Interaction of Transferrin with Solubilized Receptors from Reticulocytes

A chromatographic assay method was developed to measure solubilized transferrin receptors from reticulocytes. Binding of transferrin to its receptor was found to occur in a reversible manner involving only one type of receptor, viz. one which is readily saturated with, and has a high affinity for, transferrin. The results were consistent with a bimolecular interaction mechanism between transferrin and its receptor. A number of reticulocyte populations were examined and they contained about 100 000 receptors per cell with a receptor density of about 800 per μm 2 of cell surface area. The equilibrium constant of association ( K a ) of transferrin for its receptor was approximately 10 7 1 · mol −1 . The binding process was markedly pH‐dependent, with a pH optimum of 7.8. Binding was considerably reduced when the ionic strength or calcium ion levels were below physiological values. Analysis of the binding process at different temperatures showed that Δ H o for the interaction was −444 J · mol −1 , Δ G ° was −44 · 3 kJ · mol −1 and Δ S ° was estimated to be + 145 J · deg 1− . mol −1 . The results are consistent with an entropy‐driven reaction where polar groups establish contact surface complementarity and the specificity of the association, and hydrophobic bonding stabilises the association complex. Intact cells incubated with 59 Fe‐labelled and 125 I‐labelled transferrin showed a much greater dependence on incubation temperature than the binding of transferrin to its solubilized receptor. Arrhenius plots for the rate of transferrin uptake; the rate of iron uptake and rate of iron incorporation into haem all had similar ‘activation energies’. These plots exhibited ‘break points’ or discontinuities at 20°C and 45°C which were not seen with the solubilized receptor. Such ‘break points’ imply phase transitions in the reticulocyte membrane at these temperatures. The results in conjunction with those previously reported indicate that once membrane receptor occupation by transferrin has occurred the reaction which limits iron acquisition by reticulocytes is the endocytotic intracellular availability of the transferrin‐receptor complex.