Dynamic Complexation-Capillary Electrophoresis: An Integrative Biophysical Tool For Thermodynamic Analysis Of Biomolecular Interactions

Abstract

<p>Capillary electrophoresis is a high resolution microseparation technique that is increasingly being recognized as a physical tool to characterize biomolecular interactions, where dynamic complexation of analytes with discrete additives is used to resolve complex mixtures of solutes, including enantiomers. Despite the wide interest in developing high-throughput screening platforms for drug discovery or disease prognosis, little emphasis has been placed on enhancing "pre-analysis steps" that are often the most crucial component determining the overall performance of a method. Off-line sample pretreatment protocols for complex biological samples are often time-consuming and not amenable for automation. The major goal of this thesis is the development of a single-step analytical platform by CE for targeted metabolites that integrate several different sample pretreatment processes during separation, which can also be used to characterize the thermodynamic parameters associated with covalent and non-covalent interactions. Two distinct projects in this thesis have been examined involving boronic acid-polyol and protein-cyclic nucleotide interactions that illustrate the concept of integrating sample pretreatment with chemical analysis based on dynamic complexation-capillary electrophoresis.</p> <p>The first project consists of a new strategy for enhancing target selectivity when using 3-nitrophenylboronic acid as an electrokinetic probe in dynamic complexation-capillary electrophoresis. The differential migration of ternary boronate ester complexes permits the selective analysis of micromolar levels of UV-transparent polyol stereoisomers in urine samples that is applicable to single-step screening of in-born errors of sugar metabolism, such as galactosemia. In the second project, the impact of ligand binding on protein stability is assessed by dynamic ligand exchangeaffinity capillary electrophoresis with laser-induced native fluorescence detection. This is a convenient yet rapid format for comparative thermodynamic studies of a regulatory subunit of protein kinase involving different cyclic nucleotide analogues without off-line sample pretreatment, since ligand exchange and protein unfolding processes are integrated incapillary during electromigration.</p>