Affinity labeling of the thyroxine binding domain of human serum prealbumin with dansyl chloride

Abstract

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-

Thyroid hormone binding to human serum prealbumin and rat liver nuclear receptor: kinetics, contribution of the hormone phenolic hydroxyl group, and accommodation of hormone side-chain bulk

“…in binding due to ion pairing with the ammonium groups of protein Lys-15 residues. This was suggested from affinitylabeling studies with dansyl chloride and TV-bromoacetyl-T4 (Cheng et al, 1975(Cheng et al, , 1977b. The hormone ammonium group is believed to contribute a negative effect on binding due to electrostatic repulsion between it and the lysine ammonium groups (Andrea et al, 1978).…”

Analysis of thyroid hormone binding to human serum prealbumin by 8-anilinonaphthalene-1-sulfonate fluorescence

“…Human serum PA, obtained from Behring Diagnostics, was purified further by gel electrophoresis as described previously (Cheng et al, 1975). PA concentrations were determined spectrophotometrically at 280 nm (£']Cml% = 14.1) (Raz and Goodman, 1969).…”

Photoaffinity labeling of human thyroxine-binding prealbumin with thyroxine and N-(ethyl-2-diazomalonyl)thyroxine

“…Further evidence that photoaffinity labeling of TBPA with l-EDM-T4 was specific for this binding site was obtained from experiments employing dansyl-TBPA. Dansyl chloride competes irreversibly with T4 for the same binding site on TBPA and has been employed as an affinity reagent to label the lysine-15 residue that is located in the T4 binding domain (Cheng et al, 1975). As shown in Figure 5A, photolabeling of dansyl-TBPA with [I25I]-l-EDM-T4 produced only 10% the incorporation of radiolabel as with TBPA.…”

“…Crude rat liver nuclear extract was prepared by the method of Latham et al (1976) and it was used without further purification. TBPA was dansylated with dansyl chloride (Sigma) by using the procedure of Cheng et al (1975). Unreacted dansyl chloride was removed by dialysis against 0.1 M Tris-HCl (pH 8.0).…”