P. MISRA Duke
Superoxide sensitized by scavenging of an intermediate thus exposes hidden from dismutase has interferences
dismutases increase the rate of the aerobic photooxidation of dianisidine, riboflavin. This rate enhancement appears to be due to the catalytic 02-, which would otherwise nullify the overall photooxidation by reducing oxidation state of the dianisidine. The effect of superoxide dismutase a cyclical oxidation-reduction process, which would otherwise remain view. A sensitive and convenient augmentation assay for superoxide been devised on the basis of this effect. It appears to be remarkably free of and can be applied to crude soluble extracts of biological samples.
Superoxide dismutases are most frequently assayed by coupling a generator of O,- with an indicating scavenger for this radical. The enzyme then competes with the scavenger for the available 02- and inhibits the process being observed. The xanthine oxidase reaction, as a source of 02-, has thus been coupled with cytochrome c, as an indicating scavenger of 02- (l-4). Numerous variations on this theme, employing other sources of 02- and other indicating scavengers, have been devised (1, 5-9). Indeed, substances such as epinephrine (lo), 6-hydroxydopamine (1 l), or pyrogallol(l2) can act both as the source of O,- and as the indicating scavenger for this radical. In all of these cases, SOD’ appears to inhibit and is measured on this basis. Enzymes are usually assayed in terms of augmentation of reaction rates and attempts have been made to develop an augmentation assay for SOD. One such assay has been based upon an increase in the polarographic reduction wave for oxygen at a dropping mercury cathode (13). A spectrophotometric augmentation assay
for SOD would be desirable and one has been reported. It was based upon the ability of O,- to inhibit horseradish peroxidase and of SOD to prevent this inhibition (14). We now report a new spectrophotometric augmentation assay for SOD which is sensitive, reproducible, and applicable to crude extracts and which eliminates the need for extraneous enzymes, such as horseradish peroxidase. MATERIALS
Riboflavin was obtained from Eastman Organic Chemicals and dianisidine was from the Sigma Chemical Company. The copper-zinc-SOD was prepared from bovine erythrocytes (l), while the manganese-SOD (15) and the iron-SOD (16) were prepared from Escherichia coli. Illumination for the photochemical reactions was provided by a pair of parallel 20-W Sylvania Gro-Lux fluorescent tubes mounted on 6-in. centers in an aluminum foil-lined, open-ended box. Reaction mixtures in quartz cuvettes in cuvette holders at room temperature were placed midway between the fluorescent tubes and at intervals were transferred into a Gilford Model 2000 spectrophotometer for recording of absorbance. Dianisidine was dissolved in ethanol to 0.01 M and this ethanolic stock was added to reaction mixtures to give the desired final concentration. All other components were dissolved in 0.010 M potassium phosphate at pH 7.5.
1 This work was supported by Research Grants GM-10287 and HL-17603 from the National Institutes of Health, Bethesda, Maryland and by DAHC0474-G-0194 from the United States Army Research Ofice, Research Wangle Park. Z Abbreviations used: SOD, superoxide dismutase; BeSOD, bovine erythrocyte superoxide dismutase.
