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

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)2314-2317
Number of pages4
JournalBioorganic and Medicinal Chemistry Letters
Volume26
Issue number9
DOIs
Publication statusPublished - 2016 May 1
Externally publishedYes

Keywords

  • Coupled enzyme assay
  • Lanthanide complex
  • Oxidoreductase assay
  • Ratiometric detection
  • Time-resolved luminescence

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmaceutical Science
  • Drug Discovery
  • Clinical Biochemistry
  • Organic Chemistry

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