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It is well established that proteins and peptides can release sulfur under alkaline treatment, mainly through the β-elimination of disulfides with the concomitant formation of persulfides and dehydroalanine derivatives. In this study, 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-Vis absorbance, reaction with 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB), and cold cyanolysis, obtaining an apparent second-order rate constant of ∼10 -3  M -1  s -1 at 25 °C. The formation of GSSH and the dehydroalanine derivative was confirmed by HPLC and/or mass spectrometry. However, the mixtures did not equilibrate in a timescale of hours, and additional species, including thiol and diverse sulfane sulfur compounds were also formed, probably through further reactions of the persulfide. Cold cyanolysis is frequently used to quantify persulfides, since it measures sulfane sulfur. This method involves a step in which the sample to be analyzed is incubated with cyanide at alkaline pH. When cold cyanolysis was applied to samples containing GSSG, sulfane sulfur products that were not present in the original sample were measured. Thus, our results reveal the risk of overestimating the amount of sulfane sulfur compounds in samples that contain disulfides due to their decay to persulfides 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 GSSH from incubation of GSSG in alkali. Our study also highlights the importance of being cautious when doing and interpreting cold cyanolysis experiments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Persulfides (RSSH/RSS - ) participate in sulfur trafficking and metabolic processes, and are proposed to mediate the signaling effects of hydrogen sulfide (H 2 S). Despite their growing relevance, their chemical properties are poorly understood. Herein, we studied experimentally and computationally the formation, acidity, and nucleophilicity of glutathione persulfide (GSSH/GSS - ), the derivative of the abundant cellular thiol glutathione (GSH). We characterized the kinetics and equilibrium of GSSH formation from glutathione disulfide and H 2 S. A p K a of 5.45 for GSSH was determined, which is 3.49 units below that of GSH. The reactions of GSSH with the physiologically relevant electrophiles peroxynitrite and hydrogen peroxide, and with the probe monobromobimane, were studied and compared with those of thiols. These reactions occurred through S N 2 mechanisms. At neutral pH, GSSH reacted faster than GSH because of increased availability of the anion and, depending on the electrophile, increased reactivity. In addition, GSS - presented higher nucleophilicity with respect to a thiolate with similar basicity. This can be interpreted in terms of the so-called α effect, i.e. the increased reactivity of a nucleophile when the atom adjacent to the nucleophilic atom has high electron density. The magnitude of the α effect correlated with the Brønsted nucleophilic factor, β nuc , for the reactions with thiolates and with the ability of the leaving group. Our study constitutes the first determination of the p K a of a biological persulfide and the first examination of the α effect in sulfur nucleophiles, and sheds light on the chemical basis of the biological properties of persulfides., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Benchoam et al.)