Characterization of discrete classes of binding sites of human serum albumin by application of thermodynamic principles.

The binding interactions of four ligands differing in acid-base properties with human serum albumin (HSA) were examined as a function of temperature. Binding to HSA decreased with increasing temperature for all four ligands. The bound and free ligand concentrations obtained at different temperatures were satisfactorily fitted to a model that incorporates the effect of temperature as an independent covariable and that directly allows the estimation of the enthalpic and entropic components of the ligand-albumin interaction, along with the precision of this estimation. Using this analysis, the binding of acidic ligands could be resolved into two classes of saturable sites, with the determination of the corresponding number of sites, whereas interpretation of binding data at each isolated temperature allowed only the determination of one saturable plus one non-saturable class of site. The thermodynamic constants indicate that binding of ionizable ligands to HSA involves electrostatic plus hydrophobic interactions, whereas only hydrophobic interactions are involved in binding to a second low-affinity class of site when present. Binding of non-ionizable ligands resembles that of the second class of low-affinity sites of ionizable ligands.