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 (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.