SPIN-TRAPPING STUDIES OF PEROXYNITRITE DECOMPOSITION AND OF 3-MORPHOLINOSYDNONIMINE N-ETHYLCARBAMIDE AUTOOXIDATION - DIRECT EVIDENCE FOR METAL-INDEPENDENT FORMATION OF FREE-RADICAL INTERMEDIATES

Decomposition of peroxynitrite, the reaction product of superoxide and nitric oxide, was studied by electron paramagnetic resonance (EPR)-sp in-trapping experiments with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Proton-catalyzed decomposition of peroxynitrite at pH 7.5 resulted in the formation of the DMPO-hydroxyl radical adduct (DMPO-OH). Yields we re low as DMPO-OH decomposes by direct reactions with peroxynitrite an ion and nitrogen dioxide. The yield of DMPO-OH greatly increased in th e presence of glutathione or cysteine. Both thiols inhibited the DMPO- OH signal decay by scavenging excess peroxynitrite anion and presumabl y nitrogen dioxide yielded during peroxynitrite decomposition. In turn , the reactions of peroxynitrite with either glutathione or cysteine r esulted in the formation of thiyl radicals, detectable as the correspo nding DMPO adduct. Systematic spin-trapping studies of peroxynitrite d ecomposition in the presence of glutathione established that DMPO-hydr oxyl radical adduct formation was metal independent, occurring in a me tal-free buffer and being unaffected by diethylene-triaminepentaacetic acid. Also, quantitative competition experiments with ethanol and for mate demonstrated that the oxidant generated during peroxynitrite deco mposition reacts with rate constants similar to those expected for fre e hydroxyl radical and forming the same free radical intermediates, al pha-ethyl-hydroxy and carbon dioxide radicals, respectively. Similar s pin-trapping results were obtained in studies of the autooxidation of 3-morpholinosydnonimine, a sydnonimine which generates a flux of both superoxide and nitric oxide. The obtained results contribute for the u nderstanding of the reactivity of peroxynitrite, a transient intermedi ate of emerging biological significance. (C) 1994 Academic Press, Inc.