Chemistry, Department of


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A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy, Major: Chemistry, Under the Supervision of Professor David S. Hage. Lincoln, Nebraska: May, 2010
Copyright (c) 2010 Kathryn S. Joseph


Drug-protein binding can have a dramatic impact on the distribution and metabolism of a drug. This manuscript describes the use of high-performance affinity chromatography to examine the binding of various compounds to human serum albumin (HSA) in normal and diabetic disease states.

The first study examined the use of four coumarin compounds as possible alternatives to warfarin as a probe for Sudlow site I on HSA. High-performance affinity chromatography and immobilized HSA columns were used to compare and evaluate the binding properties of these probe candidates. It was found from this group that 4-hydroxycoumarin was the best alternative to warfarin for drug-protein binding studies on HSA.

The primary portion of this manuscript examined the binding of sulfonylurea drugs to HSA as the glycation level of HSA was increased. This work was performed by using high-performance affinity chromatography to determine the binding regions, affinities, and the number of binding sites on HSA for sulfonylureas. The first part of this study examined the binding of two sulfonylureas to non-glycated HSA. Frontal analysis and competition studies indicated that the sulfonylureas had two major classes of binding sites on HSA, with strong interactions occurring at both Sudlow sites I and II. The second part of this study examined the binding of two probe compounds, warfarin and L-tryptophan, to HSA as glycation levels of this protein increased. This study found no significant difference in the binding of warfarin to glycated HSA but observed some increases in the binding constant of L-tryptophan. The third part of the study examined the binding of the sulfonylureas to HSA with increasing levels of glycation. Minor alterations in binding were observed as the level of glycation increased. Lastly, theoretical studies were also performed to elucidate the appropriate analyte concentrations necessary for examining multi-site binding systems, such as those observed for some drug-protein interactions.