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)-spin-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 were low as DMPO-OH decomposes by direct reactions with peroxynitrite anion and nitrogen dioxide. The yield of DMPO-OH greatly increased in the presence of glutathione or cysteine. Both thiols inhibited the DMPO-OH signal decay by scavenging excess peroxynitrite anion and presumably nitrogen dioxide yielded during peroxynitrite decomposition. In turn, the reactions of peroxynitrite with either glutathione or cysteine resulted in the formation of thiyl radicals, detectable as the corresponding DMPO adduct. Systematic spin-trapping studies of peroxynitrite decomposition in the presence of glutathione established that DMPO-hydroxyl radical adduct formation was metal independent, occurring in a metal-free buffer and being unaffected by diethylene-triaminepentaacetic acid. Also, quantitative competition experiments with ethanol and formate demonstrated that the oxidant generated during peroxynitrite decomposition reacts with rate constants similar to those expected for free hydroxyl radical and forming the same free radical intermediates, alpha-ethyl-hydroxy and carbon dioxide radicals, respectively. Similar spin-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 understanding of the reactivity of peroxynitrite, a transient intermediate of emerging biological significance.