The effect of SOD on the photooxidation of dianisidine. Illumination of buffered aerobic mixtures of riboflavin and dianisi308
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0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ASSAY FOR SUPEROXIDE
dine resulted in a progressive oxidation of dianisidine to a pigment whose accumulation could be followed at 460 nm. SOD augmented this photooxidation. Figure 1 illustrates this effect. Light, dianisidine, oxygen, and riboflavin were all essential components, in that no reaction was observed in the a.bsence of any one of them. The copper-zinc, manganese and ironcontaining superoxide dismutases were equally effective in augmenting this photooxidation. Denaturing these superoxide dismutases by brief heating to 100°C completely eliminated their abilities to augment this photooxidation. Furthermore, other proteins, such as catalase and hemocyanine, which do not have SOD activity, were without effect. It thus appeared that SOD activity specifically augments this photooxidation. Optimization of conditions. Increasing the concentration of dianisidine increased the rate of photooxidation both in the absence and in the presence of SOD. Figure 2 illustrates these results. Line 1 in Fig. 2 presents the effect of dianisidine on the rate in the absence of SOD, while line 2 presents comparable data but in the presence of 1.2 pg/ml of bovine erythrocyte SOD. The dashed line is the rate in the presence of SOD corrected for that in its
FIG. 1. The effect of SOD on the photosensitized oxidation of dianisidine. Reaction mixtures containing 2 x lo+ M dianisidine, 1.3 X 10m5M riboflavin, and 0.01 M potassium phosphate at pH 7.5 were illuminated as described under Materials and Methods at room temperature and the absorbance at 460 nm was recorded. The data on line 1 were observed in the absence of SOD, while those on lines 2,3, and 4 were observed in the presence of 0.2, 0.5, and 1.0 pg/ml of BeSOD, respectively.
FIG. 2. The effect of varying the concentration of dianisidine. Reaction mixtures containing 6.7 x 10m6 M riboflavin, 0.01 M potassium phosphate, and the indicated concentrations of dianisidine with and without 1.2 pg/ml of BeSOD at pH 7.5 were illuminated for 4 min, as in Fig. 1, and the absorbance change at 460 nm was recorded. Line 1, no BeSOD; line 2, with 1.2 pglml of BeSOD; line 3, change in the presence of BeSOD corrected for that in its absence. The inset presents the data on line 3 on reciprocal coordinates.
absence. The rate in the presence of the enzyme, when corrected for the rate in its absence, was a saturable function of the dianisidine concentration, probably because one of the other reactants progressively becomes more rate limiting. This saturation behavior allowed the data to fit a straight line when plotted on reciprocal coordinates, as shown in the inset of Fig. 2. Half of the maximal rate augmentation by SOD was seen at 8.3 x lop5 M dianisidine. The rate similarly increased with the concentration of riboflavin, as shown in Fig. 3, and in this case half-maximal augmentation was seen at 6.3 x 10e6 M riboflavin. Figure 3 also demonstrates that EDTA at 1 x lOA M (dotted open circles) or catalase at 8 pg/ml (open squares) had no effect. The riboflavin-sensitized photooxidation of dianisidine and its augmentation by SOD were influenced by pH. Figure 4 presents the augmentation caused by 1.2 pg/ ml of BeSOD as a function of pH in the range of 5.5-9.0. A fairly sharp optimum was seen at pH 7 8. Specific buffer ion effects were minimal, as shown by the use of overlapping buffer ranges and by the observation that the addition of 4 mM sodium acetate or of sodium bicarbonate to the 0.10 M potassium phosphate buffer at pH 7.8 had no effect on the SOD-augmented photooxidation of dianisidine.
MISRA AND FRIDOVICH
4 6 Riboflavin , ,uM
FIG. 3. The effect of varying the concentration of riboflavin. Reaction mixtures containing 1 x lo-’ M dianisidine, 0.01 M potassium phosphate, and the indicated concentrations of riboflavin with and without 1.2 M/ml of BeSOD at pH 7.5 were illuminated for 4 mm, as in Fig. 2. Line 1, no BeSOD; line 2, with 1.2 pg/ml of BeSOD; line 3, rate in the presence of BeSOD corrected for that in its absence. The inset presents the data on line 3 on reciprocal coordinates. Data points given as dotted open circles were obtained in the presence of 1 x lO+ M EDTA, while the data points given as open squares were in the presence of catalase at 8 ~g/ml.
