Homogeneous enzyme assays in capillary electrophoresis
Abstract
The problem of heat dissipation in electrophoresis had been solved by using capillaries. Unprecedented high number of theoretical plates, more than one million, had been achieved on these narrow bore capillaries. Unfortunately, this high efficiency was not achievable for the separation of biological samples using bare fused silica capillaries. Severe adsorption of proteins caused tailing and distortion of peaks. Coating the internal surface of the silica capillary with an epoxy based neutral coating was effective in eliminating protein adsorption. The separation of model proteins using coated capillaries demonstrated the power of the CE technique in protein separations. Despite the high resolving power of CE, biochemists prefer slab gel electrophoresis, though labor intensive and low resolution, over the CE technique for the separation of proteins, nucleotides, and carbohydrates. Part of the reason is that CE can not provide biochemists the flexibility and convenience of carrying out various reactions within a homogeneous media. The purpose of this research was to develop new methods using a CE system to carry out reactions usually seen only with slab gel electrophoresis. The characteristics of mixing, as well as separation, in the CE technique were studied, and a new strategy was proposed to assay various reagents, such as enzymes and substrates. The method includes the injection of sample, the separation of an enzyme from other species in the running buffer, stop flow reaction, separation of the product from the enzyme, and transport of the product to the detector. The enzymes assayed in this research include glucose-6 phosphate dehydrogenase (G-6-PDH), acid phosphatase (AcP), leucyl aminopeptidase (LAP), and lactate dehydrogenase (LDH).
Degree
Ph.D.
Advisors
Regnier, Purdue University.
Subject Area
Analytical chemistry|Biochemistry|Pharmacology
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