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Thiol-reactive dyes are principally used to label proteins for the detection of conformational changes, assembly of multisubunit complexes and ligand-binding processes. In the case of proteins and peptides, the primary targets of thiol-reactive probes are cysteine residues. In mammalian proteins, the occurrence frequency of cysteine targets (3.3%) is less than half that of lysine targets (7.2%), which are labeled by the amine-reactive reagents described in Fluorophores and Their Amine Reactive Derivatives—Chapter 1. Some proteins and many peptides have only a single cysteine residue, enabling site-specific labeling with thiol-reactive probes. In proteins with multiple cysteine residues, the multiplicity is often small enough that it is practicable to obtain single-cysteine variants by site-directed mutagenesis without significant disruption of the structure or function of the native protein. Site-specific modification is particularly important for labeling small proteins in applications where the activity or binding affinity of the conjugate is paramount; thiol-reactive labeling is the preferred approach over amine-reactive labeling in such cases.
The relatively low abundance of cysteine residues also makes it possible to obtain saturating modification with less risk of incurring the penalties of protein precipitation and fluorescence self-quenching interactions that make high-percentage amine-reactive modification largely impracticable. In proteins with multiple cysteine residues, however, the reactivity of an individual cysteine can be very dependent on both its local environment and the hydrophobicity of the reactive dye. Site-specific modification strategies involving site-directed cysteine mutagenesis, site-dependent variations in thiol reactivity and functional group protection/deprotection have been developed to double-label proteins with donor and acceptor dyes for fluorescence resonance energy transfer (FRET) applications (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2). Thiol-reactive dyes can also be reacted with thiolated oligonucleotides for hybridization- or ligation-based nucleic acid detection applications and with thiouridine-modified tRNA for studying its association with protein synthesis machinery.
Several of the thiol-reactive probes described in this chapter are also useful for derivatizing low molecular weight thiols for various analytical assays that employ chromatographic and electrophoretic separation. An extensive review by Shimada and Mitamura describes the use of several of our thiol-reactive reagents for derivatizing thiol-containing compounds.
Thiols play a principal role in maintaining the appropriate oxidation–reduction state of proteins, cells and organisms. The susceptibility of thiols to oxidation, however, can lead to the formation of disulfides and higher oxidation products, often with loss of biological activity. Measuring the oxidation state of thiols within live cells is complicated by the high concentration of reduced glutathione in cells, which makes them difficult to assay with reagents that stoichiometrically react with the thiol (Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione—Section 15.6). Nonetheless, many useful reagents and methods have been developed for the quantitative assay of thiols and disulfides.
In proteins, thiol groups (also called mercaptans or sulfhydryls) are present in cysteine residues. Thiols can also be generated by selectively reducing cystine disulfides with reagents such as dithiothreitol (DTT, D1532) or 2-mercaptoethanol (β-mercaptoethanol), each of which must then be removed by dialysis or gel filtration before reaction with the thiol-reactive probe.
Unfortunately, removal of DTT or 2-mercaptoethanol is sometimes accompanied by air oxidation of the thiols back to the disulfides. Reformation of the disulfide bond can often be avoided by using the reducing agent tris-(2-carboxyethyl)phosphine (TCEP, T2556), which usually does not need to be removed prior to thiol modification because it does not contain thiols (Figure 2.1.1). However, there have been several reports that TCEP can react with haloacetamides or maleimides under certain conditions and that labeling in the presence of TCEP is inhibited. Carrying out thiol-reactive labeling on ammonium sulfate–precipitated proteins facilitates efficient and rapid removal of DTT after the preparatory reduction step and inhibits thiol reoxidation during the subsequent labeling reaction.
TCEP is more stable at a higher pH and at higher temperatures than is DTT and for a longer period of time in buffers without metal chelators such as EGTA; DTT is more stable than TCEP in solutions that contain metal chelators. TCEP is also more stable in the presence of Ni2+ levels that commonly contaminate proteins eluted from Ni2+ affinity columns and that rapidly oxidize DTT. Spin labels in TCEP are two to four times more stable than those in DTT, an advantage for electron paramagnetic resonance (EPR) spectroscopy. In addition, TCEP is used to stabilize solutions of ascorbic acid. TCEP is generally impermeable to cell membranes and to the hydrophobic protein core, permitting its use for the selective reduction of disulfides that have aqueous exposure. It has also been reported that TCEP can be used to deplete high-abundance plasma proteins (albumins, transferrin, etc.) prior to proteomic analysis because these proteins have a large number of disulfide bridges and are therefore particularly susceptible to reductive denaturation.
