Differed preferential iron-binding lobe in human transferrin depending on the presence of bicarbonate detected by HPLC/high-resolution inductively coupled plasma mass spectrometry

The binding of iron (Fe) to human serum transferrin (Tf) was analyzed with an HPLC system equipped with an anion exchange column and directly connected with a high-resolution inductively coupled plasma mass spectrometer for metal detection. The (56)Fe level in the eluate was monitored at resolution m/Deltam=3000. Two monoferric Tfs were assigned based on the results of urea-PAGE and desferrioxamine experiments. When Fe was added as Fe-citrate stepwise to an apo-Tf solution in the presence of bicarbonate, the N-lobe site was the preferential Fe-binding site, while the C-lobe site was preferred in the absence of bicarbonate. In both cases, the Fe-peak areas of the preferential site and Fe(2)-Tf increased up to an Fe/Tf molar ratio of 1, and then the peak area of the monoferric Tf decreased while the peak area of Fe(2)-Tf increased. When the Fe/Tf molar ratio was below 1, the amount of Fe bound to the lobe with a weaker affinity was higher in Fe(2)-Tf than in the monoferric Tf in each case. Namely, Fe(2)-Tf was the preferential binding state of Fe to human serum Tf. The preference is reasonable for transferring Fe ions effectively to Tf-receptors.