[10] Superoxide dismutase assays

[10] Superoxide dismutase assays

[10] SUPEROXIDE DISMUTASE ASSAYS 93 enzyme activity after polyacrylamide gel electrophoresis.9 Of the many assays available for SOD we find the xa...

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enzyme activity after polyacrylamide gel electrophoresis.9 Of the many assays available for SOD we find the xanthine-xanthine oxidase-cytochrome c system of McCord and Fridovich4 to be the most reliable for purposes of detection or measurement of specific activity. Activity of purified SOD is conveniently related to protein concentration measured spectrophotometrically according to Murphy and Kies j° with the correction factor of Weisiger and Fridovich tl for Mn-SOD. 9 C. O. Beauchamp and I. Fridovich, Anal. Biochem. 44, 276 (1971). l0 j. B. Murphy and M. W. Kies, Biochhn. Biophys. Acta 45, 382 (1960). u R. A. Weisiger and I. Fridovich, J. Biol. Chem. 248, 3582 (1973).

[10] S u p e r o x i d e D i s m u t a s e Assays By L. FLOHI~and F. {~TTING The term superoxide dismutase (SOD, EC shall be used for a variety of metalloproteins catalyzing the reaction 20 i- + 2 H + ~ H 2 0 2 +


The primary difficulty in assaying SOD for its enzymatic activity consists in the free radical nature of its substrate O~- which can only be supplied by generation within the assay medium. In addition, the substrate O~cannot easily be detected directly by conventional analytical tools. Routine testing of SOD therefore is performed according to the following general principle. O i- is generated enzymically or nonenzymicaily in the test medium which also contains an easily measurable indicator reacting with O~-. The SOD content of the sample is then calculated from the change of the indicator reaction. Numerous systems have been employed of which an overview is given in Table I. It is quite obvious from this list that any entity which scavenges O i- or reacts with the indicator or changes the rate of O i- formation will lead to erroneous determinations of the SOD activity. To overcome this problem different treatments of the assay sample have been proposed: extraction of the SOD with organic solvents, I acetylation of cytochrome c (cyt c), 2 addition of cyanide, 3 and dialysis. Endogenous interfering substances can also be overcome by applying the J J. M. McCord and I. Fridovich, J. Biol. Chem. 244, 6049 (1969). 2 A. Azzi, C. Montecucco, and C. Richter, Biochem. Biophys. Res. Commun. 65, 597 (1975). 3 C. Beauchamp and I. Fridovich, Anal. Biochem. 44, 276 (1971).


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TABLE l COMPONENTS OF INDIRECT ASSAYS FOR SOD Sources of OiXanthine + xanthine oxidase "-* Photoreduced flavins* Autoxidation of epinephrinee.* Autoxidation of pyrogallol~.J Autoxidation of 6-hydroxydopamine k NADH oxidation by phenazine methosulfate t Potassium superoxide" Electrochemical reduction of O2~.~

Indicators for O iReduction of cytochrome ca.b,f Reduction of nitroblue tetrazolium b.~ Reduction of tetranitromethane~ Autoxidation of epinephrineg.* Autoxidation of pyrogalloFJ Autoxidation of 6-hydroxydopamine k Oxidation of 2-ethyl- ! -hydroxy-2,5,5trimethyl-3-oxazolidine° Chemiluminescence of luminolP Nitrite formation from hydroxylammonium chloride '

