The effects of pH and temperature on the assay of superoxide dismutase A . G. BUCHANAN A N D H . LEES D~,/)or./t~rrtrr rtf'Mic~.ohiologyUtri~~cvsiry of'MrrtziroBrr, Wit~t~i/)~,,rg, Motr., Ctrtrrrtlo R372N2 Accepted August 4, 1976

B U C H A N A NA. , G . , and H. LEES. 1976. The effects of pH and temperature on the assay of superoxide dismutase. Can. J. Microbiol. 22: 1643-1646. A simple and reliable method for the measurement of superoxide dismutase (EC 1.15.1. I ) activity is described. The method is based on a linear inhibition of the reduction of acetylated cytochronie 1. by superoxide dismutase. BUCH~INAN A., G . , et H. LEES. 1976. The effects of pH and temperature on the assay of superoxitle dismut;ise. Can. J. Microbiol. 22: 1643-1646. On decllt une methode slmple et s61e poul mesulel I'.lctivrtt de la supeloxide d ~ m u t a s e( E C I ~ n e a ~ lde e la ~ e d u c t ~ odu n cytochrome 1. 1.15.1 1) La methode e\t b'lsee s u ~une rnhlb~t~on :~cetyltpal la supeloxide tl~smutase [Tladuit pal lejou~nal]

Introduction The reduction of ferricytochrome c by the superoxide anion ( 0 2 - ) has been used t o monitor various reactions involving 02- (McCord and Fridovich 19690, b ; Beauchamp and Fridovich 197 1). Interference in these reactions by peroxidases and cytochrome oxidases has recently been eliminated by the use of acetylated ferricytochrome c (ACc) in place of cytoclirome c itself. Thus the che~iiicalreduction of ACc by 0,- becomes the basis of a detection procedure for superoxide :superoxide oxidoreductase (EC (superoxide dismutase) (SOD) which limits the reduction of ACc in any given system by enzynlically destroying 0,-. This detection procedure can be used even with crude extracts of SOD (Azzi et al. 1975). The procedure has proved to be reliable, but the report of Azzi et al. did not describe the effects of temperature and p H on the sum total of the reactions. We have now done this and, on the basis of our results, would suggest what seem t o be reasonable conditions for a n SOD assay. This work was undertaken because we have reason t o believe (Buchanan and Lees, in preparation) that the well-known toxic effect of excess 0, on Azotobacter (Dalton and Postgate 1969; Hine and Lees 1976) may well be mediated by the 0,- anion and that one way the organism protects itself against this anion is by the synthesis of extra amounts of SOD. A simple assay system for SOD activity thus became a desideratum in these studies.

Materials and Methods Ferricytochrome c (type 111), xanthine oxidase (from buttermilk), and bovine SOD were obtained from the Sigma Chemical Co., xanthine from Nutritional Biochemicals Corporation. A Beckman Acta I11 fitted with a Thermocirc~~lator thermostatted water bath was used for spectrophotometric measurements. The ~nolarityof reduced ACc at any point in an experiment was deterniined by measuring the absorbance of the solution of 550 nnl (E,,,,, reduced ACc a t 550 nni = 27.7). T h e total ACc present was determined spectrophoton~etrically after conlplete reduction by dithionite (Minakami et 01. 1958). Percentage acetylation of the ferricytochrome c was determined according to Azzi et ti/. (1975). A xanthine - xanthine-oxidase system was used to generate 02-.Experinients involving variations in pH were carried out in 5 x lo-' M phosphate buffer while the final experiments of Fig. 4, run specifically at pH 9, were carried out in M borate buffer (unadj~~sted Na2B.+0,). Ethylenediaminetetraacetic acid (EDTA) (10-'Mfinal concentration) was added to both buKers. Experiments were carried out by adding to a cuvette the following solutions (final concentrations in parenM); xanthine 0.05 rnl theses): ACc 0.2 ml (25-50 x M); b ~ ~ K e0.55 r mi (I o r 5 x M);SOD (1 x 0.1 mi (according to experiment). The reaction was initiated by adding 0.1 ml xanthine-oxidase solution containing 40 ~g of enzyme. The increase in absorbance a t 550 nnl was recorded as the experiment progressed. The inhibition of the rate of red~lctionof ACc (i.e. the measurement of SOD acti iity under any given conditions) was calculated as (R,-R2)/R, x 100% where R , was the rate of reduction of ACc in the absence of SOD and R2 the rate of reduction in the presence of SOD.

