Oxidative modification and inactivation of Cu,Zn-superoxide dismutase by 2,2′-azobis(2-amidinopropane) dihydrochloride

We have investigated oxidative modification of human Cu,Zn-superoxide dismutase (SOD) by alkylperoxyl radicals and alkylperoxides. To generate free radicals, we used the hydrophilic azocompound, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). When Cu,Zn-SOD was incubated with AAPH, the enzyme activity was decreased gradually in a time-dependent manner. The oxidative damage to Cu,Zn-SOD by AAPH-derived radicals led to protein fragmentation which is associated with the inactivation of enzyme. Incubation with AAPH resulted in the release of copper ions from Cu,Zn-SOD and the generation of protein carbonyl derivatives. Catalase did not protect the fragmentation of Cu,Zn-SOD whereas azide, glutathione and a metal chelator, diethylenetriamine pentaacetic acid inhibited the protein fragmentation. When Cu,Zn-SOD that has been exposed to AAPH was subsequently analyzed by amino acid analysis, lysine, histidine, proline, and valine residues were particularly sensitive. It is suggested that oxidative damage of Cu,Zn-SOD by AAPH-derived radicals may induce the perturbation of cellular antioxidant defense systems and subsequently lead to the deleterious condition in cells. (C) 2000 Elsevier Science B.V. All rights reserved.