Superoxide dismutase pulse radiolysis

O Neill, P., Fielden, E. M. Pulse radiolysis investigation of the interaction of bovine superoxide dismutase with organic free radicals. In Chemical and Biochemical Aspects of Superoxide and Superoxide Dismutase (Bannister, J. V., Hill, H. A. O., eds.). New York-Amsterdam-Oxford, Elsevier/North-Holland, 1980, pp. 357-363... 

Wardman, P. Specificity of superoxide dismutase in catalysing redox reactions A pulse radiolysis study. In Radiation Biology and Chemistry. Research Developments (Edwards, H. E. et al., eds.), Amsterdam-Oxford-New York, Elsevier, 1979, pp. 189-196... 

Klug D, Rabani J, Fridovich I (1972) A direct demonstration of the catalytic action of superoxide dismutase through the use of pulse radiolysis. J Biol Chem 247 4839-4842 Koppenol WH (1983) Thermodynamic of the Fenton-driven Haber-Weiss and related reactions. In Cohen G, Greenwald RA (eds) Oxy radicals and their scavenger systems. Vol I Molecular aspects. Elsevier Biomedical, New York, pp 84-88... 

Pick M, Rabani J, Yost F, Fridovich I (1974) The catalytic mechanism of the manganese-containing superoxide dismutase of Escherichia coli studied by pulse radiolysis. J Am Chem Soc 96 7329-7333... 

Rotilio G, Bray RC, Fielden EM. (1972) A pulse radiolysis study of superoxide dismutase. Riochim Riophys Acta 26%-. 605-609. 

McAdam ME, LaveUe F, Fox RA, Fielden EM. (1977) A pulse-radiolysis study of the manganese-containing superoxide dismutase from bacillus stearother-moplnlus. Riochem/165 81-87. 

Goto JJ, Gralla EB, Valentine JS, Cabelli DE. (1998) Reactions of hydrogen peroxide with FALS mutant human copper-zinc superoxide dismutases studied by pulse radiolysis. J Biol Chem 273 30104-30109. 

Manganese compounds of biologic importance are examined by pulse radiolysis e.g., the rate of dismutation of radiation-generated Of is catalyzed by Escherichia coli, Mn-containing superoxide dismutase involving electron transfer in which enzymes with Mn(IV), Mn(IIl), Mn(II) and Mn(I) oxidation states are involved. A kinetic model for the reaction mechanism of an Mn dismutase from Bacillus stearothermophilus accounts for the variation of the rate of decay on the concentrations of Oj, enzyme, HjOj, NaNj, KCN and H+. 

F. Lavelle, M.E. McAdam, E.M. Fielden, E. Martin, P.B. Roberts, K. Puget, A.M. Michelson (1977). A pulse-radiolysis study of the catalytic mechanism of the iron-containing superoxide dismutase from Photobacterium leiognathi. J. Biochem., 161, 3-11. 

Another possibility for therapeutic application of polymer vesicles has been presented recently [242], Superoxide dismutase, an antioxidant enzyme, was encapsulated in the vesicular cavity and shown to remain functional in neutralizing superoxide radicals in situ. The polymer membranes were proven permeable to superoxide radicals by pulse radiolysis, and the encapsulation of the enzyme prolongs its lifetime (which is only minutes in the bloodstream, when non-shielded). 

R5. Rotilio, G., Bray, R., and Fielden, E. M., A pulse radiolysis of superoxide dismutase. Biochim. Biophys. Acta 268, 605-609 (1972). 

From fluorescence emission and other physicochemical studies strong evidence was obtained that unlike in the case of bovine erythrocuprein tryptophan was found in human erythrocuprein (204). In a comprehensive pulse radiolysis study Klug, Rabani and Fridovich (205) determined the rate constant of superoxide dismutase to be 2.3 X 109 M-1s 1. They confirmed the results of Rotilio, Bray and Fielden (182) who measured somewhat earlier a constant of 1.6 0.3 X 109 M-1s-1. Both groups were able to demonstrate the constancy of this rate constant in the approximate pFL region from 5 to 10. 

Solutions of dicyclohexyl-18 crown 6 in DMSO have been used to prepare pale yellow 0.15 mol P solutions of KO2 which contain the O2 anion in approximately the same concentration. The specificity of the superoxide dismutase enzyme was used to show that the solution did indeed contain the superoxide ion in solution. I hc redox potentials of the superoxide and hydroperoxy free radicals, O2 and HO2, have been measured by the fast reaction technique of pulse radiolysis and kinetic absorption spectrophotometry. A d.t.a. study has shown that, during the heating of LiC104-K02 mixtures containing <30% KO2, a eutectic melt occurred at 100—250 C with the loss of superoxide oxygen. At 250—300 °C the mixture melted with loss of peroxide oxygen and at 360— 500 °C the perchlorate decomposed with the loss of all the perchlorate oxygen. 

Forman and Fridovich (1973) using an indirect assay whereby O2 was generated either by the action of xanthine oxidase on xanthine or by the mechanical infusion of potassium superoxide in tetrahydrofuran. The generated OJ was allowed to react with ferricytochrome c or with tetra-nitromethane and the product formation was monitored spectroscopically. Details of the two assays are given in Section 11.3. Addition of superoxide dismutase inhibits the formation of products. A rate constant of 2 X 10 M sec was determined for all three enzymes. This value agreed with the rate constant determined by pulse radiolysis for the copper/zinc enzyme (Klug-Roth et al., 1973 Fielden et al., 1974). The mechanism of action of the superoxide dismutases has been investigated by the technique of pulse radiolysis which is described in Section II.2. The bovine erythrocyte copper/zinc enzyme is the most studied form as far as the molecular and catalytic properties are concerned (Rotilio and Fielden,... 

