AN SOD-MIMICRY MECHANISM UNDERLIES THE ROLE OF NITROXIDES IN PROTECTING PAPAIN FROM OXIDATIVE INACTIVATION

Nitroxide stable free radicals have previously been found to afford pr otection in various biological systems against diverse types of oxidat ive stress, including, ischemia/reperfusisn, hyperoxia, mechanical tra uma, toxic xenobiotics, ionizing radiation, gastric and colonic irrita nts or strong oxidants, Dismutation of superoxide has originally been suggested to be one of the mechanisms that underlie the anti-oxidant e ffect of nitroxides. However, no direct evidence has been found, so fa r, to support this assumption. In the present study, superoxide and H2 O2, generated enzymatically, were used to directly inactivate papain, a sulfhydryl enzyme, in vitro. The rate of papain inactivation served to assess the damage. The reaction mixtures contained a chelate in ord er to prevent the effect of adventitious redox-active metal ions, pre- empt the Fenton reaction and avoid hydroxyl-induced damage. Catalase o r SOD alone partially protected the papain from inactivation. The prot ective effect of nitroxides resembled that of SOD in several aspects: a) nitroxides provided partial protection; b) the protective effect of nitroxides did not increase with the elevation of their concentration (above 0.5 mM); c) the combined addition of SOD and the nitroxide did not provide greater protection than that demonstrated by nitroxides o r SOD separately; d) the effects of catalase with the nitroxide were a dditive; e) the nitroxide, like SOD itself, did not protect papain fro m H2O2-induced inactivation; f) the nitroxide was found not to be cons umed in the course of the reaction but rather to be recycled. The resu lts indicate that: (a) the main species responsible for the papain ina ctivation in a system in which the effect of transition metals is pre- empted, are O2(.-) and H2O2; (b) nitroxides inhibit the oxidative dama ge by removing superoxide not stoichiometrically, but rather catalytic ally as SOD-mimics; (c) nitroxides do not afford protection when the o xidative damage is induced directly by H2O2 land not mediated by redox -active metals). (C) 1998 Elsevier Science Inc.