79, 553-560 (1977)
Superoxide Dismutase: “Positive” Spectrophotometric Assays’ HARA The Department
P. MISRA AND IRWIN FRIDOVICH of Biochemistry,
Duke University Medical Center, Durham, North Carolina 27710
Received August 6, 1976; accepted January 11, I!,77 O,- is known to react with heme peroxidases, yielding the relatively inactive oxyperoxidase or compound III. The rate of peroxidation of dianisidine by horse radish peroxidase should, therefore, be inhibited by a source of O*-, and this inhibition should be relieved by superoxide dismutase. This was the case, and superoxide dismutases thus increased the rate of peroxidation of dianisidine in the presence of an enzymic or a photochemical flux of 02-. This increased rate of peroxidation, followed spectrophotometrically, provided the basis for assays which were sensitive, convenient, and precise.
Superoxide dismutases are unique among enzymes in that their substrate is an unstable free radical. This complicates the measurement of their catalytic activity. Enzymes are ordinarily assayed in terms of their ability to accelerate some reaction. We may call these positive assays. Convenient assays of superoxide dismutases have necessarily been of the negative type, in which the enzyme decreases the rate of some measurable reaction, For example, the xanthine oxidase reaction has been used as a source of 02-, and cytochrome c has been used as an indicating scavenger for this radical (1). Superoxide dismutases diminish the rate of cytochrome c reduction by competing for the available 02-, and 1 unit of activity was defined as that concentration of enzyme which caused a 50% inhibition under specified conditions. This classical negative assay has been modified and improved. Cyanide has been used to eliminate interference from cytochrome c peroxidase and oxidase (2), and its sensitivity has been augmented by performing the assay at elevated pH and at diminished levels of cytochrome c (3). Acetylation of cytochrome c diminishes its ability to react with both cytochrome c reductases and oxidases, but has little effect on its reactivity with O1-. These facts have been exploited in minimizing interferences with this assay for SOD (4).2 Ultimate sensitivity can be achieved by measuring the concentration of O,- which accumulates in the absence of the cytochrome c (5). 1 This work was supported by Research Grants GM-10287 and HL- 17603 from the National Institutes of Health, Bethesda, Maryland and by DAHC-0474-6-0194 from the United States Army Research Office, Research Triangle Park, North Carolina. * Abbreviations used: SOD, superoxide dismutase; HRP, horse radish peroxidase. 553 Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
Indicating scavengers for 02-, other than cytochrome c, have been useful in negative assays for SOD. These include nitroblue tetrazolium which is reduced to the formazan (6); tetranitromethane which is reduced to nitroform (1); and hydroxylamine which is oxidized to nitrite (7). Sources of 02-, other than the xanthine oxidase reaction, have also been used to good advantage. Thus, a photochemical source of 02- can be coupled with nitroblue tetrazolium to give an assay which is applicable to solutions or to polyacrylamide gel electropherograms (6, S-10). Free radical autoxidations in which O,- serves as a chain propagator or chain initiator provide negative assays of great simplicity. Thus, the autoxidations of epinephrine (1 l), pyrogallol ( 12)) 6-hydroxydopamine (13), or sulfite (14) are all inhibited by SOD and can all be used to assay SOD. All of these negative assays are unappealing, because the enzyme appears to act like an inhibitor, the inhibitory response is necessarily a nonlinear function of the enzyme concentration, and the useful range is necessarily restricted by the upper limit of 100% inhibition. Positive assays are preferable, and several have been devised, but they all suffer from inconvenience or insensitivity. Thus, 02- could be generated photochemically and measured by EPR after freeze-quenching (15). Alternatively, 02- could be generated by pulse radiolysis and measured by its absorbance in the ultraviolet range (16-20). These methods have been useful in studies of the mechanism of SOD, but are far too cumbersome for routine assays. The reduction of oxygen at a cathode coated with triphenyl phosphine oxide is a univalent process. SOD increases the height of the polarographic oxygen-reduction wave by dismuting O,- into 0, + HzOz, in the vicinity of the cathode surface (21). This is potentially a convenient positive assay for SOD, but its precision is limited, because the maximum increase in wave height by SOD is small. The elements of an apparently positive spectrophotometric assay for SOD have long been available. Thus, in 1963, Yamazaki and Piette (22) proposed that HRP reacts with 02- to yield compound III. In 1970, this proposal was repeated (23), and, in 1972, it was definitely established (24). In 1974, the reaction of HRP with 02- was studied by pulse radiolysis (25), and its rate constant was measured (26). It is known that compound III is less reactive than the other forms of peroxidase (27). The catalytic action of this peroxidase should, therefore, be inhibited by a flux of 02-, and this inhibition should be relieved by SOD. In the presence of 02-, SOD, therefore, would give the appearance of accelerating peroxidations due to HRP. These expectations have been affirmed and used as the basis of convenient new assays for SOD. MATERIALS
Superoxide dismutases were purified from bovine erythrocytes and Escherichia coli as previously described (1,28,29). Xanthine oxidase was
FIG. 1. Inhibition of HRP by an enzymic source of O,-. ‘Ihe reaction mixture, containing I00 rnkf acetaldehyde, 0.4 mM dianisidiRe, 0.5 mM imid~ole, 0.2 &ml of HRP, and 50 mM sodium carbonate at pH 10.0, was prepared as described under Materials and Methods. The reaction was started by the addition of 0.1 mM H,Oz, and the rate depicted by line I was recorded. Lines 2 and 3 were obtained when the reaction mixtures contained 1 or 2 x IO-* m xanthine oxidase, respectively. Lime 4 demonstrates that the inhibition caused by 2 x IO+ M xanthine oxidase was largely eliminated by 2 &ml of SOD.
