Sulfhydryl-reactive chemical groups in biomolecular probes for labeling and crosslinking cysteines and other sulfhydryls include maleimides, haloacetyls and pyridyl disulfides. This article describes the reaction chemistries and biology research applications of this class of reagents.


Sulfhydryl-reactive crosslinker reactive groups

Besides amine-reactive compounds, those having chemical groups that form bonds with sulfhydryls (–SH) are the most common crosslinkers and modification reagents for protein and other bioconjugate techniques. Sulfhydryls, also called thiols, exist in proteins in the side-chain of cysteine (Cys, C) amino acids. Pairs of cysteine sulfhydryl groups are often linked by disulfide bonds (–S–S–) within or between polypeptide chains as the basis of native tertiary or quaternary protein structure. Typically, only free or reduced sulfhydryl groups (–SH) [rather than sulfur atoms in disulfide bonds] are available for reaction with thiol-reactive compounds.

Sulfhydryl groups are useful targets for protein conjugation and labeling. First, sulfhydryls are present in most proteins but are not as numerous as primary amines; thus, crosslinking via sulfhydryl groups is more selective and precise. Second, sulfhydryl groups in proteins are often involved in disulfide bonds, so crosslinking at these sites typically does not significantly modify the underlying protein structure or block binding sites. Third, the number of available (i.e., free) sulfhydryl groups can be easily controlled or modified; they can be generated by reduction of native disulfide bonds, or they can be introduced into molecules through reaction with primary amines using sulfhydryl-addition reagents, such as 2-iminothiolane (Traut’s Reagent), SATA, SATP, or SAT(PEG)4. Finally, combining sulfhydryl-reactive groups with amine-reactive groups to make heterobifunctional crosslinkers provides greater flexibility and control over crosslinking procedures.

Sulfhydryl-reactive chemical groups include haloacetyls, maleimides, aziridines, acryloyls, arylating agents, vinyl sulfones, pyridyl disulfides, TNB-thiols and disulfide reducing agents. Most of these groups conjugate to sulfhydryls by either alkylation (usually the formation of a thioether bond) or disulfide exchange (formation of a disulfide bond).


Maleimide reaction chemistry

The maleimide group reacts specifically with sulfhydryl groups when the pH of the reaction mixture is between 6.5 and 7.5; the result is formation of a stable thioether linkage that is not reversible (i.e., the bond cannot be cleaved with reducing agents). In more alkaline conditions (pH >8.5), the reaction favors primary amines and also increases the rate of hydrolysis of the maleimide group to a non-reactive maleamic acid. Maleimides do not react with tyrosines, histidines or methionines.

Thiol-containing compounds, such as dithiothreitol (DTT) and beta-mercaptoethanol (BME), must be excluded from reaction buffers used with maleimides because they will compete for coupling sites. For example, if DTT were used to reduce disulfides in a protein to make sulfhydryl groups available for conjugation, the DTT would have to be thoroughly removed using a desalting column before initiating the maleimide reaction. Interestingly, the disulfide-reducing agent TCEP does not contain thiols and does not have to be removed before reactions involving maleimide reagents.

Excess maleimides can be quenched at the end of a reaction by adding free thiols. EDTA can be included in the coupling buffer to chelate stray divalent metals that otherwise promote oxidation of sulfhydryls (non-reactive).

Maleimide reaction scheme for chemical conjugation to a sulfhydryl. R represents a labeling reagent or one end of a crosslinker having the maleimide reactive group; P represents a protein or other molecule that contains the target functional group (i.e., sulfhydryl, –SH).

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Applications for maleimide crosslinking

1. Create irreducible linkages between cysteines

Homobifunctional maleimide crosslinkers (i.e., those which have a maleimide group at each end) can be used to replace cystine disulfide bonds in protein structures with permanent, non-reducible linkages between cysteines. First, the native disulfide bonds must be cleaved using TCEP or another reducing agent. Then addition of the maleimide crosslinker will conjugate pairs of cysteine sulfhydryls by thioether bonds. Although any two sulfhydryl groups can become conjugated, one expects most crosslinks to form between cysteines that naturally associate in the native secondary and tertiary structures.

Sulfhydryl-to-sulfhydryl crosslinking with homobifunctional maleimide crosslinkers is useful in certain specific situations with particular pairs of proteins or sulfhydryl molecules. However, this approach is not used very frequently.

