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

BIOORGANIC & MEDICINAL CHEMISTRY LETTERS

卷:26

Article

Terai, Takuya^1,2  Ito, Hiroki^1,2  Hanaoka, Kenjiro¹  Komatsu, Toru^1,3  Ueno, Tasuku^1,2  Nagano, Tetsuo⁴  Urano, Yasuteru^1,2,5 

[1] Univ Tokyo, Grad Sch Pharmaceut Sci, Bunkyo Ku, 7-3-1 Hongo, Tokyo 1130033, Japan

[2] AMED CREST, Chiyoda Ku, 1-7-1 Otemachi, Tokyo 1000004, Japan

[3] JST PRESTO, Chiyoda Ku, 7 Gobancho, Tokyo 1020076, Japan

[4] Univ Tokyo, Drug Discovery Initiat, Bunkyo Ku, 7-3-1 Hongo, Tokyo 1130033, Japan

[5] Univ Tokyo, Grad Sch Med, Bunkyo Ku, 7-3-1 Hongo, Tokyo 1130033, Japan

关键词: Lanthanide complex;    Time-resolved luminescence;    Oxidoreductase assay;    Ratiometric detection;    Coupled enzyme assay;   

DOI  :  10.1016/j.bmcl.2016.03.038

来源: Elsevier

【 摘 要 】

NAD(P)H-dependent oxidoreductases play important roles in biology. Recently, we reported that the luminescence lifetime of some Tb3+ 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. (C) 2016 Elsevier Ltd. All rights reserved.

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