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

We have used C-13, Ga-69, and Ga-71 NMR spectroscopy to probe the bind ing of Ga3+ to ovotransferrin in the presence of C-13-enriched carbona te and oxalate. When carbonate is the synergistic anion, two C-13 and Ga-71 signals appear sequentially, corresponding to (CO32-)-C-13 and G a3+ bound to the two metal ion binding sites of the protein. In combin ation with C-13 NMR studies of the proteolytic half-molecules of ovotr ansferrin, we found that the metal ion interacts preferentially with t he 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 wer e again assigned using the N- and C-terminal half-molecules of the pro tein, and we found substantially broader Ga-71 signals for the isolate d lobes compared to the intact protein. By exploiting the fact that th e bound Ga-71 signals are due to the central (m = 1/2 --> -1/2) transi tion of this quadrupolar (I = 3/2) nucleus and using the observed chem ical 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 isotro pic chemical shift (delta(i)), and the correlation time (tau(c)) of th e bound metal ion in each case. In three of the four cases, we also sh owed that one can obtain Ga-69 chi values from detectable (though extr emely broad) ovotransfenin-bound(69)Ga peaks and their corresponding G a-71 signals at one field (B-0 11.7 T). Moreover, the Ga-71 chi data f or protein-bound Ga3+ and a model complex, Ga(acac)(3), were used to c ompute 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).