The binding of thyroxine (T4) and 8-anilino-1-naphthalenesulfonic acid (ANS) to human serum prealbumin was measured by equilibrium dialysis at pH 7.4 in 0.05 M phosphate-0.10 M NaCl at 25 degrees. The data were analyzed for the binding constants based on equations for (1) two independent sites and (2) two identical sites with negative interaction. Evaluation by the independent site model gave the following association constants: for T4 binding, KT1 = 1.0 x 10-8 M-1, KT2 = 9.5 x 10-5 M-1; for ANS binding, KA1 = 9.5 x 10-5 M-1, KA2 = 2.1 x 10-5 M-1. The interactive model gave constants kT = 5.5 x 10-7 M-1 and kA = 5.5 x 10-5 M-1. Interaction factors, alpha, defined such that -RT in alpha is the energy of interaction, were: alpha T = 0.041 AND ALPHA A = 0.62 for T4 and ANS, respectively. The "best fit" values for the number of sites were 2.0 and 1.6 for T4 and ANS, respectively. The binding of T4 to human prealbumin was competitive with ANS, and the binding constants evaluated from competition experiments were in agreement with those found for each ligand when studied separately. On the basis of analysis of X-ray data of human prealbumin (Blake et al.) there appear to be two identical T4 sites. It is therefore evident that the binding of T4 represents a case of negative cooperativity which is presumably due to interaction between ligands.

The binding of thyroxine to prealbumin was almost completely abolished when the latter had been treated with 2 mol of dansyl chloride/mol. When dansyl chloride was replaced with dansylglycine, the latter bound noncovalently to prealbumin ( n = 2.0; k = 2.9 X lo5 M-I; CY = 0.80). Competitive equilibrium dialysis as well as spectrophotometric titration showed that dansylglycine and thyroxine compete for the same binding domain on prealbumin. Dansyl chloride is therefore an affinity label for the 10-P A 1 is involved in the transport of the thyroid hormones and of vitamin A (retinol) in plasma. While PA binds T4 and triiodothyronine directly, its binding to vitamin A is mediated by another transport protein, RBP. Vitamin A is bound to RBP which in turn is bound to PA (Kanai et al., 1968). The latter has two binding sites for T4 (Ferguson et al., 1975) and four for RBP (Van Jaarsveld et al., 1973).PA is a tetramer composed of four identical subunits of molecular weight -1 3,700 whose complete amino acid sequence has recently been elucidated (Kanda et al., 1974). The subunits have the overall shape of elongated cylinders and are tetrahedrally arranged in such a manner that a central channel is formed which penetrates the entire molecule (Blake et al., 1974). This structure is unusual since other carrier proteins or enzymes whose structures have been elucidated have crevasses rather than channels. The four binding sites for RBP on PA are best explained by assuming that each subunit carries one ligand. The fact that PA has only two binding sites for T4 suggests that these are located inside the central channel. This has indeed been confirmed by X-ray analysis (Blake et al., 1974).The aim of our studies is to localize the site of attachment of T4 within the channel by the method of affinity labeling. Since it was found in our laboratory that ANS binds to the same two sites as Td (Ferguson et al., 1975) we selected a structurally closely related compound, Dns-C1, as a possible affinity label. Dns-C1, in contrast to ANS, has a reactive sulfonyl chloride group and is therefore capable of establishing a covalent bond with a nearby nucleophile. We established first by spectrophotometric titration and by equilibrium dialysis experiments that the DNS group, like ANS, binds to the same sites as T4. We could then show by various fragmentation and fractionation procedures that t From the Clinical Endocrinology Branch, National Institute of Ar-