2. Prepare specific protein-protein conjugates

Heterobifunctional maleimide crosslinkers (i.e., such as those having a maleimide group at one end and an amine-reactive group at the other end) are commonly used to conjugate any two different purified proteins together in a controlled manner (i.e., without polymerizing or self-conjugating the respective proteins). The most popular of these reagents are NHS-ester/maleimide compounds, such as Sulfo-SMCC and its pegylated analogs, SM(PEG)n.

Chemical structure of Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC). It is a non-cleavable and membrane impermeable crosslinker. Because it contains the hydrophilic sulfonyl moiety, Sulfo-SMCC is soluble up to ~10 mM in water and many commonly used buffers, thus avoiding the use of organic solvents which may perturb protein structure.

For example, with Sulfo-SMCC, a reporter enzyme like horseradish peroxidase (HRP) can be conjugated to an antibody or other protein using a sequential, two-step procedure to create a detectable affinity probe for use in assays:

  • First, HRP is reacted in isolation with the crosslinker to allow the NHS-ester end of Sulfo-SMCC to attach at several available primary amines (side chain of lysine) on the protein surface. This creates what is called maleimide-activated HRP, namely HRP molecules that are labeled with several sulfhydryl-reactive maleimide groups.
  • Concurrently, the antibody is treated in isolation to create available sulfhydryl groups on its surface. It is either gently reduced with 2-mercaptoethylamine (2-MEA) to cleave hinge region disulfides, or it is modified with Traut's Reagent (2-Iminothiolane, 2-IT) or SATA to add sulfhydryl groups onto primary amine sites.
  • Secondly, the two activated proteins are mixed together, allowing specific, one-way linking between HRP and antibody molecules. In typical reaction conditions, one to three HRP molecules conjugate to each antibody molecules, and essentially no HRP-HRP or antibody-antibody conjugates result.

With many pairs of proteins one might wish to conjugate in this manner, the crosslinking can be done in either orientation (HRP to sulfhydryls created on the antibody or the antibody to sulfhydryls created on HRP), although the NHS-ester end must always be reacted first. Commonly needed labels like HRP are commercially available already prepared in maleimide-activated form. Nevertheless, the strategy can be used to conjugate nearly any two purified proteins needed for specialized custom procedures and experiments.

3. Prepare immunogen with cysteine-terminated peptide antigens

A particular, but very important example of NHS-ester/maleimide heterobifunctional crosslinking with Sulfo-SMCC and similar reagents, is the conjugation of synthesized cysteine-containing peptide antigens to immunogenic carrier proteins for use in antibody production. Large carrier proteins, such as BSA and KLH, can be maleimide-activated using Sulfo-SMCC to bear tens to hundreds of maleimide groups per protein molecule. This enables many peptide antigens to be conjugated to each carrier protein molecule. Commonly used carrier proteins are commercially available in maleimide-activated form, but nearly any particular protein and spacer-length variant of SMCC-like crosslinker could be used to make "carriers" for peptides or other small sulfhydryl molecules for specialized research applications.

4. Prepare specific protein-glycoprotein conjugates

Heterobifunctional maleimide crosslinkers whose opposing end contains a carbonyl-reactive hydrazide group are useful for conjugating proteins to carbohydrates of glycoproteins or other polysaccharides. See the page on Carbonyl-reactive Crosslinker Chemistry for information on the hydrazide group. The sulfhydryl-to-carbohydrate conjugation application is mentioned here, because maleimides are one of the few groups that can be paired opposite hydrazides in a single reagent. This is because the hydrazide group contains a primary amine, which is a target for NHS esters. The following image shows the chemistry involved with periodate oxidation to aldehyde for reactions with hydrazides. Although, this does not mean that conjugation to carbohydrates is limited to proteins that have native sulfhydryl groups. It simply means that whatever macromolecule one wishes to attach to a carbohydrate must first be modified to contain a sulfhydryl group. As described above, Traut's Reagent (2-iminothiolane, 2-IT) and SATA are modification reagents that add sulfhydryl groups onto primary amine sites.

Sodium periodate chemistry. A reaction with sugar residues yields aldehydes for conjugation reactions. R and R' represent connecting sugar monomers of the polysaccharide. Red asterisks indicate sites of diol cleavage. Sialic acid is also called N-acetyl-D-neuraminic acid. 

