Generation of thiyl radical by nitric oxide: a spin trapping study

Abstract

The free radical intermediates formed during the nitrosation of thiols including glutathione (GSH), L-cysteine (CystSH), captopril (CapSH) and N-acetylpenicillamine (NAPenSH) by NO˙ at physiological pH have been investigated using EPR spectroscopy combined with the spin trap DMPO. We have found that NO˙ in the presence of oxygen reacted with GSH, CystSH and CapSH to generate the corresponding thiyl radicals, which are spin trapped by DMPO to give the spin trapped adducts DMPO-SG, DMPO-SCyst and DMPO-SCap respectively. In the case of NAPenSH no spin trapped adduct is detected. Desferrioxamine, an inhibitor of peroxynitrite (ONOO^–), has no effect on the formation of either DMPO-SG or DMPO-SCyst, suggesting that ONOO^– is not involved in the formation of GS˙ and Cyst˙. In contrast, desferrioxamine inhibits the detection of DMPO-SCap, pointing to a role for ONOO^– in the formation of CapS˙. Our results demonstrate that ˙NO₂ is the most likely reactive species in the nitrosation of GSH and CystSH by oxygenated NO˙, thereby generating the corresponding thiyl radicals. Furthermore, our data indicate that the scavenging of NO˙ by thiols may lead to the production of superoxide (O₂^˙–) via the formation of thiyl radicals.