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59Fe uptake by rabbit reticulocytes from human transferrin-bound iron was studied by using transferrin solutions (35, 50, 65, 80 and 100% saturated with iron) whose only common characteristic was their content of diferric transferrin. During the early incubation period, 59Fe uptake from each preparation by reticulocytes was identical despite wide variations in amounts of total transferrin, total iron, monoferric transferrin and apotransferrin in solution. During the later phase of incubation, rate of uptake declined and was proportional to each solution's monoferric transferrin content. Uptake was also studied in a comparative experiment which used two identical, partially saturated transferrin preparations, one uniformly 59Fe-labelled and the other tracer-labelled with [59Fe]diferric transferrin. In both experiments, iron uptake by reticulocytes corresponded to utilization of a ferric ion from diferric transferrin before utilization of iron from monoferric transferrin.

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Iron, once absorbed from the gastrointestinal tract, is distributed throughout the body by the specific iron-binding protein (IBP, transferrin, siderophilin) (1). Iron binding by IBP is presumed to be of a chelate type in which the hexacoordination capacity of the ferric ion is satisfied by linkage with three phenolic oxygens, one carboxyl group, and a bicarbonate ion on the remaining coordination position (2-4). Since IBP has been shown to have a highly selective ability to deliver iron to the immature red cell (5), we have utilized various synthetic iron chelates in an attempt to delineate the characteristics for iron binding essential to carry out this function of the protein. MethodsPreparation of the reticulocyte-rich blood. Reticulocytosis was produced in adult albino rabbits (3 to 5 kg) by removing approximately 5 to 10%v of the total blood volume (30 to 40 ml) daily for 3 or 4 days by repeated cardiac puncture. Two days thereafter, a final cardiac puncture delivered all available blood, which was immediately placed into iron-free, heparinized glass containers. Reticulocyte count using the new methylene blue stain technique (6) indicated 10 to 25%, immature cells.The red cells were separated from the plasma and leukocytes by centrifugation at 1,000 X g for 15 minutes at 40 C. The harvested cells were washed and centrifuged three times with 5 vol of iron-free, phosphate

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