Sulfhydrylation, reductive

The reduction of the keto groups to enol groups occurs with many reducing compounds. Of particular interest are the various sulfhydryl compounds (H2S, GSH, homocysteine used to convert dehydroascorhic acid to ascorbic acid for analysis by an oxidation method. Similar compounds can potentially reduce dehydroascorhic acid in biological systems. The redox potential of GSH is now believed to be considerably lower than was once thought (Eo at pH 7, 30° C = —0.32) (B28), considerably below that of ascorbic acid (-(-0.058). This in part accounts for the inefficiency of sulfhydryl reductions of dehydroascorhic acid. Mapson (M6) estimates that Ae half-time of reduction of dehydro-ascorbic acid by GSH is about 15 minutes under physiological conditions of pH, temperature and concentrations. 

Disulfides. As shown in Figure 4, the and h-chains of insulin are connected by two disulfide bridges and there is an intrachain cycHc disulfide link on the -chain (see Insulin and other antidiabetic drugs). Vasopressin [9034-50-8] and oxytocin [50-56-6] also contain disulfide links (48). Oxidation of thiols to disulfides and reduction of the latter back to thiols are quite common and important in biological systems, eg, cysteine to cystine or reduced Hpoic acid to oxidized Hpoic acid. Many enzymes depend on free SH groups for activation—deactivation reactions. The oxidation—reduction of glutathione (Glu-Cys-Gly) depends on the sulfhydryl group from cysteine. 

The special properties of lipoic acid arise from the ring strain experienced by oxidized lipoic acid. The closed ring form is approximately 20 kj higher in energy than the open-chain form, and this results in a strong negative reduction potential of about —0.30 V. The oxidized form readily oxidizes cyanides to isothiocyanates and sulfhydryl groups to mixed disulfides. 

Cysteine sulfhydryls and cystine disulfides may undergo a variety of reactions, including alkylation to form stable thioether derivatives, acylation to form relatively unstable thioesters, and a number of oxidation and reduction processes (Figure 1.10). Derivatization of the side chain sulfhydryl of cysteine is one of the most important reactions of modification and conjugation techniques for proteins. 

The following protocol is useful for the modification of proteins with cystamine with subsequent reduction to create the free sulfhydryl. 

One of the most convenient ways of generating sulfhydryl groups is by reduction of indigenous disulfides. Many proteins contain cystine disulfides that are not critical to structure or activity. 

The presence of two sulfhydryl groups in DTT and DTE, however, allows the formation of a favored cyclic disulfide during the course of target protein reduction (Figure 1.75). This drives the equilibrium toward the reduction of target disulfides. Therefore, complete reduction is possible with much lower concentrations of DTT or DTE than when using monothiol systems. 

To overcome these issues, the water-soluble TCEP was synthesized and successfully used to cleave organic disulfides to sulfhydryls in water (Burns et al., 1991). The advantage of using this phosphine derivative in disulfide reduction as opposed to previous ones is its excellent stability in aqueous solution, its lack of reactivity with other common functionalities in biomolecules, and its freedom from odor. 

Regenerate the sulfhydryl containing support by following steps 2 and 3 above. Such columns can be regenerated and reused at least 10 times without any significant decrease in the reductive capacity. 

Ethylenimine may be used to introduce additional sites of tryptic cleavage for protein structural studies. In this case, complete sulfhydryl modification is usually desired. Proteins are treated with ethylenimine under denaturing conditions (6-8 M guanidine hydrochloride) in the presence of a disulfide reductant to reduce any disulfide bonds before modification. Ethylenimine may be added directly to the reducing solution in excess (similar to the procedure for Aminoethyl-8 described previously) to totally modify the —SH groups formed. 

Figure 5.3 SMPT can form crosslinks between an amine-containing molecule and a sulfhydryl-containing compound through amide and disulfide linkages, respectively. The hindered nature of the disulfide group provides better stability toward reduction and cleavage.

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