Comparative study of the iron-binding properties of human transferrins. I. Complete and sequential iron saturation and desaturation of the lactotransferrin

Human lactotransferrin binds 2 Fe3+ tightly at two specific sites. In order to demonstrate differences between the stability of the two iron-binding sites, the removal of iron was studied in buffers in the pH range 8-3 varying the ionic strength and with or without metal chelators such as phosphate ions and EDTA. The results show that in the presence of formate and acetate buffers of ionic strength 0.1–0.4 and in a pH range of 5–3, the two Fe3+ from human lactotransferrin are removed stimultaneously. Addition of 4 mM EDTA to buffers of ionic strength 0.1 and in the pH range 8–3 shows that between pH 5–4.3 the iron from only one of the binding sites, called the ‘acid labile’ site, is removed. Addition of 0.2 M phosphate ions to buffers of ionic strength 0.2 and in pH range 8–3 containing 4 mM EDTA shows that Fe3+ from the ‘acid labile’ site may be completely removed at pH 6. Removal of Fe3+ from the ‘acid stable’ site is obtained at pH 4. The differential behavior of the two iron binding sites was also shown by saturation experiments in the presence of citrate/bicarbonate buffers at different pH values. In a pH range 6.2–4.8, 50% saturation was obtained, but at pH 6.35 complete saturation was achieved. When saturation of partially saturated samples of human lactotransferrin was performed with 59Fe it was demonstrated that in the pH range 6.2–4.8 iron is bound only to the ‘acid labile’ site.