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

Abstract

Derivatives of apotransferrin were prepared in which Eu replaces Fe at either one or both of the 2 metal ion binding sites. At low temperature (77 K), pH 7.0, 2 sharp absorption lines are seen by means of laser-induced fluorescence of the bound Eu. 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 .mu.s for the A and B sites, respectively. The energy difference between the 2 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 inequivalence may be explained by a charge difference of the liganting groups at sites A and B.