TAMRA/TAMRA Fluorescence Quenching Systems for the Activity Assay of Alkaline Phosphatase
AbstractWe 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
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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.
Shiba A, Kinoshita-Kikuta E, Kinoshita E, Koike T. TAMRA/TAMRA Fluorescence Quenching Systems for the Activity Assay of Alkaline Phosphatase. Sensors. 2017; 17(8):1877.Chicago/Turabian Style
Shiba, Akio; Kinoshita-Kikuta, Emiko; Kinoshita, Eiji; Koike, Tohru. 2017. "TAMRA/TAMRA Fluorescence Quenching Systems for the Activity Assay of Alkaline Phosphatase." Sensors 17, no. 8: 1877.
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