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

Kamonwan Chamchoy,Pornpan Pumirat,Usa Boonyuen,Ubolsree Leartsakulpanich,Danaya Pakotiprapha

doi:10.1186/s12858-019-0108-1

Abstract

BackgroundThe 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.ResultsThe 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.ConclusionThe 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.

Highlights

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
This demonstrated that a minimum concentration of 100 μM of water-soluble tetrazolium salts (WST)-8 was required in the glucose dehydrogenase (GDH) assay
Our results suggested that the concentration of WST-8 could be minimized for purposes of economy; at least 100 μM of WST-8 is required in the reaction to warrant the optimal sensitivity of the assay

Summary

Introduction

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. The application of a WST-8 based assay for the quantitative measurement of dehydrogenase enzyme activity has not been described before. We reported the application of an assay based on the tetrazolium salt WST-8 for the quantitative measurement of dehydrogenase activity. Various methods are used to assay NAD(P)H [4,5,6]. Both NADH and NADPH can absorb light at 340 nm and have intrinsic fluorescence. The activity of enzymes producing or consuming NAD(P)H (dehydrogenases and oxidoreductases) is (2019) 20:4 absorption on the electrode, causing fouling, and leading to reduced sensitivity, reproducibility, and stability [12]. The bioluminescent assay is very sensitive for NAD(P)H detection but is quite expensive [14]

Results

Discussion

Conclusion