title: Detection of NAD(P)H-dependent enzyme activity by time-domain ratiometry of terbium luminescence.
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.
authors.affiliation: 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.
authors.forename: Takuya
authors.initials: T
authors.lastname: Terai
authors.affiliation: 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.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Ito
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Kenjiro
authors.initials: K
authors.lastname: Hanaoka
authors.affiliation: 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.
authors.forename: Toru
authors.initials: T
authors.lastname: Komatsu
authors.affiliation: 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.
authors.forename: Tasuku
authors.initials: T
authors.lastname: Ueno
authors.affiliation: Drug Discovery Initiative, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Nagano
authors.affiliation: 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.
authors.forename: Yasuteru
authors.initials: Y
authors.lastname: Urano
mesh_terms: D049449:Luminescence; D009249:NADP / Q000378:metabolism*; D013725:Terbium / Q000378:metabolism*
chemical_list: D013725:Terbium; D009249:NADP
keywords: Coupled enzyme assay; Lanthanide complex; Oxidoreductase assay; Ratiometric detection; Time-resolved luminescence