Peroxynitrite (ONOO؊ ) has been shown in studies on vascular relaxation and guanylate cyclase activation to react with glutathione (GSH), generating an intermediate product that promotes a time-dependent production of nitric oxide (NO). In this study, reactions of ONOO ؊ with GSH produced a new substance, which was characterized by liquid chromatography, ultraviolet spectroscopy, and electrospray tandem mass spectrometry. The mass spectrometric data provided evidence that the product of this reaction was S-nitroglutathione (GSNO 2 ) and that S-nitrosoglutathione (GSNO) was not a detectable product of this reaction. Further evidence was obtained by comparison of the spectral and chromatographic properties with synthetic standards prepared by reaction of GSH with nitrosonium or nitronium borofluorates. Both the synthetic and ONOO ؊ /GSH-derived GSNO 2 generated a protonated ion, GSNO 2 H ؉ , at m/z 353, which was unusually resistant to decomposition under collision activation, and no fragmentation was observed at collision energy of 25 eV. In contrast, an ion at m/z 337 (GSNOH ؉ ), generated from the synthetic GSNO, readily fragmented with the abundant loss of NO at 9 eV. Reactions of ONOO ؊ with GSH resulted in the generation of NO, which was detected by the head space/ NO-chemiluminescence analyzer method. The generation of NO was inhibited by the presence of glucose and/or CO 2 in the buffers employed. Synthetic GSNO 2 spontaneously generated NO in a manner that was not significantly altered by glucose or CO 2 . Thus, ONOO ؊ reacts with GSH to form GSNO 2 , and GSNO 2 decomposes in a manner that generates NO.Exposure of vascular tissue to peroxynitrite (ONOO Ϫ ) 1 results in a prolonged relaxation (1) that appears to be mediated through a glutathione (GSH)-dependent regeneration of NO (2). Peroxynitrite has also been observed to stimulate guanylate cyclase activity in a thiol-dependent manner in vascular endothelial and smooth muscle preparations (3, 4). Whereas the reaction of ONOO Ϫ with GSH has been reported to form small amounts of S-nitroso-GSH (GSNO) (3, 5), our previous studies detected a different product of this reaction, which was isolated and demonstrated to possess potent vascular relaxant activity (2). Examination of the reaction of nitrogen dioxide (NO 2 ) with GSH detected the formation of what appears to be the same product as that observed in the reaction with ONOO Ϫ (6). Because the biologically active metabolite of these reactions co-migrated on HPLC with a the product of a reaction between nitrosonium borofluorate (NO 2 BF 4 ) and GSH, the vascular relaxant detected was suggested to be a nitrated product of GSH (GSNO 2 ) (6). Thus, additional studies are needed to identify the biologically active substances derived from the reaction of GSH with ONOO Ϫ . Peroxynitrite is also known to undergo additional reactions in the presence of physiological buffered systems and GSH. One of the first observed actions of ONOO Ϫ on thiols was that it caused oxidation reactions, and an analysis of products o...
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