Journal
ANALYTICAL BIOCHEMISTRY
Volume 509, Issue -, Pages 33-40Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2016.06.026
Keywords
Mono-sulfonated tetrazolium salt; NAD(P)H assay; Dehydrogenase; Cell proliferation; Cell toxicity; High through-put screening
Funding
- Zhejiang University of Technology [414800129]
- Science and Technology planning project of Zhejiang Province [2015C33096]
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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.
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