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Sensors 2017, 17(8), 1877; https://doi.org/10.3390/s17081877

TAMRA/TAMRA Fluorescence Quenching Systems for the Activity Assay of Alkaline Phosphatase

Department of Functional Molecular Science, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan
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Received: 24 July 2017 / Revised: 7 August 2017 / Accepted: 13 August 2017 / Published: 15 August 2017
(This article belongs to the Special Issue State-of-the-Art Sensors Technology in Japan 2017)
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Abstract

We introduce two types of fluorescence-quenching assay for alkaline phosphatases (APs) by using a carboxytetramethyl-rhodamine (TAMRA)-labeled phosphate-binding tag molecule (TAMRA-Phos-tag). In the first assay, TAMRA-labeled O-phosphorylethanolamine (TAMRA-PEA) was used as an artificial AP-substrate. TAMRA-Phos-tag specifically captured TAMRA-PEA to form a 1:1 complex at pH 7.4; the intensity of the fluorescence peak of the complex at 580 nm (λex = 523 nm) was significantly reduced to 32% of the average value for the two individual components as a result of the mutual approach of the TAMRA moieties. As TAMRA-PEA was dephosphorylated by AP, the resulting TAMRA-labeled ethanolamine dissociated and the fluorescence increased in a manner dependent on the AP dose and the time. In the second assay, pyrophosphate (PP), a natural AP-substrate, was used as a bridging ligand to form a dimeric TAMRA-Phos-tag complex. The dimerization reduced the fluorescence intensity to 49% of that in the absence of PP. As pyrophosphate was hydrolyzed to two orthophosphate moieties by AP, the 580-nm fluorescence recovered in a time-dependent manner. By examining the initial slope of this time-dependent fluorescence recovery, we succeeded in evaluating the 50% inhibitory concentrations of orthovanadate toward two AP isozymes under near-physiological conditions. View Full-Text
Keywords: alkaline phosphatase; fluorometric analysis; fluorescence quenching; tetramethylrhodamine; O-phosphorylethanolamine; pyrophosphate alkaline phosphatase; fluorometric analysis; fluorescence quenching; tetramethylrhodamine; O-phosphorylethanolamine; pyrophosphate
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Shiba, A.; Kinoshita-Kikuta, E.; Kinoshita, E.; Koike, T. TAMRA/TAMRA Fluorescence Quenching Systems for the Activity Assay of Alkaline Phosphatase. Sensors 2017, 17, 1877.

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