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首页> 外文期刊>Analytical Biochemistry: An International Journal of Analytical and Preparative Methods >Mono-sulfonated tetrazolium salt based NAD(P)H detection reagents suitable for dehydrogenase and real-time cell viability assays
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Mono-sulfonated tetrazolium salt based NAD(P)H detection reagents suitable for dehydrogenase and real-time cell viability assays

机译：单磺化四唑鎓盐的NAD（P）H检测试剂适用于脱氢酶和实时细胞活力测定法

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摘要

Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and is important for several biological processes. For GDH inhibitor screening, we developed a novel mono-sulfonated tetrazolium salt (EZMTT), which can be synthesized using H2O2 oxidation and purified easily on silica gel in large quantities. The EZMTT detection method showed linear dose responses to NAD(P)H, dehydrogenase concentration and cell numbers. In E. coli GDH assay, the EZMTT method showed excellent assay reproducibility with a Z factor of 0.9 and caused no false positives in the presence of antioxidants (such as BME). Using the EZMTT-formazan-NAD(P)H system, we showed that EGCG is a potent E. coli GDH inhibitor (IC50 45 nM) and identified that Ebselen, a multifunctional thioredoxin reductase inhibitor, inactivated E. coli GDH (IC50 213 nM). In cell-based assays at 0.5 mM tetrazolium concentration, EZMTT showed essentially no toxicity after a 3-day incubation, whereas 40% of inhibition was observed for WST-8. In conclusion, EZMTT is a novel tetrazolium salt which provides improved features that are suitable for dehydrogenases and real-time cell-based high-throughput screening (HTS). (C) 2016 Elsevier Inc. All rights reserved.
机译：谷氨酸脱氢酶（GDH）催化L-谷氨酸的氧化脱氨基，对几种生物方法很重要。对于GDH抑制剂筛选，我们开发了一种新型单磺化四唑鎓盐（EZMTT），其可以使用H 2 O 2氧化合成，并在大量的硅胶上容易纯化。 EZMTT检测方法显示线性剂量响应NAD（P）H，脱氢酶浓度和细胞数。在大肠杆菌GDH测定中，EZMTT方法显示出优异的测定再现性，Z因子为0.9，并且在存在抗氧化剂（例如BME）中没有引起的误报。使用EZMTT-FORMAZAN-NAD（P）H系统，我们表明EGCG是一种有效的大肠杆菌GDH抑制剂（IC50 45nm），并鉴定出EBSELEN，多功能硫醚素还原酶抑制剂，灭活大肠杆菌GDH（IC50 213nm ）。在0.5mM四济碱浓度下的基于细胞的测定中，在孵育3天后，EZMTT显示出基本上没有毒性，而WST-8观察到40％的抑制。总之，EZMTT是一种新型四唑鎓盐，其提供适用于脱氢酶和基于实时细胞的高通量筛选（HTS）的改进特征。 （c）2016年Elsevier Inc.保留所有权利。

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