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Degradation of protein disulphide bonds in dilute alkali.

ABSTRACT: The degradation of S--S bonds in 0.2 M-NaOH at 25 degrees C was studied for a series of proteins and simple aliphatic disulphide compounds, by using cathodic stripping voltammetry, ion-selective-electrode potentiometry, spectrophotometry and ultrafiltration. The disulphide bonds that dissociated in 0.2 M-NaOH were usually those that are solvent accessible and that can be reduced by mild chemical reductants. Some unexpected differences were found between similar proteins, both in the number of S--S bonds dissociated and in their rates of decomposition. Chymotrypsin has one S--S bond attacked, whereas chymotrypsinogen and trypsinogen have two. Ribonuclease A has two S--S bonds dissociated, but ribonuclease S and S-protein have three. Denaturation in 6 M-guanidine hydrochloride before alkaline digestion caused the loss of an additional S--S bond in ribonuclease A and insulin, and increased the rate of dissociation of the S--S bonds of some other proteins. The initial product of S--S bond dissociation in dilute alkali is believed to be a persulphide intermediate formed by a beta-elimination reaction. This intermediate is in mobile equilibrium with bisulphide ion, HS-, and decomposes at a mercury electrode or in acid solution to yield a stoichiometric amount of sulphide. Rate constants and equilibrium constants were measured for the equilibria between HS- and the intermediates involved in the alkaline dissociation of several proteins. Elemental sulphur was not detected in any of the protein digests. It is suggested that formation of HS- from a persulphide intermediate involves a hydrolysis reaction to yield a sulphenic acid derivative. The small polypeptides glutathione and oxytocin gave only a low yield of persulphide, and their alkaline decomposition must proceed by a mechanism different from that of the proteins.

SUBMITTER: Florence TM

PROVIDER: S-EPMC1162031 | biostudies-other | 1980 Sep

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: Disulphide bridges are well known to play key roles in stability, folding and functions of proteins. Introduction or deletion of disulphides by site-directed mutagenesis have produced varying effects on stability and folding depending upon the protein and location of disulphide in the 3-D structure. Given the lack of complete understanding it is worthwhile to learn from an analysis of extent of conservation of disulphides in homologous proteins. We have also addressed the question of what structural interactions replaces a disulphide in a homologue in another homologue. RESULTS: Using a dataset involving 34,752 pairwise comparisons of homologous protein domains corresponding to 300 protein domain families of known 3-D structures, we provide a comprehensive analysis of extent of conservation of disulphide bridges and their structural features. We report that only 54% of all the disulphide bonds compared between the homologous pairs are conserved, even if, a small fraction of the non-conserved disulphides do include cytoplasmic proteins. Also, only about one fourth of the distinct disulphides are conserved in all the members in protein families. We note that while conservation of disulphide is common in many families, disulphide bond mutations are quite prevalent. Interestingly, we note that there is no clear relationship between sequence identity between two homologous proteins and disulphide bond conservation. Our analysis on structural features at the sites where cysteines forming disulphide in one homologue are replaced by non-Cys residues show that the elimination of a disulphide in a homologue need not always result in stabilizing interactions between equivalent residues. CONCLUSION: We observe that in the homologous proteins, disulphide bonds are conserved only to a modest extent. Very interestingly, we note that extent of conservation of disulphide in homologous proteins is unrelated to the overall sequence identity between homologues. The non-conserved disulphides are often associated with variable structural features that were recruited to be associated with differentiation or specialisation of protein function.

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Degradation of protein disulphide bonds in dilute alkali.

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