Capillary Electrophoresis for Measuring Biological Interactions: Binding Affinity and Enzyme Kinetics

Abstract

This dissertation is based upon research in the use of capillary electrophoresis for the measurement of biological interactions. The studies presented here research two classes of analytes, steroids and oligosaccharides, both of which have profound importance to biological systems. Capillary electrophoresis separation methods were utilized to determine binding affinity and enzyme kinetic constants. The second chapter describes the first use of a semi-permanent phospholipid coating that is utilized in capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) for the purpose of monitoring enzymatic hydrolysis and the detection of maltooligosaccharides. Although phospholipids in free solution are generally unused in ESI, they did not interfere with the detection of linear and branched oligosaccharides using ESI operated in negative mode. The CE and ESI were coupled using a coaxial sheath flow interface. The separation was operated in reversed polarity, and the electroosmotic flow was effectively suppressed by the phospholipid coating. The method was characterized with linear oligosaccharides and used to monitor the enzymatic hydrolysis of maltooligosaccharides with alpha-amyloglucosidase. Branched oligosaccharides were separated and detected with the system. The enzyme beta1-4 galactosidase was used to distinguish branched isomeric oligosaccharides derived from asialofetuin. The third chapter of this dissertation presents the measurement of the binding affinity of 17beta-estradiol with an immobilized DNA aptamer by using capillary electrophoresis. Estradiol captured by the immobilized aptamer was injected into the separation capillary using pH-mediated sample stacking. Stacked 17beta-estradiol was then separated using micellar electrokinetic capillary chromatography and detected with UV-visible absorbance. Following incubation with immobilized DNA, analysis of free and bound estradiol yielded a dissociation constant of 70 +/- 10 microM. The method was also used to screen binding affinity of the aptamer for other steroids. Chapter three demonstrates the effectiveness of capillary electrophoresis to assess the binding affinity of DNA aptamers.