Capillary electrophoretic analysis of alkaline phosphatase inhibition by theophylline.
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 microM, and 8.6 amole of alkaline phosphatase are required for each assay. The calculated K(i) for theophylline is 90 microM for the capillary electrophoretic enzyme-inhibitor assays.
Whisnant
A R
AR
Department of Chemistry, The University of Tennessee, Knoxville 37996-1600, USA.
Johnston
S E
SE
Gilman
S D
SD
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors*; D019075:Electrophoresis, Capillary / Q000379:methods; D005456:Fluorescent Dyes; D009943:Organophosphorus Compounds; D013050:Spectrometry, Fluorescence / Q000379:methods; D013806:Theophylline / Q000494:pharmacology*; D013997:Time Factors
D005456:Fluorescent Dyes; D009943:Organophosphorus Compounds; C075487:AttoPhos; D013806:Theophylline; D000469:Alkaline Phosphatase