Results Figure 1 shows the effect of pH o n the rate of reduction of ACc by 02-in the presence and absence of SOD a t 29 "C. The effect of tempera-


CAN. J. MICROBIOL. VOL. 22, 1976

?, FIG.I . The rate of reduction of ACc (AA550 nm/min) by 0,- in the presence (A) and absence ( 0 ) of SOD at different pH values. Total ACc concentration: 40 pM. SOD: 0.5 pg. Temperature 29 "C.



12 p H









FIG.3. Percentage inhibition of ACc reduction by 0 2 in the presence of 0.5 pg SOD at different pH values ( 0 , temperature = 29 "C) and different temperatures (0, pH = 9). Total ACc concentration = 40 pM.

ture on the reduction at pH 9 are shown in Fig. 2 (these experiments were carried out in phosphate buffer but exactly the same results were obtained with borate buffer). T h e inhibition of ACc reduction by SOD, calculated from Figs. 1 and 2, is shown in Fig. 3. It is clear from this figure that while the inhibition is little affected by temperature, it is markedly dependent o n p H , being maximal at about p H 9. The effects of different ACc concentrations o n the rates of inhibition of ACc reduction by varying amounts of SOD at pH 9 are shown in Fig. 4. The effect of varying the xanthine-oxidase concentration o n the curves shown in Fig. 4 was minimal. An S O D concentration that gave 3 3 z inhibition with 2 x lo-' g of xanthine oxidase showed 34% inhibition with 12 x lo-' g xanthine oxidase when the ACc concentration was maintained at 25 pM. FIG.2. The rate of reduction of ACc (AA55O nm/min) by 0 2 -in the presence (A) and absence ( 0 ) of SOD at different temperatures. Total ACc concentration: 40 P M . SOD: 0.5 pg. pH = 9.

Discussion It Seems clear from the results (Figs. 1, 2 a n d 3) that SOD activity, when measured as a n inhibition of ACc reduction by enzymically gener-


80 -


, ,

, ,



wished to compare the activity of one SOD preparation with another, providing that the plots of S O D concentration against inhibition of ACc reduction were always linear. But clearly they are not. This is in general agreement with the adrenochrome Fridovich (1971) formation 011 the by findings effect of of SOD Misra on and

= G o ;


