The utility of N,N-biotinyl glutathione disulfide in the study of protein S-glutathiolation

Brennan, J. P., Miller, J. I.A., Fuller, W. , Wait, R., Begum, S., Dunn, M. J. and Eaton, P. (2005) The utility of N,N-biotinyl glutathione disulfide in the study of protein S-glutathiolation. Molecular and Cellular Proteomics, 5(2), pp. 215-225. (doi: 10.1074/mcp.M500212-MCP200) (PMID:16223748)

Abstract

Glutathione disulfide (GSSG) accumulates in cells under an increased oxidant load, which occurs during neurohormonal or metabolic stimulation as well as in many disease states. Elevated GSSG promotes protein S-glutathiolation, a reversible post-translational modification, which can directly alter or regulate protein function. We developed novel strategies for the study of protein S-glutathiolation that involved the simple synthesis of N,N-biotinyl glutathione disulfide (biotin-GSSG). Biotin-GSSG treatment of cells mimics a defined component of oxidative stress, namely a shift in the glutathione redox couple to the oxidized disulfide state. This induces widespread protein S-glutathiolation, which was detected on non-reducing Western blots probed with streptavidin-horseradish peroxidase and imaged using confocal fluorescence microscopy and ExtrAvidin-FITC. S-Glutathiolated proteins were purified using streptavidin-agarose and identified using proteomic methods. We conclude that biotin-GSSG is a useful tool in the investigation of protein S-glutathiolation and offers significant advantages over conventional methods or antibody-based strategies. These novel approaches may find widespread utility in the study of disease or redox signaling models where GSSG accumulation occurs.

• Item Type: Articles
• Additional Information: The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Fuller, Professor Will
• Authors: Brennan, J. P., Miller, J. I.A., Fuller, W., Wait, R., Begum, S., Dunn, M. J., and Eaton, P.
• College/School: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
• Journal Name: Molecular and Cellular Proteomics
• Publisher: American Society for Biochemistry and Molecular Biology
• ISSN: 1535-9476
• ISSN (Online): 1535-9484