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).įor 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: EDTA can be included in the coupling buffer to chelate stray divalent metals that otherwise promote oxidation of sulfhydryls (non-reactive).
#THIOL FUNCTIONAL GROUP FREE#
Interestingly, the disulfide-reducing agent TCEP does not contain thiols and does not have to be removed before reactions involving maleimide reagents.Įxcess maleimides can be quenched at the end of a reaction by adding free thiols. 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. 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. Maleimides do not react with tyrosines, histidines or methionines. 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. 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). 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). Sulfhydryl-reactive chemical groups include haloacetyls, maleimides, aziridines, acryloyls, arylating agents, vinylsulfones, pyridyl disulfides, TNB-thiols and disulfide reducing agents. Finally, combining sulfhydryl-reactive groups with amine-reactive groups to make heterobifunctional crosslinkers provides greater flexibility and control over crosslinking procedures. 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. 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. 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. Sulfhydryl groups are useful targets for protein conjugation and labeling. Typically, only free or reduced sulfhydryl groups (–SH) are available for reaction with thiol-reactive compounds. 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. Sulfhydryls, also called thiols, exist in proteins in the side-chain of cysteine (Cys, C) amino acids.
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.
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