as determined by the Lowry method. Closed dots (0) represent the average of four separate determinations and bars (=) indicate the range of experimental values. Open squares (0) refer to assays with boiled samples.

540

PAOLETTI

tT

AL.

addition it is worth mentioning that NADPH reacts as well as NADH in the system (data not shown) without serving as a substrate for NADH-oxidase. This fact confirms the flexibility of our method and may turn out very useful when assaying for SOD in fractions containing high levels of NADH-oxidase. Changes of temperature, pH and oxygen tension in the system may influence the absolute rate of NADH oxidation, but are without effect on the relative degree of inhibition observed. Each set of assays must refer to its own control and best measurements are obtained when the values of .&4+,,, of the control. over an S-min interval, are within the range of 0.150-0.400. Reactivity of reagent 3 is increased by storage: therefore aged solutions immediately yield maximal rates of NADH oxidation without the initial delay shown by fresh-made preparation of the complex. Catalase does not interfere with the assay, while hydrogen peroxide inhibits it at a millimolar concentration level.
DISCUSSION

Any reaction inhibitable by superoxide dismutase could potentially provide the basis for an indirect assay of the enzyme and, according to that principle, several methods have been developed over the years. However. as also pointed out by Oberley (15). only a few procedures permit the sensitive and reliable determination of enzymatic activity in tissue extracts with low SOD levels. Our chemical assay seems particularly suitable to that purpose since it allows the measurement of minute amounts of SOD, such as 2 ng, which are far below the detection limit of most published methods. In addition. we have found that fifty percent inhibition, i.e., one unit of the enzyme, is produced by 15 ng of pure protein, while values of catalytic activity. as determined by the xanthine-oxidase/ cytochrome c ( 1), NADH-diaphorase/hydroxylamine ( 13) and xanthine-oxidase/nitro blue tetrazolium (NBT) ( 12) systems, are ca. 200,626, and 630 ng, respectively. A substantial improvement of the NBT assay has been obtained by Buettner (16), whose procedure

yields exactly the same sensitivity reported here. However. with his method, saturation levels are not attainable and different values for half-maximal inhibition are obtained for pure and crude SOD preparations. These inconveniences are frequently observed in assays involving the reduction of NBT. cytochrome (. or other suitable detector and ascribed to the action of aspecific electron donors on chromogenic substrates. Our method. on the contrary, relies on the oxidation of the detector (NADH in this case) and therefore will not be affected by the presence of reductants which are known to occur in tissue extracts ( 12). This lack of interference is clearly shown by the fact that calibration curves for pure SOD and rat liver cytosol are almost identical and saturation levels (99%) are attainable in both cases. The latter result implies that in our system the same value for catalytic activity is obtained by using either 50% or half-maximal inhibition for calculation. This avoids the necessity of running a full calibration curve each time and is of valuable practical importance, especially when dealing with samples having low SOD levels. From our data. a specific activity of ca. 66.000 unit/mg protein and ca. 100 unit/mg protein can be calculated for SOD in pure beef erythrocytes preparations (Diagnostic Data Inc.: see Materials and Methods) and rat liver cytosol, respectively. In addition to the experiments with rat liver cytosol, the present method has been applied successfully to the measurement of SOD in a variety of other cell extracts and body fluids (data not shown). So far, the only major inconvenience encountered comes from hemoglobin; thus, for reliable determination of SOD in hemolysates. this molecule must be removed from the sample before assaying. An important problem in SOD analysis is the discrimination between the cuprozinc- and manganese-form of the enzyme. Cyanide. at concentrations used for inhibiting the cuprozinc enzyme, does not interfere with our assay and manganese-SOD (Mn-SOD) can be easily determined by differential measurement. In addition, this method has proved ofgreat value in determining traces of Mn-SOD separated

SUPEROXIDE

DISMUTASE

DETERMINATION

541

from rat liver cytosol by means of gel filtration (data not shown). In this regard it is worth recalling that our assay is carried out at pH 7.4 which favors the detection of Mn-SOD. In fact, physiological pH is the most suitable for optimal activity of Mn-SOD, which is not reliably assayed at elevated pH values as required by other sensitive methods ( 17,18). On the whole, the present procedure for SOD determination involves stable and inexpensive reagents and consists of a single spectrophotometric step, easily performed on a time scale of minutes. It appears particularly suitable for application in the field of biochemistry. plant physiology, and clinical chemistry.
ACKNOWLEDGMENTS
We would like to thank Professor A. Fonnesu. &airman of the Institute. and Dr. V. Boddi for his continuous suggestions and statistical analyses. This research was supported by a grant from the Minister0 della Pubblica Istruzione (6Og).

REFERENCES
I. McCord. 2. McCord. (1971) 3. Fridovich. J. M., and Fridovich, 1. ( 1969) J. Viol C11crn. I.

4. Ballou. D., Palmer. G.. and Massey. V. (1969) Biochrm. Bioqlzm R~.L Cornrnm 36, 898-904. 5. Rotilio. G.. Bray, R. C., and Fielden. E. M. (1972) Birxhirn. BiuphJ,.r. .1ctu 268. 605-609. 6. Klug. D.. Rabani, 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 (Michelson A. M.. McCord. J. M.. and Fridovich, I.. eds.), pp. I l- 17. Academic Press. New York. 9. Flohe. L.. and Gtting, F. ( 1984) in Methods in Enzymology (Colowick. S. P.. and Kaplan. N. 0.. eds.). Vol. 105, pp. 93-104. Academic Press. New York. IO. Bensinger. 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. Beuchamp. C.. and Fridovich. I. ( 197 I ) .,lr~ul. B,nchrtn. 44, 276-187. 13. Elstner, E. F.. Youngman. R. J.. and OBwald, W. ( 1383) irz Methods of Enzymatic Analysis (Bergmmeyer, H. U.. ed.). Vol. III. pp. 293-302, Verlag Chemie, Weinheim. 14. Lowry. 0. H.. Rosebrough. H. J.. Farr. A. L., and Randall. R. J. ( 195 I ) J. Blol. 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. 105. pp. 457-464. Academic Press. New York. 16. Buettner. G. R.. Oberley, L. W.. and Leuthauser. S. W. H. C. ( 1978) Phr~toc~hcr,?. Photohid 28, 693695.

244,6049-6055.

J. M.. Keele. B. B.. Jr.. and Fridovich. Proi,. Nar. .-I(& SC,; C51 68, 1024-1027. I. (1978) Sc~rc~nc~c 201, 875-880.

17. Marklund, S. (1976) J Bird 18. Misra. H. P.. and Fridovich, 247, 3170-3175.

Chn.

251, 7504-7507.

I. (1972)

d Bid

Chc177