Comprehensive characterization of the interaction between prototypical drug-site markers and multiple sites on human serum albumin by microbore frontal gel chromatography.

Abstract

Human serum albumin (HSA) has three major binding sites for drugs, site I, site II, and FA1 site. Dansyl amino acids (Dans-AAs) have long been used as convenient markers to judge whether a low molecular weight molecule of interest (ligand) binds to sites I or II. However, crystal structures of HSA-Dans-AA complexes have revealed that Dans-AAs with strict site specificity are also bound to non-marker sites. To characterize the multiple binding of Dans-AAs in detail, the average number of the bound ligands per HSA molecule were obtained in a free ligand concentration of 1-400 μM for dansylate (DA) and seventeen Dans-AAs using microbore frontal gel filtration chromatography. Analysis of the binding curves indicated that there are three specific binding sites for Dans-AAs. Four Dans-AAs with hydrophobic sidechain bind to all the sites with identical affinity, whereas DA and four Dans-AAs bind equally to two of them. Nine Dans-AAs bind to one of the three sites with the maximum occupancy ranging from 72% to 94%. The UV-vis absorption spectrum of HSA-ligand complex was obtained for DA and ten Dans-AAs, revealing that the dansyl moiety is in hydrophobic environment and conformational changes in the binding site residues are induced.