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A Phos-Tag-Based Fluorescence Quenching System for Activity Assay and Inhibitor Screening for Alkaline Phosphatase

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http://www.scirp.org/journal/PaperInformation.aspx?PaperID=49377#.VI5zDcnQrzE

Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules. Here we introduce a novel FRET-based fluorescence quenching system for assaying the activity of alkaline phosphatase (AP) by using a phos-phate-binding tag molecule, Phos-tag {1,3-bis[bis(pyridine-2-ylmethyl)amino]propan-2-olato dizinc(II) complex}, attached to a nonfluorescent 4-{[4-(dimethylamino)phenyl]diazenyl}benzoyl (Dabcyl: λmax 475 nm) dye group. The fluorogenic biomolecule riboflavin 5’-phosphate (FMN: λem 525 nm) was used as an AP substrate. The Dabcyl-labeled Phos-tag specifically captured FMN to form a stable 1:1 complex, resulting in efficient fluorescence quenching. The quenching efficiency was more than 95% for a mixture of 12 μM FMN and 13.5 μM Dabcyl-labeled Phos-tag in aqueous solution at pH 7.4 and 25°C. When FMN was dephosphorylated with AP, riboflavin was released into the solution and fluorescence from the flavin moiety appeared. By using this quenching system, we succeeded in detecting time- and dose-dependent dephosphorylation of FMN by AP under near-physiological conditions.
Cite this paper
Kinoshita-Kikuta, E. , Kurosaki, H. , Kunisada, N. , Kinoshita, E. and Koike, T. (2014) A Phos-Tag-Based Fluorescence Quenching System for Activity Assay and Inhibitor Screening for Alkaline Phosphatase. American Journal of Analytical Chemistry, 5, 796-804. doi: 10.4236/ajac.2014.512088.
 

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