Time-resolved fluorescence-based assay for the determination of alkaline phosphatase activity and application to the screening of its inhibitors.
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 (Eu(3)TC). 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 Eu(3)TC 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 micromol 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 micromol L(- 1) of theophylline.
Schrenkhammer
Petra
P
Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Regensburg, Germany.
Rosnizeck
Ina C
IC
Duerkop
Axel
A
Wolfbeis
Otto S
OS
Schäferling
Michael
M
D000469:Alkaline Phosphatase / Q000032:analysis* / Q000037:antagonists & inhibitors*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005063:Europium; D005456:Fluorescent Dyes; D006801:Humans; D066298:In Vitro Techniques; D049449:Luminescence; D008163:Luminescent Measurements / Q000379:methods; D009942:Organometallic Compounds; D010710:Phosphates; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013754:Tetracyclines; D013806:Theophylline / Q000494:pharmacology
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D009942:Organometallic Compounds; D010710:Phosphates; D013754:Tetracyclines; C488317:europium(III) tetracycline; D005063:Europium; D013806:Theophylline; D000469:Alkaline Phosphatase