Thyroxine-protein interactions: Binding constants for interaction of thyroxine analogues with the thyroxine binding site on human thyroxine-binding globulin

Abstract

The binding constants for interaction of various thyroxine analogues with the thyroxine binding site on human thyroxine-binding globulin have been determined. Equilibrium dialysis, at pH 7.4 and 37°C, was used to measure the competitive effects of different iodothyronine compounds on the binding of 125I-labeled thyroxine to highly purified thyroxine-binding globulin. Relative to L-thyroxine, K = 6 · 10 9 M −1, the association constants of some important analogues were D-thyroxine, 1.04 · 10 9 M −1, 3,5-diiodo-3′-isopropyl- L-thyronine, 4.9 · 10 8 M −1; L-triiodothyronine, 3.3 · 10 8 M −1, 3,3′,5′- DL-triiodothyronine (reverse triiodothyronine), 3.1 · 10 8 M −1; tetraiodothyropropionic acid, 2.7 · 10 8 M −1; tetraiodothyroacetic acid, 2.6 · 10 8 M −1; 3′,5′-diiodo- DL-thyronine, 8.3 · 10 7 M −1; and 3,5-diiodo- DL-thyronine, 7.1 · 10 7 M −1. Calculation of the ΔG 0 values for binding of the analogues indicates that a major contribution to the free energy favoring binding is made by the alanine side chain of thryroxine. A change in configuration of the α-amino group from the L to D form causes an unfavorable change of 1 kcal/mol in the free energy of binding. Removal of the α-amino group as in tetraiodothyropropionic acid causes an unfavorable change of 1.9 kcal/mol in the free energy of binding. With regard to ring substituents, the results indicate that the two inner 3,5-iodines make about the same contribution to binding as the two outer 3′,5′-iodines.