isolatedfrom cream asdescribedby Waudet al. (30).Dianisidineand horse radish peroxidase were obtained from the Sigma Chemical Co. Acetaldehyde was a product of Eastman Organic Chemic~s and was distilled prior to use. Riboflavin was obtained from Eastman, and methionine from General Biochemicals, Inc. Illumination for the photochemicalreactionswas provided by a pair of parallel 20-WSylvania Gro-Lux fluorescenttubes, mountedon 6 in. centersin an ~uminum foillined open-endedbox. Reaction mixtures in quartz cuvettes, in cuvette holdersat room temperature,wereplacedmidway betweenthe fluorescent tubes, and, at I-min intervals, they were transferredto a Gilford Model 2000spectrophotometerfor recording of absorbance.All reactions were pe~o~ed at 25°C. Stock acetaldehydecontainedperoxide which was not removed by a single distillation. This was eliminated by preparing a reaction mixture, containing 100mM acetaldehyde,0.4 mM dianisidine, 0.5 mM imidazole, and 4 &g/ml of HRP in 0.05 M sodium carbonate at pH 10.0, and by incubating this mixture at 25°C for I5 min. During this incubation, the endogenousHz02 was consumed in the HRP-catalyzedperoxidation of dianisidine. Only the 0.04 mM dianisidinewas usedup, indicating that the acetaldehydeinitially contained0.04 moI% HzOz.The mixture was then filtered throughWhatmanNo. 1paperto eliminate the insolubleproduct of the peroxidationof dianisidine, andit was then storedat room temperature until usedin the SOD assays.When exogenousHZ02was added,the level of HRP was reducedto 0.2 pg/ml. This was the casein Fig. 1.
MISRA AND FRIDOVICH
4 5 6 Oxldose, CM X IO?
FIG. 2. Effect of varying the concentration of xanthine oxidase. The reaction mixture was prepared as described in Fig. 1, except that 4 &ml of HRP was used. The reactions were initiated by adding the indicated concentration of xanthine oxidase in the absence (line 1) and in the presence (line 2) of 0.5 &ml of bovine erythrocyte SOD.
RESULTS Inhibition of the Peroxidation Enzymic Source of 02-
The aerobic xanthine oxidase reaction generates both Oz- and H202, and the relative proportions of these products of oxygen reduction depend upon pH, oxygen concentration and, to a lesser degree, upon substrate concentration (31). The production of O,- by xanthine oxidase is increased by raising the pH. For this reason, we chose to work at pH 10.0, at which 80% of the electrons traversing the xanthine oxidase were employed in the univalent reduction of oxygen (3 1). If 02- inhibits peroxidase, as 0
SuperoxIde 2 I
dismutase , fig /ml 3 4 5 I I I
(-1 6 I
7 I .
0.2 dwnutase . fig/ml
FIG. 3. Response of the xanthine oxidase-HRP system to SOD. The reaction mixture was prepared as described in Fig. 2 with varying levels of SOD, and the reaction was initiated by adding xanthine oxidase to 3 x lo-* M. SOD augmented the reaction rate as shown.
FIG. 4. 0.033 mM riboflavin, Materials 3,2 &ml enzyme.
Inhibition of HRP by a photochemical source of OZ-. Reaction mixtures, containing EDTA, 3.3 mM methionine, 0.3 mM dianisidine, 3.3 @ml of HRP, 0.33 pg/ml of and 16.6 mM potassium phosphate at pH 7.8, were illuminated as described under and Methods. Additional components were: line 1, none; line 2, 1 &ml of SOD; line of SOD; and line 4,5 pg/ml of SOD. The SOD used was the bovine erythrocyte
anticipated, then the peroxidation of dianisidine by HRP should be inhibited by a concurrent xanthine oxidase reaction, and this inhibition should be relieved by SOD. A pH of 10.0, which is optimal for the production of O,- by xanthine oxidase, is well above the pH optimum for HRP. This problem was circumvented by the use of imidazole, since such nitrogenous compounds markedly broaden the pH range for the effective peroxidation of dianisidine by HRP (32). Xanthine and urate are effectively peroxidized by HRP and, thus, would compete with the chromogenic Superoxide 0
100 , ng /ml
FIG. 5. Responsiveness of the photochemical-HRP system to SOD. Reaction mixtures were prepared and illuminated as described in Fig. 4. The augmentation in rate is presented as a function of SOD concentration.
