Capillary electrophoresis-integrated immobilized enzyme reactor for kinetic and inhibition assays of beta-secretase as the Alzheimer's disease drug target
| Authors | |
|---|---|
| Year of publication | 2019 |
| Type | Article in Periodical |
| Magazine / Source | Journal of Separation Science |
| MU Faculty or unit | |
| Citation | |
| web | Full Text |
| Doi | http://dx.doi.org/10.1002/jssc.201800947 |
| Keywords | beta-secretase; capillary electrophoresis; immobilized enzyme reactor; inhibition; magnetic particles |
| Description | Capillary electrophoresis-integrated immobilized enzyme reactors are becoming an increasingly popular alternative for enzyme kinetic and inhibition assays thanks to their unique set of features including cost effectiveness, repeated use of the enzyme, minuscule sample consumption, rapid analysis time and easy automation. In this work we present the development and application of a CE-integrated immobilized enzyme reactor based on magnetic particles for kinetic and inhibition studies of beta secretase, a key enzyme in the development of Alzheimer’s disease and a promising drug target. We document the optimization of the immobilization procedure, characterization of immobilized beta secretase, optimization of a mutually compatible incubation protocol and separation method as well as the production of the CE-integrated immobilized enzyme reactor. The applicability of the CE-integrated immobilized enzyme reactor was demonstrated by kinetic assay with an unlabelled substrate and by inhibition assays using three structurally different reference inhibitors. The resulting kinetic and inhibition parameters clearly support the applicability of the herein presented method as well as document the fundamental phenomena which need to be taken in account when comparing the results to other methods. |
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