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The binding of iron to transferrin was studied by loading iron (III) onto apotransferrin in a chloride and a nitrilotriacetate form. When magnesium was added, a marked increase occured in both the rate of iron binding and the maximum level of iron loaded on transferrin utilizing either iron salt. In the absence of magnesium the amount of iron required to achieve 50 percent saturation of the binding sites was 1.6 × 10−4M, whereas when magnesium was added, only about one-third as much iron (0.54 × 10−4M) was required. These data suggest an allosteric effect on transferrin by magnesium which potentiates iron (III) binding.

Measurement of the distribution of the four species of transferrin, viz, apotransferrin, diferric transferrin and the two monoferric transferrin, before and after incubation of iron-rich rabbit transferrin with rabbit reticulocytes showed that not all transferrin released from the cells were in the form of apotransferrin. Instead, a mixture of all four species of the protein was released with apotransferrin and C-terminal monoferric transferrin being the major fractions. The buffer solution containing 125I-labelled transferrin showed a continuous gain in percentages in apotransferrin and C-terminal monoferric transferrin after each incubation with reticulocytes. The N-terminal monoferric transferrin, however, remained unchanged suggesting that in the process of transferrin uptake by cells, the diferric transferrin releases its iron from the acid-labile site at N-domain first before the other iron from the acid-stable site.