Masters Theses

Date of Award

5-2004

Degree Type

Thesis

Degree Name

Master of Science

Major

Chemistry

Major Professor

S. Douglass Gilman

Committee Members

James Q. Chambers, David C. Baker

Abstract

Reversible inhibition, irreversible inhibition, and activation of calf intestinal alkaline phosphatase (EC 3.1.3.1) have been studied by capillary electrophoresis. The capillary electrophoretic enzyme-inhibitor assays were performed by electrophoretically mixing zones of inhibitor and enzyme in a substrate-filled capillary. Enzyme inhibition was indicated by a decrease in product formation detected in the capillary by laser-induced fluorescence. Reversible enzyme inhibitors could be quantified by Michaelis-Menten treatment of the electrophoretic data. Reversible, competitive inhibition of alkaline phosphatase by sodium vanadate and sodium arsenate has been examined. The calculated Ki value for the capillary electrophoretic enzyme-inhibitor assays was 2.1 µM for sodium vanadate and 21 µM for sodium arsenate. The limit of detection for sodium vanadate was 3 µM, and the limit of detection for sodium arsenate was 10 µM. Reversible, non-competitive inhibition of alkaline phosphatase by theophylline has been studied. The calculated Ki value for the capillary electrophoretic enzyme-inhibitor assays for theophylline was 102 µM, and the limit of detection was 3 µM. Irreversible inhibition of alkaline phosphatase by EDTA at concentrations of 1.0 mM or higher has been observed. Activation of alkaline phosphatase has also been observed for EDTA at concentrations from 20 to 400 µM.

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