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Journal of Biomolecular Screening
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Time-Resolved Fluorescence-Based Assay for the Determination of Alkaline Phosphatase Activity and Application to the Screening of Its Inhibitors

Petra Schrenkhammer

Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Regensburg, Germany, michael.schaeferling{at}chemie.uni-regensburg.de

Ina C. Rosnizeck

Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Regensburg, Germany

Axel Duerkop

Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Regensburg, Germany

Otto S. Wolfbeis

Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Regensburg, Germany

Michael Schäferling

Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Regensburg, Germany

A single-step end point method is presented for determination of the activity of the enzyme alkaline phosphatase (ALP) using the effect of enhancement of fluorescence of the easily accessible europium(III)-tetracycline 3:1 complex (Eu3TC). Its luminescence, peaking at 616 nm if excited at 405 nm, is enhanced by a factor of 2.5 in the presence of phosphate. Phenyl phosphate was used as a substrate that is enzymatically hydrolyzed to form phenol and phosphate. The latter coordinates to Eu3TC and enhances its luminescence intensity as a result of the displacement of water from the inner coordination sphere of the central metal. The assay is performed in a time-resolved (gated) mode, which is shown to yield larger signal changes than steady-state measurement of fluorescence. The limit of detection for ALP is 4 µmol L—1. Based on this scheme, a model assay for theophylline as inhibitor for ALP was developed with a linear range from 14 to 68 µmol L— 1 of theophylline. (Journal of Biomolecular Screening 2008:9-16)

Key Words: alkaline phosphatase • phosphate probe • europium • enzyme inhibition • screening assay

Journal of Biomolecular Screening, Vol. 13, No. 1, 9-16 (2008)
DOI: 10.1177/1087057107312031
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