A Flash-Photometric Method for Determination of Reactivity of Superoxide: Application to Superoxide Dismutase Assay

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Abstract
Pulse-generation of O 2− by a flash was used to determine the reactivity of O 2− . O 2− 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 O 2− generated by flash yielded the reaction rate constant between cytochrome c and O 2− and the spontaneous disproportionation rate constant of O 2− . 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 O 2− . The catalytic rate constant and activation energy at pH 7.3 of bovine liver Cu,Zn-superoxide dismutase were found to be 1.75 × 10 9 M −1 ·S −1 at 25°C and 26.9 kJ·M −1 , respectively. The kinetics of O 2− decay can be also monitored at 240 nm in this flash-photometric system and gave the spontaneous disproportionation rate constant of O 2− and the catalytic rate constant of superoxide dismutase.