Spectroscopic and thermodynamic studies on the binding of gadolinium(III) to human serum transferrin.
A wide variety of thermodynamic, kinetic, and spectroscopic studies have demonstrated differences between the two metal-binding sites of transferrin. In the present investigation, we have further assessed these differences with respect to the binding of gadolinium, evaluated by UV difference spectrophotometry, electron paramagnetic resonance (EPR) titration, EPR difference spectroscopy in conjunction with urea gel electrophoresis, and equilibrium dialysis. Combinations of these studies establish that only one site of the protein binds Gd(III) sufficiently firmly to be characterized. In order to reveal which of the two sites accepts Gd(III), we made use of monoferric transferrins preferentially loaded with Fe(III) at either site in EPR spectroscopic studies. Because of the overlap of signals, difference spectroscopy was required to distinguish resonances arising from Fe(III) and Gd(III) specifically complexed to the protein. When iron is bound to the C-terminal site, leaving the N-terminal site free for binding of gadolinium, the difference spectrum shows no evidence of specific binding. However, when iron is bound to the N-terminal site, the difference spectrum shows a resonance line at g' = 4.1 indicative of specific binding, thus implicating the C-terminal site in the binding of Gd(III). The effective stability constant for the binding of Gd(III) to this site of transferrin at pH 7.4 and ambient pCO2 is 6.8 X 10(6) M-1. At physiological pCO2, the formation of nonbinding carbonato complexes of Gd(III) precludes a substantial role for transferrin in the transport of the lanthanide in vivo.
Zak
O
O
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461.
Aisen
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P
D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D005682:Gadolinium / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D014168:Transferrin / Q000378:metabolism*
D014168:Transferrin; D005682:Gadolinium