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Reactions of active oxygen and nitrogen species studied by EPR and spin trapping
A J Carmichael1, L Steel-Goodwin, B Gray
1Radiation Biophysics Department, Armed Forces Radiobiology Research Institute, Bethesda, Maryland 20889-5603.
Abstract:
The reactions of hydrogen peroxide (H2O2) with nitrite (NO2-) and of superoxide (O2-.) with nitric oxide (NO.) were studied using EPR and spin trapping techniques. These reactions reportedly have a common peroxynitrite (OONO-) intermediate. It has been suggested that this intermediate when protonated rapidly decomposes producing hydroxyl radicals (.OH) and the nitrogen dioxide radical (NO2.). The production of .OH in the reaction between H2O2 and NO2- was confirmed in spin trapping experiments using the spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). H2O2 and NO2- were mixed at neutral pH and then the pH was decreased to pH 3-3.5 in the presence of DMPO or DMPO and ethanol. In these experiments, the EPR spectrum of the DMPO-OH adduct was obtained in addition to a weak EPR spectrum consisting of a triplet of triplets (a N = 1.415 mT and a N beta = 0.35 mT) indicating the addition of a nitrogen centered radical to DMPO. The formation of .OH was confirmed using ethanol as an .OH scavenger. The DMPO-hydroxyethyl adduct was produced from the reaction of .OH with ethanol. However, in experiments using an excess of ethanol, the formation of DMPO-OH was not prevented. This suggests that the DMPO-OH formed in the decomposition of HOONO does not entirely originate from a direct addition of .OH to DMPO. The reaction of O2-. with NO. was carried out in deaerated and air-saturated solutions at pH 12.3 where the dismutation of O2-. is minimal. The pH was then decreased to pH 3-3.5 in the presence of DMPO or DMPO and ethanol. In these experiments, the most prominent EPR spectrum obtained was a triplet of triplets (aN = 1.415 mT and a beta N = 0.35 mT) suggesting the addition of a nitrogen centered radical to DMPO. The formation of DMPO-OH was minimal and there was no formation of DMPO-hydroxyethyl adducts in the presence of ethanol. The results suggest that NO. in solution yields additional reactive species which act as nitrating agents in the presence of DMPO.