The study of oxidative stress has attracted considerable interest and has been the focus of much research in recent years. Cumulative oxidative damage to tissues has been implicated in a number of disease states, e.g. the aging process, cancer, and ischemia reperfusion. The study of oxidative stress in the mitochondria has shown that hydrogen peroxide is produced via the incomplete reduction of oxygen during oxidative phosphorylation. Hydrogen peroxide levels are kept relatively low under normal physiological conditions. Under certain conditions, such as inflammation, excessive amounts of hydrogen peroxide are produced. The production of excess hydrogen peroxide is thought to precede several occurrences, such as lipid peroxidation, DNA and/or protein damage, and glutathione depletion, that are characteristic of oxidative stress. We have begun to probe the role of cytochrome c free-radicals in oxidative stress by probing the anaerobic reaction between horse heart cytochrome c (HHCC) and hydrogen peroxide using the spin-trapping reagent 3,5- dibromo-4-nitrosobenzenesulfonic acid (DBNBS). The ESR spectrum of the reaction showed the presence of one major free-radical product with possibly a minor species also being formed, while the MS clearly showed the presence of at least four distinct DBNBS adducts. The identity of these adducts is being determined via isolation, proteolysis, and MALDI/MS identification. We have also begun to investigate the possibility that the oxidatively-induced free-radical on HHCC can initiate free-radical oxidation of other proteins that do not, by themselves, undergo ree-radical oxidation under identical reaction conditions. Initial results indicate that proteins such as Rnas A, oxidized insulin B and renin substrate tetradecapeptide, are readily oxidized by the HHCC free-radical. Proteolysis combined with MALDI/MS indicates that the DBNBS is located on a tyrosine residue of oxidized insulin B.
