New insights into the S-nitrosothiol-ascorbate reaction. The formation of nitroxyl.

Although the ascorbate-dependent reduction of S-nitrosothiol to the corresponding thiol function is frequently used for analyzing S-nitrosocysteinyl residues in proteins, it proceeds with low yields. Our re-investigation of the ascorbate-S-nitrosothiol reaction demonstrates now the intermediacy of nitroxyl (HNO/3NO-) that is highly effective in oxidizing thiols. The occurrence of the HNO reporter molecule, i.e., N2O, during and after reaction was unequivocally demonstrated by 15N NMR. The yield of HNO from the S-nitrosoglutathione-ascorbate reaction was determined with the aid of a Mn(III)-complex to 60%, a value that was significantly higher than the one of nitric oxide formation (48%) at physiological pH. The same HNO yield was observed with a S-nitrosothiol bound to a protein (i.e., S-nitroso-papain). With the known chemistry of nitroxyl, it was possible to optimize the experimental conditions so that the GSNO-ascorbate reaction yielded stoichiometric amounts of glutathione after a reaction period of 1 min.