title: Conversion of S-phenylsulfonylcysteine residues to mixed disulfides at pH 4.0: utility in protein thiol blocking and in protein-S-nitrosothiol detection.
abstract: A three step protocol for protein S-nitrosothiol conversion to fluorescent mixed disulfides with purified proteins, referred to as the thiosulfonate switch, is explored which involves: (1) thiol blocking at pH 4.0 using S-phenylsulfonylcysteine (SPSC); (2) trapping of protein S-nitrosothiols as their S-phenylsulfonylcysteines employing sodium benzenesulfinate; and (3) tagging the protein thiosulfonate with a fluorescent rhodamine based probe bearing a reactive thiol (Rhod-SH), which forms a mixed disulfide between the probe and the formerly S-nitrosated cysteine residue. S-Nitrosated bovine serum albumin and the S-nitrosated C-terminally truncated form of AdhR-SH (alcohol dehydrogenase regulator) designated as AdhR*-SNO were selectively labelled by the thiosulfonate switch both individually and in protein mixtures containing free thiols. This protocol features the facile reaction of thiols with S-phenylsulfonylcysteines forming mixed disulfides at mild acidic pH (pH = 4.0) in both the initial blocking step as well as in the conversion of protein-S-sulfonylcysteines to form stable fluorescent disulfides. Labelling was monitored by TOF-MS and gel electrophoresis. Proteolysis and peptide analysis of the resulting digest identified the cysteine residues containing mixed disulfides bearing the fluorescent probe, Rhod-SH.
authors.affiliation: Department of Chemistry and Biochemistry, Montana State University, PO Box 173400, Bozeman, MT 59717-3400, USA. grieco@chemistry.montana.edu.
authors.forename: B D
authors.initials: BD
authors.lastname: Reeves
authors.forename: N
authors.initials: N
authors.lastname: Joshi
authors.forename: G C
authors.initials: GC
authors.lastname: Campanello
authors.forename: J K
authors.initials: JK
authors.lastname: Hilmer
authors.forename: L
authors.initials: L
authors.lastname: Chetia
authors.forename: J A
authors.initials: JA
authors.lastname: Vance
authors.forename: J N
authors.initials: JN
authors.lastname: Reinschmidt
authors.forename: C G
authors.initials: CG
authors.lastname: Miller
authors.forename: D P
authors.initials: DP
authors.lastname: Giedroc
authors.forename: E A
authors.initials: EA
authors.lastname: Dratz
authors.forename: D J
authors.initials: DJ
authors.lastname: Singel
authors.forename: P A
authors.initials: PA
authors.lastname: Grieco
mesh_terms: D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*; D004220:Disulfides / Q000737:chemistry* / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D015394:Molecular Structure; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000032:analysis* / Q000037:antagonists & inhibitors*
chemical_list: D004220:Disulfides; D013438:Sulfhydryl Compounds; D012710:Serum Albumin, Bovine; D000426:Alcohol Dehydrogenase; D003545:Cysteine