Figure 2.1.1 Reduction of a disulfide using TCEP (tris-(2-carboxyethyl)phosphine, hydrochloride; T2556). Unlike DTT (dithiothreitol, D1532), TCEP does not itself contain thiols, and therefore downstream thiol labeling reactions do not require preliminary removal of the reducing reagent.
The primary thiol-reactive reagents, including iodoacetamides, maleimides, benzylic halides and bromomethylketones, react by S-alkylation of thiols to generate stable thioether products. Arylating reagents such as NBD halides react with thiols or amines by a similar substitution of the aromatic halide by the nucleophile. Because the thiolate anion is a better nucleophile than the neutral thiol, cysteine is more reactive above its pKa (~8.3, depending on protein structural context). However, as in the case of amine modification by succinimidyl esters (Fluorophores and Their Amine-Reactive Derivatives—Chapter 1), reagent stability also decreases with increasing pH (e.g., maleimide hydrolysis to unreactive maleamic acid; see below), and therefore a compromise pH of 7.0–7.5 is typically used for protein modifcation reactions. It has been reported that iodoacetamide and maleimide adducts with intracellular proteins have different degrees of stability and toxicity. Analysis of the intracellular reactivity and toxicity of haloacetyl and maleimido thiol-reactive probes in HEK 293 cells indicates that maleimides are less stable and iodoacetamides are more toxic (putatively because maleimide adducts degrade before they are able to trigger damage-signaling pathways).
Also available are the TS-Link series of reagents for reversible thiol modification (Thiol-Reactive Probes Excited with Visible Light—Section 2.2). The TS-Link reagents are water-soluble thiosulfates that react stoichiometrically with thiols to form mixed disulfides.
Thiols also react with many of the amine-reactive reagents described in Fluorophores and Their Amine-Reactive Derivatives—Chapter 1, including isothiocyanates and succinimidyl esters. However, the reaction products appear to be insufficiently stable to be useful for routine modification of thiols in proteins. Although the thiol–isothiocyanate product (a dithiocarbamate) can react with an adjacent amine to yield a thiourea, the dithiocarbamate is more likely to react with water, consuming the reactive reagent without forming a covalent adduct.
In addition to insertion or deletion of cysteine residues by site-directed mutagenesis, several reagents have been developed for introducing thiols into proteins, nucleic acids and lipids. Because the selective introduction of thiols is particularly important for crosslinking two biomolecules, these reagents are discussed in Crosslinking and Photoactivatable Reagents—Chapter 5.
A method for the site-specific double-labeling of a protein containing at least one vicinal diol and another distal thiol has been reported. In this labeling protocol, the vicinal diol is first protected with phenylarsine oxide (PAO) to allow labeling of the unprotected distal thiol with Oregon Green 488 maleimide (O6034, Thiol-Reactive Probes Excited with Visible Light—Section 2.2). The blocked vicinal diol is then deprotected with dithiothreitol (DTT) and labeled with Alexa Fluor 350 maleimide (A30505, Thiol-Reactive Probes Excited with Ultraviolet Light—Section 2.3). Target proteins may need to be engineered to contain a vicinal diol and distal thiol in order to employ this labeling strategy.
In a similar double thiol-labeling method, instead of PAO protection/deprotection, the protein's tetracysteine tag was labeled using FlAsH-EDT2 reagent (T34561, Thiol-Reactive Probes Excited with Visible Light—Section 2.2), and the Alexa Fluor 568 maleimide (A20341, Thiol-Reactive Probes Excited with Visible Light—Section 2.2) was used to label a distal cysteine. Fluorescence resonance energy transfer (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2) between the FlAsH label and Alexa Fluor 568 dye was then used to detect ligand-induced conformational changes in the C-terminal domain of the β2-adrenoreceptor in SF9 cells.
