Spin-Trapping of Free Radicals Formed during the Oxidation of Glutathione by Tetramethylammonium Peroxynitrite

Abstract

Glutathionyl radical (GS•) formed during the oxidation of glutathione by tetramethylammonium peroxynitrite ([NMe4][ONOO]) was spin-trapped with 5,5′-dimethyl-1-pyrrolineN-oxide (DMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrrolineN-oxide (DEPMPO). This radical reacted with ammonium formate to form the carbon dioxide anion radical (CO•−2). The superoxide anion formed during oxidation of GSH by peroxynitrite salt was trapped with DMPO and detected as the DMPO-hydroxyl adduct. Addition of SOD mimic completely abolished the spectrum of the hydroxyl adduct but not the spectrum of the DMPO-glutathionyl radical adduct. Addition of seleno-dl-cystine or its reduced form caused a dramatic inhibition in the formation of spin adducts, suggesting that seleno-dl-cysteine is a more effective scavenger of peroxynitrite. The oxygen uptake observed during oxidation of GSH by peroxynitrite salt was inhibited by spin traps. In the presence of catalase, approximately 50% of the oxygen consumed was restored, indicating stoichiometric conversion of O2to H2O2during oxidation of GSH by peroxynitrite salt. Results indicate that nitrite and glutathione disulfide are formed as the major products during oxidation of GSH by peroxynitrite.