Premium
Capillary electrophoretic analysis of alkaline phosphatase inhibition by theophylline
Author(s) -
Whisnant Angela R.,
Johnston Stephen E.,
Gilman S. Douglass
Publication year - 2000
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/(sici)1522-2683(20000401)21:7<1341::aid-elps1341>3.0.co;2-9
Subject(s) - alkaline phosphatase , chemistry , capillary electrophoresis , detection limit , substrate (aquarium) , electrophoresis , enzyme , chromatography , theophylline , non competitive inhibition , enzyme assay , biochemistry , phosphatase , biology , ecology , endocrinology
An analytical method for studying enzyme inhibition has been developed using capillary electrophoresis with laser‐induced fluorescence detection. This technique is based on electrophoretic mixing of zones of enzyme and inhibitor in substrate‐filled capillaries. Enzyme catalytic activity is measured by detecting the fluorescent reaction product as it migrates past the detector. Reversible enzyme inhibition is indicated by a transient decrease in product formation. The enzyme, alkaline phosphatase, has been studied using the fluorogenic substrate AttoPhos ([2,2′‐bibenzothiazol]‐6‐hydroxy‐benzthiazole phosphate). This assay has been used to quantify theophylline, a noncompetitive, reversible inhibitor of alkaline phosphatase. The detection limit for theophylline is estimated at 3 μ M , and 8.6 amole of alkaline phosphatase are required for each assay. The calculated K i for theophylline is 90 μ M for the capillary electrophoretic enzyme‐inhibitor assays.