Ligand binding to natural and modified human serum albumin

This paper reports evaluation of ligand binding constants for unmodified or biotinylated HSA (HSA_B) for two well-known HSA binding ligands, naproxen and bromocresol green. Results demonstrate differential scanning calorimetry (DSC) is a reliable quantitative method for straight-forward and rapid evaluation of ligand binding constants for HSA and modified derivatives. DSC measured the thermodynamic stability of free and ligand-bound HSA and HSA_B at pH = 6.0, 7.4 and 8.0. DSC analysis provided a quantitative gauge of responses of HSA and HSA_B thermodynamic stability to ligand binding. The influence of different levels of biotinylation of HSA_B on ligand binding, and how ligand binding varied as a function of pH for these molecules was also examined. In the three pH environments, biotinylation increased stability of HSA_B alone compared to free HSA at pH 7.4. Stabilities of free protein and ligand-bound complexes varied with pH in the order, pH = 6.0>7.4>8.0. Our analytical approach provided very accurate estimates for known binding constants of these ligands for HSA. Results revealed, for both ligands, extent of biotinylation of HSA_B affected binding, reducing binding constants from three to 100-fold. DSC analysis was able to delineate inter-relationships between molecular structure and thermodynamic stability of HSA and HSA_B bound by ligands; and their variations with pH.