5. Precisely label antibodies or other proteins with biotin or fluorophores

Most biotin reagents and fluorescent reagents for labeling macromolecules at sulfhydryl groups are based on the maleimide chemistry. Usually, protein labeling is directed at primary amines using NHS-ester reagents, but certain proteins can become inactivated for intended applications when labeled at primary amines. By contrast, sulfhydryl-bearing cysteines are usually less abundant than amine-bearing lysines and can provide for more specific, uniform labeling. For example, antibodies (e.g., IgG) contain disulfide bonds at the hinge region that can be selectively reduced to expose just those sulfhydryl groups for labeling using maleimide compounds. The result is monovalent "half"-antibodies, each of which is labeled with exactly one biotin or fluorophore tag. The diagram below illustrates how a Maleimide-PEG solid-phase biotinylation kit may be used to efficiently biotinylated an antibody. 

Steps involved in on-column sulfhydryl-targeted antibody Biotinylation.  Using the approach illustrated in the diagram, the Thermo Scientific EZ-Link Maleimide-PEG Solid-Phase Biotinylation Kit facilitates sulfhydryl-targeted antibody biotinylation that provides greater control over the labeling reaction and clean-up process.  This innovative antibody-labeling system uses nickel-chelated agarose to temporarily immobilize antibody molecules via their histidine-rich Fc regions. Once held in place on the resin, the antibody can be gently reduced at the hinge-region to produce free sulfhydryls that are then biotinylated with the Maleimide-PEG2-Biotin reagent. Excess labeling reagent and byproducts are then washed away before recovering the labeled and purified antibody from the resin using a mild imidazole solution. No gel filtration or dialysis is needed. 


Haloacetyl reaction chemistry

The most commonly used haloacetyl crosslinkers contain an iodoacetyl or a bromoacetyl group. Haloacetyls react with sulfhydryl groups at physiologic pH. The reaction of the iodoacetyl group proceeds by nucleophilic substitution of iodine with a sulfur atom from a sulfhydryl group, resulting in a stable thioether linkage. Using a slight excess of the iodoacetyl group over the number of sulfhydryl groups at pH 8.3 ensures sulfhydryl selectivity. In the absence of free sulfhydryls, or if a large excess of iodoacetyl group is used, the iodoacetyl group can react with other amino acids. Imidazoles can react with iodoacetyl groups at pH 6.9 to 7.0, but the incubation must proceed for longer than one week.

Histidyl side chains and amino groups react in the unprotonated form with iodoacetyl groups above pH 5 and pH 7, respectively. To limit free iodine generation, which has the potential to react with tyrosine, histidine, and tryptophan residues, perform iodoacetyl reactions and preparations in the dark. Avoid exposure of iodoacetyl compounds to reducing agents. Once the reaction is complete, the activated molecules no longer need to be protected from light. 

Iodoacetyl reaction scheme for chemical conjugation to a sulfhydryl. R represents a labeling reagent or one end of a crosslinker having the iodoacetyl or bromoacetyl reactive group; P represents a protein or other molecule that contains the target functional group (i.e., sulfhydryl, –SH).

Applications for haloacetyl crosslinking

1. Haloacetyl crosslinking

In principle, homobifunctional and heterobifunctional iodoacetyl or bromoacetyl crosslinkers can be used for most of the same sorts of applications as their maleimide counterparts. In practice, however, fewer haloacetyl crosslinking compounds are commercially available or commonly used in bioconjugation techniques. One difference from maleimides is that haloacetyls do not contain a ring structure; therefore, they make it possible to create very short crosslinks.

2. Iodoacetyl labeling reagents

Several iodoacetyl biotinylation reagents are still commercially available and frequently used to label antibodies and various proteins. Those familiar with fluorescent labeling will recognize the fluorescein reagent 5-IAF (5-iodoacetamido-fluorescein).

Although maleimide reagents are more popular, only empirical testing and comparison can confirm whether an iodoacetamide or maleimide reagent will label more consistently and produce a better functioning probe for any particular protein.

3. Iodoacetyl immobilization supports

One application where haloacetyls remain the more popular sulfhydryl-reactive chemistry is protein immobilization to beaded agarose resin. Iodoacetyl-activated agarose is the basis for SulfoLink Coupling Resin and Kits. The resin can be used to immobilize half-antibodies (see above) or protein subunits whose cysteine disulfide bonds have been reduced. However, the most important and common application of iodoacetyl-activated agarose is to immobilize cysteine-containing peptides for antibody purification after having used the same peptide as antigen for immunization and antibody production (see above discussion about immunogen preparation with Sulfo-SMCC). The representative protein gel below provides a description of results obtained a micro peptide coupling kit.  

