Chemistry, Department of


First Advisor

David S. Hage

Date of this Version

Spring 2019


P. Tao, Analysis of drug-protein interactions during diabetes by high-performance affinity chromatography, M.S. Thesis, University of Nebraska-Lincoln, 2019


A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Chemistry, Under the Supervision of Professor David S. Hage. Lincoln, Nebraska: May, 2019

Copyright 2019 Pingyang Tao


High-performance affinity chromatography (HPAC) is a type of liquid chromatography in which solutes are separated based on their binding to a stationary phase that is a biologically-related agent. Sulfonylurea drugs have significant binding to proteins in blood, with most of this binding is believed to occur with human serum albumin (HSA). HSA is known to be modified as a result of the high serum levels of glucose that is present during diabetes, which may also alter the function of HSA as a binding agent for many drugs in circulation. Some modifications of HSA that can occur during diabetes involves early stage glycation and advanced glycation. Drugs that may be affected by these changes include various sulfonylurea drugs, as are commonly used to treat type II diabetes. This thesis describes the development of tools and techniques based on HPAC to examine the effects of glycation on the binding of HSA. A major portion of this research describes the use of HPAC to examine the effect of early stage glycation on the binding of two first-generation sulfonylurea drugs. Frontal analysis and zonal elution studies were used to provide information on association equilibrium constants and the number of binding sites for these interactions. Significant levels of both increases and decreases in affinity were observed for these drugs at the levels of glycation that were examined. A second portion of this research describes the utilization of HPAC to investigate the effect of advance glycation on the binding of two first-generation sulfonylurea drugs and one second-generation sulfonylurea drug. Two major advanced glycation end-products (AGEs) of HSA, glyoxal- and methylglyoxal-modified HSA, were studied in this work. Immunoextraction and zonal elution were used to estimate the global affinity of these drugs with normal or in vitro modified HSA, while competition studies based on zonal elution were used to measure the binding strengths of these drugs at specific sites on these preparations of HSA.

Advisor: David S. Hage