The Reaction of Ferric Salts with Transferrin

The use of Fe3+ salts to saturate apotransferrin in buffer solutions and serum is often found in basic research and clinical procedures, despite a lack of adequate documentation. We have investigated the reaction of Fe3+ salts with apotransferrin using the absorption peak of Fe3+-transferrin at 470 nm to monitor the reaction. While titration of apo-transferrin with Fe3+-nitrilotriacetic acid gives a linear function and clear end point, titration with FeCl3 results in a sigmoidal-shaped curve and no clear end point is reached. The spectral data indicate that only 5 to 25% of the iron becomes bound when 1 eq of FeCl3 is added to apotransferrin at neutral pH. The remaining sites of the protein are vacant and available for reaction with Fe3+-nitrilotriacetic acid. Several methods for separating unbound FeCl3 from transferrin were attempted. They were, however, all unsuccessful and often gave misleading results. A stoichiometric reaction of Fe3+ salts with apotransferrin is only obtained when the reactants are initially mixed at a low pH and then carefully adjusted to neutrality. A method is also described for obtaining fully saturated, chelate-free Fe3+-transferrin, using Fe3+-nitrilotriacetic acid as the iron reagent. In view of the poor reactivity of Fe3+ salts with apotransferrin at neutral pH, proton release studies have been re-examined with Fe3+-nitrilotriacetic acid. A value close to 2.6 H+ released per Fe3+ bound was obtained by two methods.