Sulfenylthiosulfate

Figure 1.121 Sodium tetrathionate reacts with thiols to form reactive sulfenylthiosulfate intermediates. Another sulfhydryl-containing molecule may couple to this active group to create a disulfide linkage.

To remove the sulfenylthiosulfate blocking group, add a 300-fold excess of DTT over the amount of blocked sulfhydryls present. Alternatively, add DTT to obtain a 0.01-0.1M final concentration. Cysteine also may be utilized to regenerate some enzymes to full activity. 

A final method of forming disulfide crosslinks between toxins and targeting molecules is the use of S-sulfonate formation using sodium sulfite (Na2SC>3) in the presence of sodium tetrathion-ate (Na2S40g). Tetrathionate reacts with sulfhydryls to form sulfenylthiosulfate intermediates (section 1.1.5.2). These derivatives are reactive toward other thiols to create disulfide linkages... 

Sodium tetrathionate (Na2S406) is a redox compound that under the right conditions can facilitate the formation of disulfide bonds from free sulfhydryls. The tetrathionate anion reacts with a sulfhydryl to create a somewhat stable active intermediate, a sulfenylthiosulfate (Fig. 102). Upon attack of the nucleophilic thiolate anion on this activated species, the thiosulfate (S203 =) leaving group is removed and a disulfide linkage forms (Pihl and Lange, 1962). The reduction of tetrathionate to thiosulfate in vivo was a subject of early study (Theis and Freeland, 1940 Chen et al., 1934). 

Figure 102 Sodium tetrathionate reacts with thiols to form reactive sulfenylthiosulfate intermediates.

The remainder of this chapter deals, in the main, with the preparation of a number of derivatives of the cysteinyl residue such as the acidic compounds, cysteic acid, S-carboxymethylcysteine, S-sulfo-cysteine, and the S-sulfenylthiosulfate of cysteine, the basic derivative S-aminoethylcysteine and S-carboxamidomethylcysteine. Procedures are also presented for the application of N-ethylmaleimide, p-mercuribenzoate, 5,5 -dithiobis(2-nitrobenzoate), and azobenzene-2-sulfenyl bromide to the spectrophotometric determination of sulfhydryl groups. 

It should be noted that the formation of a stable sulfenylthiosulfate is a phenomenon observed with native proteins (Pihl and Lange 1962). When simple thiols such as cysteine are reacted with an excess of tetrathionate, S-sulfocysteine is formed (Inglis and Liu 1970). If thiol is in excess, the sulfenylsulfonate can react with a second molecule of thiol to form a disulfide. 

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