Effects of anion binding on the conformations of the two domains of ovotransferrin.

A previous paper (Harris (1985) Biochemistry 24, 7412-7418) reported the occurrence of two classes of anion binding sites in transferrin. To evaluate the locations of the two anion binding sites in relation to the two major domains of transferrin we determined the binding constants of whole ovotransferrin and its two half-molecules by means of the difference UV spectroscopic technique. Anions induced strong negative absorbance at 245 nm in the order: citrate greater than phosphate greater than bicarbonate for whole ovotransferrin and the N-terminal half-molecule; and: phosphate greater than citrate greater than bicarbonate for the C-terminal half-molecule. The anion dissociation constants of the N-terminal half-molecule were consistent with lower dissociation constants, and those of the C-terminal half-molecule, with higher dissociation constants of whole ovotransferrin, indicating that the two classes of anion binding sites correspond to the binding sites in individual structural domains. Anion binding markedly protected the N-terminal half-molecule, but not the C-terminal half-molecule from digestion with trypsin and disulfide reduction with dithiothreitol. As to the far and near ultraviolet CD spectra data, however, there was no significant difference between in the presence and absence of an anion. Therefore, the binding of an anion would induce some conformational changes which were not reflected by the CD spectrum.