Improving Coupled Enzyme Assays With Capillary Electrophoresis

The goal of this project is to address limitations of coupled enzyme assays using capillary electrophoresis (CE). In some enzyme assays, it is ineffective to directly detect products and substrates spectroscopically (absorption or fluorescence). Coupling the enzyme-catalyzed reaction of interest with another enzyme-catalyzed reaction allows for indirect detection of product formation or substrate depletion for the target enzyme. While conventional coupled enzyme assays are facile for kinetic measurements, they can be problematic for inhibition screening due to spectral and inhibitor interferences. To address these limitations of coupled assays, CE has been used to develop enzyme assays for acetyl-coenzyme A carboxylase (ACC). The first approach is to utilize CE to separate assay components (substrates, products, and inhibitors) and avoid using coupling enzymes. In Chapter 2, a previously developed bacterial holo-ACC assay was optimized and applied to study the inhibition of bacterial holo-ACC, BC, and CT by five flavonols (myricetin, quercetin, anrantine osage orange, galangin, and DHF). The IC50 values for these inhibitors were determined using offline CE assays. A new offline assay developed for a human form of ACC, human ACC2, is presented in Chapter 3. This assay was used to detect inhibition of the human enzyme. The second approach is to use coupling enzymes, but in combination with CE separation to reduce spectral interference and inhibitor interference with the coupled reactions. The envisioned CE coupled enzyme assay will use a light emitting diode (LED) as the excitation source for fluorescence detection of NADH consumption. Although LEDs have been widely used as alternatives to lasers for fluorescence detection, studies on the stability and noise associated with LEDs for CE detection are limited. In Chapter 4, a 470-nm LED was quantitatively compared to argon ion laser lines as a source for fluorescence detection with CE. Appendix 1 presents progress in the development of the CE online coupled assay for ACC. This assay will use magnetic beads as the solid support for immobilized the coupling enzymes, and the fluorescence detection of NADH with LED excitation