Disulfides reductive cleavage

It has been generally accepted that the extreme-pressure performance of disulfides is better than that of monosulfides (Allum Ford 1965 Forbes 1970). The difference was explained simply with the anion radical lubrication model (Kajdas 1994). Monosulfides are reduced less readily than disulfides. Reductive cleavage of disulfides with the generation of active species RS and RS proceeds more easily than that of monosulfides. Accordingly, disulfides exhibit more efficient load-carrying properties. 

FIGURE 5.18 Methods for cleavage of disulfide bonds in proteins, (a) Oxidative cleavage by reaction with performic acid, (b) Reductive cleavage with snlfliydryl compounds. Disulfide bridges can be broken by reduction of the S—S link with snlfliydryl agents such as 2-mercaptoethanol or dithiothreitol. Because reaction between the newly reduced —SH groups to re-establish disulfide bonds is a likelihood, S—S reduction must be followed by —SH modification (1) alkylation with iodoac-etate (ICH,COOH) or (2) modification with 3-bromopropylamine (Br— (CH,)3—NH,). 

U. T. Ruf gg u. J. Rudinger, Reductive Cleavage of cystine disulfides with tributylphosphins, Methods Enzymol. 47, 111 (1977). 

Figure 4-3. Oxidative cleavage of adjacent polypeptide chains linked by disulfide bonds (shaded) by per-formic acid (left) or reductive cleavage by 3-mercap-toethanol (right) forms two peptides that contain cysteic acid residues or cysteinyl residues, respectively.

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. 

SMPT often is used in place of SPDP for the preparation of immunotoxin conjugates. The hindered disulfide of SMPT has distinct advantages in this regard. Thorpe et al. (1987) showed that SMPT conjugates had approximately twice the half-life in vivo as SPDP conjugates. Antibody-toxin conjugates prepared with SMPT possess a half-life in vivo of up to 22 hours, presumably due to the decreased susceptibility of the hindered disulfide toward reductive cleavage. 

Note Some protocols do not call for a reduction step. The addition of borohydride at this level may result in disulfide bond cleavage and loss of protein activity in some cases. As an alternative to reduction, add 50pi of 0.2M lysine in 0.5M sodium carbonate, pH 9.5 to each ml of the conjugation reaction to block excess reactive sites. Block for 2 hours at room temperature. Other amine-containing small molecules may be substituted for lysine—such as glycine, Tris buffer, or ethanolamine. 

Reductive cleavage of disulfides is very easy and can be accomplished without affecting some other readily reducible groups such as nitro groups. 

Other dc and ac polarographic assays have been reported for warfarin [86], tetramethylthiuram disulfide employing the reduction of the S-S bond [87], and hexachlorophene using reductive cleavage of the halogram atom [88]. Another report dealt with dc polarographic assay of the p-fluoro-substituted butyro-phenone derivatives triperidol, fluanisone, and haloperidol [89]. 

The nitrogen-sulfur bond in 161 (X = S, R = NPhth X = S02, R = OMe) is easily broken under certain conditions. For instance, attempts to obtain a palladium-catalyzed carbonylation reaction with the iodide gave only the ring-opened disulfide 162 and the sulfonic acid 163, presumably by reductive cleavage of the N-S bond by the triphenylphosphine in the reaction mixture <2000T5571>. 

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