TL-205 AND C-13 NMR-STUDIES OF HUMAN SEROTRANSFERRIN AND CHICKEN OVOTRANSFERRIN

We have examined the binding of Tl3+ to human serotransferrin and chicken ovotransferrin in the presence of carbonate and oxalate by Tl-205 and C-13 NMR spectroscopy. With carbonate as the synergistic anion, one observes two Tl-205 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 C-13-labeled carbonate, one finds two closely spaced doublets in the carbonyl region of the C-13 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 (2)J(Tl-205-C-13) approximate to 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 Tl-205 and C-13 NMR signals due to the bound metal ion and anion in both proteins. From titration studies, we found that Tl3+ 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 Tl-205 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 (C2O42-)-C-13 in the C-13 NMR spectra of both proteins. From studies of the Tl3+/(C2O42-)-C-13 adducts of the half-molecules of ovotransferrin and the N-terminal lobe of serotransferrin at two magnetic fields, we have shown that the C-13 NMR signals for the carbonyl carbons due to bound oxalate in each site are split into a doublet of doublets by carbon-carbon [(1)J(C-13-C-13) approximate to 70-75 Hz] and thallium-carbon [(2)J(Tl-205-C-13) approximate to 15-30 Hz) spin-spin couplings. These results suggest that oxalate binds to Tl3+ in a 1,2-bidentate manner in both transferrins. Finally, from field dependence studies we found that the line widths of the Tl-205 NMP signals for the Tl3+/carbonate forms of ovo- and serotransferrin increase dramatically with increasing external magnetic field strength (B-o). 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 Tl3+ of Delta sigma = 680 ppm. These findings have important ramifications concerning the potential of Tl-205 and other heavy I = 1/2 metal nuclei to study metalloproteins by NMR spectroscopy.