Use of chromatography to investigate biological recognition of a ligand by human serum albumin

Chromatographic temperature studies and differential scanning calorimetry have been used to elucidate the mechanism of retention of dansyl amino acids by human serum albumin (HSA) as stationary phase in reversed-phase liquid chromatography (RPLC) (Guillaume et al.,Anal. Chem. 1997,69, 4979). A novel mathematical development is proposed for description of HSA-dansyl amino acid association when HSA is used as mobile-phase modifier and C18 as stationary phase. The solute retention factor depends on the concentration of HSA in the bulk mobile phase and on the binding constant for the guest-HSA complex. The degree of complexationnc (the amount, %, of guest complexed) was determined. Enthalpy-entropy compensation was also analyzed in relation to this mathematical model to confirm the complexation behavior of the solute with HSA. Variation of the binding constant with the concentration of sodium phosphate, i.e. with the electrostatic force controlling HSA-solute association, is described. This theoretical model enabled estimation of the surface charge density (σ/F) and the accessible solvent surface area of the site II binding cavity; these were found to be approximately 8.7×10−7 mol m−2 and 2 nm2.