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  2. Conversion of S-phenylsulfonylcysteine residues to mixed disulfides at pH 4.0: utility in protein thiol blocking and in protein-S-nitrosothiol detection

Conversion of S-phenylsulfonylcysteine residues to mixed disulfides at pH 4.0: utility in protein thiol blocking and in protein-S-nitrosothiol detection

  • Org Biomol Chem. 2014 Oct 28;12(40):7942-56. doi: 10.1039/c4ob00995a.
B D Reeves 1 N Joshi G C Campanello J K Hilmer L Chetia J A Vance J N Reinschmidt C G Miller D P Giedroc E A Dratz D J Singel P A Grieco
Affiliations

Affiliation

  • 1 Department of Chemistry and Biochemistry, Montana State University, PO Box 173400, Bozeman, MT 59717-3400, USA. grieco@chemistry.montana.edu.
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.

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