First Name:
Taylor
Last Name:
Jones
Major Department:
Chemistry
Thesis Director:
Libby G. Puckett
Date of Thesis:
Aug 2014
This work is focused on the indirect measurement of enzyme activity using a multicomponent instrument that interfaces capillary electrophoresis (CE) with chemiluminescence (CL) detection. The CE-CL system was validated by injecting various concentrations of luminol to react with hydrogen peroxide in a post-column reactor, where chemiluminescence was detected using a photon counter. Research was ultimately focused on determining the Michaelis-Menton constant for the model enzyme glucose oxidase by using electrophoretically mediated microanalysis (EMMA) and luminol to detect the production of hydrogen peroxide as a byproduct. This method of indirectly studying the kinetics of enzymes will allow other enzymes that generate hydrogen peroxide to be studied. Some advantages of this system are its speed, reduced reagent consumption and reduced waste generation.
Much of initial work involved troubleshooting the instrument and finding optimal conditions and solution concentrations for consistent operation. Instrument validation was successful, and a linear (R2 = 0.9922) calibration curve was made after injecting various concentrations of luminol. After injecting various concentrations of glucose oxidase, it was concluded that an optimal enzyme concentration of 20 U/mL should be used for kinetic studies. Kinetic studies began with various substrate concentrations, and a glucose oxidase concentration of 20 U/mL. A Km value of 220 mM was found for glucose oxidase, but more work will be necessary.