Joe V. Bannister

Aspects of the Structure, Function, and Applications of Superoxide Dismutas

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.

Isolation and properties of the complex nonheme-iron-containing cytochrome b557 (bacterioferritin) from pseudomonas aeruginosa

A nonheme-iron-containing cytochrome b557, also known as bacterioferritin, was isolated from Pseudomonas aeruginosa. Gel electrophoresis showed that the protein subunits had a molecular weight of 18 kD and it is suggested that the intact molecule contained 24 subunits. The isolated protein contained 8.7% by weight Fe and 8% by weight phosphate. Most of the Fe was contained in the nonheme-iron core and could be readily removed by dialysis against 0.12 M thioglycollic acid. The resulting apobacterioferritin contained approximately one protoporphyrin IX group for every five subunits and, in addition, an unidentified fluorophor. The nonheme-iron core was found to be amorphous with a mean core size of 60-65 A.

Application of the Electrochemistry of Cytochrome C to the Measurement of Superoxide Radical Production

An electrochemical sensor for the measurement of the superoxide anion produced as a result of the respiratory burst of neutrophils is described. The reduction of cytochrome c by the superoxide ion was monitored using a surface-modified gold electrode. The current produced was shown to be superoxide-specific and proportional to the number of neutrophils in both purified neutrophil preparations and plasma.

Amperometric NADH determination via both direct and mediated electron transfer by NADH oxidase from Thermus aquaticus YT-1

Using NADH oxidase extracted from Thermus aquaticus YT-1, the determination of NADH by the direct oxidation of labile FADH2 and by mediated electron transfer using the Ru(III/IV) redox couple is described. A scheme for enzyme amplification is outlined using alcohol dehydrogenase (ADH) and NADH oxidase in solution, and enables 1–10 μM NADH to be determined. Detection is performed at + 150 mV using the RuCl62−/RuCl63− couple. Additionally, an electrode is modified by an ADH/NADH oxidase bilayer, enabling ethanol determination (20–200 μ M) at 0 mV via the oxidation of ruthenium red.

Evolutionary Aspects of Superoxide Dismutase: The Copper/Zinc Enzyme

Copper/zinc superoxide dismutase is typically an enzyme of eukaryotes. The presence of the enzyme in the ponyfish symbiont Photobacterium leiognathi and some free living bacteria does not have an immediate explanation. Amino acid sequence alignment of 19 Cu/Zn superoxide dismutases shows 21 invariant residues in key positions related to maintenance of the beta-barrel fold, the active site structure including the electrostatic channel loop, and dimer contacts. Nineteen other residues are invariant in 18 of the 19 sequences. Thirteen of these nearly invariant residues show substitutions in Photobacterium Cu/Zn superoxide dismutase. Copper/zinc superoxide dismutase from the trematode Schistosoma mansoni shows an N-terminal sub-domain with a hydrophobic leader peptide, as in human extracellular superoxide dismutase which is a Cu/Zn enzyme. The latter also has a C-terminal sub-domain with preponderance of hydrophilic and positively charged residues. The amino acid sequence of this superoxide dismutase between the N-terminal and C-terminal regions shares many features of cytosolic Cu/Zn superoxide dismutase, including 20 of the 21 invariant residues found in 19 Cu/Zn enzymes, suggesting a similar type of beta-barrel fold and active site structure for the extracellular enzyme.

A kinetic stidy of the catalysed oxidation of 1',3-dimethylferrocene ethylamine by cytochrome c peroxidase

Abstract The kinetics of the catalysed oxidation of 1,3-dimethylferrocene ethylamine by cytochrome c peroxidase was studied in the presence of two hydroperoxide substrates using cyclic voltammetry. The second order rate constants for the reduction of oxidised CcP were determined as 3.57 × 10 6 dm 3 mol −1 s −1 in the presence of hydrogen peroxide and 5.00 × 10 5 dm 3 mol −1 s −1 in the presence of m -chloroperoxybenzoic acid. Hydrogen peroxide therefore proved to be a more efficient electron acceptor than m -chloroperoxybenzoic acid. The relative rates of reaction of the two substrates were found to be similar to those for the immobilised enzyme. In addition we have also presented some preliminary results for the coupling of a range of other ferrocene derivatives with CcP.

