A flash-photometric method for determination of reactivity of superoxide: application to superoxide dismutase assay.
Pulse-generation of O2- by a flash was used to determine the reactivity of O2-, O2- was produced within 10 ms by a flash of light through the excitation of FMN in the presence of N,N,N',N'-tetramethylethylenediamine and oxygen. Kinetic analysis of cytochrome c reduction by O2- generated by flash yielded the reaction rate constant between cytochrome c and O2- and the spontaneous disproportionation rate constant of O2-. We applied it for superoxide dismutase assay using a linear relation between superoxide dismutase concentration and the apparent rate constant of cytochrome c reduction by O2-. The catalytic rate constant and activation energy at pH 7.3 of bovine liver Cu,Zn-superoxide dismutase were found to be 1.75 x 10(9) M-1 . s-1 at 25 degrees C and 26.9 kJ . M-1, respectively. The kinetics of O2- decay can be also monitored at 240 nm in this flash-photometric system and gave the spontaneous disproportionation rate constant of O2- and the catalytic rate constant of superoxide dismutase.
D000818:Animals; D002417:Cattle; D003574:Cytochrome c Group / Q000378:metabolism; D005486:Flavin Mononucleotide; D006736:Horses; D007700:Kinetics; D008027:Light; D008099:Liver / Q000201:enzymology; D009206:Myocardium; D010100:Oxygen / Q000032:analysis*; D010782:Photolysis; D013053:Spectrophotometry / Q000379:methods; D013482:Superoxide Dismutase / Q000378:metabolism*; D013481:Superoxides / Q000032:analysis*
D003574:Cytochrome c Group; D013481:Superoxides; D005486:Flavin Mononucleotide; D013482:Superoxide Dismutase; D010100:Oxygen