Hara P. Misra

Superoxide dismutase: a photochemical augmentation assay.

Superoxide dismutases increase the rate of the aerobic photooxidation of dianisidine, sensitized by riboflavin. This rate enhancement appears to be due to the catalytic scavenging of O2−, which would otherwise nullify the overall photooxidation by reducing an intermediate oxidation state of the dianisidine. The effect of superoxide dismutase thus exposes a cyclical oxidation-reduction process, which would otherwise remain hidden from view. A sensitive and convenient augmentation assay for superoxide dismutase has been devised on the basis of this effect. It appears to be remarkably free of interferences and can be applied to crude soluble extracts of biological samples.

Evidence for superoxide-dependent reduction of Fe3+ and its role in enzyme-generated hydroxyl radical formation☆

This report describes studies yielding additional evidence that superoxide anion (O2) production by some biological oxidoreductase systems is a potential source of hydroxyl radical production. The phenomenon appears to be an intrinsic property of certain enzyme systems which produce superoxide and H2O2, and can result in extensive oxidative degradation of membrane lipids. Earlier studies had suggested that iron (chelated to maintain solubility) augmented production of the hydroxyl radical in such systems according to the following reaction sequence: O2 + Fe3+ leads to O2 + Fe2+ Fe2+ + H2O2 leads to Fe3+ + HO-+OH-. The data reported below provide additional support for the occurrence of these reactions, especially the reduction of Fe3+ by superoxide. Because the conditions for such reactions appear to exist in animal tissues, the results indicate a mechanism for the initiation and promotion of peroxidative attacks on membrane lipids and also suggest that the role of antioxidants in intracellular metabolism may be to inhibit initiation of degradative reactions by the highly reactive radicals formed extraneously during metabolic activity. This report presents the following new information: (1) Fe3+ is reduced to Fe2+ during xanthine oxidase activity and a significant part of the reduction was oxygen dependent. (2) Mn2+ appears to function as an efficient superoxide anion scavenger, and this function can be inhibited by EDTA. (3) The O2-dependent reduction of Fe3+ to Fe2+ by xanthine oxidase activity is inhibited by Mn2+, which, in view of statement 2 above, is a further indication that the reduction of the iron involves superoxide anion. (4) Free radical scavengers prevent or reverse the Fe3+ inhibiton of cytochrome c3+ reduction by xanthine oxidase. (5) The inhibition of xanthine oxidase-catalyzed reduction of cyt c3+ by Fe3+ does not affect uric acid production by the xanthine oxidase system. (6) The reoxidation of reduced cyt c in the xanthine oxidase system is markedly enhanced by Fe3+ and is apparently due to enhanced HO-RADICAL formation since the Fe3+-stimulated reoxidation is inhibited by free radical scavengers, including those with specificity for the hydroxyl radical.

Superoxide dismutase and the oxygen enhancement of radiation lethality

Abstract Escherichia coli B were more susceptible to radiation lethality and showed a greater oxygen enhancement ratio when exposed in dilute suspension (1 × 105 cells/ml) than when exposed in dense suspensions (1 × 109 cells/ml). The oxygen enhancement, seen with dilute suspensions, was diminished by superoxide dismutase, catalase, mannitol, or histidine. Heat-denatured superoxide dismutase was without effect. The results are interpreted as indicating a role for O2− plus H2O2 in the oxygen enhancement of radiation lethality, and a scheme is proposed which is consistent with the observations.

Inhibition of superoxide dismutases by azide.

Abstract Iron-containing Superoxide dismutases are more sensitive to inhibition by azide than are the corresponding manganese containing enzymes, while the copper-zinc Superoxide dismutases are least sensitive. Thus, at pH 7.8, 10 m m azide inhibited Cu-Zn, Mn, and Fe enzymes by ~10%, ~30% and ~73%, respectively. Stated differently, the concentrations of N 3 − required to cause 50% inhibition of the Cu-Zn, Mn, and Fe enzymes was ~32 m m , ~20 m m and ~4 m m , respectively. These inhibitions by azide, which were imposed and reversed rapidly, appear to provide a useful criterion for distinguishing among the classes of these enzymes. Sensitivity towards inhibition by N 3 − can be applied to the Superoxide dismutases in crude extracts, for the purpose of deciding to which class they belong.

Superoxide dismutase: “Positive” spectrophotometric assays☆

Abstract O 2 − 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 2 − , 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 O 2 − . This increased rate of peroxidation, followed spectrophotometrically, provided the basis for assays which were sensitive, convenient, and precise.

Superoxide dismutase and peroxidase: A positive activity stain applicable to polyacrylamide gel electropherograms☆

Abstract The oxidation of dianisidine, photosensitized by riboflavin, is accelerated by superoxide dismutase. Polyacrylamide gel electropherograms soaked in riboflavin plus dianisidine and subsequently illuminated develop stable brown bands at positions bearing superoxide dismutase activity. This constitutes a new, convenient, and advantageous activity stain for this class of enzymes. Peroxidases are also stained by this procedure due to the photochemical production of H 2 O 2 . This does not constitute an interference with the specificity of the stain, since peroxidase bands develop more slowly than superoxide dismutase bands and can be further identified through the use of inhibitors or of independent staining for peroxidase. The new, positive activity stain for superoxide dismutases can be applied to crude extracts of cells.

A peroxide-dependent reduction of cytochrome c by NADH

Abstract 1. (a) Ethyl hydrogen peroxide, fatty acid peroxides, hydrogen peroxide or p-dioxane peroxide caused the reduction of ferricytochrome c by NADH. Benzoyl peroxide was much less effective in causing this reduction. 2. (b) O2 had no effect on the rates of oxidation of NADH or reduction of cytochrome c; but the presence of O2 did change the mechanism of the reaction. This was demonstrated through the use of superoxide dismutase which inhibited the reduction of cytochrome c in the presence of O2 but had no effect in the absence of O2. 3. (c) Ethyl hydrogen peroxide was converted to acetaldehyde in the course of this reaction. Indeed, ethyl hydrogen peroxide was converted to acetaldehyde by reaction with ferricytochrome c, methemoglobin or hemin but not with ferrocytochrome c or with ferricyanide. 4. (d) A mechanism has been proposed which satisfactorily accounts for these observations.

Cyanide Catalyzes the Oxidation of Alpha-Hydroxyaldehydes and Related Compounds: Monitored as the Reduction of Dioxygen, Cytochrome c, and Nitroblue Tetrazolium,

Abstract Cyanide catalyzed the oxidation of α-hydroxycarbonyls and of related compounds. In the cases of glyceraldehyde 3-phosphate and of dihydroxyacetone phosphate the tautomeric enediol was the obligatory intermediate which reacted with cyanide yielding the active reductant. Cytochrome c , nitroblue tetrazolium, and dioxygen were all reduced by this reductant. In the case of dioxygen the product was the superoxide radical which could then secondarily reduce cytochrome c or nitroblue tetrazolium. In air-equilibrated reaction mixtures, at 25 °C, approximately 35% of cytochrome c reduction and 95% of nitroblue tetrazolium reduction was mediated by superoxide, as judged from susceptibilities to inhibition by superoxide dismutase. Since the oxidations observed were univalent, carbon-centered radicals appear to be necessary intermediates, and their secondary reactions generated a multiplicity of products, seen as smears on thin-layer chromatograms. Free cyanide must be regenerated during these secondary reactions, since cyanide functioned catalytically in the overall process. A partial mechanism has been proposed in explanation of these observations.