title: 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.
authors.affiliation: Institut für Physiologische Chemie, Universitätsklinikum Essen, Hufelandstrasse 55, 45 122 Essen, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Wiesweg
authors.forename: U
authors.initials: U
authors.lastname: Berchner-Pfannschmidt
authors.forename: J
authors.initials: J
authors.lastname: Fandrey
authors.forename: F
authors.initials: F
authors.lastname: Petrat
authors.forename: H
authors.initials: H
authors.lastname: de Groot
authors.forename: M
authors.initials: M
authors.lastname: Kirsch
mesh_terms: D001205:Ascorbic Acid / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D003545:Cysteine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008763:Methylhydrazines / Q000737:chemistry* / Q000378:metabolism; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism; D015538:Nitrosation; D010084:Oxidation-Reduction; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D026403:S-Nitrosothiols / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism
chemical_list: D008763:Methylhydrazines; D011506:Proteins; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D026403:S-Nitrosothiols; D013438:Sulfhydryl Compounds; D009569:Nitric Oxide; D001710:Biotin; C021315:S-nitrosocysteine; D005978:Glutathione; D003545:Cysteine; D001205:Ascorbic Acid