Studies on thyroid hormone-binding proteins. II. Binding of thyroid hormones, retinol-binding protein, and fluorescent probes to prealbumin and effects of thyroxine on prealbumin subunit self association.

Vitamin A in human plasma is transported by its specific carrier protein, the retinol-binding protein. Under physiological conditions the protein forms a stable protein-protein complex with the tetrameric plasma protein, the thyroxine-binding prealbumin. Human prealbumin was shown to interact with the fluorescent probes 1,8-anilinonaphthalene sulfonate (ANS) and 2-p-toluidinylnaphthalene-6-sulfonate (TNS). ANS bound to the protein at two independent sites with the apparent association constant 3 X 10(5) M-1, whereas TNS interacted with a single site with the binding constant 5 X 10(k) M-1. The fluorescent yield of protein-bound ANS was 0.95, a more than 200-fold enhancement compared with that of ANS in aqueous solutions. TNS enhanced its quantum yield nearly 500-fold to 0.37. On addition of thyroid hormones the fluorescent probes could be quantitatively displaced from the protein. This finding suggested that triiodthyronine, thyroxine, and the probes bound to a common site in prealbumin, which is likely to have a strongly hydrophobic character. The association constants for the interaction between prealbumin and the thyroid hormones could be calculated by using the hormones as competitive inhibitors in the TNS-prealbumin titrations. The data from the competition experiments together with those obtained from equilibrium dialysis revealed one major hormone binding site on the protein. The calculated association constant were 9 X 10(6) M-1 and 1 X 10(8) M-1 for triiodothyronine and thyroxine, respectively. Prealbumin monomers were bound to Sepharose by covalent attachment, and their properties were examined. Evidence was obtained demonstrating that the retinol-binding protein could interact with a single subunit of prealbumin. The estimated apparent association constant for the interaction of the protein and the matrix-bound monomeric prealbumin was 3 X 10(4) M-1, approximately 250-fold lower than that measured for protein and matrix-bound tetrameric prealbumin. The data, however, strongly suggest that there are four retinol-binding protein sites per prealbumin molecule. Using the technique of sedimentation equilibrium ultracentrifugation the prealbumin subunit self-association has been studied. The energy of the interaction for the prealbumin subunits is very high, and various concentrations of guanidine hydrochloride has to be used to perturb the equilibrium. All experiments indicated that prealbumin dissociates directly into monomers without the presence of intermediate forms. Thyroxine perturbed the chemical equilibrium of the prealbumin monomer-tetramer system by strengthening the interaction between the subunits.