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

Generation of Superoxide Radical during Autoxidation of Hydroxylamine and an Assay for Superoxide Dismutase

Publisher Summary The primary difficulty in assaying Superoxide dismutase (SOD) for its enzymatic activity consists in the free radical nature of its substrate O 2 · - which can only be supplied by generation within the assay medium. The substrate O 2 · - cannot easily be detected directly by conventional analytical tools. Routine testing of SOD, therefore, is performed according to a general principle, which is explained in the chapter. If an absolute measure of physiological levels of SOD is intended, a direct immunochemical method is suggested in addition to activity measurements. This chapter focuses on measuring SOD in biological media. It presents a qualitative test for SOD activity based on the reduction of nitro blue tetrazolium (NBT) by O 2 · - , a simple immunological determination of the SOD molecule of sufficient sensitivity and avoiding labeled reagents, and an indirect measure of SOD activity to be used in purified samples.

Superoxide dismutase assays.

The current status of superoxide dismutase (SOD) is that it is an enzyme with diverse ramifications. This review attempts an understanding of SOD as a structural, functional, and biological entity. Accordingly, the review is in three parts. The first part discusses SOD in terms of protein structure, proceeding from primary to secondary and three-dimensional structure for the three forms of SOD: copper/zinc SOD, manganese SOD, and iron SOD. This is the order of structural knowledge of the enzyme. Iron SOD is an enzyme of prokaryotes and some higher plants. Manganese SOD is an enzyme of prokaryotes and eukaryotes. Copper/zinc SOD is an enzyme of eukaryotes and certain prokaryotes. The evolutionary relationships of the three forms of SOD, the status of the copper/zinc SOD gene in prokaryotes, and the cloning and sequencing of SOD genes are discussed. The second part of the review deals with the catalytic mechanism of SOD in the three forms of the enzyme. Structural and mechanistic conclusions from various spectroscopic studies are critically considered. A detailed picture is given of the active site of copper/zinc SOD. The third part is a review of SOD in the general context of oxygen toxicity. After consideration of the question of superoxide toxicity and superoxide pathology, several areas in which SOD has been investigated or used as a tool in a biochemical, pharmacological, or clinical context are discussed, including population genetics; trisomy 21; development and senescence; the nutritional copper, zinc, and manganese status; hemolysis and anemia; oxygen toxicity in the lung and nervous system; inflammation, autoimmune disease and chromosome breakage, ischemia and degenerative changes; radiation damage; and malignancy. A comprehensive picture is given of measurements of SOD activity in disease states, and the question of superoxide-related disease is considered at several points.

Aspects of the structure, function, and applications of superoxide dismutase

Potato tuber tissue discs, which were aged after wounding in order to acquire hypersensitive reactivity, reduced extracellular cytochrome c and nitroblue tetrazolium (NBT) following inoculation with an incompatible, but not compatible, race of Phytophthora infestans . The cytochrome c -reducing activity rapidly increased from l to 4 h after inoculation along with an increase in the percentage of hypersensitively dead cells, and then decreased from the time when most of the penetrated cells had died. A localized activation of NBT reduction around invading hyphae of the incompatible, but not those of the compatible, race was observed at early stages of penetration before cell death. The reductive activity of the discs was also elicited by treatment with a hypersensitivity-eliciting substance, hyphal wall components of the fungus. Superoxide dismutase (SOD), an enzyme catalysing the conversion of the superoxide anion (O 2 − ) to H 2 O 2 and O 2 inhibited the enhanced reducing activity of the discs when added to the assay solution, indicating that cytochrome c and NBT may be reduced by O 2 − generated from the discs. Pre-infectional, vacuum infiltration of the discs with a solution containing SOD significantly delayed the occurrence of hypersensitive cell death caused by infection with the incompatible race as well as the accumulation of phytoalexin. Application of SH-binding reagents and NADP + , but not respiratory inhibitors, inhibited the elicitation of the reducing activity caused by infection with the incompatible race. These results indicate that an O 2 − -generating system may be activated in potato tissues during the incompatible interaction induced by invading fungi or fungal wall components, and also that the generation of O 2 − may be involved during hypersensitive cell death as a trigger of the sequence of resistance reactions.

Involvement of superoxide anion generation in the hypersensitive response of potato tuber tissues to infection with an incompatible race of Phytophthora infestans and to the hyphal wall components

Photoreduced flavins react very rapidly with molecular oxygen to give the superoxide anion. This radical is highly reactive with cytochrome c and p-nitro blue tetrazolium salts. The reduction of th...

Reactions of superoxide anion, catechols, and cytochrome c

Superoxide dismutase catalyzes the conversion of the single electron reduced species of molecular oxygen to hydrogen peroxide and oxygen. The widely distributed copper enzyme has been purified from two fungi. Identical chromatographic and electrophoretic behavior of the enzyme isolated from different sources indicates great similarity in the molecular properties of the enzyme from eucaryotic organisms. A photosensitized recording assay procedure for the enzyme was developed which elminates the use of a second enzyme system for generating the substrate, superoxide anion. Kinetic data indicate that the reaction between enzyme and superoxide anion shows a logarithmic dependence on concentration under the conditions of the method.The fungal enzyme contains 2 mol of zinc and 2 mol of copper per mole of holoenzyme. The reaction between the enzyme-bound copper and several catechol derivatives has been examined through the use of electron spin resonance spectroscopy. It was concluded from these studies that these...

Purification of superoxide dismutase from fungi and characterization of the reaction of the enzyme with catechols by electron spin resonance spectroscopy.

An L-dihydroorotate–ubiquinone reductase system was obtained from beef liver mitochondria in the form of a high molecular weight complex. Whole mitochondrial preparations of mouse liver and electron-transport particles of beef heart exhibited some cytochrome-linked oxidation of the pyrimidine precursor. Solubilization of the complex from beef liver by deoxycholate and subsequent purification largely removed cytochrome components and yielded a complex which was incapable of reducing oxygen. Non-heme iron, flavin, ubiquinone, and lipids were present in the complex. The exact role of each of these components in the linkage of dihydroorotate oxidation with parts of the respiratory chain is not known.A convenient and sensitive spectrophotometric assay for dihydroorotate oxidation based on the coupled reduction of 2,6-dichlorophenolindophenol by ubiquinone and possibly, to a much lesser extent, by other redox components of the complex was developed. This assay procedure was applicable to the whole particles as ...

Richard W. Miller

Papers

Reactions of superoxide anion, catechols, and cytochrome c

Purification of superoxide dismutase from fungi and characterization of the reaction of the enzyme with catechols by electron spin resonance spectroscopy.

Mammalian dihydroorotate - ubiquinone reductase complex'

The Oxidation of Catechols by Reduced Flavins and Dehydrogenases: AN ELECTRON SPIN RESONANCE STUDY OF THE KINETICS AND INITIAL PRODUCTS OF OXIDATION

Biosynthesis of the Purines XXVII. N-(5-AMINO-1-RIBOSYL-4-IMIDAZOLYLCARBONYL)-l-ASPARTIC ACID 5'-PHOSPHATE KINOSYNTHETASE