A quantitative test for superoxide radicals produced in biological systems.
The preparation and properties of a partially succinoylated cytochrome c, suited for the detection of superoxide anion radicals in liver microsomes, is reported. By succinoylation of 45% of the primary amino groups of horse heart cytochrome c the activity towards solubilized NADPH--cytochrome P-450 reductase was diminished by 99% compared with native cytochrome c. The capacities of cytochrome b5 and cytochrome c oxidase to reduce the succinoylated ferricytochrome c and oxidize succinoylated ferrocytochrome c respectively were decreased to a similar extent. However, the bimolecular rate constant for the reduction of the partially succinoylated ferricytochrome c by O2-. was estimated to be one-tenth of the value for the reaction of O2-. with native ferricytochrome c a pH 7.7. On this basis the quantification of O2-. generated by NADPH-supplemented liver microsomes became possible. The initial rates of succinoylated ferricytochrome c reduction determined at various finite concentrations of the cytochrome c derivative can be extrapolated to obtain true rates of O2-. generation in a homogeneous system. The problems encountered in the quantitative determination of O2-. produced  in biological membranes, e.g. microsomes, are discussed.
D000818:Animals; D003574:Cytochrome c Group / Q000378:metabolism; D005609:Free Radicals; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008722:Methods; D008862:Microsomes, Liver / Q000032:analysis; D010084:Oxidation-Reduction; D010100:Oxygen / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000032:analysis*; D014969:Xanthine Oxidase / Q000378:metabolism
D003574:Cytochrome c Group; D005609:Free Radicals; D013481:Superoxides; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D010100:Oxygen