Reactions Using Non-Enzymically Prepared

Or From Tetrabutyl-Ammonium Superoxide. Tetrabutyl-ammonium superoxide was found convenient for the superoxide dismutase assay since it dissolved readily without decomposition in N,N-dimethyl form-amide (70). Infusions of this solution into a cuvette containing aqueous oxidized cytochrome c reduced the available cytochrome c. In the presence of different erythrocuprein concentrations the reduction rate was progressively diminished. According to McCord and Fridovich (70) an enzyme unit was defined as 50% inhibition of the rate of reduction of cytochrome c. Alternatively tetranitromethane was found appropriate for monitoring Or (70, 136—148) where the stable nitroform anion C(N02)3 is being formed (Equ. (a))  

Using this reaction, McCord and Fridovich evaluated the rate constant for the reaction of superoxide dismutase with Or as approximately 5 X 1011 M 1 sec 1. The corresponding value for the spontaneous disproportionation of Oi- near neutral / H (Eq. (b)) is assumed to be 8.5 x 107 M 1 sec 1 (second-order rate constant) (139, 140). Fridovich et al. concluded from these data that erythrocuprein accelerates the Or disproportionation by 3—4 orders of magnitude. 

Or Prepared by Pulse Radiolysis. Pulse radiolysis proved most convenient to prepare Or in relatively high concentrations without the use of dimethylsulphoxide or N,N-dimethyl formamide as possible interfering solvents (139, 182, 193, 194). Moreover, the time resolution was as low as 1, itsec which allowed the determination of the kinetics and mechanism of the enzyme. Oxygen-saturated aqueous solutions containing trace amounts of ethanol (85 mM) as a scavenger for OH radicals were irradiated by 4 MeV electrons. The Oi- concentration was monitor- 

Oi- Generated by Flavin-dependent OzActivation. It was shown above that artificially produced Oi- is a convenient tool for the study, of the enzymic reaction of erythrocuprein in a simple model system avoiding undesired interference from other sources. However, Oi- formation, including all known intermediate states, should also be studied in the light of its possible biological relevance. Thus, attention will be focused upon the formation of Oi- using a biochemically most important compound, namely the flavin moiety (for comprehensive studies see Refs. 183-185). 

Experimental evidence for the participation of the flavin moiety in Oi- formation in nonenzymic reactions was presented by Ballou et al. (151). During the oxidation of different reduced flavins by molecular oxygen substantial yields of Oi- were obtained. Tetra-acetyl riboflavin proved most appropriate as a model compound (153). The respective EPR parameters for Oi- were in close agreement with those obtained in the KIO4—H2O2 system, in the xanthine oxidase reaction (149), and in the oxidation of the reduced tetra-acetyl riboflavin. They also agree well with the EPR data obtained earlier (154—156). The time course of the appearance and decay of Oi- produced by the reaction of tetra-acetyl riboflavin is shown in Fig. 24. 

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