Application of WST-8 based colorimetric NAD(P)H detection for quantitative dehydrogenase assays
Open Access
- 8 April 2019
- journal article
- research article
- Published by Springer Science and Business Media LLC in BMC Biochemistry
- Vol. 20 (1), 1-14
- https://doi.org/10.1186/s12858-019-0108-1
Abstract
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. 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. 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.Keywords
Funding Information
- Institution for the Promotion of Teaching Science and Technology (18/2557)
- Faculty of Tropical Medicine, Mahidol University
- Thailand Graduate Instituted of Science and Technology (TG-22-14-58-050D)
This publication has 57 references indexed in Scilit:
- The role of short-chain dehydrogenase/oxidoreductase, induced by salt stress, on host interaction of B. pseudomalleiBMC Microbiology, 2014
- Tools for mass screening of G6PD deficiency: validation of the WST8/1-methoxy-PMS enzymatic assay in UgandaMalaria Journal, 2013
- Bioanalytical method validation: An updated reviewPharmaceutical Methods, 2010
- Bioanalytical method validation: An updated reviewPharmaceutical Methods, 2010
- Colorimetric microbial viability assay based on reduction of water-soluble tetrazolium salts for antimicrobial susceptibility testing and screening of antimicrobial substancesAnalytical Biochemistry, 2009
- Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell levelJournal of Photochemistry and Photobiology B: Biology, 2009
- Marked decrease in specific activity contributes to disease phenotype in two human glucose 6-phosphate dehydrogenase mutants, G6PDUnionand G6PDAndalusHuman Mutation, 2005
- Benzothiazole-Containing Tetrazolium Salts That Produce Water-Soluble Formazan Dyes Absorbing at a Long Wavelength upon NADH ReductionAnalytical Sciences, 1996
- Haemoglobin Interference in the Bichromatic Spectrophotometry of NAD(P)H at 340/380 nmcclm, 1993
- A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry, 1976