Discriminating formation of HNO from other reactive nitrogen oxide species

Nitroxyl (HNO) exhibits unique pharmacological properties that often oppose those of nitric oxide (NO), in part due to differences in reactivity toward thiols. Prior investigations suggested that the end products arising from the association of HNO with thiols were condition-dependent, but were inconclusive as to product identity. We therefore used HPLC techniques to examine the chemistry of HNO with glutathione (GSH) in detail. Under biological conditions, exposure to HNO donors converted GSH to both the sulfinamide [GS(O)NH2] and the oxidized thiol (GSSG). Higher thiol concentrations generally favored a higher GSSG ratio, suggesting that the products resulted from competitive consumption of a single intermediate (GSNHOH). Formation of GS(O)NH2 was not observed with other nitrogen oxides (NO, N2O3, NO2, or ONOO−), indicating that it is a unique product of the reaction of HNO with thiols. The HPLC assay was able to detect submicromolar concentrations of GS(O)NH2. Detection of GS(O)NH2 was then used as a marker for HNO production from several proposed biological pathways, including thiol-mediated decomposition of S-nitrosothiols and peroxidase-driven oxidation of hydroxylamine (an end product of the reaction between GSH and HNO) and NG-hydroxy-l-arginine (an NO synthase intermediate). These data indicate that free HNO can be biosynthesized and thus may function as an endogenous signaling agent that is regulated by GSH content.