Oxidative damage to catalase induced by peroxyl radicals: functional protection by melatonin and other antioxidants.

Thermal decomposition by the azo initiator 2,2' azobis(2-amidinopropane) dihydrochloride (AAPH) has been widely used as a water-soluble source of free radical initiators capable of inducing lipid peroxidation and protein damage. Here, in a lipid-free system, AAPH alone (40 mM) rapidly induced protein modification and inactivation of the enzyme catalase (EC 1.11.1.6). Using SDS-PAGE, it was shown that protein band intensity is dramatically reduced after 4 h of incubation with AAPH, leading to protein aggregation. Several antioxidants including melatonin, glutathione (GSH) and trolox prevented catalase modification when used at a 250 microM concentration whereas ascorbate was only effective at 1 mM concentration. All the antioxidants tested reduced carbonyl formation although melatonin was the most effective in this regard. Enzyme inactivation caused by AAPH was also significantly reduced by the antioxidants and again melatonin was more efficient than the other antioxidants used in this study. Results shown here demonstrate that alkyl peroxyl radicals inactivate catalase and reduce the effectiveness of cells to defend against free radical damage; the damage to catalase can be prevented by antioxidants, especially melatonin.