Nuclear magnetic resonance studies of the acid–base chemistry of amino acids and peptides. IV. Mixed disulfides of cysteine, penicillamine, and glutathione

Abstract

The acid–base chemistry of the ammonium groups of penicillamine–glutathione mixed disulfide and cysteine–glutathione mixed disulfide has been characterized by 13C nmr and that for the ammonium groups of penicillamine–cysteine mixed disulfide by 1H nmr. The mixed disulfides were formed by thiol/disulfide exchange. Chemical shift titration data were obtained simultaneously for the mixed disulfides, the thiols, and the symmetrical disulfides in the mixture. Since the fractional titration of individual ammonium groups can be obtained directly from chemical shift data, it was not necessary to isolate the mixed disulfides. The chemical shift data indicate that the two ammonium groups of each of the mixed disulfides are titrated over the same pH range, with the acidity of the ammonium group of the penicillamine and cysteine parts of the penicillamine–glutathione and cysteine–glutathione mixed disulfides 4.4 and 3.9 times as acidic as those of the glutathione part. In the penicillamine–cysteine mixed disulfide, the penicillamine ammonium group is 1.7 times as acidic as that of the cysteine part. Macroscopic and microscopic acid dissociation constants are reported for each of the mixed disulfides. The advantages of nmr as a method for characterizing the acid–base chemistry of mixed disulfides are discussed.