Use of entrapment and high-performance affinity chromatography to compare the binding of drugs and site-specific probes with normal and glycated human serum albumin

Abstract

Protein entrapment and high-performance affinity chromatography were used with zonal elution to examine the changes in binding that occurred for site-specific probes and various sulfonylurea drugs with normal and glycated forms of human serum albumin (HSA). Samples of this protein in a soluble form were physically entrapped within porous silica particles by using glycogen-capped hydrazide-activated silica; these supports were then placed into 1.0 cm × 2.1 mm inner diameter columns. Initial zonal elution studies were performed using (R)-warfarin and L-tryptophan as probes for Sudlow sites I and II (i.e., the major drug binding sites of HSA), giving quantitative measures of binding affinities in good agreement with literature values. It was also found for solutes with multisite binding to the same proteins, such as many sulfonylurea drugs, that this method could be used to estimate the global affinity of the solute for the entrapped protein. This entrapment and zonal approach provided retention information with precisions of ±0.1-3.3% (± one standard deviation) and elution within 0.50-3.00 min for solutes with binding affinities of 1 × 10(4)-3 × 10(5) M(-1). Each entrapped-protein column was used for many binding studies, which decreased the cost and amount of protein needed per injection (e.g., the equivalent of only 125-145 pmol of immobilized HSA or glycated HSA per injection over 60 sample application cycles). This method can be adapted for use with other proteins and solutes and should be valuable in high-throughput screening or quantitative studies of drug-protein binding or related biointeractions.

Fig. 1

Entrapment of a protein by reaction of hydrazide-activated silica with mildly oxidized glycogen. Details on this approach are provided in the text

Fig. 2

Chromatograms obtained for the injection of a (R)-warfarin or b L-tryptophan as a site-selective probe onto a control column containing no entrapped protein or columns containing entrapped normal human serum albumin (HSA) or glycated HSA. These results were obtained at 0.50 mL/min. Other conditions are given in the text

Fig. 3

Basic structure of a sulfonylurea drug and the structures of the specific sulfonylurea drugs that were examined in this study

Fig. 4

Chromatograms obtained for acetohexamide on a control column containing no entrapped protein or on columns containing entrapped normal HSA or glycated HSA. These results were obtained at 0.50 mL/min. Other conditions are given in the text