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tooxidation. Lines 1 and 2 refer to concentrations of SOD shown on the lower abscissa, whereas line 3 refers to the effects of the higher range of concentrations shown on the upper abscissa. It is evident that the augmentation of this photooxidation was a linear function of SOD, in the range of O-O.5 pg/ml, under the conditions chosen. The data in lines 1 and 3 were obtained after 4 min and those in line 2 were obtained after 8 min of illumination. Interferences. Ethanol, which was used to dissolve the dianisidine, was without obvious effect in that its concentration could be increased from 0.067 to 0.50 M without modifying the rates of photooxidation seen in the absence or in the presence of SOD. The assay could be used equally well to measure the purified copper-zincSOD from bovine erythrocytes, the manganese-SOD from E. coli and from the red alga (Prophyridium cruentum), or the iron-SOD from E. coli. It could also be applied to crude soluble extracts. Thus, a soluble extract of E. coli B with was found to contain 200 units/ml of SOD by the classical cytochrome c reduction assay (1)
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FIG. 4. The effect of pH. Reaction mixtures containing 6.7 x 10ep M riboflavin, 1 x 10m4M dianisidine, 1.2 pg/ml of BeSOD, and buffers (0, 0.01 M sodium acetate; 0, 0.10 M potassium phosphate; A, 0.02 M Tris-glycinate; W, 0.05 M sodium borate; A, 0.05 M sodium carbonate at the indicated pH) were illuminated for 4 min as described in Fig. 2. The data presented have been corrected for the rate seen in the absence of SOD. Addition of 4 mM sodium acetate or sodium carbonate to the 0.10 M potassium phosphate at pH 7.8 had no effect on rate.
An augmentation assay for SOD. The ability of SOD to increase the rate of oxidation of dianisidine, sensitized by riboflavin, provides the basis for a sensitive and convenient assay for SOD. Figure 5 presents the effects of SOD upon this pho-
.4 , w/ml
FIG. 5. The effect of superoxide dismutase. Reaction mixtures containing 1.3 X 10e5 hf riboflavin, 2 X lo-’ M dianisidine, 0.01 M potassium phosphate, and the indicated concentrations of BeSOD were illuminated for 4 or 8 mm as described in Fig. 2. The data shown have been corrected for changes seen in the absence of SOD. Lines 1 and 3 present changes at 460 nm after 4 min of illumination, while line 2 presents changes seen after 8 min of illumination. Lines 1 and 2 refer to the lower range of SOD concentration shown on the lower abscissa, while line 3 refers to the higher range of concentration on the upper abscissa.
was found to contain the identical amount of activity by the photochemical augmentation assay described above. Soluble extracts of bovine liver were also found to have the same level of activity by the two assays. If the sample to be assayed is intensely colored, as in the case of erythrocyte lysates, there is an interference due to masking of the light. This could easily be overcome, in the case of hemolysates, by employing the Tsuchihashi procedure to selectively remove the hemoglobin (1). Mechanism. Assuming that SOD is specific for 02-, we propose that the photooxidation of dianisidine generates both O,and a radical intermediate of dianisidine, which can react to reverse the overall effect of the photooxidation. Thus: Rb + hv-tRb* Rb” + DHz 4 RbH + DH. RbH + 02 --, Rb + H+ + OzH++02-+DH.+DH2+02 DH.+DH.-+D+DH,
(3) (4) (5)
