Abstract
This study evaluates how the polarity of the medium affects the binding efficiency of hydrophobic ligands with human serum albumin (HSA). The polarity of the aqueous medium was changed by adding 1,4-dioxane in concentrations of 0%, 10%, and 20% w/w, resulting in solvent mixtures with decreasing dielectric constants (ε = 80, 72, and 63). The addition of 1,4-dioxane did not affect the integrity of the protein, as confirmed by Far-UV-CD, Rayleigh scattering, and time-resolved fluorescence experiments. The impact of medium polarity on the binding constants was evaluated using 1,6-diphenyl-1,3,5-hexatriene (DPH), octyl gallate (OG), quercetin, and rutin as ligands. The association constants of DPH decreased as the medium hydrophobicity increased: at 0%, Ka = 19.8 × 10(5) M(-1); at 10%, Ka = 5.3 × 10(5) M(-1); and at 20%, Ka = 1.7 × 10(5) M(-1). The decrease was still higher using OG: at 0%, Ka = 5.2 × 10(6) M(-1); and at 20%, Ka = 2.2 × 10(5) M(-1). The results in the same direction were obtained using quercetin and rutin as ligands. Molecular dynamics simulations illustrated the hydrophobic effect at the molecular level. The energy barrier for DPH to detach from the protein's hydrophobic site and to move into the bulk solution was higher at 0% (9 kcal/mol) than at 20% 1,4-dioxane (7 kcal/mol). The difference was higher for OG, with 14 and 6 kcal/mol, respectively. Based on these findings, it was shown that the difference in hydrophobicity between the protein's microenvironment and the surrounding solvent is an essential component for the effectiveness of the interaction. These results shed light on albumin-ligand complexation, a molecular interaction that has been extensively studied.