Studies of Anion Binding by Transferrin Using Carbon-13 Nuclear Magnetic Resonance Spectroscopy

The ¹³C NMR spectra of apotransferrin and the Co³⁺ and Fe³⁺ complexes of transferrin with bound ¹³C-enriched (bi)carbonate have been studied at 68 MHz. Information has been obtained about the mechanism of metal binding, the spatial relationship of the metal and the anion binding sites, the ionization state of the anion, the protein ligation of the anion, and differences in the properties of the two anion binding sites. The spectrum of the Co³⁺2 complex contains a doublet resonance due to nonexchanging anion and three resonances due to exchanging anions. The nonexchanging anion is bound at the B site, and on the basis of its chemical shift value and its pH behavior we concluded that it is carbonate. The exchanging anions are bound at the A site, and they are assigned to bound bicarbonate and a protein- carbamino adduct. In the spectra of the Fe³⁺2 complex, no resonances corresponding to specifically bound anion are observed since these ¹³C resonances are broadened beyond detection by interaction with the paramagnetic Fe³⁺. In a previous study at 25 MHz, it was similarly observed that the ¹³C resonances were broadened beyond detection, but due to limitations of the signal to noise ratio it was only determined that the metal-anion distance is less than 9Å [Harris, D. C., Gray, G. A., & Aisen, P. (1974) J. Biol. Chem. 249, 5261-5264]. In the present study, it is calculated that the Fe³⁺-C distance must be less than 4.9Å, which implies that the anion is directly bound to the Fe³⁺ in the physiologically important Fe³⁺-transferrin-carbonate complexes. Two resonances are observed in the spectra of apotransferrin which appear to be due to specific anion binding. This indicates that anion binding to transferrin occurs prior to metal binding and that anion binding may be a preliminary step for metal binding. On the basis of the spectra and their pH behavior, it is concluded that the anion binding ligand at the B site is probably a guanidino group of arginine, while that at the A site may be either a guanidino group of arginine or an e-amino group of lysihe. The difference in the properties of the two anion sites and the state of the anion bound at the two sites could explain the functional difference in their iron-donating properties.