substrate, dianisidine. Therefore, acetaldehyde was used as the xanthine oxidase substrate. Figure 1 presents the rates of peroxidation of dianisidine in the absence (line 1) and presence (lines 2 and 3) of a concurrent xanthine oxidase reaction. SOD relieved this inhibition (line 4). It is evident that the 02- generated by the xanthine oxidase reaction inhibited the peroxidation of dianisidine. The xanthine oxidase reaction itself produces HzOz, both directly by the divalent reduction of oxygen and indirectly by dismutation of 02-. It should be possible, therefore, to observe the peroxidation of dianisidine by HRP, dependent only upon the H,O, generated by a concurrent xanthine oxidase reaction, and SOD should augment this peroxidation. If such a reaction system were to be used as the basis of a positive assay for SOD, it would be important to establish the optimum concentration of xanthine oxidase. Figure 2, line 1, presents the rate of the peroxidation of dianisidine in the absence of exogenous H202, as a function of the concentration of xanthine oxidase, and line 2 illustrates the augmentation in rate caused by SOD. This augmentation of the rate of peroxidation can provide the basis of a positive assay for SOD. Thus, Fig. 3 presents this augmentation as a function of the concentration of SOD. In the range of O-O.25 &ml of SOD, the response was linear. Inhibition of the Peroxidation Source of 02-
by a Photochemical
Illumination of riboflavin in the presence of oxygen and such electron donors as methionine or EDTA generates OZ-, and this has been used as the basis of assays of SOD (6). Such a photochemical source of 02- also inhibited the HRP-catalyzed peroxidation, and SOD prevented this inhibition. SOD thus gave the appearance of augmenting this peroxidation of dianisidine. Figure 4 illustrates the very slow peroxidation observed when the inhibition of HRP by the photochemical O,- was unopposed (line 1) and the progressive relief of this inhibition by increasing amounts of SOD (lines 2,3, and 4). Figure 5 presents the augmentation of rate as a function of SOD. The response was linear in the range of O-0.08 &ml. Assays of Crude Extracts
The peroxidation of dianisidine by HRP is entirely dependent upon H202, which was either added to the reaction mixture as in Fig. 1 or generated in situ by the xanthine oxidase reaction (Figs. 2 and 3) or by the photochemical oxidation of methionine (Figs. 4 and 5). Catalase must, therefore, be expected to interfere. There was sufficient catalase present in a crude soluble extract of bovine liver or human erythrocyte lysate to interfere significantly. This was overcome by using 3-amino-1,2,4-triazole to inhibit catalase selectively and irreversibly (33, 34). Crude tissue
extracts (3.0 ml) were treated with 0.05 mM aminotriazole and were then dialyzed against 0.05 mM aminotriazole, 0.1 mM H202, and 50 mM potassium phosphate, at pH 7.0 and 25°C for 4 hr. This was followed by overnight dialysis against the neutral phosphate buffer in the cold. Extracts so treated were devoid of catalase activity and could be assayed for SOD activity by the spectrophotometric assays described before. This treatment with aminotriazole and H,O, did not perceptibly modify the level of SOD present in these extracts, as assessed by the classical assay (1). Furthermore, pure bovine erythrocyte SOD, added to the crude extracts of liver or erythrocytes as an internal standard, was completely recovered. DISCUSSION
Two positive spectrophotometric assays for SOD have been described. These are dependent upon the inhibition of HRP by 02- and the relief of this inhibition by SOD. In the first of these assays, the xanthine oxidase reaction was used as the source of 02-, whereas, in the second assay, a photochemical source of O,- was employed. The assay based upon xanthine oxidase must be performed at an elevated pH in order to realize the maximum production of O,- by this enzyme (3 1). A further advantage may be gained by working with solutions equilibrated with 100% 0, (31). The necessity for a high pH makes this assay relatively insensitive toward the manganese and the iron-containing superoxide dismutases, since these enzymes, unlike the copper and zinc superoxide dismutases, are less active at pH 10 than at pH 7 (35). The assay based upon the photochemical flux of 02- avoided these problems, since it could be performed at pH 7.8. It was, however, discontinuous in the sense that illumination had to be interrupted at intervals, so that changes in absorbance could be monitored. All of these assays appear to be specific for superoxide dismutases. Thus, boiled superoxide dismutases were inactive as was the unrelated copper-protein limulus hemocyanine. These assays were linear within limited ranges of concentration of SOD. The response to SOD was, in all cases, an increase in the rate of the reaction being followed. The assays were convenient and were sensitive in that 0.050 pg/ml of SOD could easily be measured. Furthermore, they could be applied to crude extracts once the possible interference by catalase was eliminated. REFERENCES 1. 2. 3. 4.
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