EQUILIBRIUM DIALYSIS STUDIES OF THE BINDING OF THYROXINE BY HUMAN SERUM ALBUMIN*

Abstract

Equilibrium dialysis has been employed to investigate the binding of thyroxine by human serum albumin. Various temperatures and buffers were used, including 38[degree]C, pH 7.4, and 0.15 ionic strength (isotonic). Detailed studies of various albumin preparations yielded results consistent with four primary binding sites per molecule, with an apparent association constant of approximately 100,000. Studies exploring variation in pH and behavior of acetylated albumin preparations suggested that four cationic groups on the protein molecule interact with the anionic phenolate groups of thyroxine and its analogs. The evidence is compatible with the hypothesis that the four primary binding sites are epsilon-amino groups of lyslne residues of albumin, but the nature of the binding sites is not considered proven. With the observation that 15% of the total serum thyroxine is bound to albumin, it was possible to employ the binding constants obtained to compute the theoretical free thyroxine concentration of serum; the value calculated was 0.6 x 10-10 M.