Resolution of the two metal binding sites of human serum transferrin by low-temperature excitation of bound europium (III).

Derivatives of apotransferrin have been prepared in which europium replaces iron at either one or both of the two metal ion binding sites. At low temperature (77 K), pH 7.0, two sharp absorption lines are seen by means of laser-induced fluorescence of the bound europium. The one at 579.88 nm (17 245 cm-1) is assigned to the C-terminal region A site, and the other at 579.26 nm (17 263 cm-1) is assigned to the N-terminal region B site. The lifetimes of the excited 5D0 states are 210 +/- 20 and 310 +/- 30 mus for the A and B sites, respectively. The energy difference between the two peaks is a function of pH, with the splitting decreasing from 0.62 nm (18.5 cm-1) at pH 7.0 to 0.15 nm (4.5 cm-1) at pH 8.0. This spectroscopic unequivalence may be explained by a charge difference of the liganding groups at sites A and B.