Thyroxine-protein interactions: VI. Structural requirements for binding of benzene derivatives to thyroxine-binding sites on human serum albumin

Abstract

Structural requirements for binding to thyroxine-binding sites on human serum albumin were studied by measuring the relative abilities of various substituted benzene derivatives, such as phenols and benzoates, to displace thyroxine from albumin in equilibrium dialysis experiments performed at pH 7.4 and 30 °. Relatively strong binding of benzene derivatives to thyroxine-binding sites on albumin depended upon two structural requirements. These were (1) a requirement for an anionic group, and (2) the presence on the phenyl ring of at least one highly polarizable substituent such as an iodine atom in the case of benzoates, or the presence of two highly polarizable substituents in the case of phenols. The effect of different substituents on the binding of phenols was in the order: alkyl group (CH 3-, or (CH 3) 2C-) < NO 2, Cl < Br, I. The diphenyl ether structure of thyroxine was not an obligatory requirement for strong binding since compounds with a single benzene ring were bound as tightly as thyroxine as shown by the competition experiments and confirmed by fluorescence quenching. Bulky halogen atoms ortho to the carboxylate group caused large reductions in relative binding affinities of benzoates. This ortho effect indicated that coplanarity of the carboxylate anion with the phenyl ring was required for optimal binding. Based on the steric requirement for coplanarity, a model for binding to the primary thyroxine-binding site was suggested which visualized the outer diiodophenolate portion of thyroxine as fitting into a cleft in a predominantly hydrophobic region of albumin.