Speciation of reactive sulfur species and their reactions with alkylating agents: do we have any clue about what is present inside the cell?

• Published in: British journal of pharmacology Vol. 176; no. 4; pp. 646 - 670
• Main Authors: Bogdándi, Virág, Ida, Tomoaki, Sutton, Thomas R, Bianco, Christopher, Ditrói, Tamás, Koster, Grielof, Henthorn, Hillary A, Minnion, Magda, Toscano, John P, Vliet, Albert, Pluth, Michael D, Feelisch, Martin, Fukuto, Jon M, Akaike, Takaaki, Nagy, Péter
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.02.2019; John Wiley and Sons Inc
• Subjects: Adult; Alkylating agents; Alkylating Agents - chemistry; Alkylation; Blood plasma; Cysteine; Glyceraldehyde-3-phosphate dehydrogenase; Humans; Iodoacetamide - chemistry; Labeling; Lysis; Maleimides - chemistry; Methyl Methanesulfonate - analogs & derivatives; Methyl Methanesulfonate - chemistry; Oxidation; Research Paper; Speciation; Species; Sulfenic acid; Sulfides; Sulfur; Sulfur Compounds - analysis; Sulfur Compounds - blood; Sulfur Compounds - chemistry; Themed Section: Research Papers
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1111/bph.14394

  Background and Purpose Posttranslational modifications of cysteine residues represent a major aspect of redox biology, and their reliable detection is key in providing mechanistic insights. The metastable character of these modifications and cell lysis‐induced artifactual oxidation render current state‐of‐the‐art protocols to rely on alkylation‐based stabilization of labile cysteine derivatives before cell/tissue rupture. An untested assumption in these procedures is that for all cysteine derivatives, alkylation rates are faster than their dynamic interchange. However, when the interconversion of cysteine derivatives is not rate limiting, electrophilic labelling is under Curtin–Hammett control; hence, the final alkylated mixture may not represent the speciation that prevailed before alkylation. Experimental Approach Buffered aqueous solutions of inorganic, organic, cysteine, GSH and GAPDH polysulfide species were used. Additional experiments in human plasma and serum revealed that monobromobimane can extract sulfide from the endogenous sulfur pool by shifting speciation equilibria, suggesting caution should be exercised when interpreting experimental results using this tool. Key Results In the majority of cases, the speciation of alkylated polysulfide/thiol derivatives depended on the experimental conditions. Alkylation perturbed sulfur speciation in both a concentration‐ and time‐dependent manner and strong alkylating agents cleaved polysulfur chains. Moreover, the labelling of sulfenic acids with dimedone also affected cysteine speciation, suggesting that part of the endogenous pool of products previously believed to represent sulfenic acid species may represent polysulfides. Conclusions and Implications We highlight methodological caveats potentially arising from these pitfalls and conclude that current derivatization strategies often fail to adequately capture physiological speciation of sulfur species. Linked Articles This article is part of a themed section on Chemical Biology of Reactive Sulfur Species. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.4/issuetoc