Electron paramagnetic resonance measurement of the distance between the metal binding sites of transferrin

The distance between the two metal binding sites of human serum transferrin is calculated from measurements made of the paramagnetic broadening of the EPR spectrum of Cu²⁺ bound at the NH₂-terminal (B) site from Fe³⁺ bound at the COOH-terminal (A) site. Complexes of monoferric(A)-monocupric(B) transferrin, monocupric(B) transferrin, and monogallium(A)-monocupric(B) transferrin were prepared under identical conditions. Computer simulation and direct measurement of the EPR spectra from these complexes demonstrate a paramagnetic broadening of 0.6 ± 0.1 G in the line width of the Cu²⁺ spectrum due to the paramagnetic Fe³⁺ bound at the other site. The distance between the two metal binding sites is calculated to be 41.6 ± 2.8 Å.