Abstrakt
This thesis is focused on the applications of capillary electrophoresis in two important areas of life sciences, proteomics and enzyme assays. In the first part, Pluronic F-127 copolymer was studied as a sieving matrix for proteomic applications of capillary gel electrophoresis.
The effect of thermoassociation of Pluronic F-127 on the separation selectivity was investigated. The performance of Pluronic F-127 in capillary gel electrophoresis was compared with dextran as a commonly used sieving matrix.
The second part focuses on electrophoretic enzyme assays. A new method for the separation of N,N',N''-triacetylchitotriose, N,N'-diacetylchitobiose and N-acetylglucosamine, as a substrate and products of β-N-acetylhexosaminidase enzyme, was developed.
After the optimization of pH and concentration of the tetraborate-based background electrolyte, the developed method was statistically evaluated. The repeatability of measurements was determined as well as limits of detection and quantification for all three analytes.
Afterwards, other possible approaches to the separation of saccharides found in the literature were tested and critically compared with our method. The method of separation was successfully used for offline monitoring of enzymatic hydrolysis of N,N',N''-triacetylchitotriose with β-N-acetylhexosaminidase.
Using the same background electrolyte and separation conditions, an online assay of the enzyme with N,N'- diacetylchitobiose as a substrate has been developed employing the 'Transverse Diffusion of Laminar Flow Profiles' model in order to predict and optimize mixing of the reagents inside the separation capillary. Using this method, the inhibition of β-N-acetylhexosaminidase with dimethylformamide could be observed.
The enzyme assay was applicable also for N,N',N''- triacetylchitotriose as a substrate after adjusting the pH of background electrolyte.