Comprehensive characterization of the interaction between prototypical drug-site markers and multiple sites on human...

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 17 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 10 Dans-AAs, revealing that the dansyl moiety is in hydrophobic environment and conformational changes in the binding site residues are induced.