TY - JOUR
T1 - Detection of NAD(P)H-dependent enzyme activity by time-domain ratiometry of terbium luminescence
AU - Terai, Takuya
AU - Ito, Hiroki
AU - Hanaoka, Kenjiro
AU - Komatsu, Toru
AU - Ueno, Tasuku
AU - Nagano, Tetsuo
AU - Urano, Yasuteru
N1 - Funding Information:
This work was supported in part by the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Specially Promoted Research Grant 22000006 to T.N., Grants 24689003 and 24659042 to K.H., 24655147 to K.T., 25104506 and 15K05529 to T.T., and 23249004 to Y.U.), by SENTAN , JST (K.H.), Grant-in-Aid for JSPS Fellows (H.I.), Astellas Foundation for Research on Metabolic Disorders (T.T.), and by the Uehara Memorial Foundation (T.T.).
Publisher Copyright:
© 2016 Elsevier Ltd. All rights reserved.
PY - 2016/5/1
Y1 - 2016/5/1
N2 - 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.
AB - 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.
KW - Coupled enzyme assay
KW - Lanthanide complex
KW - Oxidoreductase assay
KW - Ratiometric detection
KW - Time-resolved luminescence
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U2 - 10.1016/j.bmcl.2016.03.038
DO - 10.1016/j.bmcl.2016.03.038
M3 - Article
C2 - 27013390
AN - SCOPUS:84961282382
SN - 0960-894X
VL - 26
SP - 2314
EP - 2317
JO - Bioorganic and Medicinal Chemistry Letters
JF - Bioorganic and Medicinal Chemistry Letters
IS - 9
ER -