Schrenkhammer, Petra and Rosnizeck, Ina C. and Duerkop, Axel and Wolfbeis, Otto S. and Schaeferling, Michael (2008) Time-resolved fluorescence-based assay for the determination of alkaline phosphatase activity and application to the screening of its inhibitors. JOURNAL OF BIOMOLECULAR SCREENING, 13 (1). pp. 9-16. ISSN 1087-0571,
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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 mu mol 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 mu mol L-1 of theophylline.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | PROTEIN-TYROSINE PHOSPHATASES; FLOW-INJECTION DETERMINATION; HYDROGEN-PEROXIDE; EUROPIUM-TETRACYCLINE; FLUOROMETRIC-DETERMINATION; INORGANIC-PHOSPHATE; LUMINESCENT PROBE; METAL-IONS; THEOPHYLLINE; DISCOVERY; alkaline phosphatase; phosphate probe; europium; enzyme inhibition; screening assay |
| Subjects: | 500 Science > 540 Chemistry & allied sciences |
| Divisions: | Chemistry and Pharmacy > Institut für Analytische Chemie, Chemo- und Biosensorik > Chemo- und Biosensorik (Prof. Antje J. Bäumner, formerly Prof. Wolfbeis) |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 18 Nov 2020 06:52 |
| Last Modified: | 18 Nov 2020 06:52 |
| URI: | https://pred.uni-regensburg.de/id/eprint/31676 |
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