J. McCord and I. Fridovich, J. Biol. Chem. 244, 6049 (1969). b M. L. Salin and J. M. McCord, J. Clin. Invest. 54, 1005 (1974). c C. Beauchamp and I. Fridovich, Anal. Biochem. 44, 276 (1971). a D. D. Tyler, Biochenl. J. 147, 493 (1975). e E. Elstner and A. Heupel, Anal. Biochenl. 70, 616 (1976). I V. Massey, S. Strickland, S. Mayhew, L. Howell, P. Engei, R. Matthews, M. Schuman, and P. Sullivan, Biochenl. Biophys. Res. Commun. 36, 891 (1969). g H. P. Misra and I. Fridovich, J. Biol. Chem. 247, 3170 (1972). t, M. Sun and S. Zigman, Anal. Biochem. 90, 81 (1978). ~K. Puget and A. M. Michelson, Biochimie 56, 1255 (1974). J S. Marklund and G. Markland, Eur. J. Biochem. 47, 469 (1974). t R. E. Heikkila and F. Cabbat, Anal. Biochem. 75, 356 (1976). i R. Fried, Biochhnie 57, 657 (1975). " S. Marklund, J. Biol. Chem. 251, 7504 (1976). " J . McCord and I. Fridovich, J. Biol. Chem. 243, 5753 (1968). o G. M. Rosen, E. Finkelstein, and E. J. Rauckman, Arch. Biochem. Biophys. 215, 367 (1982). P R. E. Bensinger and Ch. M. Johnson, Anal. Biochem. 116, 142 (1981). q H. P. Misra and I. Fridovich, Anal. Biochem. 79, 553 (1977). ' Y. Kono, Arch. Biochem. Biophys. 186, 189 (1978). ' Y. Kobayashi, S. Okahata, K. Tanabe, and T. Usui, J. immnnol. Methods 24, 75 (1978).

technique of parallel line analysis of variance. 4 As a note of general precaution it should therefore be stated that all these indirect procedures provide little more than a possibility of estimating relative SOD concentrations in samples of comparable composition. SOD assays based on direct monitoring of O~- by sophisticated techniques (Table II) require an even higher degree of sample purity and shall not be dealt with here further. If an absolute measure of physiological levels of SOD is intended, we suggest a direct immunochemical method in addition to activity measurements. If the ratio of enzymatic activity to concentration of SOD antigen is unchanged independently of the composition of the test medium 4 G. E. EIdred and J. R. Hoffert, Anal. Biochem. 110, 137 (1981).




TABLE 1I DIRECTASSAYSOF SOD Pulse radiolysis ".b Rapid-freeze EPW .a Stopped-flow spectroscopy e.f Polarographic techniques s 19F NMR spectroscopy h

D. Klug, J. Rabani, and 1. Fridovich, J. Biol. Chem. 247, 4839 (1972). b G. Rotilio, R. C. Bray, and E. M. Fielden, Biochim. Biophys. Acta 268, 605 (1972). ~ D. Ballou, G. Palmer, and V. Massey, Biochem. Biophys. Res. Commun. 36, 898 (1969). a W. H. Orme-Johnson and H. Beinert, Biochem. Biophys. Res. Commun. 36, 905 (1969)." G. J. McClune and J. A. Fee, FEBS Len. 67, 294 (1976).l S. Marklund, J. Biol. Chem. 251, 7504 (1976). g A. Rigo, P. Viglino, and G. Rotilio, Anal. Biochem.

68, 1 (1975). h A. Rigo, P. Viglino, E. Argese, M. Terenzi, and G. Rotilio, J. Biol. Chem. 254, 1759 (1979).

(for example during purification) and further, if recovery rates of known amounts of e n z y m e added to the test medium are invariably 100%, we consider the description o f SOD activity properly done. Following this reasoning we therefore will focus on measuring SOD in biological media. We present (1) a qualitative test for SOD activity based on the reduction of nitro blue tetrazolium (NBT) by Oi-; (2) a simple immunological determination o f the SOD molecule o f sufficient sensitivity and avoiding labeled reagents; and (3) an indirect measure of SOD activity to be used in purified samples.

N B T Test This assay was originally reported by Beauchamp and Fridovich, 3 The color reaction is ideally suited to serve as a fast and sensitive test for monitoring SOD in polyacrylamide or agarose gels. Principle

Flavins, e.g., riboflavin, can be photochemically reduced in the presence o f an oxidizable substance, e.g., T E M E D . Upon reoxidation in air




reduced flavins will generate O~-, 5 and the superoxide radical in turn will reduce the colorless nitro blue tetrazolium to a blue formazan 6,7 which is practically insoluble. SOD by scavenging 0 i - will inhibit color formation and appear as a colorless spot.

