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Molecular & Cellular Proteomics 5:215-225, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

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

Jonathan P. Brennan, Jonathan I. A. Miller, William Fuller, Robin Wait^¶, Shajna Begum^¶, Michael J. Dunn^|| and Philip Eaton^,**

From the Departments of Cardiology and Cardiac Physiology, Cardiovascular Division, King’s College London, The Rayne Institute, St. Thomas’ Hospital, London SE1 7EH, United Kingdom, ^¶ Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College London, 1 Aspenlea Road, Hammersmith, London W6 8LH, United Kingdom, and ^|| Proteome Research Centre, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland

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.

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