Application of WST-8 based colorimetric NAD(P)H detection for quantitative dehydrogenase assays

被引:100
作者
Chamchoy, Kamonwan [1 ]
Pakotiprapha, Danaya [2 ,3 ]
Pumirat, Pornpan [4 ]
Leartsakulpanich, Ubolsree [5 ]
Boonyuen, Usa [1 ]
机构
[1] Mahidol Univ, Fac Trop Med, Dept Mol Trop Med & Genet, Bangkok 10400, Thailand
[2] Mahidol Univ, Fac Sci, Dept Biochem, Bangkok 10400, Thailand
[3] Mahidol Univ, Fac Sci, Ctr Excellence Prot & Enzyme Technol, Bangkok 10400, Thailand
[4] Mahidol Univ, Fac Trop Med, Dept Microbiol & Immunol, Bangkok 10400, Thailand
[5] Natl Sci & Technol Dev Agcy, Natl Ctr Genet Engn & Biotechnol, Pathum Thani 12120, Thailand
关键词
WST-8; Tetrazolium salts; Dehydrogenase activity; WATER-SOLUBLE FORMAZAN; TETRAZOLIUM SALT; ELECTROCATALYTIC OXIDATION; GLUCOSE-DEHYDROGENASE; NADH; VIABILITY; PURIFICATION; INDICATOR; CONFORMATION; VALIDATION;
D O I
10.1186/s12858-019-0108-1
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Background: The reduction of tetrazolium salts by NAD(P)H to formazan product has been widely used to determine the metabolic activity of cells, and as an indicator of cell viability. However, the application of a WST-8 based assay for the quantitative measurement of dehydrogenase enzyme activity has not been described before. In this study, we reported the application of an assay based on the tetrazolium salt WST-8 for the quantitative measurement of dehydrogenase activity. The assay is performed in a microplate format, where a single endpoint is measured at 450 nm. Results: The optimized dehydrogenase-WST-8 assay conditions, the limit of detection (LOD), accuracy, and precision for measuring NAD(P)H, were demonstrated. The sensitivity of the WST-8 assay for detecting NAD(P)H was 5-fold greater than the spectrophotometric measurement of NAD(P)H absorption at 340 nm (LOD of 0.3 nmole vs 1.7 nmole, respectively). In the dehydrogenase assay, the colorimetric WST-8 method exhibits excellent assay reproducibility with a Z' factor of 0.9. The WST-8 assay was also used to determine dehydrogenase activity in biological samples, and for screening the substrate of uncharacterized short-chain dehydrogenase/oxidoreductase from Burkholderia pseudomallei. Conclusion: The results suggest that the WST-8 assay is a sensitive and rapid method for determining NAD(P)H concentration and dehydrogenase enzyme activity, which can be further applied for the high-throughput screening of dehydrogenases.
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页数:14
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