Reagents 4-Nitro blue tetrazolium chloride (NBT, Serva) Riboflavin (Sigma) N,N,N',N'-Tetramethylethylenediamine (TEMED, Merck) 5,5-Diethylbarbituric acid sodium salt (barbital, Merck) Agarose Type A (Pharmacia) Gel bond film (Marine Colloids) Solution A: 25 mg NBT and 10 mg riboflavin are dissolved in 100 mi doubly distilled water and kept absolutely dark in the cold. Under these conditions the reagent is stable at least for 6 weeks Solution B: 1 g TEMED in 100 ml doubly distilled water

Procedure Immediately after termination of the electrophoresis or electrofocusing, the gel is soaked in solution A by pipetting - 1 ml onto a 100-cm ~ gel surface. After 20 min solution B is allowed to soak into the gel by applying the same procedure. Then, the gel is exposed to an appropriate light source (sun, lightbox etc.). SOD bands will appear colorless against a blue background. As the formazan dye is insoluble and stays in the gel matrix, excess reagents can be removed by soaking the gel in water. After drying the original can be kept as a record. An example is shown in Fig. 1.

Comments The detection limit of this method is around 2 ng. Though quantitative densitometry has been described, s we consider the test as a qualitative one: a fast identification of the enzyme. Complete inhibition of cuprozinc superoxide dismutases by 2 mM cyanide may be utilized to distinguish these from mangano- and iron-type e n z y m e s ) V. Massey, S. Strickland, S. G. Mayhew, L. G. Howell, P. C. Engel, R. G. MaUhews, M. Schuman, and P. A. Sullivan, Biochem. Biophys. Res. Commun. 36, 891 (1969). 6 K. V. Rajagopalan and P. Handler, J. Biol. Chem. 239, 2022 (1964). 7 R. W. Miller, Can. J. Biochem. 48, 935 (1970). s W. Bohnenkamp and U. Weser, Hoppe Seylers Z. PhyMol. Chem. 356, 747 (1975). 9 C. Beauchamp and I. Fridovich, Biochim. Biophys. Acta 317, 50 (1973).




FIG. I. NBT staining of bovine (lane 4, 60 ng) and human (lane 5, 80 ng) Cu,Zn-SOD after electrophoresis in agarose. Electrophoresis conditions: 250 V, 20 min in 150 mM glycine, 20 mM TRIS, pH 8.5. The agarose film was from Coming, as was the uncooled electrophoresis cell.

The NBT stain described above has been widely used for screening of SOD patterns after electrophoresis of crude tissue homogenates. Myeloperoxidase, like other peroxidases, will also mimic SOD activity in this test.t° We can conclude that the NBT staining technique in order to be specific has to be combined with a SOD-specific means of recognition and/or separation, e.g., mobility in an electric field or binding to antibodies.

Electroimmunoassay for Cu,Zn-SOD Numerous immunological SOD assays have been used in the past to monitor SOD levels in tissue samples.t~-2° Because of regulatory restrictions to many laboratories, work with radioactive tracers, however, is a t0 p. Patriarca, P. Dri, and M. Snidero, J. Lab. Clin. Med. 90, 289 (1977). it j. W. Harts, S. Funakoshi, and H. F. Deutsch, Clin. Chim. Acta 46, 125 (1973). t~ U. Reiss and D. Gershon, Biochem. Biophys. Res. Commun. 73, 255 (1976). J3 A. W. Eriksson, R. R. Frants, P. H. Jongbloet, and J. B. Bijlsma, Clin. Genet. 10, 355 (1976). " J . D. Crapo and J. M. McCord, Am. J. Physiol. 231, 1196 (1976). 's K. Kelly, C. Barefoot, A. Sehon, and A. Petkau, Arch. Biochem. Biophys. 190, 531 (1978). ,6 B. C. Del Villano and J. A. Tischfield, J. lmmunol. Methods 29, 253 (1979). t7 A. Baret, P. Michel, M. R. lmbert, J. L. Morcellet, and A. M. Michelson, Biochem. Biophys. Res. Commun. 88, 337 (1979). ta H. Joenje, R. R. Frants, F. Arwert, G. J. de Bruin, P. J. Kostense, J. J. van den Kamp, J. de Koning, and A. W. Eriksson, Scand. J. CIbl. Lab. Invest. 39, 759 (1979). 19 A. Petkau, T. P. Copps, and K. Kelly, Biochhn. Biophys. Acta 645, 71 (1981). 20 G. Bartosz, M. Soszynski, and W. Retelewska, Mech. Ageing Dev. 17, 237 (1981).




