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.

Analysis of reduced monoclonal antibodies using size exclusion chromatography coupled with mass spectrometry

“…The mass spectra of peaks a to c are shown in Figure 4b. The theoretical molecular weights of antibody-A heavy chain without C-terminal Lys and with a core-fucosylated biantennary complex oligosaccharide structure without terminal galactose ( linkage has been well documented in the literature [31][32][33] and reported for monoclonal antibodies [17,28]. No interpretable mass spectrum was obtained for the shoulder that eluted in front of peak a.…”

Bacterial RNA Extraction and Purification from Whole Human Blood Using Isotachophoresis

“…Although NaOH can reduce these S−S bonds, the half-life of RNase in 0.2 M NaOH is ∼30 min. 43 Meanwhile, as we have mentioned, long incubation in alkali conditions degrades RNA. However, RNase degradation can be greatly accelerated during alkali lysing by adding detergents (e.g., SDS), or reducing agents (e.g., DTT).…”

Fragmentation of peptide disulfides under conditions of negative ion mass spectrometry: Studies of oxidized glutathione and contryphan

“…The proposed fragmentation pathways for the formation of dehydroalanine, thioaldehyde, and reduced glutathione are reminiscent of chemical cleavages occurring at the cystine site in the peptide under base catalyzed condition in solutions [23][24][25]. We have attempted to obtain experimental support for these proton abstraction processes by subjecting a deuterated sample of glutathione, in which all exchangeable hydrogens have been replaced by deuterium, to collision induced dissociation in an ion trap mass spectrometer.…”