Mono-sulfonated tetrazolium salt based NAD(P)H detection reagents suitable for dehydrogenase and real-time cell viability assays.
Anal Biochem
; 509: 33-40, 2016 09 15.
Article
en En
| MEDLINE
| ID: mdl-27387057
ABSTRACT
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).
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Sales de Tetrazolio
/
Proteínas de Escherichia coli
/
Glutamato Deshidrogenasa
/
NADP
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
Idioma:
En
Revista:
Anal Biochem
Año:
2016
Tipo del documento:
Article
País de afiliación:
China