burden and costly. Enzyme-labeled immunoassays for SOD would probably circumvent these restrictions, though the preparation of appropriate reagents again needs experience. In case a picogram-detection limit for SOD is not essential one might therefore consider one of the classical immunochemicai determinations, e.g., an electroimmunoassay (EIA) according to Laurell and McKay2j as described below.

Principle In an electric field SOD as a charged antigen moves in an agarose matrix which contains the respective antibody. Under appropriate conditions the antibody-antigen complex precipitates and forms a rocketshaped loop, the area of which is directly proportional to the amount of antigen present.

Reagents Agarose Type A (Pharmacia) Gel bond film (Marine Colloids) 5,5-Diethylbarbituric acid sodium salt (Barbital, Merck) Staining solution (filter before use): 2.5 g Coomassie Brillant Blue G250 (Serva), in 225 ml ethanol (96%), 225 ml water, 50 ml acetic acid Destaining solution: as above but without dye Antisera: the antisera were raised in rabbits or rats according to standard protocols. From the rabbit antisera the immunoglobulin fraction was isolated by threefold precipitation with ammonium sulfate (33% saturation)

Cu,Zn-SOD Pure preparations (->98% as judged by SDS-gradient gel electrophoresis) which were derived from liver (bovine) or placenta (human) were used as antigens or standards. Solutions of SOD below 10 p.g/ml were protected against adsorption losses by addition of 0.01% ovalbumin (w/v).

Equipment A horizontal electrophoresis chamber Desaga Multiphor equipped with a water-cooled plate was used. The power supply was from LKB, type 2197. 21 C. B. Laureil and E. J. McKay, this series, Voi. 73, p. 339.




FIG. 2. Typical EIA for bovine Cu,Zn-SOD. Plate size 12 x 10 cm; antibody concentration: 4 mg lgG in 24 ml of 1.2% agarose in 17 mM sodium barbital buffer, pH 8.6. Duration of electrophoresis 16 hr, voltage 2.5 V/cm; loops: 5-30 standards in/zg/ml; 16 and 25: controls; a-i: unknowns.

Procedure Twenty-four milliliters of 1.2% agarose in 17 mM barbital buffer, pH 8.6, is molten in a boiling waterbath. The solution is allowed to cool down to 55-57 °. At that temperature the antibody solution (100-500 t~l, depending on the titer) is carefully admixed and then quickly poured onto a cut polyester plastic support matirx (10 × 12 cm) which is held on a horizontal leveling tray by four rectangular aluminum bars, thus forming an edge for the liquid agarose-antibody mixture. After cooling a row of 16 holes of diameter 3 mm is punched into the agar 3 cm off the rim and 3 mm apart (Fig. 2). The wells are filled with I0/xl each of the standards and unknowns. For electrophoresis we use 17 mM Na barbital buffer pH 8.6 and 8.5 V/cm (3 hr) or 2.5 V/cm (16 hr). Voltage is measured directly in the gel with two electrodes through holes in the cover lid of the electrophoresis cell. The temperature of the cooling system is 5° . After termination of the electrophoresis the gel is dried: first, several layers of filter paper are kept under pressure on the gel (10-15 min) and the remaining moisture is evaporated in a hot air oven (50°) or with a hair drier. Staining is performed for 15 min, followed by several changes of the destaining solution. Drying yields a film for permanent record. The calibration curve is produced by plotting the peak heights of the standards, which is correct as long as the morphology of the "rockets" is









