Irwin Fridovich

Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels☆

Nitro blue tetrazolium has been used to intercept O2− generated enzymically or photochemically. The reduction of NBT by O2− has been utilized as the basis of assays for superoxide dismutase, which exposes its presence by inhibiting the reduction of NBT. Superoxide dismutase could thus be assayed either in crude extracts or in purified protein fractions. The assays described are sensitive to ng/ml levels of super-oxide dismutase and were applicable in free solution or on polyacrylamide gels. The staining procedure for localizing superoxide dismutase on polyacrylamide electrophoretograms has been applied to extracts obtained from a variety of sources. E. coli has been found to contain two superoxide dismutases whereas bovine heart, brain, lung, and erthrocytes contain only one.

Assaying for Superoxide Dismutase Activity: Some Large Consequences of Minor Changes in Conditions*,'

Most assays for superoxide dismutase depend upon competition between the enzyme and some indicating scavenger for O-2. We have investigated the effects of experimental variables on assays based upon the use of either ferricytochrome c or nitro blue tetrazolium. Our results should help investigators to avoid the numerous potential pitfalls which necessarily surround these assay methods.

Biological effects of the superoxide radical

Can the superoxide radical exert deleterious effects independent of participating with H2O2 in the production of the hydroxyl radical? Examination of the superoxide-related literature reveals data suggesting an affirmative answer to this question.

SUPEROXIDE ANION RADICAL (O2.-), SUPEROXIDE DISMUTASES, AND RELATED MATTERS

A field of inquiry may be said to have come of age when conclusions initially viewed as remarkable or even unbelievable are accepted as commonplace. Study of the biology of the superoxide anion radical and of related free radicals, and the defenses thereto, has now reached this happy state of maturity. Superoxide and even hydroxyl radicals are now known to be produced in living systems, and elaborate systems of defense and repair, which minimize the ravages of these reactive species, have been described. New members of the superoxide dismutase, catalase, and peroxidase families of defensive enzymes are being found, as are new targets that are modified by O2 .. In addition, the involvement of O2 . in both physiological and pathological processes is being established. A weighty tome would be needed to encompass a comprehensive coverage of this field of study. This review will describe only aspects of the biology of oxygen radicals that currently engage the interest of the writer. Hopefully they will also be of interest to the reader. Other recent reviews may serve to fill the gaps in this one (1–6).

Subcellular Distribution of Superoxide Dismutases (SOD) in Rat Liver Cu,Zn-SOD IN MITOCHONDRIA

Rat liver was homogenized in isotonic buffer, fractionated by differential centrifugation, and then subfractionated by equilibrium sedimentation in Nycodenz gradients. Fractions were assayed for both Cu,Zn-superoxide dismutase (SOD) and Mn-SOD by exploiting the cyanide sensitivity of the former activity and by the use of specific antibodies. As expected, the cytosol and lysosomal fractions contained Cu,Zn-SOD; while the mitochondrial matrix contained Mn-SOD. In mitochondria, Cu,Zn-SOD was found in the intermembrane space and Mn-SOD in the matrix and also on the inner membrane. The Mn-SOD associated with the inner membrane was solubilized by 0.5 m NaCl. Surprisingly the intracellular membrane fraction (microsomes) contained bound Cu,Zn-SOD that could be solubilized with a detergent, and to lesser degree with 0.5m NaCl. Both the cytosolic and mitochondrial Cu,Zn-SODs were isolated and compared. They have identical molecular mass, cyanide sensitivity, SDS sensitivity, heat stability, and chloroform + ethanol stability. Tissue from Cu,Zn-SOD knockout mice was entirely devoid of Cu,Zn-SOD; indicating that the cytosolic and the intermembrane space Cu,Zn-SODs are coded for by the same gene. The significance of this distribution of the SODs is discussed.

[41] Preparation and assay of superioxide dismutases

Publisher Summary This chapter discusses the preparation and assay of superoxide dismutase (SOD). SODs are found in all oxygen-utilizing organisms and constitute a defense against oxygen toxicity. SODs were first isolated from erythrocytes as a copper protein of unknown function. Thus, some SODs contain copper and zinc, others contain manganese, and still others contain iron. Assay techniques for each of these enzymes are similar, but distinct isolation procedures are used in their purification. Most mammalian tissues contain both a cuprozinc and a mangano superoxide dismutase. SODs are unique among enzymes in that their substrate is an unstable free radical. This complicates the measurement of their catalytic activity. Convenient assays of SODs have necessarily been of the indirect type. Such assays consist of two components: a superoxide generator and a superoxide detector. The control reaction rate can be completely inhibited by large amounts of SOD if a xanthine oxidase of high quality is being used. Partially degraded xanthine oxidase can, to a small extent, reduce cytochrome c by a nonsuperoxide mediated mechanism.

