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

We have examined the binding of Tl3+ to human serotransferrin and chic ken ovotransferrin in the presence of carbonate and oxalate by Tl-205 and C-13 NMR spectroscopy. With carbonate as the synergistic anion, on e observes two Tl-205 NMR signals due to the bound metal ion in the tw o high-affinity iron-binding sites of each protein. When the same addu cts are prepared with C-13-labeled carbonate, one finds two closely sp aced doublets in the carbonyl region of the C-13 NMR spectrum of serot ransferrin; these correspond to the labeled anion directly bound to th e 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 m etal 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 ovotransfer rin 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 serotransferr in, whereas no site preference can be noted for ovotransferrin. When o xalate is used as the anion instead of carbonate, the Tl-205 NMR signa ls arising from the bound metal ion in the sites of ovotransferrin are shifted downfield and become almost degenerate. A very complex patter n of resonances is observed for bound (C2O42-)-C-13 in the C-13 NMR sp ectra of both proteins. From studies of the Tl3+/(C2O42-)-C-13 adducts of the half-molecules of ovotransferrin and the N-terminal lobe of se rotransferrin 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) ap proximate to 70-75 Hz] and thallium-carbon [(2)J(Tl-205-C-13) approxim ate to 15-30 Hz) spin-spin couplings. These results suggest that oxala te binds to Tl3+ in a 1,2-bidentate manner in both transferrins. Final ly, from field dependence studies we found that the line widths of the Tl-205 NMP signals for the Tl3+/carbonate forms of ovo- and serotrans ferrin increase dramatically with increasing external magnetic field s trength (B-o). We have determined that these effects can be attributed to nuclear relaxation via the chemical shift anisotropy (CSA) mechani sm and calculated a value of the chemical shift anisotropy for serotra nsferrin-bound Tl3+ of Delta sigma = 680 ppm. These findings have impo rtant ramifications concerning the potential of Tl-205 and other heavy I = 1/2 metal nuclei to study metalloproteins by NMR spectroscopy.