Quantification of thiols and disulfides

• Published in: Biochimica et biophysica acta Vol. 1840; no. 2; pp. 838 - 846
• Main Authors: Winther, Jakob R., Thorpe, Colin
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.02.2014
• Subjects: Animals; biophysics; Detection; disulfide bonds; Disulfides - analysis; Disulfides - chemistry; Exchange; homeostasis; Humans; membrane proteins; Modification; Nucleophile; oxidation; Oxidation-Reduction; post-translational modification; Proteins - chemistry; reactive oxygen species; Redox; Sulfhydryl Compounds - analysis; Sulfhydryl Compounds - chemistry; sulfides; thiols; Modification; TNB; ABD-F; CDAP; ME; 4-DPS; GSH; THP; HMD; GSSG; MBBr; SDS; DTNB; AMS; Redox; PAGE; EDTA; ER; Exchange; PEG; MMTS; Detection; TCEP; SBD-F; Nucleophile; tris(2-carboxyethyl) phosphine; 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole; 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid; glutathione disulfide; glutathione; tris(2-hydroxyethyl) phosphine; polyethyleneglycol; 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate; polyacrylamide gel electrophoresis; 1-cyano-4-dimethylamino-pyridinium; heavy maleimide derivative; 2-mercato ethanol; Monobromobimane; sodium dodecylsulfate; 5-thio-2-nitrobenzoic acid; 5,5′-dithiobis-(2-nitrobenzoic) acid; 4,4′-dithiodipyridine; endoplasmic reticulum; S-methyl methanethiosulfonate; ethylenediamine tetraacetic acid
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2013.03.031

Disulfide bond formation is a key posttranslational modification, with implications for structure, function and stability of numerous proteins. While disulfide bond formation is a necessary and essential process for many proteins, it is deleterious and disruptive for others. Cells go to great lengths to regulate thiol-disulfide bond homeostasis, typically with several, apparently redundant, systems working in parallel. Dissecting the extent of oxidation and reduction of disulfides is an ongoing challenge due, in part, to the facility of thiol/disulfide exchange reactions. In the present account, we briefly survey the toolbox available to the experimentalist for the chemical determination of thiols and disulfides. We have chosen to focus on the key chemical aspects of current methodology, together with identifying potential difficulties inherent in their experimental implementation. While many reagents have been described for the measurement and manipulation of the redox status of thiols and disulfides, a number of these methods remain underutilized. The ability to effectively quantify changes in redox conditions in living cells presents a continuing challenge. Many unresolved questions in the metabolic interconversion of thiols and disulfides remain. For example, while pool sizes of redox pairs and their intracellular distribution are being uncovered, very little is known about the flux in thiol-disulfide exchange pathways. New tools are needed to address this important aspect of cellular metabolism. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. •Efficient quenching is essential for accurate determination of thiols and disulfides.•It is necessary to carefully consider rates of alkylation and reduction in the context of pH.•Some alkylation reactions are subject to reversibility.•Many alkylation reagents have side-reactions which are incompatible with down-stream procedures.