A SC-45 NMR-STUDY OF OVOTRANSFERRIN AND ITS HALF-MOLECULES

The binding of Sc3+ to chicken ovotransferrin has been investigated by Sc-45 and C-13 NMR spectroscopy. In the presence of carbonate, one ob serves two Sc-45 and C-13 signals which can be assigned using the prot eolytic half-molecules of ovotransferrin to bound Sc3+ and (CO32-)-C-1 3 in both metal ion binding sites of the protein. When the synergistic anion is changed to oxalate, two overlapping Sc-45 resonances are aga in detected. Several properties of the transferrin-bound Sc-45 signals , such as their dependence on pulse length, magnetic field, protein si ze, and temperature, are consistent with the detection of only the cen tral (m = 1/2 --> -1/2) transition of a quadrupolar nucleus under far from extreme narrowing conditions. From Sc-45 chemical shift and line width data for the Sc3+/carbonate form of ovotransferrin at four magne tic fields, we have calculated values for the quadrupole coupling cons tant ((chi)) and rotational correlation time (tau(c)) for the bound me tal ion in each site of the protein. In addition, from chemical shift information at two fields, we have obtained estimates of chi for the S c3+/oxalate form of ovotransferrin, as well as for the Sc3+/carbonate derivative of human serotransferrin. The results in each case are comp arable to the chi values we have determined for two octahedral Sc3+ or ganometallic complexes. From the chi data, we have calculated values f or the electric field gradient (\eq(ionic)\) at the metal nucleus for transferrin-bound Sc3+, by taking into account the nuclear quadrupole moment for Sc-45 and the Sternheimer antishielding factor for Sc3+. Th ese results are compared to our previous Al-27 NMR data for the analog ous Al3+ forms of the transferrins [Aramini, J. M.; Vogel, H. J. J. Am . Chem. Sec. 1993, 115, 245-252. Aramini, J. M.; Germann, M. W.; Vogel , H. J. J. Am. Chem. Sec. 1993, 115, 9750-9753]. This report represent s the first Sc-45 NMR study of a metalloprotein and is another example of the applicability of quadrupolar metal ion NMR to the investigatio n of metal ion binding sites in large proteins.