Low-level chemiluminescence of hydroperoxide-supplemented cytochrome c

Abstract

Ferricytochrome c showed low-level chemiluminescence with a light-emission measured of about 1 .times. 103-3 .times. 103 counts/s, when supplemented with organic hydroperoxides. Tertiary hydroperoxides (cumene hydroperoxide and tert-butyl hydroperoxide) showed a saturation behavior at about 5 mM-hydroperoxide, whereas primary hydroperoxides showed a quadratic dependence on the hydroperoxide concentration. Chemiluminescence depended linearly on cytochrome c concentration, and optimal light emisson was observed at [tert-butyl hydroperoxide]/[ferricytochrome c] ratios of 160-500. Hydroperoxide-supplemented ferricytochrome c consumed 02 at a rate of 1.0 .mu.mol/min per .mu.mol of cytochrome c; the rate of 02 uptake was linearly related to concentration of cytochrome c. The Soret absorption band of ferricytochrome c decreased about 64% after incubation with tert-butyl hydroperoxide, whereas the 530 nm band was almost totally abolished. Light emission was inhibited competitively by cyanide, inhibited by singlet-oxygen quenchers (e.g., .beta.-carotene), scavengers (e.g., dimethylfuran) and traps (e.g., histidine and tryptophan) and increased by singlet-oxygen-chemiluminescence enhancer 1,4-diazabicyclo[2.2.2]-octane. Superoxide dismutase had no effect on the present system. The participation of free radicals is suggested by the effect of the radical trap 2,5-di-tert-butylquinol. Singlet-oxygen dimol emission seems to be mainly responsible for the observed light emission; a mechanism that can account for the major part of the present experimental observations is proposed.