Laboratorio de Enzimología, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay; Laboratorio de Fisicoquímica Biológica, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay; Centro de Investigaciones Biomédicas (CEINBIO), Universidad de la República, Montevideo, Uruguay.
Department of Chemistry and Macromolecules Innovation Institute, Virginia Tech, Blacksburg, Virginia, USA.
J Biol Chem. 2022 Oct;298(10):102402. doi: 10.1016/j.jbc.2022.102402. Epub 2022 Aug 19.
Hydrogen sulfide (HS) is produced endogenously by several enzymatic pathways and modulates physiological functions in mammals. Quantification of HS in biochemical systems remains challenging because of the presence of interferents with similar reactivity, particularly thiols. Herein, we present a new quantification method based on the formation of pyrene excimers in solution. We synthesized the probe 2-(maleimido)ethyl 4-pyrenylbutanoate (MEPB) and determined that MEPB reacted with HS in a two-step reaction to yield the thioether-linked dimer (MEPB)S, which formed excimers upon excitation, with a broad peak of fluorescence emission centered at 480 nm. In contrast, we found that the products formed with thiols showed peaks at 378 and 398 nm. The difference in emission between the products prevented the interference. Furthermore, we showed that the excimer fluorescence signal yielded a linear response to HS, with a limit of detection of 54 nM in a fluorometer. Our quantification method with MEPB was successfully applied to follow the reaction of HS with glutathione disulfide and to quantify the production of HS from cysteine by Escherichia coli. In conclusion, this method represents an addition to the toolkit of biochemists to quantify HS specifically and sensitively in biochemical systems.
硫化氢（HS）由几种酶促途径产生，调节哺乳动物的生理功能。由于存在与反应性相似的干扰物，特别是硫醇，因此在生化系统中定量 HS 仍然具有挑战性。在此，我们提出了一种基于溶液中芘激基缔合物形成的新定量方法。我们合成了探针 2-（马来酰亚胺基）乙基 4-芘丁酸酯（MEPB），并确定 MEPB 与 HS 发生两步反应生成硫醚键连接的二聚体（MEPB）S，该二聚体在激发时形成激基缔合物，荧光发射的宽峰位于 480nm 处。相比之下，我们发现与硫醇形成的产物在 378nm 和 398nm 处出现峰。产物之间的发射差异可以防止干扰。此外，我们表明，激基缔合物荧光信号对 HS 呈现线性响应，在荧光计中检测限为 54nM。我们使用 MEPB 的定量方法成功地用于跟踪 HS 与谷胱甘肽二硫化物的反应，并定量大肠杆菌中半胱氨酸产生的 HS。总之，该方法代表了生物化学家和生物学家在生化系统中特异性和灵敏地定量 HS 的工具包的补充。
