Detection of NAD(P)H-dependent enzyme activity by time-domain ratiometry of terbium luminescence

Takuya Terai; Hiroki Ito; Kenjiro Hanaoka; Toru Komatsu; Tasuku Ueno; Tetsuo Nagano; Yasuteru Urano

doi:10.1016/j.bmcl.2016.03.038

Detection of NAD(P)H-dependent enzyme activity by time-domain ratiometry of terbium luminescence

Bioorg Med Chem Lett. 2016 May 1;26(9):2314-7. doi: 10.1016/j.bmcl.2016.03.038. Epub 2016 Mar 11.

Authors

Takuya Terai¹, Hiroki Ito², Kenjiro Hanaoka³, Toru Komatsu⁴, Tasuku Ueno², Tetsuo Nagano⁵, Yasuteru Urano⁶

Affiliations

¹ Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; AMED CREST, 1-7-1 Otemachi, Chiyoda-ku, Tokyo 100-0004, Japan. Electronic address: t-terai@mol.f.u-tokyo.ac.jp.
² Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; AMED CREST, 1-7-1 Otemachi, Chiyoda-ku, Tokyo 100-0004, Japan.
³ Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
⁴ Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; JST PRESTO, 7 Gobancho, Chiyoda-ku, Tokyo 102-0076, Japan.
⁵ Drug Discovery Initiative, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
⁶ Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; AMED CREST, 1-7-1 Otemachi, Chiyoda-ku, Tokyo 100-0004, Japan; Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Electronic address: uranokun@m.u-tokyo.ac.jp.

PMID: 27013390
DOI: 10.1016/j.bmcl.2016.03.038

Abstract

NAD(P)H-dependent oxidoreductases play important roles in biology. Recently, we reported that the luminescence lifetime of some Tb(3+) complexes is sensitive to NAD(P)H, and we used this phenomenon to detect activities of these enzymes. However, conventional time-resolved luminescence assays are susceptible to static quenchers such as ATP. Herein we describe a detection methodology that overcomes this issue: the intensity of the sample is measured twice with different delay times and the intensity ratio value is used as an index of NAD(P)H concentration. The method is more robust than single-point measurement, and is compatible with high-throughput assays using conventional microplate readers.

Keywords: Coupled enzyme assay; Lanthanide complex; Oxidoreductase assay; Ratiometric detection; Time-resolved luminescence.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Luminescence
NADP / metabolism*
Terbium / metabolism*

Substances

Terbium
NADP