Monitoring of enzymatic reactions using capillary electrophoresis with conductivity detection

Capillary electrophoresis combined with contactless conductivity detection allows to
separate and detect the ionic species, which are neither UV absorbing nor fluorescent.
This thesis focuses on the applications of this method on enzymatic reactions in
different analytical tasks.
First, the non-ionic species ethanol, glucose, ethyl acetate and ethyl butyrate were
made accessible for analysis by capillary electrophoresis via charged products or
byproducts obtained in enzymatic conversions using hexokinase, glucose oxidase,
alcohol dehydrogenase and esterase. The conversion of glucose and that of
ethylacetate were also successfully demonstrated on a microchip-device.
Quantification of ethyl acetate, was found possible with a detection limit of
approximately 7 μM. Then the model of urea catalyzed by urease was chosen for the
study of the enzymatic mechanism, the effect of substrate concentration and pH value
as well as the Michaelis-Menten constant. The determination of urea in human blood
as clinical application of this enzymatic reaction was tested. 10 human blood samples
were collected from a hospital and analyzed. The results were comparable with the
established methods. The method was then extended to proteome analysis;
identification of proteins is generally achieved through proteolytic digestion with
enzymes such as pepsin and trypsin. Protein digestion with pepsin and trypsin was
successfully monitored by capillary electrophoresis. Minigastrin I, myoglobin,
cytochrome C, human serum albumin and bovine serum album were the model
proteins digested by pepsin, cytochrome C and myoglobin were the model proteins
digested by trypsin. Electrophoretically mediated micro-analysis (EMMA) technique
is employed for the tryptic digestion of cytochrome C and apomyoglobin.
Finally, the enantioselective hydrolysis of esters of amino acids with lipase was
monitored. Porcine pancreas lipase was found to have a better efficiency on
hydrolysis and enantioselectivity than wheat germ lipase. L-threonine methyl ester
demonstrated stronger enantioselectivity than L-serine methyl ester.
Acetylcholinesterase inhibitors can be used as drug against Alzheimer disease or
nerve agents. Three compounds, namely galantamine, paraoxon and Huperzine-A,
were the model inhibitors to study the behavior and kinetics of the inhibitors. The
values of IC50 were obtained through graphical plot. Their dependence on the time
course was monitored and graphically illustrated.