the 0,- ion. Clearly a linear plot is obtained only with low ACc and low SOD concentrations. Z Nevertheless, at concentrations of 0 to 0.5 pg 5 40SOD/niI and 25 x l o p 6 M ACc in borate buffer 1 U at pH 9, the plot of S O D concentration against a 30inhibition of ACc reduction by 0,- is linear at I / 29 "C. The kinetics of the reaction are probably 20complicated (Rigo et (11. 1975) and beyond the /lo scope of our current investigations into theaction 10 of the 0,- ion, of which the present results form I only a part. However, we would suggest that the I conditions we have outlined do form the basis of 0 05 10 15 2 0 25 30 a reliable assay for S O D activity and that 1 unit fig /mi SOD of S O D activity be defined as the amount of FIG.4. Percentage inhibition of ACc reduction by 0 2 - S O D that gives 2 5 2 inhibition (limit of assay a t pH 9, 29 'C, at different total ACC concentrations in being 0-50z inhibition) of the rate of 25 pM the presence of different amounts of SOD. Total ACc A C reduction ~ at p~ 9 by 02-generated by a concentration: 25 1111.1(O), 40 IIM (U), 50 p M (A). xanthine - xanthine-oxidase system. This seems ated 0 , - , is maximal a t a b o ~ l pH t 9. This is con- to be a genuine assay system in which the activity of one SOD preparation can be compared venlent because sodlum tetraborate (Na,B,O,, borax) has a pH of 9 when dissolved 111 water and directly with another without tlie necessity of can thus be used directly as an assay buffer. bringing tlie inhibition of cytochrome reduction Values higher than pH 9 give lower assay values to a predetermined value as demanded by the for a given SOD solut~on,possibly because of current assay system (McCord and Fridovich competition between SOD and O H - ions for the 1 9 6 9 ~ ) . 0,- ion (Rigo ct rrl. 1975); it IS not llkely that Ack~lowledgement radical attack of tlie lysyl I-esldues of SOD, One of us (H.L.) thanks the National Research known to occur at pH 7.4, would be marked at pH 9 (Barra et rrl. 1975). The temperature used Council of Canada for a grant to support this for the assay does not seem to be very ~mportant. work. We used 29 "C simply because ~t was just above A z z ~ A , , , C. MONTECUCCO.and C . RICHTER. 1975. The use of acetylated ferricytochrome C for the detection of maximum laboratory te~nperature and could superoxide radicals produced in biological rnernbl-anes. therefore be maintained by a simple heating Biochem. Biophys. Res. Comrnun. 65: 597-603. thermostat. The reason why tlie SOD activlty BARRA,D., F. BOSSA,L . CACABREESE. G. ROTILLO,P. B. fell markedly above 45 "C (Fig. 2) is not clear ROBERTS. and E. FIELDER. 1975. Selective destruction of amino acid residues in irradiated solutions of superoxsince SOD is thermostable up to 70 "C (McCord ide dismutase. Biochem. Biophys. Res. Cornrnun. 64: and Fridovich 19696). 1303- 1309. The SOD assay is a competition between ACc BEAUCHAMP,C . , and I. FRIDOVICH. 1971. Superoxide and SOD for 0,-. One might therefore reasondisrnutase: improved assays and assay applicable to acrylamidegels. Anal. Biochem. 44: 276287. ably expect that the ACc concentration would 1969. Effect of oxygen have some effect on the assay result. That this is DALTON,H., and J . R. POSTGATE. on growth of Azorohncler chroococcrrtn in batch and so IS shown in Fig. 4; the higher the ACc concontinuous cultures. J . Gen. Microbial. 54: 463-473. centration, the lower the effect of the SOD. This H I N E , P. W., and H. LEES. 1976. T h e growth of effect would be of n o importance if one merely nitrogen-fixing Aiorobncter cl~roococcrrn~ in continuous k









CAN. J. MICROBIOL. VOL. 22. 1976

culture under intense aeration. Can. J. Microbial. 22: 611-618. MCCORD,J . M., and I. FRIDOVICH. 1969lr. Superoxide dismutase. An enzymic function for erythrocuprein hemocuprein. J. Biol. Chem. 244: 6049-6055. 1969b. The utility of superoxide dismutase in studying free radical reactions. 1. Radicals generated by the interaction of sulfite, dirnethyl sulfoxide, and oxygen. J. Biol. Chem. 244: 605G6063. M I N A K A MSI ., , K . TITAMI,and H. ISHIKURA. 1958. The

structure of cytochrome c. 11. Properties of acetylated cytochrome C. J . Biochem. (Tokyo), 45: 341-352. MISRA,H. P.. and I. FRIDOVICH. 1971. The generation of superoxide radical during the autoxidation of ferredoxins. J . Biol. Chem. 246: 688G6890. RIGO, A , , P. V I G L L I N Oand , G. ROTILLO.1975. Kinetic study of 0 , dismutation by bovine superoxide dismutase. Evidence for saturation of the catalytic sites by OZ-. Biochern. Biophys. Res. Cornrnun. 63: 1013-1018.

The effects of pH and temperature on the assay of superoxide dismutase.

The effects of pH and temperature on the assay of superoxide dismutase A . G. BUCHANAN A N D H . LEES D~,/)or./t~rrtrr rtf'Mic~.ohiologyUtri~~cvsiry o...
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