Mössbauer studies of electrophoretically purified monoferric and diferric human transferrin.

Electrophoretically purified 57Fe-enriched monoferric and diferric human transferrins and selectively labeled complexes ([C-56Fe,N-57Fe]transferrin and [C-57Fe,N-56Fe]transferrin) were studied by Mössbauer spectroscopy. The data were recorded at 4.2 K over a wide range of applied magnetic fields (0.05-6 T) and were analyzed by a spin-Hamiltonian formalism. Characteristic hyperfine parameters were found and the obtained zero-field splitting parameters (D = 0.25 +/- 0.05 cm-1 and E/D = 0.30 +/- 0.02) agree with previous electron paramagnetic resonance (EPR) findings. The weak-field spectra of the [N-57Fe]transferrin are slightly broader than those of the [C-57Fe]transferrin, indicating that the N-terminal iron site may be more heterogeneous. However, the absorption line positions and the relative intensities of the subspectra originating from the three Kramers doublets of each Fe3+ site are identical. Thus the electronic structures of the two iron sites can be described by the same set of spin-Hamiltonian parameters, indicating that the ligand environments for the two sites are the same, as suggested by the recent X-ray crystallographic studies. This suggestion is further supported by the observation that the strong-field spectra of the two monoferric transferrins are indistinguishable. The selectively labeled mixed-isotope transferrins exhibit spectra that are identical to those of the corresponding monoferric 57Fe-enriched transferrins, implying that the occupation of one iron site has little or no effect on the immediate environment of the other site, a finding that is not surprising since the two sites are separated by approximately 4.2 nm.