Ratiometric measurement of hydrogen sulfide and cysteine/homocysteine ratios using a dual-fluorophore fragmentation strategy.
Ratiometric measurement of hydrogen sulfide and cysteine/homocysteine ratios using a dual-fluorophore fragmentation strategy.
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DOI：
10.1021/ac501680d
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发表时间：
2014-07-15
影响因子：
7.4
通讯作者：
Pluth, Michael D.
中科院分区：
文献类型：
--
作者：
Hammers, Matthew D.;Pluth, Michael D.
硫化氢（H2S）是生物学中一个不可缺少的信号分子，具有复杂的生成、易位和代谢过程，与细胞硫醇相互交织。然而，区分H2S和生物硫醇之间复杂的相互作用仍然具有挑战性，因为在复杂的氧化还原环境中很难同时监测H2S和硫醇的水平。作为揭开硫化氢和硫醇在硫氧化还原稳态中的复杂性的一步，我们提出了一种双荧光团碎片化策略，允许相对H2S和半胱氨酸（Cys）或同型半胱氨酸（Hcy）浓度的比例测定，这是H2S生物合成中的两种重要代谢物。关键的设计原理是基于硝基苯并呋喃-香豆素（NBD-Coum）结构，该结构在亲核芳香族取代H2S或Cys/Hcy时分裂成光谱可微分的产物。香豆素和NBD-Cys或NBD-Hcy加合物的荧光强度之比的测量产生一个动态范围为3个数量级的s型响应。所开发的支架显示出快速反应（<1分钟），并且对含有巯基的亲核试剂具有选择性，而不是其他活性硫、氧和氮物质，包括醇和胺功能化氨基酸、多原子阴离子硫物质、NO和HNO。此外，NBD-Coum被证明可以区分并报告含H2S、Cys和胱氨酸的模拟硫池中不同的氧化应激刺激。
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.
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