Time-resolved fluorescence-based assay for the determination of alkaline phosphatase activity and application to the screening of its inhibitors

被引：30

作者：

Schrenkhammer, Petra ^{[1
]}

Rosnizeck, Ina C. ^{[1
]}

Duerkop, Axel ^{[1
]}

Wolfbeis, Otto S. ^{[1
]}

Schaeferling, Michael ^{[1
]}

机构：

[1] Univ Regensburg, Inst Analyt Chem, D-93040 Regensburg, Germany

来源：

JOURNAL OF BIOMOLECULAR SCREENING | 2008年 / 13卷 / 01期

关键词：

alkaline phosphatase; phosphate probe; europium; enzyme inhibition; screening assay;

D O I：

10.1177/1087057107312031

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：9 / 16

页数：8