Widespread sulfenic acid formation in tissues in response to hydrogen peroxide.
A principal product of the reaction between a protein cysteinyl thiol and hydrogen peroxide is a protein sulfenic acid. Because protein sulfenic acid formation is reversible, it provides a mechanism whereby changes in cellular hydrogen peroxide concentration may directly control protein function. We have developed methods for the detection and purification of proteins oxidized in this way. The methodology is based on the arsenite-specific reduction of protein sulfenic acid under denaturing conditions and their subsequent labeling with biotin-maleimide. Arsenite-dependent signal generation was fully blocked by pretreatment with dimedone, consistent with its reactivity with sulfenic acids to form a covalent adduct that is nonreducible by thiols. The biotin tag facilitates the detection of protein sulfenic acids on Western blots probed with streptavidin-horseradish peroxidase and also their purification by streptavidin-agarose. We have characterized protein sulfenic acid formation in isolated hearts subjected to hydrogen peroxide treatment. We have also purified and identified a number of the proteins that are oxidized in this way by using a proteomic approach. Using Western immunoblotting we demonstrated that a highly significant proportion of some individual proteins (68% of total in one case) form the sulfenic derivative. We conclude that protein sulfenic acids are widespread physiologically relevant posttranslational oxidative modifications that can be detected at basal levels in healthy tissue, and are elevated in response to hydrogen peroxide. These approaches may find widespread utility in the study of oxidative stress, particularly because hydrogen peroxide is used extensively in models of disease or redox signaling.
Saurin
Adrian T
AT
Department of Cardiology, Cardiovascular Division, The Rayne Institute, St. Thomas' Hospital, King's College London, London SE1 7EH, United Kingdom.
Neubert
Hendrik
H
Brennan
Jonathan P
JP
Eaton
Philip
P
D000818:Animals; D018053:Arsenites / Q000737:chemistry; D001671:Biochemistry / Q000379:methods; D001710:Biotin / Q000737:chemistry; D015153:Blotting, Western; D003512:Cyclohexanones / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000494:pharmacology*; D008297:Male; D008301:Maleimides / Q000737:chemistry; D013058:Mass Spectrometry; D008954:Models, Biological; D009206:Myocardium / Q000378:metabolism / Q000473:pathology; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010455:Peptides / Q000737:chemistry; D010477:Perfusion; D011499:Protein Processing, Post-Translational; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D013434:Sulfenic Acids / Q000737:chemistry* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry
D018053:Arsenites; D003512:Cyclohexanones; D008301:Maleimides; D010455:Peptides; D013434:Sulfenic Acids; D013438:Sulfhydryl Compounds; C043592:maleimide; D001710:Biotin; C004774:dimedone; D006861:Hydrogen Peroxide; C015001:arsenite