Widespread sulfenic acid formation in tissues in response to hydrogen peroxide

Abstract

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.heart ͉ oxidative stress ͉ myocardium ͉ redox signaling

Oxidoreductase regulation of Kv currents in rat ventricle

“…For example, diamide can directly cause the formation of thiyl radical (-S . ) or thiolate anion (-S − ) intermediates [37], whereas H 2 O 2 forms sulfenic acid (-SOH) intermediates in target proteins [37]. Whether such mechanisms explain the electrophysiological differences between diamide and H 2 O 2 in our study warrants further experimentation on a molecular level.…”

“…2B). Although H 2 O 2 increases GSSG formation via glutathione peroxidase [1,2,11,22,23], it can also directly attack protein thiols to produce oxidized intermediates [37]. Our data thus suggest that the different effects of diamide and H 2 O 2 on I ss may be due to different thiol intermediates produced by these oxidants.…”

Morphology and DNA degeneration during autoschizic cell death in bladder carcinoma T24 cells induced by ascorbate and menadione treatment

“…The plasma membrane peroxidations favor an unregulated increase in intracellular calcium ion levels (Orrenius et al, 1989;Farber et al, 1990;Farber, 1994;Sakagami and Satoh, 1997), which, along with thiol oxidations, poison mitochondria. That damage decreases the output of ATP (Saurin et al, 2004) necessary to maintain the homeostatic cell energy charge (Atkinson, 1968) and to assist in repairing the ongoing damage associated with the peroxidations (Stadtman, 1992). Mitochondrial alterations have already been reported following VC, VK3, and VC:VK 3 treatment (Gilloteaux et al, 1995(Gilloteaux et al, , 2001a(Gilloteaux et al, , 2001b(Gilloteaux et al, , 2001cJamison et al, 1997).…”

“…This cycling process generates intracellular H 2 O 2 and other reactive oxygen species (ROS). While hydrogen peroxide is also known to alter thiol residues of specific macromolecules (Saurin et al, 2004), it primarily initiates numerous membrane lipid peroxidations (Desai and Tappel, 1963;Sawant et al, 1964;Edgar, 1970). The plasma membrane peroxidations favor an unregulated increase in intracellular calcium ion levels (Orrenius et al, 1989;Farber et al, 1990;Farber, 1994;Sakagami and Satoh, 1997), which, along with thiol oxidations, poison mitochondria.…”

Oxidation of proteins: Basic principles and perspectives for blood proteomics

“…The same methodology can be applied for the specific enrichment of oxidised-thiol containing proteins: free thiols are first blocked, oxidised thiols are reduced chemically and further reacted with a probe containing an affinity bait, such as biotin [108][109][110][111][112]. When isotope-coded-affinitytags (ICAT) reagents are used in this way, relative quantification between two differentially oxidised samples can be obtained [113,114].…”