Chemistry, Department of


First Advisor

David S. Hage

Date of this Version



Woolfork, Ashley G.; Hage, David S. 2020. Evaluation of the Overall Binding of Acetohexamide and Tolbutamide with Methyl Glyoxal-Modified HSA by High Performance Affinity Chromatography. Poster presentation, UCARE Research Fair, Spring 2020, University of Nebraska - Lincoln


Copyright 2020 by the authors.


High performance affinity chromatography (HPAC) was used in a zonal competition format to investigate the overall binding of two sulfonylurea drugs with modified human serum albumin (HSA). The in vitro modifications of this protein that were examined included glycation, as occurs in the presence of elevated glucose levels during diabetes, and the formation of advanced glycation end-products (AGEs) due to the reaction of HSA with methylglyoxal (MGo). The sulfonylurea drugs acetohexamide and tolbutamide, which are used to treat type II diabetes, were each injected onto 10 mm x 2.1 mm i.d. affinity microcolumns that contained normal HSA and HSA modified with MGo. Competition studies based on a zonal elution format were carried out by using R-warfarin as a site –specific probe for Sudlow site I of HSA and L-tryptophan as a site-specific probe for Sudlow site II. The studies were performed with eight mobile phase concentrations of the drugs, ranging from 0-25 µM, and at a flowrate of 0.5 mL/min and a temperature of 37°C. The resulting information made it possible to determine the site-specific association equilibrium constants for each drug with normal and modified HSA. A decrease of approximately 0.4-fold to 0.8-fold in binding strength was found when comparing normal HSA vs control MGo-modified HSA and normal HSA vs diabetic MGo-modified HSA. These experiments illustrated how affinity microcolumns and zonal competition could be used in detailed studies of interactions by sulfonylurea drugs with normal HSA and modified forms of this protein. Such information may be useful in future applications within the field of personalized medicine for the development of customized treatments for patients with type II diabetes.