Ligand binding to natural and modified Human serum albumin.

Eskew, Matthew W; Koslen, Megan M; Benight, Albert S

This paper reports evaluation of ligand binding constants for unmodified or biotinylated HSA (HSAB) 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 HSAB at pH = 6.0, 7.4 and 8.0. DSC analysis provided a quantitative gauge of responses of HSA and HSAB thermodynamic stability to ligand binding. The influence of different levels of biotinylation of HSAB 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 HSAB 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 HSAB affected binding, reducing binding constants from three to 100-fold. Naproxen binding was less sensitive to increased levels of biotinylation compared to bromocresol green. DSC analysis was able to delineate inter-relationships between molecular structure and thermodynamic stability of HSA and HSAB bound by ligands; and their variations with pH. This study provides another example of the general utility of differential scanning calorimetry (DSC) for evaluating ligand binding constants for Human Serum Albumin (HSA).