The utility of N,N-biotinyl glutathione disulfide in the study of protein S-glutathiolation.
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.
Brennan
Jonathan P
JP
Cardiovascular Division, Department of Cardiology, King's College London, The Rayne Institute, St. Thomas' Hospital, London SE1 7EH, United Kingdom.
Miller
Jonathan I A
JI
Fuller
William
W
Wait
Robin
R
Begum
Shajna
S
Dunn
Michael J
MJ
Eaton
Philip
P
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D019921:Biotinylation; D019803:Glutathione Disulfide / Q000737:chemistry*; D008297:Male; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000378:metabolism; D011506:Proteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D051381:Rats; D017208:Rats, Wistar
D000911:Antibodies, Monoclonal; D011506:Proteins; D019803:Glutathione Disulfide