The goal of the research proposed in this application is to advance the study of enzyme function through1 the development and application of capillary electrophoresis-based approaches for studying enzyme1 inhibition. Enzyme inhibitors are primary therapeutic targets for a wide range of human diseases. This research takes advantage of the unique capabilities of capillary electrophoretic methods for controlling1 the mixing and separation of zones of molecules with different electrophoretic mobilities to study the kinetics of enzyme catalyzed reactions. Work with alkaline phosphatase will focus on continuing the development of quantitative models of the capillary electrophoretic enzyme inhibition assays and understanding the role that affinity interactions between enzymes and inhibitors play in these1 experiments. The unexpected apparent activation of alkaline phosphatase by EDTA at micromolar concentrations will be explored, and assays will be developed for attomole quantities of enzyme sampled1 directly from non-denaturing slab gels. Currently, high sensitivity capillary electrophoretic enzyme1 inhibition assays are limited to enzymes for which fluorogenic substrates are available. This limitation will be overcome through the development of optically gated vacancy capillary electrophoresis in capillaries1 and microfabricated devices. This advance will enable the study of any enzyme at very low concentrations for which a fluorescent (as opposed to fluorogenic) substrate can be synthesized, greatly expanding the potential applicability of this capillary electrophoretic approach for enzyme inhibition analysis. Capillary electrophoretic enzyme inhibition assays will be developed for enzymes, which use NAD+tNADH and NADP+/NADPH as cofactors to further expand the range of enzymes that can be1 investigated using this methodology. Finally, specific enzymes of biomedical significance and related inhibitors will be investigated. Potent synthetic inhibitors of adenosine deaminase will be studied using these assays. Kynureninase and related inhibitors will also be investigated.
| Henken, Rachel L; Chantiwas, Rattikan; Gilman, S Douglass (2012) Influence of immobilized biomolecules on magnetic bead plug formation and retention in capillary electrophoresis. Electrophoresis 33:827-33 |
| Yan, Xiaoyan; Gilman, S Douglass (2010) Improved peak capacity for CE separations of enzyme inhibitors with activity-based detection using magnetic bead microreactors. Electrophoresis 31:346-52 |
