Identifier

etd-05232013-110656

Degree

Doctor of Philosophy (PhD)

Department

Chemistry

Document Type

Dissertation

Abstract

New methods for studying enzymes and enzyme inhibition using capillary electrophoresis (CE) were developed and applied. Because CE is a separation technique, spectral interference is minimized by separation of substrates, products, inhibitors, and sample matrix components. Two types of CE enzyme assays were explored in this dissertation research. The first assay was based on optically gated vacancy capillary electrophoresis (OGVCE) with laser-induced fluorescence detection (LIF). This approach involves periodic photobleaching of fluorescent substrates and products. The initial goal of the study was to develop an assay for adenosine deaminase (ADA). A fluorescent ADA substrate was synthesized; however, the substrate was unusually photostable and could not be photobleached. In Chapter 2, a comparative study is presented that quantified the photostability of the fluorescent ADA substrate relative to other fluorophores that have been used for OGVCE in order to obtain a better understanding of the ideal properties of a dye for OGVCE assays. The development of an off-column CE assay for measuring acetyl coenzyme A carboxylase holoenzyme (holo-ACC) activity and inhibition is presented in Chapter 3. The two reactions catalyzed by the holo-ACC components, biotin carboxylase (BC) and carboxyltransferase (CT), were simultaneously monitored in the assay, which required successful separation of two substrates and two products using micellar electrokinetic chromatography (MEKC). Additionally, a previously reported off-column CE assay for only the CT component of ACC was optimized, and an off-column CE assay for only the BC component of ACC was developed. Finally, the CE-based enzyme assays developed in Chapter 3 were used for screening of botanical extracts against holo-ACC, BC and CT (Chapter 4). Compounds known to be present in the botanical extracts that inhibited holo-ACC were selected based on a detailed literature search and tested for inhibition of holo-ACC, BC and CT. Synthetic compounds selected based on ligand homology studies were also tested for inhibition of holo-ACC. These CE assays were simple, off-column methods, where spectral interference and other limitations of previous methods were greatly reduced. The development of methods that can directly monitor reactants and products for multiple enzyme-catalyzed reactions will have impact beyond the enzymes presented in this dissertation.

Date

2013

Document Availability at the Time of Submission

Release the entire work immediately for access worldwide.

Committee Chair

Gilman, Samuel D.

Included in

Chemistry Commons

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