The amino acid cysteine has unique chemical properties, endowing it with the ability to engage in a wild variety of redox reactions and to coordinate metals. These properties make cysteine a key residue in enzymatic catalysis, protein oxidative folding and trafficking, reactive oxyten species (ROS) and reactive nitrogen species (RNS) sensing and signaling (1-3). Its unique properties also make this residue vulnerable to the reaction with and modifications by a wide
John T. Hancock (ed.), Methods in Molecular Biology, Redox-Mediated Signal Transduction, vol. 476 © 2008 Humana Press, a part of Springer Science + Business Media, Totowa, NJ DOI: 10.1007/978-1-59745-129-1_13
spectrum of electrophiles, especially ROS and RNS, potentially leading to protein-loss of function.
The cysteine residue exists in vivo in the fully reduced free thiol form (-SH or -S-) and in different oxidation forms, the thiyl radical (-S' ), the disulfide bond (Cys-S-S-Cys), the sulfenic (-SOH), sulfinic (SO2 H), and sulfonic (-SO3 H) acid forms, and the S-nitrosylated form (-S-NO) (4). Cysteine-thiyl radical and cysteine-sulfenic acid are very unstable because of their highly reactive nature and thus cannot be easily identified biochemically. In contrast, cysteine-sulfinic and sulfonic acid are irreversible forms of protein oxidation, although the cysteine-sulfinic acid that forms in peroxiredoxins is enzymati-cally retro-reduced by sulfiredoxin (5, 6). Disulfide bonds are relatively stable, reversing to the reduced state by thiol-disulfide exchange with kinetics depending on the protein context and the redox nature of the milieu. Disulfide bonds can be formed catalytically by specific thiol oxidases systems, such as the oxygen-dependent FAD-sulfhydryl oxidases Erol (7) and Ervl that drive endoplasmic reticulum (ER)-protein oxidative folding during ER secretion and intermembrane mitochondrial space protein import, respectively (8). Disulfide bonds can also form upon reaction of thiols with peroxide or RNS as, for instance, at the reactive cysteine residues of thiol-based peroxidases and of redox sensors (1, 9, 10).
Disulfide bonds are formed as part ofthe catalytic cycle ofspecific enzymes such as ribonucleotide reductase (11). In the cytoplasm, disulfide bonds are also often found in the form of mixed disulfides between protein-thiols and GSH also named S-thiolation. Protein S-thiolation emanates from the condensation between reduced GSH and oxidized protein thiols in the thiyl or sulfenic acid forms that are generated by reaction with peroxides and other intracel-lular oxidants. S-thiolation protects these oxidized cysteine residues from further oxidation and may also regulate the function of specific proteins (12). Two potent NADPH-dependent thiol-reducing systems, the thioredoxin and glutathione (GSH) pathways, are present in the cytoplasm and assist protein-thiol reduction (13). Establishing the in vivo redox state of cysteine residues is thus an important task given the many important cellular responses that rely on cysteine redox modifications.
During cell breakage, reduced cysteine residues can undergo oxidation by O2--derived H2O2 and, conversely, oxidized residues can be reduced by thiol-disulfide exchange with cellular reduct-ases, potentially making difficult to evaluate their true in vivo redox state. Acidic quenching of thiol groups, which can circumvent this problem (see Notel), is best achieved by breaking cells in the presence of trichloroacetic acid (TCA) (pH < 1), which also precipitates soluble cellular proteins. TCA-based acidic quenching is common to and the first step of the methods presented here
Oxidized protein-thiol labeling
Oxidized protein-thiol purification
Mobility shift visible
Mobility shift not visible
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