Albumin binding sites studied by high-performance liquid affinity chromatography

Abstract

High performance liquid affinity chromatography with chiral stationary phase is used to characterize the binding sites in human serum albumin. For that purpose the possibility to use both phenylbutazone and diazepam as markers for binding sites I and II, respectively, is investigated. Their binding is characterized by affinity constants and binding capacity, determined using a mathematical method modified for more than one type of sites. The results obtained suggest that both phenylbutazone and diazepam have two types of binding sites: high- and low-affinity sites. Diazepam as an analyte binds to the high-affinity sites of the marker phenylbutazone. Phenylbutazone as an analyte binds to the low-affinity binding sites of the marker diazepam. A conclusion is derived that the binding sites for the both markers are overlapping and so they cannot be specific and entirely differentiated. Probably these are binding areas with common subsites.