Inactivation of glutathione peroxidase by superoxide radical.

The selenium-containing glutathione peroxidase, when in its active reduced form, was inactivated during exposure to the xanthine oxidase reaction. Superoxide dismutase completely prevented this inactivation, whereas catalase, hydroxyl radical scavengers, or chelators did not, indicating that O2 was the responsible agent. Conversion of GSH peroxidase to its oxidized form, by exposure to hydroperoxides, rendered it insensitive toward O2. The oxidized enzyme regained susceptibility toward inactivation by O2 when reduced with GSH. The inactivation by O2 could be reversed by GSH; however, sequential exposure to O2 and then hydroperoxides caused irreversible inactivation. Reactivity toward CN- has been used as a measure of the oxidized form of GSH peroxidase, whereas reactivity toward iodoacetate has been taken as an indicator of the reduced form. By these criteria both O2 and hydroperoxides convert the reduced form to oxidized forms. A mechanism involving oxidation of the selenocysteine residue at the active site has been proposed to account for these observations.

Intracellular production of superoxide radical and of hydrogen peroxide by redox active compounds

Several compounds have been found capable of diverting the electron flow in Escherichia coli and thus causing increased intracellular production of O2− and H2O2. One indication of this electron-shunting action was increased cyanide-resistant respiration and one cellular response was increased biosynthesis of the manganese-containing superoxide dismutase and of catalase. Blocking cytochrome oxidase with cyanide or azide increased the electron flow available for reduction of paraquat and presumably of the other exogenous compounds tested and thus increased their biological effects. Paraquat, pyocyanine, phenazine methosulfate, streptonigrin, juglone, menadione, plumbagin, methylene blue, and azure C were all effective in elevating intracellular production of O2− and H2O2. The effect of alloxan appeared paradoxical in that it increased cyanide-resistant respiration without significantly increasing the cell content of the manganese-superoxide dismutase and with only a small effect on the level of catalase. The alloxan effect on cyanide-resistant respiration was artifactual and was due to an oxygen-consuming reaction between alloxan and cyanide, rather than to a diversion of the intracellular electron flow. With paraquat as a representative electron-shunting compound, the increase in biosynthesis of the manganese-superoxide dismutase was prevented by inhibitors of transcription or of translation, but not by an inhibitor of replication. The increase in this enzyme activity, caused by paraquat and presumably by the other compounds, was thus due to de novo enzyme synthesis activated or derepressed at the level of transcription.

Critical evaluation of the use of hydroethidine as a measure of superoxide anion radical.

The fluorogenic oxidation of hydroethidine (HE) to ethidium (E+) has been used as a measure of O2-. Evaluation of this method confirms that O2-, but not O2 or H2O2, rapidly oxidizes HE to E+. However the ratio of E+ produced per O2- introduced decreased as the flux of O2- was increased. This suggested that HE can catalyze the dismutation of O2- and this was affirmed. HE was oxidized to a red product, distinct from E+ by ferricytochrome c and a similar oxidation may occur within Escherichia coli. HE inhibited the growth and killed a SOD-null strain to a greater extent than the SOD-replete parental strain and these effects were much diminished under anaerobic conditions. This indicated that E+ was responsible for the toxicity of HE and indeed E+ was seen to be toxic under both aerobic and anaerobic conditions. In view of the data presented HE can be recommended as a qualitative but not as a quantitative measure of O2(-1).

Requirement for superoxide in excitotoxic cell death.

We tested the pathogenic role of O2-) radicals in excitotoxic injury. Inactivation of the TCA cycle enzyme, aconitase, was used as a marker of intracellular O2- levels, and a porphyrin SOD mimetic was used to scavenge O2-. The selective, reversible, and SOD-sensitive inactivation of aconitase by known O2- generators was used to validate aconitase activity as a marker of O2- generation. Treatment of rat cortical cultures with NMDA, KA, or the intracellular O2- generator PQ2+ produced a selective and reversible inactivation of aconitase, which closely correlated with subsequent cell death produced by these agents. The SOD mimetic, but not its less active congener, attenuated both aconitase inactivation and cell death produced by NMDA, KA, and PQ2+. These results provide direct evidence implicating O2(-) generation in the pathway to excitotoxic injury.