Design, Validation, and Application of an Enzyme-Coupled Hydrogen Sulfide Detection Assay.
Design, Validation, and Application of an Enzyme-Coupled Hydrogen Sulfide Detection Assay.
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DOI：
10.1021/acs.biochem.8b01083
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发表时间：
2018-12
期刊：
影响因子：
2.9
通讯作者：
M. Lynch;B. Crane
中科院分区：
文献类型：
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作者：
M. Lynch;B. Crane
硫化氢（H2S）是生物合成中的一种关键代谢物，作为一种重要的气体传递物越来越被人们所认识。由于H2S具有扩散性和反应性，因此很难量化，特别是在原位。虽然有几种可用的检测方案，但它们都有缺点。为了量化鞭毛蛋白FlgE交联反应中硫化物的释放，我们开发了一种酶偶联硫化物检测方法，使用大肠杆菌o -乙酰丝氨酸巯基水解酶CysM。通过CysM将HS-转化为l-半胱氨酸，然后与巯基特异性荧光染料7-二乙基氨基-3-（4-马来酰亚苯基）-4-甲基香豆素衍生，可以通过荧光高效液相色谱法方便地检测和定量H2S。通过与完善的亚甲基蓝硫化物检测方法进行比较，验证了该方法的有效性，并通过干扰试验证明了该方法在常见硫醇（如谷胱甘肽、2-巯基乙醇、二硫苏糖和l-蛋氨酸）以及一系列阴离子存在下的稳定期。然后，我们将该实验应用于上述由密螺旋体鞭毛钩蛋白FlgE交联的赖氨酸丙氨酸。总的来说，与之前报道的H2S检测方法不同，该方法提供了一个生物兼容的平台，可以准确、专门地原位测量硫化氢，即使是在长时间尺度上产生的硫化氢。
Hydrogen sulfide (H2S) is a key metabolite in biosynthesis and is increasingly being recognized as an essential gasotransmitter. Owing to its diffusible and reactive nature, H2S can be difficult to quantify, particularly in situ. Although several detection schemes are available, they have drawbacks. In efforts to quantify sulfide release in the cross-linking reaction of the flagellar protein FlgE, we developed an enzyme-coupled sulfide detection assay using the Escherichia coli O-acetylserine sulfhydrylase enzyme CysM. Conversion of HS- to l-cysteine via CysM followed by derivatization with the thiol-specific fluorescent dye 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin enables for facile detection and quantification of H2S by fluorescent HPLC. The assay was validated by comparison to the well-established methylene blue sulfide detection assay and the robustness demonstrated by interference assays in the presence of common thiols such as glutathione, 2-mercaptoethanol, dithiothreitol, and l-methionine, as well as a range of anions. We then applied the assay to the aforementioned lysinoalanine cross-linking by the Treponema denticola flagellar hook protein FlgE. Overall, unlike previously reported H2S detection methods, the assay provides a biologically compatible platform to accurately and specifically measure hydrogen sulfide in situ, even when it is produced on long time scales.