Rocket fuel for the quantification of S-nitrosothiols. Highly specific reduction of S-nitrosothiols to thiols by methylhydrazine

Abstract

Reduction of S-nitrosothiols to the corresponding thiol function is the key step in analyzing S-nitrosocysteinyl residues in proteins. Though it has been shown to give low yields, ascorbate-dependent reduction is commonly performed in the frequently used biotin-switch technique. We demonstrate that the compound methylhydrazine can act as a specific and efficient reducing agent for S-nitrosothiols. The corresponding thiol function is exclusively generated from low molecular weight and proteinaceous S-nitrosothiols while methylhydrazine failed to reduce disulfides. It was possible to optimize the experimental conditions so that thiol autoxidation is excluded, and high reaction yields (> 90%) are obtained for the thiol function. The biotin-switch technique performed with methylhydrazine-dependent reduction shows remarkably improved sensitivity compared to the ascorbate-dependent procedure.