Capillary Electrophoretic Method for Kinetic and Inhibition Studies of β-Secretase in Nanoliter-Scale Format
| Title in English | Capillary Electrophoretic Method for Kinetic and Inhibition Studies of in Nanoliter-Scale Format |
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| Authors | |
| Year of publication | 2016 |
| Type | Conference abstract |
| MU Faculty or unit | |
| Citation | |
| Description | Alzheimer’s disease (AD) represents the most common cause of the dementia. Development of a cure for AD, however, is hindered by limited knowledge about its cause; currently available medications may only temporarily improve accompanying symptoms. Since amyloid plaques, deposits which were found in a brain tissue of the most AD patients, cause the inflammatory reaction leading to degradation of neurons, specific inhibition of ß-secretase (BACE), enzyme responsible for formation of the amyloid plaques, appears to be a promising way for slowing down or even stopping the progression of the disease. Capillary electrophoresis (CE) represents a suitable technique to assess the enzyme activity in vitro. It provides high separation efficiency, requires minuscule amounts of sample and reagents and offers high throughput via automation. Furthermore, a fused-silica capillary can serve as a nanoliter-scale reaction vessel from which formed products can be directly analyzed. Such procedures are referred to as on-line CE methods that comprise incubation of an enzymatic reaction, separation of reaction products, and their detection and quantitation in a fully automated way. For these reasons, the main goal of this study was to develop an on-line CE method for kinetic and inhibition studies of BACE. A principle of transverse diffusion of laminar flow profiles technique was adopted for mixing of enzyme and model substrate, decapeptide DAEFRSEVNL, inside the capillary. Conceptually, solutions of enzyme and substrate were injected by hydrodynamic pressure as a series of consecutive plugs with parabolic profiles allowing rapid mixing of the enzyme and substrate by transverse diffusion. After the incubation was finished, method enabled separation of all products in 6 min. The fragment DAEFR was detected and quantified using UV-Vis detector in this study, however, 5% acetic acid used as a BGE also enables utilization MS detection. The final method was validated and used for kinetic and inhibition study of ß-secretase. Results obtained were in good agreement with literature conducted control off-line study. |
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