Evidence for functional differences between the two sites of rabbit transferrin: effects of serum and carbon dioxide

A recently developed technique combining urea gel electrophoresis with Western immunoblotting has been modified for assessing the relative ability of each iron binding site of rabbit transferrin in delivering iron to rabbit reticulocytes. The two sites can be made to release iron at the same or differing rates, depending on the experimental conditions. In Hanks' balanced salts solution in an atmosphere of room air or 5% CO2, the acid-labile site in the N-terminal lobe of the protein was found to be 1.4- and 2.9-times more effective than its acid-stable counterpart in providing iron to reticulocytes after 90 min incubation. Both sequential and simultaneous release of iron from the two sites was observed, but sequential release was initiated only from the N-terminal site. The same site also proved to be a better iron donor by a factor of 2 when incubations were conducted in Hanks' medium enriched with 20% serum in 5% CO2. Only in 20% serum in air were the two sites found to be equivalent iron suppliers to reticulocytes. In the cases studied, an atmosphere of 5% CO2 increased 2-fold the effectiveness of iron donation by the acid-labile site to reticulocytes, while the presence of 20% serum enhanced the iron-donating ability of the acid-stable C-terminal site. Thus, the transferrin-reticulocyte interaction is sensitive to environmental variables, and such sensitivity may help account for apparent discrepancies in previous studies of the relative iron-donating abilities of the two sites of transferrin.