Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, Olomouc, 77515, Czech Republic.
Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, Olomouc, 77515, Czech Republic.
Free Radic Biol Med. 2021 Feb 20;164:381-389. doi: 10.1016/j.freeradbiomed.2021.01.007. Epub 2021 Jan 9.
Covalent modifications of thiol and amine groups may control the function of proteins involved in the regulatory and signaling pathways of the cell. In this study, we developed a simple cysteamine assay which can be used to study the reactivity of electrophilic compounds towards primary amine and thiol groups in an aqueous environment. The detection principle is based on the electrochemical, photometrical and mass spectrometric analyses of cysteamine (2-aminoethanethiol) as the molecular probe. This technique is useful for studying the reaction kinetics of electrophiles with thiol (SH) and amino (NH) groups. The decrease in analytical responses of cysteamine was monitored to evaluate the reactivity of three electrophilic activators of the Nrf2 pathway, which mediates the cellular stress response. The SH-reactivity under cell-free conditions of the tested electrophiles decreased in the following order: 4-hydroxy-2-nonenal ≥ nitro-oleic acid > sulforaphane. However, as shown in RAW264.7 cells, the tested compounds activated Nrf2-dependent gene expression in the opposite order: sulforaphane > nitro-oleic acid ≥ 4-hydroxy-2-nonenal. Although other factors in addition to chemical reactivity play a role in biological systems, we conclude that this cysteamine assay is a useful tool for screening potentially bioactive electrophiles and for studying their reactivity at a molecular level.
巯基和胺基的共价修饰可能会控制细胞调节和信号通路中涉及的蛋白质的功能。在这项研究中，我们开发了一种简单的半胱胺测定法，可用于研究亲电化合物在水相环境中对伯胺和巯基的反应性。检测原理基于电化学、分光光度法和质谱分析半胱胺（2-氨基乙硫醇）作为分子探针。该技术可用于研究亲电试剂与巯基 (SH) 和氨基 (NH) 基团的反应动力学。监测半胱胺分析响应的降低，以评估三种 Nrf2 途径的亲电激活剂的反应性，该途径介导细胞应激反应。在无细胞条件下，测试的亲电试剂的 SH 反应性按以下顺序降低：4-羟基-2-壬烯醛≥硝基油酸＞萝卜硫素。然而，如 RAW264.7 细胞所示，测试化合物以相反的顺序激活 Nrf2 依赖性基因表达：萝卜硫素＞硝基油酸≥4-羟基-2-壬烯醛。尽管除了化学反应性之外，还有其他因素在生物系统中起作用，但我们得出结论，这种半胱胺测定法是筛选潜在生物活性亲电试剂和研究其分子水平反应性的有用工具。