The Role of Glutathione in Protection against DNA Damage Induced by Rifamycin SV And Copper(II) Ions

Incubation of calf thymus DNA in the presence of rifamycin SV induces a decrease in the absorbance of DNA at 260 nm. The effect, was found to be proportional to the antibiotic concentration and enhanced by copper(II) ions. In the presence of rifamycin SV and copper(II), a significant increase in thiobarbituric acid-reactive (TBA-reactive) material is also observed. This effect is inhibited to different degrees by the following antioxidants: catalase 77%; thiourea 72%; glutathione (GSH) 62%; ethanol 52%; and DMSO 34%, suggesting that both hydrogen peroxide (H2O2) and hydroxyl radicals (OH.) are involved in DNA damage. Rifamycin SV-copper(II) mixtures were also found to induce the production of peroxidation material from deoxyribose and, in this case, glutathione and ethanol were the most effective antioxidant substrates with inhibition rates of 91% and 88% respectively. Electrophoretic studies show that calf thymus DNA becomes damaged after 20 min. incubation in the presence of both agents together and that the damaged fragments run with migration rates similar to those obtained by the metal chelating agent 1,10-phenanthroline. Normal DNA electrophoretic pattern was found to be preserved by catalase, and GSH at physiological concentrations and by thiourea. No protection is observed in the presence of ethanol or DMSO. The results obtained indicate the involvement of different reactive species in the degradation process of DNA due to rifamycin SV-copper(II) complex and emphasize the role of reduced glutathione as an oxygen free radical scavenger.

Amperometric determination of copper using screen-printed electrodes

Abstract An amperometric method for the determination of copper ions has been developed using disposable, screen-printed electrodes modified with the ligand bis-cyclohexanone oxaldihydrazone. The electrodes operate at +250 mV versus SCE, giving a linear response to copper across the range 0.03–0.3 mM (r = 0.983, n = 13). The effect on the electrode response of variations in pH, temperature, ligand concentration and storage time are outlined; as well as the effect of competing cations including magnesium, barium, ammonium, calcium, cobalt, iron(III), nickel and zinc.

A simple method for increasing hapten immunogenicity by a specific structural modification of the carrier

A simple procedure to bind haptens, drugs or peptides selectively through their amino or carboxylic group to a spacer arm modified non-immunogenic polypeptide is described. Gelatin, a well known non-immunogenic carrier, was modified by blocking its amino groups. Spacer arms with primary amino groups such as beta-alanine or ethylenediamine were conjugated to this protein by carbodiimide resulting in spacer modified gelatin. These modified polypeptides were tested for their ability to selectively bind haptens through their amino or carboxylic groups. Three probes were used and the results obtained confirm the hypothesis. Three conjugates obtained were further used to induce an immune response in mice. Enhancement of the immunogenicity of these spacer-arm supported haptens was observed. Overall, this study provides a rational approach to the production of well defined antigens using a simple conjugation technique.

Effect of metal ion catalyzed oxidation of rifamycin SV on cell viability and metabolic performance of isolated rat hepatocytes

The effect of rifamycin SV on metabolic performance and cell viability was studied using isolated hepatocytes from fed, starved and glutathione (GSH) depleted rats. The relationships between GSH depletion, nutritional status of the cells, glucose metabolism, lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) production in the presence of rifamycin SV and transition metal ions was investigated. Glucose metabolism was impaired in isolated hepatocytes from both fed and starved animals, the effect is dependent on the rifamycin SV concentration and is enhanced by copper (II). Oxygen consumption by isolated hepatocytes from starved rats was also increased by copper (II) and a partial inhibition due to catalase was observed. Cellular GSH levels which decrease with increasing the rifamycin SV concentration were almost depleted in the presence of copper (II). A correlation between GSH depletion and LDH leakage was observed in fed and starved cells. Catalase induced a slight inhibition of the impairment of gluconeogenesis, GSH depletion and LDH leakage in starved hepatocytes incubated with rifamycin SV, iron (II) and copper (II) salts. Lipid peroxidation measured as MDA production by isolated hepatocytes was also augmented by rifamycin SV and copper (II), especially in hepatic cells isolated from starved and GSH depleted rats. Higher cytotoxicity was observed in isolated hepatocytes from fasted animals when compared with fed or GSH depleted animals. It seems likely that in addition to GSH level, there are other factors which may have an influence on the susceptibility of hepatic cells towards xenobiotic induced cytotoxicity.