The Utility of N,N-Biotinyl Glutathione Disulfide in the Study of Protein S-Glutathiolation

• Published in: Molecular & cellular proteomics Vol. 5; no. 2; pp. 215 - 225
• Main Authors: Brennan, Jonathan P, Miller, Jonathan I A, Fuller, William, Wait, Robin, Begum, Shajna, Dunn, Michael J, Eaton, Philip
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 01-02-2006
• Subjects: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Biotinylation; Glutathione Disulfide - chemistry; Male; Molecular Sequence Data; Myocytes, Cardiac - metabolism; Proteins - analysis; Proteins - chemistry; Proteins - isolation & purification; Rats; Rats, Wistar
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.M500212-MCP200

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.