The Indirect Determination Of Enzyme Kinetics Using Capillary Electrophoresis With Chemiluminescence Detection

A custom capillary electrophoresis system with post-column chemiluminescence detection (CE-CL) was used with electrophoretically mediated microanalysis (EMMA) to indirectly determine the enzyme kinetics of glucose oxidase. Validation of the instrument was performed through injecting luminol hydrodynamically to measure the consistency of the results, as well as learning how to use the CE-CL system. Based on the results obtained from the luminol injections, there was strong linearity present, with an R2 value of 0.9925, as the concentration of luminol increased. The glucose oxidase was introduced by electrokinetically injecting the enzyme into the system to react with the substrate, B-D-glucose. The byproduct, hydrogen peroxide, was produced on column and reacted with luminol in the outlet cell, in which the production of light was detected by the photon counter. Based on the results obtained from the glucose oxidase injections, there was a fairly strong linearity present, with an R2 value of 0.976. As the concentration of glucose oxidase increased, the intensity of the plateaus also increased because there was an increase in the formation of product. CE-CL has been proven to be an effective technique to indirectly determine kinetic constants of enzymes that produce hydrogen peroxide and to be later compared to literature values.