Kinetics of superoxide-induced exchange among nitroxide antioxidants and their oxidized and reduced forms

Nitroxide stable radicals generally serve for probing molecular motion in membranes and whole cells, transmembrane potential, intracellular oxygen and pH, and are tested as contrast agents for magnetic resonance imaging. Recently nitroxides were found to protect against oxidative stress. Unlike most low molecular weight antioxidants (LMWA) which are depleted while attenuating oxidative damage, nitroxides can be recycled. In many cases the antioxidative activity of nitroxides is associated with switching between their oxidized and reduced forms. In the present work, superoxide radicals were generated either radiolytically or enzymatically using hypoxanthine/xanthine oxidase. Electron paramagnetic resonance (EPR) spectrometry was used to follow the exchange between the nitroxide radical and its reduced form; whereas, pulse radiolysis was employed to study the kinetics of hydroxylamine oxidation. The results indicate that: a) The rate constant of superoxide reaction with cyclic hydroxylamines is pH-independent and is lower by several orders of magnitude than the rate constant of superoxide reaction with nitroxides; b) The oxidation of hydroxylamine by superoxide is primarily responsible for the non-enzymatic recycling of nitroxides; c) The rate of nitroxides restoration decreases as the pH decreases because nitroxides remove superoxide more efficiently than is hydroxylamine oxidation; d) The hydroxylamine reaction with oxidized nitroxide (comproportionation) might participate in the exchange among the three oxidation states of nitroxide. However, simulation of the time-dependence and pH-dependence of the exchange suggests that such a comproportionation is too slow to affect the rate of non-enzymatic nitroxide restoration. We conclude that the protective activity of nitroxides in vitro can be distinguished from that of common LMWA due to hydroxylamine oxidation by superoxide, which allows nitroxide recycling and enables its catalytic activity.