AL-27 AND C-13 NMR-STUDIES OF ALUMINUM(3+) BINDING TO OVOTRANSFERRIN AND ITS HALF-MOLECULES

The binding of Al3+ to ovotransferrin and its half-molecules in the presence of C-13-enriched carbonate and oxalate has been investigated by Al-27 and C-13 NMR spectroscopy. With carbonate as the synergistic anion, two overlapping signals are observed in both the C-13 and Al-27 NMR spectra; these correspond to the chelated Al3+ and the labeled anion at both metal ion binding sites in the protein. In the case of oxalate and Al3+ binding, two closely spaced Al-27 signals are also observed. Two pairs of doublets are detected in the C-13 NMR spectrum, suggesting that oxalate binds via only one carboxylate moiety to the metal ion in each site. The chemical shifts of the protein-bound Al-27 signals are diagnostic of octahedrally coordinated aluminum. C-13 and Al-27 NMR experiments on the purified N- and C-terminal half-molecules of ovotransferrin have facilitated the assignment of these signals. From these assignments and titration experiments we found that in the presence of carbonate the N-terminal site of ovotransferrin binds Al3+ with a higher affinity than does the C-site. However, changing the synergistic anion to oxalate alters the specificity; under these conditions Al3+ is preferentially complexed by the C-site of the protein. These experiments demonstrate a clear difference in the behavior of the two metal ion binding sites of ovotransferrin. The ovotransferrin-bound Al-27 NMR signals have some rather unusual properties characteristic of quadrupolar nuclei bound in slow exchange to large macromolecules far from extreme narrowing conditions. First, the maximum intensity of the protein-bound Al-27 signal is substantially less than that observed for equimolar solutions of the free metal ion. Second, the signals exhibit a unique pulse angle dependence where a maximum in peak intensity is attained at pulse lengths that are less than half the 90-degrees pulse for an aqueous Al3+ solution. Third, Al-27 signals for Al3+ bound to the half-molecules of ovotransferrin are much broader than those for the intact protein, reflecting the importance of molecular motion on the detectability of quadrupolar nuclei. Fourth, an increase in the external magnetic field strength causes line narrowing and a 2-4 ppm downfield dynamic frequency shift. These data are all consistent with the idea that only the central (1/2 --> -1/2) transition of the Al-27 (spin 5/2) nucleus is observed, ThiS Al-27 NMR study exemplifies the potential of this nucleus as a probe for the nature of the iron binding sites of transferrins and possibly other metalloproteins.