A spin label study of the thyroid hormone-binding sites in human plasma thyroxine transport proteins.

Abstract

The binding site topographies of the three thyroid hormone-transporting proteins in human serum--prealbumin, thyroxine binding globulin, human serum albumin--have been studied with the aid of five spin-labeled analogs of L-thyroxine in which the distance between the phenolic hydroxyl and the nitroxide nitrogen ranged from 17 to 23 A. In the presence of prealbumin, the electron spin resonance spectrum of 3-([alpha-carboxy-4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenethyl]-carbamoyl)-2,2,5,5-tetramethyl-3-pyrrolinl-yloxy-ethyl ester revealed the presence of a highly immobilized spin label. As the chain length between the thyroxyl moiety and the pyrroline ring was increased, the mobility of the nitroxide group in the prealbumin-bound labels increased. If the spin labels bind in an extended conformation, the thyroxine-binding site was estimated to be approximately 21 A in depth. This finding is consistent with the known crystal structure of prealbumin and suggests that the solution and crystal conformations of the protein are very similar. In contrast to prealbumin, the thyroxine-binding site on thyroxine-binding globulin was found to be more open and possibly deeper. Human serum albumin has two binding sites for thyroxine, one of which has a higher affinity and is deep enough to accommodate a molecule that is 23 A in length. The lower affinity site is somewhat shallower and probably wider, as thyroxine spin labels bound to this site exhibited greater mobility.