| Popis: |
Human orosomucoid (OMD) is one of the best-characterized seromucoids whose structure and function remain unknown. A great amount of work has been done to determine a structure of this protein but none has been forthcoming. There has been considerable interest in this molecule because of its possible role in the immune response and drug transport. OMD was isolated from human serum and separated into its gene products by immobilized metal affinity chromatography (IMAC). The gene products, GPI and GPII, were characterized by fluorescence quenching, drug binding, thermal unfolding and circular dichroism (CD). The thermal unfolding data showed that GPII unfolded at the same temperature but with lower thermodynamics than GPI and unfractionated OMD (uOMD). This indicates GPII unfolds with a non-two-state transition. CD spectroscopy showed that GPII had increased amounts of a-helical structure and decreased b-sheet structure when compared to GPI. The fluorescence quenching experiments showed that the differences in tryptophan environments were small. Characterization of the cyanogen-treated uOMD, GPI and GPII showed no difference in thermal unfolding or CD spectroscopy. Furthermore, analysis of the CD spectra show that the fractions of secondary structure are essentially the same in the treated as untreated. Ligand binding of cyanogen-treated uOMD,GPI and GPII showed a decrease in binding sites to one half the untreated. A corresponding two-fold increase in the binding enthalpy and association constant was observed. These results were interpreted to mean that two conformers of OMD exist with unique salt bridge linkages. One conformer permits ligand binding with increased affinity while the second is closed to binding. The conformers and their corresponding salt bridges regulate drug binding through accessibility of the binding pocket. In addition a third conformer may exist in the presence of ligand. |