/" '







bovine SOD (.ug/mL) Fio. 3. Calibration curve of the EIA shown in Fig. 2.

identical in all of the samples. A typical experiment is presented in Fig. 2, the calibration curve of which is shown in Fig. 3. Quality control data of the EIA for bovine Cu/Zn-SOD: The lower limit of sensitivity is - 1 0 ng, i.e., 1 /~g/ml if 10-/zl samples are used; the coefficients of variation for three independent quality control standards (80, 160, and 250 ng) used in the system were 5.1, 4.9, and 3.4%, respectively, during 85 assays. No change of the recovery rate of 100%--within the variation of the assay--could be observed with 1000-fold excess bovine serum albumin or ovalbumin, 20-fold excess human Cu,Zn-SOD. Comments

In a program devoted to screening for extraction procedures from cell lysates this assay for bovine Cu,Zn-SOD proved very reliable and convenient. As at least some of the enzymatic activity of the SOD molecule is still present when the enzyme is bound to its antibody, the NBT-staining technique is also applicable in the EIA for SOD. This leads to an increase in the lower detection limit of - 1 ng (Fig. 4b). In Fig. 4a an example is given for the EIA for human SOD utilizing antisera from rats. A principle drawback is that one measures the concentration of an antigen, not the catalytic activity of the SOD. We therefore suggest correlating the antigen concentration with data on the enzymatic activity. This is supported by an observation of Reiss and Gershon. ~ They described an age-related reduction of the enzymatic activity of the cytoplasmic SOD as compared to several structural parameters such as antigenicity and molecular weight which were unchanged. Similarly, Glass and Gershon 22 22 G. A. Glass and D. Gershon, Biochem. Biophys. Res. Commun. 103, 1245 (1981).




FIG. 4. (a) Example of an E1A for human Cu,Zn-SOD. The antiserum was developed in rats, with 200 p,I used per 10 ml of 1.2% agarose; voltage, 2.5 V/cm for 16 hr. Loops 1-5, standards; 6 and 7, unknowns. (b) Example for the NBT staining of precipitin loops of bovine Cu,Zn-SOD. Loops I-6, 25 ng, 12.5, 6.25, 3.1, 1.6, and 0.8 ng, respectively. The amount of antibody was reduced to 640 p.g IgG/24 ml agarose. Electrophoresis was performed for 3 hr at 10 V/cm at pH 8.6. r e p o r t e d a d e c r e a s e o f the antigen-related specific e n z y m a t i c activity o f S O D during aging o f rats and aging o f e r y t h r o c y t e s . F e r r i c y t o c h r o m e c R e d u c t i o n Assay

Principle T h e r e d u c t i o n rate o f c y t o c h r o m e c b y s u p e r o x i d e radicals is monit o r e d at 550 n m utilizing the x a n t h i n e - x a n t h i n e oxidase s y s t e m as the




FIG. 4b. See legend on p. 101.

source for O~-. SOD will compete for superoxide and decrease the reduction rate of cytochrome c. Reagents Potassium dihydrogen phosphate p.a. Disodium hydrogen phosphate p.a. Ethylenediaminetetraacetic acid disodium salt p.a. (EDTA) Xanthine, crystalline (99-100%) (Merck) Xanthine oxidase from buttermilk grade I (Sigma) Cytochrome c from horse heart, research grade (Serva, Kat.-No. 18020) Solution A. 0.76 mg (5 p,mol) xanthine in 10 ml 0.001 N sodium hy-




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bovine 50D FIG. 5. Typical calibration curve of the cytochrome c reduction assay. The rate of reduction of cytochrome c, inhibited by SOD, is plotted as the reciprocal absorbance change per minute versus concentration of SOD standards. Each individual point represents the mean value of a triplicate determination; 355 ng SOD per assay volume (3 ml) thus meets the definition of I unit of enzyme activity. This particular sample of bovine SOD therefore has a specific activity of 2817 U/mg.

