Hammers Matthew D, Pluth Michael D
Department of Chemistry and Biochemistry, Institute of Molecular Biology, Material Science Institute, 1253 University of Oregon , Eugene, Oregon 97403, United States.
Anal Chem. 2014 Jul 15;86(14):7135-40. doi: 10.1021/ac501680d. Epub 2014 Jun 26.
Hydrogen sulfide (H2S) is an integral signaling molecule in biology with complex generation, translocation, and metabolism processes that are intertwined with cellular thiols. Differentiating the complex interplay between H2S and biological thiols, however, remains challenging due to the difficulty of monitoring H2S and thiol levels simultaneously in complex redox environments. As a step toward unraveling the complexities of H2S and thiols in sulfur redox homeostasis, we present a dual-fluorophore fragmentation strategy that allows for the ratiometric determination of relative H2S and cysteine (Cys) or homocysteine (Hcy) concentrations, two important metabolites in H2S biosynthesis. The key design principle is based on a nitrobenzofurazan-coumarin (NBD-Coum) construct, which fragments into spectroscopically differentiable products upon nucleophilic aromatic substitution with either H2S or Cys/Hcy. Measurement of the ratio of fluorescence intensities from coumarin and the NBD-Cys or NBD-Hcy adducts generates a sigmoidal response with a dynamic range of 3 orders of magnitude. The developed scaffold displays a rapid response (<1 min) and is selective for sulfhydryl-containing nucleophiles over other reactive sulfur, oxygen, and nitrogen species, including alcohol- and amine-functionalized amino acids, polyatomic anionic sulfur species, NO, and HNO. Additionally, NBD-Coum is demonstrated to differentiate and report on different oxidative stress stimuli in simulated sulfur pools containing H2S, Cys, and cystine.
硫化氢（H₂S）是生物学中一种不可或缺的信号分子，其生成、转运和代谢过程复杂，且与细胞硫醇相互交织。然而，由于难以在复杂的氧化还原环境中同时监测H₂S和硫醇水平，区分H₂S与生物硫醇之间复杂的相互作用仍然具有挑战性。作为揭示硫氧化还原稳态中H₂S和硫醇复杂性的一步，我们提出了一种双荧光团断裂策略，该策略能够对相对H₂S和半胱氨酸（Cys）或同型半胱氨酸（Hcy）浓度进行比率测定，这两种物质是H₂S生物合成中的重要代谢产物。关键设计原理基于一种硝基苯并呋喃香豆素（NBD - Coum）构建体，它在与H₂S或Cys/Hcy发生亲核芳香取代反应后会断裂成光谱上可区分的产物。通过测量香豆素与NBD - Cys或NBD - Hcy加合物的荧光强度比值，可产生一个动态范围为3个数量级的S形响应。所开发的支架显示出快速响应（<1分钟），并且对含巯基的亲核试剂具有选择性，优于其他活性硫、氧和氮物种，包括醇和胺官能化的氨基酸、多原子阴离子硫物种、NO和HNO。此外，在含有H₂S、Cys和胱氨酸的模拟硫池中，NBD - Coum被证明能够区分并报告不同的氧化应激刺激。
