Localized conformational perturbation of human serum albumin. A study of the thyroxine binding site at the amino terminus

Abstract

The principal thyroxine binding site on human serum albumin is in the region of the amino terminus. Binding at this site is accompanied by a bathochromic shift in the thyroxine spectrum which is different from the spectrophotometric titration of thyroxine in solution. No spectral change is associated with binding at the secondary sites. The primary thyroxine binding site is more sensitive to urea denaturation than the molecule as a whole. A concentration of 0.5 m, which has no discernible effect on the overall conformation of serum albumin reduced the bathochromic shift without change in the association constant. Therefore, the thyroxine-protein interactions which produce the spectral change do not contribute appreciably to the binding energy. In 2 m urea, the primary binding site begins to deteriorate. At this urea concentration overall conformational alterations can be detected in serum albumin. Of the five secondary binding sites, only one was insensitive to both 0.5 and 2 m urea. Therefore, although it was possible to account for the binding at all of the secondary sites with the assumption of a single association constant this group of binding sites is heterogeneous. The sensitivity of the region of the amino terminus to urea denaturation was also demonstrated with leucineaminopeptidase, which can cleave the amino terminal amino acid in 0.5 m urea but cannot attack native serum albumin. No thyroxine binding was observed in 4 m urea. The selective denaturation by urea of the thyroxine binding sites is considered to be due to particular urea sensitivity of specific, mutually insulated, regions of the albumin molecule.