Off-campus UNL users: To download campus access dissertations, please use the following link to log into our proxy server with your NU ID and password. When you are done browsing please remember to return to this page and log out.
Non-UNL users: Please talk to your librarian about requesting this dissertation through interlibrary loan.
Characterization of drug-protein interactions using high-performance affinity chromatography
Abstract
High-performance affinity chromatography is used to study the binding mechanisms of thyroid hormones and warfarin with immobilized human serum albumin (HSA). In Chapter 2, we investigate the mechanisms involved in the binding of thyroid hormones and related compounds to the warfarin and indole sites of HSA. It is shown that the association constants for the thyroid hormones at the warfarin and indole regions are the same as for the high affinity sites on HSA. The change in energy associated with the interactions of thyroid hormones at both regions has a significant component due to entropy. The binding of these molecules at the indole site is affected mainly by the degree of phenol group ionization and the number or position of iodides present in the hormone's structure. In Chapters 3 and 4, we characterize the thermodynamic and kinetic processes involve in the binding and separation of R- and S-warfarin on a high-performance immobilized HSA column. Frontal analysis is used to determine the strength and degree of binding for each enantiomer. R- and S-warfarin bind at the same region on HSA; however, the measured capacities are consistently higher for the R-enantiomer. The association constants at pH 7.4 and 37$\sp\circ$C are 2.1 $\times$ 10$\sp5$ and 2.6 $\times$ 10$\sp5$ M$\sp{-1}$ for R- and S-warfarin, respectively. The values of $\Delta$G for R-warfarin and S-warfarin are similar at 37$\sp\circ$C (7.5 and 7.7 kcal/mol), but the contribution due to $\Delta$S is greater for the R-enantiomer. Kinetic parameters for R- and S-warfarin are examined by measuring the broadness of their chromatographic peaks. The free energy of activation for R-warfarin is approximately five times larger than it is for S-warfarin. The entropy of activation for S-warfarin is approximately thirty times greater than it is for R-warfarin. These thermodynamic and kinetic results suggest that R-warfarin interacts mainly with the binding site interior, whereas S-warfarin interacts more with the site's outer surface. The different changes in entropy for these solutes makes it possible to vary their separation by changing column temperature. Both thermodynamic properties and column binding capacities are important in determining the degree of separation for these compounds.
Subject Area
Analytical chemistry|Biochemistry|Pharmacology
Recommended Citation
Loun, Bounthon, "Characterization of drug-protein interactions using high-performance affinity chromatography" (1994). ETD collection for University of Nebraska-Lincoln. AAI9500606.
https://digitalcommons.unl.edu/dissertations/AAI9500606