Doctoral Program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Japan.
J Toxicol Sci. 2013;38(3):477-84. doi: 10.2131/jts.38.477.
Although covalent modification of protein thiols by electrophilic metals is implicated in disruption of protein functions associated with toxicity, there are limited methods available to detect such modifications. In the present study, we established a convenient method to assess modification of protein thiols by electrophiles, referred to as a biotin-PEAC₅-maleimide (BPM)-labeling assay. In this assay, protein S-modification by electrophiles can be estimated by a decrease in protein modification by BPM, a thiol reactive probe. Using methylmercury (MeHg) as a model electrophilic metal, thiol modification of cellular proteins was detected by the BPM-labeling assay in SH-SY5Y cell lysates and primary mouse hepatocytes. The sensitivity and reliability of the assay was confirmed by atomic absorption spectrometry with recombinant Keap1 as a model thiol protein. This assay was applied to not only MeHg but also to other metals such as cadmium and lead. We also established a BPM-precipitation assay with avidin-agarose beads to separate BPM-modified cellular proteins followed by detection with the individual antibodies. This assay was available for detecting MeHg-induced S-modification of cellular Keap1 in SH-SY5Y cells. Taken together, we have developed reliable simple methods to estimate protein S-modification by electrophilic metals.
尽管亲电子金属对蛋白质巯基的共价修饰与毒性相关的蛋白质功能障碍有关，但目前可用的检测这种修饰的方法有限。在本研究中，我们建立了一种评估亲电子体修饰蛋白质巯基的简便方法，称为生物素-PEAC5-马来酰亚胺（BPM）标记测定法。在该测定法中，通过 BPM（一种巯基反应探针）对蛋白质修饰的减少，可以估计亲电子体对蛋白质 S-修饰的影响。使用甲基汞（MeHg）作为模型亲电子金属，在 SH-SY5Y 细胞裂解物和原代小鼠肝细胞中，通过 BPM 标记测定法检测细胞蛋白的巯基修饰。原子吸收光谱法证实了该测定法的灵敏度和可靠性，其中重组 Keap1 作为模型巯基蛋白。该测定法不仅适用于 MeHg，还适用于其他金属，如镉和铅。我们还建立了一种 BPM-琼脂糖珠沉淀测定法，用于分离 BPM 修饰的细胞蛋白，然后用相应的抗体进行检测。该测定法可用于检测 SH-SY5Y 细胞中 MeHg 诱导的细胞 Keap1 的 S-修饰。总之，我们已经开发了可靠的简单方法来估计亲电子金属对蛋白质巯基的修饰。
