GALLIUM(3+) BINDING TO OVOTRANSFERRIN AND ITS HALF-MOLECULES - A MULTINUCLEAR NMR-STUDY

We have used C-13, Ga-69, and Ga-71 NMR spectroscopy to probe the binding of Ga3+ to ovotransferrin in the presence of C-13-enriched carbonate and oxalate. When carbonate is the synergistic anion, two C-13 and Ga-71 signals appear sequentially, corresponding to (CO32-)-C-13 and Ga3+ bound to the two metal ion binding sites of the protein. In combination with C-13 NMR studies of the proteolytic half-molecules of ovotransferrin, we found that the metal ion interacts preferentially with the N-site of the intact protein. In the case of oxalate, one observes two pairs of doublets due to the nonequivalent carbonyl carbons of the bound anion in both sites and, again, two overlapping Ga-71 signals. Ovotransferrin exhibits no site preference for Ga3+ in the presence of oxalate. The C-13 and Ga-71 signals characteristic of this adduct were again assigned using the N- and C-terminal half-molecules of the protein, and we found substantially broader Ga-71 signals for the isolated lobes compared to the intact protein. By exploiting the fact that the bound Ga-71 signals are due to the central (m = 1/2 --> -1/2) transition of this quadrupolar (I = 3/2) nucleus and using the observed chemical shifts and line widths of the protein-bound Ga-71 signals at two magnetic fields (B-0 = 9.4 and 11.7 T), we have calculated values for the quadrupole coupling constant chi) of the Ga-71 nucleus, its isotropic chemical shift (delta(i)), and the correlation time (tau(c)) of the bound metal ion in each case. In three of the four cases, we also showed that one can obtain Ga-69 chi values from detectable (though extremely broad) ovotransfenin-bound(69)Ga peaks and their corresponding Ga-71 signals at one field (B-0 11.7 T). Moreover, the Ga-71 chi data for protein-bound Ga3+ and a model complex, Ga(acac)(3), were used to compute the electric field gradient (eq(ionic)) at the bound metal ion in each case; these values are placed in the context of our earlier Al-27 and Sc-45 NMR studies of ovotransferrin (Aramini, J. M.; Vogel, H. J. J. Am. Chem. Soc. 1991, 116, 1988-1993).