The long-term objective of this research is to correlate the structure and function of thyroxine-binding glogulin (TBG), the major transport protein of the thyroid hormones in the serum of most mammals. Photoaffinity labeling and differential labeling experiments will be performed to identify the amino acids at or near the thyroxine (T4) binding site in TBG. TBG, derivatized at the active site with a radioactive label, will degraded proteolytically, the resulting radioactive peptides purified, and the sequence of amino acids determined. The T4 analog, N-2 diazo-3,3,3-trifluoropropionyl-L-thyroxine, containing [125I]T4, will be used as a photoaffinity label. For the differential labeling experiments, several group specific reagents such as diazotized diiodosulfanilic acid, iodoacetate and phenyglyoxal will be studied to ascertain their effect on binding. Experiments will also be performed in which [125I]T4 will be attached covalently to the binding site by reaction of the TBG[125I] complex with the bifunctional reagent, difluorodinitrobenzene. TBG will be subjected to limited proteolysis to determine if an intact molecule is required for binding. Circular dichroims measurements will be made of TBG and the TBG-T4 complex to monitor confirmational changes that may occur concomitantly with loss in binding activity resulting from proteolysis. The nuclear magnetic resonance spectrum of TBG-T4 will be compared with that of free TBG to study changes that occur on complex formation with special emphasis on the spectral region corresponding to the histidine residues in the protein. In a collaborative study, X-ray diffraction analysis of TBG and the TBG-T4 complex will be carried out by Dr. Vivian Cody using highly purified TBG prepared in this laboratory. The results of these studies should provide a more thorough description of the topography of TBG in the vicinity of the binding site and should lead to a more complete understanding of the factors involved in modulating transport of the thyroid hormones in human serum.
| Tabachnick, M; Korcek, L (1986) Effect of long-chain fatty acids on the binding of thyroxine and triiodothyronine to human thyroxine-binding globulin. Biochim Biophys Acta 881:292-6 |
