Studies of Enzyme Kinetics and Inhibition Through Capillary Electrophoretic Enzyme Assays

Abstract

Enzyme assays are used throughout the biological, chemical, and medical sciences for the study of enzymes, and typically are performed by bulk solution measurements in cuvettes or microplates. The development of new enzyme assays that combine the measurement of reaction rates with separation techniques is of notable importance. This research encompasses applications of capillary electrophoresis (CE) for enzyme assays. There are a variety of CE methods for the study of enzyme-catalyzed reactions, both off-line and on-line. This dissertation introduces and highlights the advantages of CE enzyme assays by applying a simple and straightforward assay to overcome a limitation of bulk solution measurements. The dissertation then focuses on the development of on-line homogenous CE enzyme assays based on a technique called electrophoretically mediated microanalysis (EMMA). The EMMA assays in this dissertation use fluorescence detection of NADH to study enzymes that utilize NAD+/NADH as a cofactor. Several methods for studying the kinetics of two dehydrogenase enzymes as well as their inhibition by monitoring NADH fluorescence are utilized. Variations of EMMA will be presented, including transient (plug-plug), continuous engagement, and a mixed format for quantitative studies of inhibition. In the first study, the activity of ADH is monitored through NADH production in the presence and absence of inhibitors. The next two chapters explore a novel EMMA methodology in which the depletion of NADH is monitored, resulting in an electropherogram that is inverse relative to previous EMMA methods. The remainder of the dissertation focuses on the use of magnetic beads with immobilized enzymes for heterogeneous on-line enzyme assays. A study of bead surface chemistry effects on the formation and retention of magnetic bead plugs in a capillary during CE demonstrated that the type of biological molecule immobilized on the surface of these beads significantly impacted their immobilization and retention. Finally, the activity of a two enzyme microreactor utilizing enzyme coated magnetic beads, where product of the first reaction is a substrate of the second, was studied. This work examined the impact of the placement of the beads within the capillary, flow rate, and flow type on the overall enzyme-catalyzed reaction rate.