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Review
. 2014 Feb;1840(2):838-46.
doi: 10.1016/j.bbagen.2013.03.031. Epub 2013 Apr 6.

Quantification of Thiols and Disulfides

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Free PMC article
Review

Quantification of Thiols and Disulfides

Jakob R Winther et al. Biochim Biophys Acta. .
Free PMC article

Abstract

Background: 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.

Scope of review: 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.

Major conclusions: 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.

General significance: 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.

Keywords: 1-cyano-4-dimethylamino-pyridinium; 2-mercato ethanol; 4,4′-dithiodipyridine; 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole; 4-DPS; 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid; 5,5′-dithiobis-(2-nitrobenzoic) acid; 5-thio-2-nitrobenzoic acid; 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate; ABD-F; AMS; CDAP; DTNB; Detection; EDTA; ER; Exchange; GSH; GSSG; HMD; MBBr; ME; MMTS; Modification; Monobromobimane; Nucleophile; PAGE; PEG; Redox; S-methyl methanethiosulfonate; SBD-F; SDS; TCEP; THP; TNB; endoplasmic reticulum; ethylenediamine tetraacetic acid; glutathione; glutathione disulfide; heavy maleimide derivative; polyacrylamide gel electrophoresis; polyethyleneglycol; sodium dodecylsulfate; tris(2-carboxyethyl) phosphine; tris(2-hydroxyethyl) phosphine.

Figures

Fig. 1
Fig. 1
Thiol-disulfide exchange. The attack of a thiolate (the nucleophile, S-n) on a disulfide bond takes place through a linear transition state where the central sulfur atom (Sc) will participate in a new disulfide bond and resolution of a new leaving group thiolate (S-lg). Which of the two sulfur atoms participating in the disulfide bond will eventually act as leaving group is dependent on steric, electrostatic and intrinsic acidity of the thiolate species involved.
Fig. 2
Fig. 2
Addition of thiols to maleimides together with selected exchange and ring opening reactions. The reaction of thiol (R1-SH) with maleimides (in this case NEM) is reversible, albeit shifted strongly toward the adduct formation (Reaction A). In the presence of excess thiols (R2-SH), however, reversal of the initial adduct leads to the accumulation of the alternative adduct (Reactions B and C). The adduct is susceptible to ring-opening by hydrolysis of the imide (Reaction D).
Fig. 3
Fig. 3
Cyanylation using CDAP. Reaction (A) of CDAP with cysteinyl peptide results in formation of a cyanylated species. This species can react further in 1.5 M NH4OH to cleave the adjacent N-proximal peptide bond (B).
Fig. 4
Fig. 4
Reaction mechanism for the reduction of disulfide bonds by phosphines. Here, the reaction of tris-(2-hydroxyethyl) phosphine (THP) is shown. Several other water soluble phosphines are commercially available, or have been described in the literature.
Fig. 5
Fig. 5
Detection of thiols using Ellman's reagent (DTNB). Due to the low pKa of the TNB it forms an extremely efficient leaving group. The net effect of this is that the analyte efficiently forms disulfide bonds if steric and concentration issues allow. Note that for each analyte thiol reacted, a TNB is formed.
Fig. 6
Fig. 6
Hydrolysis of DTNB. Hydroxyl ions will attack the disulfide bond of DTNB forming a sulfenic acid and a thiolate. This reaction is particularly relevant for activated disulfides like that found in DTNB.
Fig. 7
Fig. 7
Structure of 4-DPS showing subsequent reaction with thiols. For each thiol oxidized a molecule of thiopyridone is made. The latter is a favored tautomer of the thiol, which absorbs strongly at 324 nm. Note that as the thiopyridone is uncharged even at low pH the absorbtion remains unaffected.
Fig. 8
Fig. 8
Reaction of benzofurazanes with thiols. The reaction products are highly fluorescent.
Fig. 9
Fig. 9
Reaction of MMTS with thiols. The formation of a methyldisulfide (Reaction A) is prone to shuffling with adjacent reactive thiols (Reaction B). This renders MMTS unreliable as a thiol quencher.

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