Iodoacetamides readily react with all thiols, including those found in peptides, proteins and thiolated polynucleotides, to form thioethers (Figure 2.1.2); they are somewhat more reactive than bromoacetamides. When a protein's cysteine residues are blocked or absent, however, iodoacetamides can sometimes react with methionine residues. They may also react with histidine or tyrosine, but generally only if free thiols are absent. Although iodoacetamides can react with the free base form of amines, most aliphatic amines, except the α-amino group at a protein's N-terminus, are protonated and thus relatively unreactive below pH 8. In addition, iodoacetamides react with thiolated oligonucleotide primers, as well as with thiophosphates.
Iodoacetamides are intrinsically unstable in light, especially in solution; reactions should therefore be carried out under subdued light. Adding cysteine, glutathione or mercaptosuccinic acid to the reaction mixture will quench the reaction of thiol-reactive probes, forming highly water-soluble adducts that are easily removed by dialysis or gel filtration. Although the thioether bond formed when an iodoacetamide reacts with a protein thiol is very stable, the bioconjugate loses its fluorophore during amino acid hydrolysis, yielding S-carboxymethylcysteine.
Figure 2.1.2 Reaction of a thiol with an alkyl halide.
Maleimides are excellent reagents for thiol-selective modification, quantitation and analysis. In this reaction, the thiol is added across the double bond of the maleimide to yield a thioether (Figure 2.1.3). Applications of these fluorescent and chromophoric analogs of N-ethylmaleimide (NEM) strongly overlap those of iodoacetamides, although maleimides apparently do not react with methionine, histidine or tyrosine. Reaction of maleimides with amines usually requires a higher pH than reaction of maleimides with thiols.
Hydrolysis of the maleimide to an unreactive product can compete significantly with thiol modification, particularly above pH 8. Furthermore, once formed, maleimide-derived thioethers can hydrolyze to an isomeric mixture of succinamic acid adducts, or they can undergo cyclization with adjacent amines to yield crosslinked products. This latter reaction is much less frequently encountered than the former. Deliberate acceleration of the hydrolytic succinimide to succinamide acid ring-opening reaction by molybdate or chromate catalysis provides a strategy for decreasing the heterogeneity of bioconjugates derived from maleimide derivatization of thiols.
Figure 2.1.3 Reaction of a thiol with a maleimide.
Several of our thiol-reactive probes can be used to form reversible bonds, including BODIPY FL L-cystine (B20340), as well as the TS-Link BODIPY thiosulfate and TS-Link DSB-X biotin C5-thiosulfate reagents (Thiol-Reactive Probes Excited with Visible Light—Section 2.2, Biotinylation and Haptenylation Reagents—Section 4.2).
Symmetric disulfides such as BODIPY FL L-cystine undergo a thiol–disulfide interchange reaction to yield a new asymmetric disulfide (Figure 2.1.4), a reaction that is freely reversible and thiol-specific. This disulfide linkage can be cleaved with reagents such as DTT or TCEP.
Thiosulfates (R–S–SO3–), including our water-soluble TS-Link reagents, are similar to disulfides in that they stoichiometrically react with thiols to form disulfides (Figure 2.1.5). However, unlike the reaction of the BODIPY FL cystine probe with a free thiol, no excess of the TS-Link reagent is required to drive the equilibrium.
Figure 2.1.4 Reaction of a thiol with a symmetric disulfide.
Figure 2.1.5 Reaction of a TS-Link reagent (R1) with a thiol (R2), followed by removal of the label with a reducing agent.
The Measure-iT Thiol Assay Kit (M30550) provides an easy and accurate method for quantitating thiols. This thiol assay has a linear range of 0.05–5 μM thiol (Figure 2.1.6), making it up to 400 times more sensitive than colorimetric methods based on Ellman’s reagent.
Each Measure-iT Thiol Assay Kit contains:
Simply dilute the reagent 1:100, load 100 μL into the wells of a microplate, add 1–10 μL sample volumes, mix, then read the fluorescence. Maximum fluorescence signal is attained within 5 minutes and is stable for at least 1 hour. The assay is performed at room temperature, and common contaminants are well tolerated in the assay. The Measure-iT Thiol Assay Kit provides sufficient materials for 500 assays, based on a 100 μL assay volume in a 96-well microplate format; this thiol assay can also be adapted for use in cuvettes or 384-well microplates.
