ReviewAn introduction to methods for analyzing thiols and disulfides: Reactions, reagents, and practical considerations
Introduction
The majority of the thiols (SH)2 and disulfides (SS) in cells are found as the amino acid cysteine and its disulfide, cystine (Fig. 1A). The thiolate anion is intrinsically one of the strongest biological nucleophiles; thus, the thiol group of cysteine is one of the most reactive functional groups found in proteins [1]. Protein disulfide bonds are typically introduced and removed through a thiol–disulfide exchange reaction (Fig. 1B). This mechanism of transferring reducing equivalents between thiol and disulfide pairs is central in redox biology and is, for example, applied by cytosolic thioredoxin with its active site in the reduced form to reduce protein disulfides and in the endoplasmic reticulum (ER) by protein disulfide isomerases in their oxidized form to generate disulfide bonds. The reaction is initiated by a nucleophilic attack of a thiolate on an existing disulfide bond, leading to oxidation of the nucleophilic thiol and reduction of the leaving group sulfur [2]. In thiol–disulfide exchange reactions, it is important to consider reaction rate and the equilibrium constants between various thiol and disulfide species. Because the thiolate anion is the reactive species, these properties are particularly sensitive to thiol pKa values. In addition, the kinetics and thermodynamics of thiol–disulfide exchange reactions are affected by electrostatic factors from neighboring charged groups as well as strain and entropy (for detailed reviews, see Refs. [3], [4]).
Cellular SH groups are implicated in the coordination of metal ions and the defense against oxidants, and the reversible formation of disulfide bonds is involved in regulation of enzyme activity, signal transduction, transcriptional activity, and protein folding [5]. Because the thiols and disulfides of proteins and low-molecular-weight compounds are involved in so many essential cellular functions, reliable and accurate methods to identify and quantify them are in high demand. For example, methods for determining the in vivo thiol oxidation state of specific oxidoreductases can be crucial for determining their functions, and the identification of proteins with redox-active cysteines can lead to elucidation of redox regulation pathways. The reactive nature of thiols is, however, often an experimental challenge. In contrast to the extracellular space, the cytosolic concentration of reduced thiols is much higher than the concentration of disulfides, and the SH group easily oxidizes during cell lysis and sample preparation. One should consider that these chemical reactions can take place rapidly and spontaneously [6], and overlooking the possibility of postlysis thiol–disulfide exchange reactions can lead to mis-interpretations of data.
This review outlines the basic issues to consider when dealing with biochemical and cellular aspects of thiol–disulfide chemistry. Considering the volume of literature on the subject, we cannot cover it comprehensively and so we apologize to the many highly qualified contributions that we, within the given scope, do not mention. The overall focus is on practical aspects, including typical biochemical experimental conditions and caveats to consider in interpreting results. Reagents for thiol derivatization and disulfide reduction are evaluated and compared, and we discuss how to avoid conflict between mutually cross-reactive thiol reagents. We consider this review to be an introduction to experimental thiol–disulfide biochemistry updated with selected contemporary knowledge on the subject.
Section snippets
Quenching cellular thiol–disulfide exchange: Trapping in vivo conditions
Probably the most critical step when working with redox biology is quenching samples to trap the cellular thiol–disulfide status. This step not only is crucial for preventing artificial oxidation during cell lysis or sample preparation but also must be so rapid that perturbation of the thiol–disulfide equilibria is avoided. This is particularly important in cell extracts that contain redox-active enzymes that, if not rapidly denatured, can very efficiently transfer disulfides between different
Preventing thiol oxidation by molecular oxygen
Thiols can be oxidized by molecular oxygen, as illustrated in a classic study by Haber and Anfinsen [22] where reduced ribonuclease regained both native disulfide bonds and enzymatic activity within 20 h at pH 8 in the absence of oxidants except dissolved oxygen. We now know that this process is catalyzed by trace metal ion contaminants in reagent solutions. Intermediates in the oxidation reaction include reactive oxygen species [23], which can also induce irreversible oxidation of proteins
Measuring thiol–disulfide ratios: Common pitfalls
The general approach for measuring thiols and disulfides is shown in Fig. 5. Although the depicted strategy initially seems simple, several issues should be considered before proceeding. Reduced sulfhydryl groups can be detected merely by incubating with a suitable thiol-specific detection reagent (Fig. 5A). Here the main challenge is to avoid perturbation of the thiol–disulfide equilibrium, that is, by applying alkylating agents that block thiols faster than disulfide exchange can occur. The
Thiol alkylating agents
Thiol alkylating agents are central to experimental redox biology and are used for a variety of applications in the characterization of thiols and disulfides. As described previously, alkylation reagents are used to quench the thiol–disulfide status on cell lysis (Fig. 2) and, as illustrated in Fig. 5B, are applied to mask free thiols so as to specifically detect disulfides. In this section, a general introduction to the most commonly used thiol alkylating agents is provided, and their
Reducing agents
Any detection and quantification of disulfide bonds requires efficient and quantitative reduction of disulfides. As illustrated in Fig. 5, disulfide reduction is typically flanked by two thiol derivatization steps, rendering the reduction step particularly complicated. In addition to being an efficient reductant, the reducing agent must be removable (e.g., by gel filtration) or unable to cross-react with the derivatization agent in the next step. Alternatively, the product of the reaction
Thiol detection agents
A wide variety of reagents is available for detection of thiols. These include active aromatic disulfide reagents with useful spectrophotometric properties and alkylating reagents modified with detection labels. This section provides examples of thiol detection agents and their applicability. When using derivatives of alkylating agents, the experimental considerations described in the previous sections for reaction conditions, reactivity, specificity, and stability apply. After labeling
Concluding remarks
The past decade has been an exciting time in the field of cellular thiol–disulfide biochemistry. Considerable insight into many aspects of cellular redox regulation has been obtained, and the field is gaining the interest of a wider audience. Nevertheless, the reactive character of the SH group provides researchers in the field with quite a challenge. Reliable measurements of thiols and disulfides are largely dependent on proper sample treatment, and the relevant controls should always be
Acknowledgments
Colin Thorpe, Kristine Steen Jensen, Jonas Nielsen, and Christine Tachibana are thanked for critically reading the manuscript. The technical assistance of Svetlana Hansen and Berit Schultz is gratefully acknowledged. This work was supported by the Danish Natural Science Research Council and a generous donation from Ib Henriksens Fond for HPLC equipment.
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