SPIN TRAP STUDIES ON THE DECOMPOSITION OF PEROXYNITRITE

The observation of the hydroxyl radical spin adduct of 5,5-dimethyl-1- pyrroline-N-oxide (DMPO) is evaluated as a probe for the production of hydroxyl radicals from the decomposition of peroxynitrite, Although a weak signal corresponding to the DMPO-hydroxyl radical spin adduct (( .)DMPO-OH) is observed when peroxynitrite is allowed to decompose in t he presence of DMPO, it is concluded that this does not constitute pro of of the presence of free hydroxyl radicals. The observed rate consta nt for the decay of peroxynitrite increases from 0.35 to 0.51 s(-1) (4 6% increase) when the concentration of DMPO is increased from 0 to 75 mM. This strongly suggests there is a reaction between DMPO and HOONO, or between DMPO and an activated intermediate of HOONO, to produce th e hydroxyl radical spin adduct, The addition of glutathione or cystein e produces a large increase in the intensity of the (.)DMPO-OH spin ad duct signal; experiments employing superoxide dismutase suggest that t he increases in the amounts of (.)DMPO-OH adduct are produced from the decomposition of the spin adduct of the superoxide radical ((.)DMPO-O OH). The superoxide adduct arises as a result of the autoxidation of t hiols, a process known to produce superoxide. The results presented he re are incompatible with the formation of free hydroxyl radicals but c an be explained in terms of an intermediate of HOONO that is less reac tive and more selective than the free hydroxyl radical. (C) 1995 Acade mic Press, Inc.