Figure 2.1.6 Linearity and sensitivity of the Measure-iT thiol assay. Triplicate 10 µL samples of glutathione were assayed using the Measure-iT Thiol Assay Kit (M30550). Fluorescence was measured using excitation/emission of 490/520 nm and plotted versus glutathione concentration. The variation (CV) of replicate samples was <2%.
Ultrasensitive colorimetric quantitation of both protein and nonprotein thiols can be achieved using the Thiol and Sulfide Quantitation Kit (T6060). In this assay, which is based on a method reported by Singh, thiols or sulfides reduce a disulfide-inhibited derivative of papain, stoichiometrically releasing the active enzyme (Figure 2.1.7). Activity of the enzyme is then measured using the chromogenic papain substrate L-BAPNA via spectrophotometric detection of p-nitroaniline release at 412 nm.
Although thiols and inorganic sulfides can also be quantitated using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB or Ellman's reagent, D8451), the enzymatic amplification step in the Thiol and Sulfide Quantitation Kit enables researchers to detect as little as 0.2 nanomoles of thiols or sulfides—a sensitivity that is about 100-fold better than that achieved with DTNB. Thiols in proteins can be detected indirectly by incorporating the disulfide cystamine into the reaction mixture. Cystamine undergoes an exchange reaction with protein thiols, yielding 2-mercaptoethylamine (cysteamine), which then releases active papain. Thiols that are alkylated by maleimides, iodoacetamides or other reagents are excluded from detection and can therefore be assayed subtractively.
The Thiol and Sulfide Quantitation Kit contains:
Sufficient reagents are provided for approximately 50 assays using standard 1 mL cuvettes or 250 assays using a microplate format.
Figure 2.1.7 Chemical basis for thiol detection using the Thiol and Sulfide Quantitation Kit (T6060): A) The inactive disulfide derivative of papain, papain–SSCH3, is activated in the presence of thiols; B) active papain cleaves the substrate L-BAPNA, releasing the p-nitroaniline chromophore; C) protein thiols, often poorly accessible, exchange with cystamine to generate 2-mercaptoethylamine (cysteamine), which is functionally equivalent to the thiol R–SH in step A.
Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid) or DTNB; D8451) remains an important reagent for spectrophotometric quantitation of protein thiols and, by extension, the analysis of thiol–disulfide exchange reactions and oxidative thiol modifications. It readily forms a mixed disulfide with thiols, liberating the chromophore 5-mercapto-2-nitrobenzoic acid (absorption maximum 410 nm, EC ~13,600 cm-1M-1). Only protein thiols that are accessible to this water-soluble reagent are modified. Inaccessible thiols can usually be quantitated by carrying out the titration in the presence of 6 M guanidinium chloride. DTNB conjugates of glutathione and other thiols can be separated by HPLC and quantitated based on their absorption.
Several maleimides—including 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM, D346; Thiol-Reactive Probes Excited with Ultraviolet Light—Section 2.3) and N-(7-dimethylamino-4-methylcoumarin-3-yl)maleimide (DACM, D10251; Thiol-Reactive Probes Excited with Ultraviolet Light—Section 2.3)—are not appreciably fluorescent until after conjugation with thiols, and are therefore useful for thiol quantitation. Similarly, fluorescein-5-maleimide (F150, Thiol-Reactive Probes Excited with Visible Light—Section 2.2) exhibits an analytically useful 10-fold fluorescence enhancement upon reaction with thiols. Monobromobimane (M1378, M20381; Thiol-Reactive Probes Excited with Ultraviolet Light—Section 2.3) is also essentially nonfluorescent until it reacts with thiols and can be used to determine thiol levels in cells.
In addition, most of the fluorescent thiol-reactive reagents in this chapter can be used as derivatization reagents for analyzing thiols by techniques such as HPLC that utilize a separation step. 5-(Bromomethyl)fluorescein is the reagent with the greatest intrinsic sensitivity for this application. See Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione—Section 15.6 for a further discussion of methods to quantitate reduced glutathione in cells.
For a detailed explanation of column headings, see Definitions of Data Table Contents
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