In Reaction (1) the riboflavin absorbs a photon and becomes electronically excited. In Reaction (21, the excited riboflavin oxidizes dianisidine, yielding the flavin semiquinone and a dianisidine radical which, in the absence of competing reactions, would dismute as in Reaction (5) to yield the divalently oxidized dianisidine, which absorbs at 460 nm. However, the flavin semiquinone produced in Reaction (2) can reduce O2 to 02- [Reaction (311 and the O,can, in turn, reduce the dianisidine radical [Reaction (4)1, thus preventing formation of the colored product by Reaction (5). SOD intercepts the 02-, by catalyzing its dismutation, and thus diminishes the extent of Reaction (4) relative to Reaction (5). The possibility that 02- could reduce the final product of dianisidine oxidation and thus actually reverse the change in absorbance at 460 nm was tested and was excluded. Thus, horseradish peroxidase plus H,Oz was used to oxidize dianisidine to the 460-rim-absorbing product. This product is not very soluble in water and precipitates if the reaction mixture is incubated for 20 min or longer. The precipitate
was collected by centrifugation, dissolved in ethanol, and then exposed to a flux of 02- generated by the action of 2 x 10eg M xanthine oxidase on 4 x 10e5 M xanthine in 0.05 M potassium phosphate, 1 x lop4 M EDTA at pH 7.8. The final concentration of ethanol in this reaction mixture was less than 5% and did not interfere with the flux of 02- as judged by the rate of SOD-inhibitable reduction of cytochrome c. Exposure to this enzymatic source of 02- was without effect on the 460-nm absorbance of the oxidized dianisidine. DISCUSSION
The aerobic photooxidation of dianisidine, sensitized by riboflavin, appears to be a slow process. Superoxide dismutases augment the net photooxidation and appear to act with great specificity. Thus, catalase and Limulus sp. hemocyanin were without effect and the effects of crude extracts of bovine liver or of E. coli were entirely accounted for in terms of their known contents of SOD. Thus, the hundreds or thousands of proteins in these crude extracts, other than SOD, were without effect. Heat-denatured superoxide dismutases were also without effect. We conclude that the superoxide dismutases augmented the observed photooxidation by virtue of their ability to catalytically scavenge 02-. One can then imagine a mechanism, outlined by Reactions (l)-(5), in which the intermediates O,- and a dianisidine radical react to nullify the original photooxidation event. In this view, the aerobic photooxidation of dianisidine, as sensitized by riboflavin, is actually more rapid than meets the eye and the apparent slowness of the overall process was due to a back reaction in which 02- was an essential participant. In any case, the rate augmentation caused by SOD was shown to provide an assay which is simple, inexpensive, reproducible, and free of serious interferences. REFERENCES 1. MCCORD, J. M., AND FRIDOVICH, I. (1969)5. Biol. Chem. 244, 6049-6055. 2. WEISIGER, R. A., AND FRIWVICH, I. (1973) J. Bid. Chem. 248, 3582-3592.
3. SALIN, M. L., AND MCCORD, J. M. (197415. C&z. Znuest. 54, 1005-1009. 4. Azzr, A., MONTECUCCO, C., AND RICHTER, C. (1975) Biochem. Biophys. Res. Commun. 65, 597-603. 5. BEAUCHAMP, C., AND FRIDOVICH, I. (1971)Anal. B&hem. 44, 276-287. 6. ELSTNER, E. F., AND HEUPEL, A. (1976) Anal. B&hem. 70, 616-620. 7. NELSON, N., NELSON, H., AND RACKER, E. (1972) Photochem. Photobiol. 16, 481-489. 8. RAPP, U., ADAMS, W. C., AND MILLER, R. W. (1973) Canud. J. B&hem. 51, 158-171. 9. WINTERBOURN, C. C., HAWKINS, R. E., BRIAN, M., AND CARROLL, R. W. (1975) J. Lab. Clin.
FRIDOVICH Med. 85, 337-341. 10. MISRA, H. P., AND FRIDOVICH, I. (1972) J. Biol. Chem. 247, 3170-3175. 11. COHEN, G., AND HEIKKILA, R. (1974) J. Biol. Chem. 249, 2447-2452. 12. MARKLUND, S., AND MARKLUND, G. (1974) Eur. J. Biochem. 47, 469-474. 13. RIGO, A., VIGLINO, P., AND ROTILIO, G. (1975) Anal. Biochem. 68, l-8. 14. MISRA, H. P., AND FRIDOVICH, I. (1977) Anal. B&hem., in press. 15. KEELE, B. B., JR., MCCORD, J. M., AND FRIWVICH, I. (1970) J. Biol. Chem. 245, 6176-6181. 16. YOST, F. J., AND FRIDOVICH, I. (1973) J. Biol. Chem 248, 4905-4908.