Efficient purification of anti-peptide antibody from crude serum using the Micro Peptide Coupling Kit. Insulin peptide (245 µg) coupled in a kit spin column was used to purify anti-insulin from 40 µg of bovine insulin anti-serum. Eluted protein was separated by SDS-PAGE (4-20% gel) and stained with Thermo Scientific GelCode Blue Stain Reagent (Product No. 24590).

Lane 1: 40 µg bovine insulin anti-serum (crude serum).
Lane 2-7: Alternating non-reduced and reduced fractions (A = whole IgG; B = heavy chain; C = light chain).
Lanes 2 & 3: Eluted fraction 1 (10 µL each).
Lanes 4 & 5: Eluted fraction 2 (10 µL each).
Lanes 6 & 7: Eluted fraction 3 (10 µL each).
Lane 8: MW Marker (Part No. 26681).

Pyridyl disulfides

Pyridyl disulfide reaction chemistry

Pyridyl disulfides react with sulfhydryl groups over a broad pH range (the optimum is pH 4 to 5) to form disulfide bonds. During the reaction, a disulfide exchange occurs between the molecule's –SH group and the reagent's 2-pyridyldithiol group. As a result, pyridine-2-thione is released and can be measured spectrophotometrically (Amax = 343nm) to monitor the progress of the reaction. These reagents can be used as crosslinkers and to introduce sulfhydryl groups into proteins. The disulfide exchange can be performed at physiologic pH, although the reaction rate is slower in acidic conditions.

Pyridyldithiol reaction scheme for cleavable (reversible) chemical conjugation to a sulfhydryl. R represents a labeling reagent or one end of a crosslinker having the pyridyl disulfide reactive group; P represents a protein or other molecule that contains the target functional group (i.e., sulfhydryl, –SH).


Applications for pyridyl disulfide crosslinking

Because pyridyldithiol compounds form disulfide bonds with target sulfhydryls, conjugates prepared using these crosslinkers are cleavable with familiar disulfide reducing agents, such as dithiothreitol (DTT) or sample buffer for protein electrophoresis (SDS-PAGE). Thus, pyridyldisulfide crosslinkers and labeling reagents are useful alternatives to maleimide and haloacetyl reagents when there is a need to reverse the sulfhydryl-conjugation step later in an experimental procedure (i.e., to exactly recover the original sulfhydryl-containing molecule).

For example, in experimental situations where the protein conjugate becomes part of the sample that will be electrophoresed, it may be preferable to allow the conjugate to be cleaved and separated into original components. In some experiments, comparison of electrophoresis or Western blot results from use of cleavable vs. noncleavable crosslinkers may be instructive.

As a second example, one could label a purified protein with HPDP-Biotin and then allow the protein to bind putative interactors in a cell lysate sample. Next, the intact protein complex could be captured by the biotin group to streptavidin agarose, at which point the disulfide bond could be cleaved to release all components of the protein interaction complex for subsequent analysis.

Chemical Structure of HPDP-Biotin. This molecule is a pyridyldithiol-biotin compound for labeling protein cysteines and other molecules that contain sulfhydryl groups. This reagent specifically reacts with reduced thiols (-SH) in near-neutral buffers to form reversible disulfide bonds. HPDP-Biotin is useful for labeling and affinity-purification applications that require recovery of the original, unmodified molecule.

Finally, one could create a reversible sulfhydryl immobilization resin by using Sulfo-LC-SPDP to pyridyldithiol-activate DADPA resin.

Recommended reading

  1. Ishikawa, E., et al. (1983). Enzyme-labeling of antibodies. J Immunoassay 4:209-327.
  2. Brinkley, M.A. (1992). A survey of methods for preparing protein conjugates with dyes, haptens and cross-linking reagents. Bioconjugate Chem 3:2-13.
  3. Hashida, S., et al. (1984). More useful maleimide compounds for the conjugation of Fab to horseradish peroxidase through thiol groups in the hinge. J Appl Biochem 6:56-63.
  4. Mattson, G., et al. (1993). A practical approach to cross-linking. Molecular Biology Reports 17:167-83.
  5. Partis, M.D., et al. (1983). Cross-linking of proteins by omega-maleimido alkanoyl N-hydroxysuccinimide esters. J Protein Chem 2:263-77.
  6. Yoshitake, S., et al. (1982). Mild and efficient conjugation of rabbit Fab and horseradish peroxidase using a maleimide compound and its use for enzyme immunoassay. J Biochem 92:1413-24

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