Pulse radiolysis xanthine oxidase

Many transition metal complexes have been considered as synzymes for superoxide anion dismutation and activity as SOD mimics. The stability and toxicity of any metal complex intended for pharmaceutical application is of paramount concern, and the complex must also be determined to be truly catalytic for superoxide ion dismutation. Because the catalytic activity of SOD1, for instance, is essentially diffusion-controlled with rates of 2 x 1 () M 1 s 1, fast analytic techniques must be used to directly measure the decay of superoxide anion in testing complexes as SOD mimics. One needs to distinguish between the uncatalyzed stoichiometric decay of the superoxide anion (second-order kinetic behavior) and true catalytic SOD dismutation (first-order behavior with [O ] [synzyme] and many turnovers of SOD mimic catalytic behavior). Indirect detection methods such as those in which a steady-state concentration of superoxide anion is generated from a xanthine/xanthine oxidase system will not measure catalytic synzyme behavior but instead will evaluate the potential SOD mimic as a stoichiometric superoxide scavenger. Two methodologies, stopped-flow kinetic analysis and pulse radiolysis, are fast methods that will measure SOD mimic catalytic behavior. These methods are briefly described in reference 11 and in Section 3.7.2 of Chapter 3. 

The production of ethylene from methional (3-thiomethylpropanal) was induced by the oxidation of xanthine by dioxygen catalysed by xanthine oxidase The second-order rate constant for the reaction of hydroxyl radicals with methional was estimated by pulse radiolysis to amount to 8.2 x lO s while the superoxide anion reacted more slowly The short lag period of the ethylene production induced by the oxidation of xanthine could be overcome by the addition of small amounts of hydrogen peroxide. The reaction was inhibited by SOD or by catalase, and by scavengers of hydroxyl radicals, so that the Haber-Weiss reaction was implicated... 

Hille, R., and Anderson, R. F., 1991, Electron Transfer in Milk Xanthine Oxidase as Studied by Pulse Radiolysis, J. Biol. Chem. 266 5608n5615. 

As stated above, there was a rapid realization among different groups that pulse radiolysis was an ideal method to examine the details of the enzyme function. This is because the natural substrate, superoxide, is one of the secondary radicals that can be formed rapidly and efficiently upon the radiolysis of water, as discussed in Sec. 2, over a very wide pH range. The classic method for superoxide generation, using the xanthine/xanthine oxidase system, has pH range... 

In 1993 Weiss, Riley, and co-workers reported a study on purported SOD mimics by stopped-flow UV-vis spectroscopy (428) in which they assessed reactivity by following the decay of the superoxide absorption at 245 nm. Two of the earlier techniques that had been used to assess SOD activity included observation by UV-vis spectroscopy of the oxidation of nitroblue tetrazolium (NBT) (68) or the oxidation of a cytochrome c by superoxide (52). Both systems used superoxide from an in situ generator, frequently xanthine oxidase, wherein the complex being analyzed was compared to a calibrated oxidation of the chromophore alone and in the presence of MnSOD. The direct observation of the decrease in the superoxide signal with time by UV-vis is also possible, and superoxide may be introduced as a solution (428) or generated, in some cases, by pulsed radiolysis (79, 80). In these direct observation experiments, the rate of decay of superoxide in the presence of the complex is compared to the rates of decay of superoxide alone and in the presence of one unit of activity of MnSOD. In all cases, the systems are usually referenced, or calibrated, against the same set of conditions with MnSOD. Due to interactions with cytochrome c with components of assay mixtures other than superoxide, false readings of activity were often observed for some early SOD mimics. The NBT, stopped-flow, or pulsed radiolysis techniques have tended to provide the more accurate answers on the ability of reputed MnSOD mimics. To be considered active in any manner with respect to the decay of superoxide in the stopped-flow analyses, Weiss et al. stated that compounds based on their analyses needed to exhibit kcat values in excess of 10B 5 M 1 s 1 (428). 

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,... 

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-... 

Several quinones are used clinically in the chemotherapy of cancer [61,62]. Some examples are adriamycin (doxorubicin), daunomycin (daunorubicin), mitomycin C and more recent diaziridinyl benzoquinones and diamino anthraquinones [5]. Physiological enzyme based reduction of these quinones caused by xanthine oxidase, cytochrome P450 reductase etc.[63], leads to the formation of semiquinone and hydroquinone forms. Pulse radiolysis can generate and characterise these intermediates and products [10]. 

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. 

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