Thallium-205 and carbon-13 NMR studies of human sero- and chicken ovotransferrin
| Autor: | Peter H. Krygsman, James M. Aramini, Hans J. Vogel |
|---|---|
| Rok vydání: | 1994 |
| Předmět: |
Magnetic Resonance Spectroscopy
Analytical chemistry Carbonates Biochemistry Oxalate Adduct Ion Metal chemistry.chemical_compound Serotransferrin Animals Humans Thallium Carbon Isotopes Oxalates biology Chemistry Transferrin Nuclear magnetic resonance spectroscopy Ovotransferrin Carbon-13 NMR Crystallography visual_art visual_art.visual_art_medium biology.protein Chickens Conalbumin |
| Zdroj: | Biochemistry. 33(11) |
| ISSN: | 0006-2960 |
| Popis: | We have examined the binding of TP+ to human serotransferrin and chicken ovotransferrin in the presence of carbonate and oxalate by 205Tl and 13C NMR spectroscopy. With carbonate as the synergistic anion, one observes two 2osTl NMR signals due to the bound metal ion in the two high-affinity iron-binding sites of each protein. When the same adducts are prepared with 13C-labeled carbonate, one finds two closely spaced doublets in the carbonyl region of the 13C NMR spectrum of serotransferrin; these correspond to the labeled anion directly bound to the metal ion in both sites of the protein. The analogous resonances in ovotransferrin are completely degenerate, and only one doublet can be detected. The magnitudes of the spin-spin coupling between the bound metal ion and carbonate range from 2J(205Tl-13C) = 270 to 290 Hz. We have used the proteolytic half-molecules of ovotransferrin and the recombinant N-terminal half-molecule of serotransferrin to assign the 205Tl and 13C NMR signals due to the bound metal ion and anion in both proteins. From titration studies, we found that T13+ is bound with a greater affinity at the C-terminal site of serotransferrin, whereas no site preference can be noted for ovotransferrin. When oxalate is used as the anion instead of carbonate, the 205Tl NMR signals arising from the bound metal ion in the sites of ovotransferrin are shifted downfield and become almost degenerate. A very complex pattern of resonances is observed for bound 13C2042- in the 13C NMR spectra of both proteins. From studies of the T13+/13C20d2- adducts of the half-molecules of ovotransferrin and the N-terminal lobe of serotransferrin at two magnetic fields, we have shown that the 13C NMR signals for the carbonyl carbons due to bound oxalate in each site are split into a doublet of doublets by carbon-carbon (1J(13C-13C) = 70-75 Hz) and thallium-carbon (2J(20STl- 13C) = 15-30 Hz) spin-spin couplings. These results suggest that oxalate binds to T13+ in a 1,2-bidentate manner in both transferrins. Finally, from field dependence studies we found that the line widths of the 205Tl NMR signals for the T13+/carbonate forms of ovo- and serotransferrin increase dramatically with increasing external magnetic field strength (BO). We have determined that these effects can be attributed to nuclear relaxation via the chemical shift anisotropy (CSA) mechanism and calculated a value of the chemical shift anisotropy for serotransferrin-bound T13+ of Au = 680 ppm. These findings have important ramifications concerning the potential of *05Tl and other heavy Z = '/z metal nuclei to study metalloproteins by NMR spectroscopy. |
| Databáze: | OpenAIRE |
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