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