droxide and 24.8 mg (2/zmol) cytochrome c are admixed with 100 ml 50 mM phosphate buffer pH 7.8 containing 0. I mM EDTA. The solution is stable for 3 days at 4 °. Solution B. Freshly prepared solution of xanthine oxidase in 0.1 mM EDTA, ~0.2 U/ml. As the activity of the xanthine oxidase may vary, one should use sufficient enzyme to produce a rate of cytochrome c reduction of 0.025 absorbance units/min in the assay without SOD. Procedure One unit of SOD is defined as that amount of enzyme which inhibits the rate of cytochrome c reduction, under the conditions specified, by 50% I (see Fig. 5). To be able to extrapolate accurately to this value of 50% inhibition one should use several dilutions of one enzyme solution. For relative activity measurements we relate the data to a standard preparation utilizing a plot I/AE min -~ versus standards. Solution B is kept on ice; solution A is thermostated at 25° as is the cell compartment of the spectrophotometer. To adjust the proper wavelength at 550 nm cytochrome c is reduced with some crystals of sodium dithionite and the maximum (550 nm) used as wavelength calibration. (1) Pipet 2.9 ml of solution A into a 3 ml cuvette; (2) add 50 ~1 of sample (water, SOD-standards or unknowns); (3) start the reaction with 50/xl solution B; (4) after mixing record the




absorbance change at 550 nm; (5) plot I / A E min-l--derived from the linear part of the reaction--versus concentration of SOD standards. Comments

The assay works well in pure systems, the within-assay variation being below 10% (triplicates). The sensitivity of the assay defined as the lowest amount of SOD that can be significantly distinguished (p < 0.05) from the blank is - 1 5 ng/ml. A typical calibration curve is presented in Fig. 5. It should be noted that the concentration of ferricytochrome c is crucial. The apparent SOD activity is inversely related to the cytochrome c concentration. Commercial lots may be quite variable in this respect. This difficulty can be overcome by adequate standardization of the cytochrome c solution or by calibration of the test system with a standard SOD preparation. If one intends to use the assay in nonpure systems the following controls and additions should be considered. Interfering reactants in the assay medium have to be checked in recovery experiments by addition of known amounts of a pure SOD preparation to the test sample. Each sample has to be dialyzed. This will eliminate smaller, free molecules like ascorbate, reduced glutathione, catecholamines, etc. To block peroxidases the addition of 2/xM potassium cyanide into the assay medium has been recommended. This concentration is claimed not to affect bovine Cu,Zn-SOD. 3 However, more recently Rigo et al. 23 had determined polarographicaily the Ki values for cyanide and azide to be 1.77 x 10 -6 and 1.43 x 10-2 M, respectively (pH 9.8 and 25°). It seems evident from these data that cyanide will not only affect peroxidases but also affect SOD. Azide, 10-5 M, is obviously more suitable for blocking peroxidases specifically. 24 A different approach takes advantage of the fact that acetylated cytochrome c though still reducible by 0 i- is not susceptible to oxidases or reductases which use cytochrome c as a substrate. 2,25 As the reduction rate of acetylated cytochrome c is decreased, the sensitivity of the assay utilizing acetylated cytochrome c is increased twofold. To distinguish mangano- or iron-type enzymes from the cuprozinc type, one makes use of the inhibition of the latter by 2 mM cyanide in the assay medium) 23 A. Rigo, P. Viglino, and G. Rotilio, Anal. Biochem. 68, 1 (1975). ,4 H. Theorell, #z "The Enzymes" (J. B. Sumner and K. Myrback, eds.), Vol. II. Pt. 1, p. 397. Academic Press, New York, 1951. 25 E. Finkelstein, G. M. Rosen, S. E. Patton, M. S. Cohen, and E. J. Rauckman, Biochem. Biophys, Res. Commun. 102, 1008 (1981).