Masters Theses

Date of Award

5-2005

Degree Type

Thesis

Degree Name

Master of Science

Major

Chemistry

Major Professor

S. Douglass Gilman

Committee Members

Kelsey D. Cook, Bin Zhao

Abstract

Alkaline phosphatase inhibition by metal chelators has been studied using capillary electrophoresis. The enzyme-inhibition assays were performed by electrophoretically mixing enzyme and inhibitor zones in a substrate filled capillary. Enzyme inhibition could be seen as a decrease in product formation as detected using laser-induced fluorescence. The enzyme-inhibition assays were adapted so they could be performed using a commercial CE system. Use of a commercial system is desirable for these assays due to ease of use and system features such as autosamplers and capillary and sample cooling. This technique could prove useful for pharmaceutical industries as a screening tool for new drug therapies.

Six metal chelators were studied as enzyme inhibitors using CE enzyme-inhibition assays. EDTA inhibition assays performed previously on a laboratory constructed CE system were compared to those using a commercial system. The assays performed on the commercial system showed irreversible inhibition at concentrations of 1.0 mM or less and activation at 2.0 mM. The opposite trend was seen with the laboratory constructed system. One hypothesis to explain this discrepancy is that two different water sources contained different concentrations of metal contaminants. The two water sources were used due to equipment failure making one source unavailable. The common metal chelators EGTA, NTA, and 1,10-phenanthroline all exhibited reversible inhibition of alkaline phosphatase at concentrations of 0.08 to 2.0 mM. Crown ethers, which are not commonly used as enzyme inhibitors, were also studied. 12-crown-4 and 18-crown-6 both reversibly inhibited alkaline phosphatase at concentrations of 0.08 to 0.3 mM. The reversible inhibition exhibited by the various chelators examined (excluding EDTA) could be due to a time dependent mechanism in which Zn2+, which is required by ALP, is slowly removed, rendering the enzyme inactive.

Alkaline phosphatase inhibition was also studied using on-column reagent addition. The enzyme was added on-column, through a gap reactor and mixed with the fluorescent enzyme substrate, Attophos, and zones of inhibitor. Theophylline, a reversible noncompetitive inhibitor, and sodium vanadate, a reversible competitive inhibitor were used. Enzyme inhibition could be easily seen as a decrease in product formation detected by laser-induced fluorescence.

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