Pulse radiolysis investigation of the manganese superoxide dismutase for... 

This scheme has been proposed for the manganese superoxide dismutase from Paracoccus denilrificans (Teresch et al., 1983). This enzyme has been shown to exhibit saturation behavior by pulse radiolysis. 

All the inhibitions reported so far indicate that there is no known specific inhibitor for the manganese superoxide dismutase. The enzyme from B. stearothermophilus was found to be inhibited by cacodylate (Thornalley et al., 1982) however, the inhibition could only be observed in the xanthine/xanthine oxidase assay and not in the pulse radiolysis assay. No evidence was obtained that cacodylate could be competing with the enzyme for because little or no inhibition was observed when the copper/zinc rather than the manganese enzyme was assayed in the presence of cacodylate. Further investigations revealed that the inhibitory effect is due to a cacodylate anion radical produced by the interaction of hydroxyl radicals (generated by the xanthine/xanthine oxidase reaction) and cacodylate anions. A radical of pamoic acid (4,4 -methylenebis-(3-... 

Rapid freeze epr can be used as a direct assay of superoxide dismutase, as described in the earlier work by Ballou et al., (1969) who studied the effect of superoxide dismutase on the decay of the signal of O2. Superoxide was trapped by rapid freezing during the reaction with oxygen of anaerobically reduced tetraacetyl riboflavin. The O2 available for reaction with superoxide dismutase was ca. 10 M, and under these experimental conditions, it was possible to estimate that the turnover rate number of superoxide dismutase was at least 3 X 10 min . As a consequence of the difficulty inherent in the method, rapid freeze epr has not resulted in routine assay of superoxide dismutase. It has been used in a different approach for mechanistic studies (Fielden et al., 1974). In this case O2 was generated by pulse radiolysis, and the valence state of the enzyme estimated by epr. 

Fig. 16. Effect of ionic strength on dismutation rate of O2 by superoxide dismutase as measured experimentally by pulse radiolysis at pH 8. Ionic strengths were adjusted using NaCl. O, Bovine SOD , human SOD expressed in yeast , mutant Cys-6Ala, Cys-lllSer of human SOD expressed in yeast , Thr-137Ile SOD A, Arg-143Lys.

The direct evaluation of the catalytic activity of superoxide dismutases by pulse radiolysis was firstly developed in 1972 A recent overview is provided by... 

Pulse radiolysis has met many advantages. The reaction constant can be determined directly and the enzymic activity is linear dependent on the enzyme concentration. The yield of superoxide is extremely high and the method is highly sensitive. The distinction between catalytic and non-catalytic reactions is possible. For example, the reactivity of superoxide dismutase was found to be catalytically and of second order. Rate constants between 1.5 and 2.6x W M s at neutral pH are reported, thus being near the diffusion control By way of contrast, caerulo-... 

Another advantage of pulse radiolysis lies in the evaluation of the catalytic scavenging of superoxide by low molecular mass complexes. Unlike the polarographic method, another direct superoxide dismutase assay, the reaction parameters of such compounds can be easily obtained. 

Recently, the superoxide dismutase activity of low molecular mass copper chelates in the indirect coupled assay systems has been dispute It was demonstrated that copper in CuSO and Cu(II)(gly)2 prevents the ferricytochrome c and nitroblue tetrazolium reduction. This is not virtually new, as it is a well known phenomenon that Cu(II)-salts lead to a reoxidation of ferrocytochrome c and that they are potent inhibitors of xanthine oxidase which is often used as Oj" -generator in indirect SOD assay systems Although the indirect assays may be sometimes inadequate for the measurement of the SOD-activity, there are no doubts that low molecular mass copper chelates have their superoxide dismutase during pulse radiolysis. 

The kinetics of formation of the intermediate complex between catalase and HgOa have been re-examined, and reports of pulse radiolysis experiments with superoxide dismutase and its manganese-containing form have appeared. Bovine superoxide dismutase is known to contain two Cu and two Zn atoms per molecule and it has been shown that the copper site, which is directly involved in the catalytic activity, comprises three nitrogens and a water molecule bound to the metal in a field with less than axial symmetry. Less is known of the zinc site but it has recently been shown, by e.p.r. spectroscopy with the cobalt-copper form of the enzyme, that the... 

The stopped-flow technique has been used to study the anaerobic reduction of fungal laccase B by hydroquinone, ascorbate, and ferrocyanide, for which a model is formulated, and the reduction of caeruloplasmin by the hydrated electron has been followed by pulse radiolysis. The latter technique has also been used to study the inhibition of another copper-containing enzyme, superoxide dismutase, by cyanide ions. Comparison of the rates and activation parameters for the reduction by chromous ion of blue copper in laccase, stellacyanin, and spinach and French-bean plasto-cyanins indicates that reduction of the Cu(614) site in laccase may occur by intramolecular electron transfer from one of the Cu(330) sites. The value of 17.4 kcal mol for the activation enthalpy associated with the reduction of cytochrome c by the same species is consistent with a mechanism in which major conformational changes in the protein accompany electron transfer. 

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