Speciation of reactive sulfur species and their reactions with alkylating agents: do we have any clue about what is present inside the cell?
Speciation of reactive sulfur species and their reactions with alkylating agents: do we have any clue about what is present inside the cell?
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DOI：
10.1111/bph.14394
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发表时间：
2019-02-01
影响因子：
7.3
通讯作者：
Nagy, Peter
中科院分区：
文献类型：
--
作者：
Bogdandi, Virag;Ida, Tomoaki;Nagy, Peter
背景与目的半胱氨酸残基的翻译后修饰是氧化还原生物学的一个重要方面，其可靠的检测是提供机械性见解的关键。这些修饰的亚稳特性和细胞裂解诱导的人工氧化使目前最先进的方案依赖于细胞/组织破裂前不稳定的半胱氨酸衍生物的烷基化稳定。在这些过程中，一个未经检验的假设是，对于所有半胱氨酸衍生物，烷基化速度快于它们的动态交换。然而，当半胱氨酸衍生物的相互转化不受速率限制时，亲电标记受Curtin-Hammett控制；因此，最终的烷基化混合物可能不代表烷基化之前占优势的物种。在人体血浆和血清中的其他实验表明，单溴双胺可以通过改变形态平衡从内源硫库中提取硫化物，这表明在使用该工具解释实验结果时应谨慎。KEY结果在大多数情况下，烷基化多硫化物/硫醇衍生物的形态取决于实验条件。烷基化以浓度和时间依赖的方式干扰硫的形态形成，强烷基化剂断裂多硫链。此外，用双酮标记磺酸也影响了半胱氨酸的形态形成，这表明以前被认为代表亚磺酸物种的部分内源产物池可能代表多硫化物。结论和实施我们强调了这些缺陷可能产生的方法学警告，并得出结论，当前的衍生化策略往往不能充分捕捉硫物种的生理性形态形成。
BACKGROUND AND PURPOSEPosttranslational 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 APPROACHBuffered 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 RESULTSIn 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 IMPLICATIONSWe highlight methodological caveats potentially arising from these pitfalls and conclude that current derivatization strategies often fail to adequately capture physiological speciation of sulfur species.