Disulfides form persulfides at alkaline pH leading to potential overestimations in the cold cyanolysis method

BENCHOAM, DAYANA; CUEVASANTA, ERNESTO; SEMELAK, JONATHAN A.; MASTROGIOVANNI, MAURICIO; ESTRIN, DARÍO A.; MÖLLER, MATÍAS N.; ALVAREZ, BEATRIZ

FREE RADICAL BIOLOGY AND MEDICINE

ELSEVIER SCIENCE INC

Año: 2023 vol. 207 p. 63 - 71

0891-5849

It is well established that proteins and peptides can release sulfur under alkaline treatment, mainly through theβ-elimination of disulfides with the concomitant formation of persulfides and dehydroalanine derivatives. In thisstudy, we evaluated the formation of glutathione persulfide (GSSH/GSS - ) by exposure of glutathione disulfide(GSSG) to alkaline conditions. The kinetics of the reaction between GSSG and HO - was investigated by UV–Visabsorbance, reaction with 5,5 ′ -dithio-bis-(2-nitrobenzoic acid) (DTNB), and cold cyanolysis, obtaining anapparent second-order rate constant of ~10 -3 M -1 s -1 at 25 ◦ C. The formation of GSSH and the dehydroalaninederivative was confirmed by HPLC and/or mass spectrometry. However, the mixtures did not equilibrate in atimescale of hours, and additional species, including thiol and diverse sulfane sulfur compounds were alsoformed, probably through further reactions of the persulfide. Cold cyanolysis is frequently used to quantifypersulfides, since it measures sulfane sulfur. This method involves a step in which the sample to be analyzed isincubated with cyanide at alkaline pH. When cold cyanolysis was applied to samples containing GSSG, sulfanesulfur products that were not present in the original sample were measured. Thus, our results reveal the risk ofoverestimating the amount of sulfane sulfur compounds in samples that contain disulfides due to their decay topersulfides and other sulfane sulfur compounds at alkaline pH. Overall, our study highlights that the β-elimi­nation of disulfides is a potential source of persulfides, although we do not recommend the preparation of GSSHfrom incubation of GSSG in alkali. Our study also highlights the importance of being cautious when doing andinterpreting cold cyanolysis experiments.