Two pathways of iron uptake in bovine spleen apoferritin dependent on ironconcentration

Iron incorporation by bovine spleen apoferritin either with ferrous ammoniu m sulfate in different buffers or with ferrous ammonium sulfate and phospha te was studied. Iron uptake and iron autoxidation were recorded spectrophot omerically. The buffers [4-(2-hydroxyethyl)-1-piperazinyl]ethanesulphonic a cid (Hepes) and tris(hydroxymethyl)aminoethane (Tris) exhibited pH-dependen t iron autoxidation, with Tris showing less iron autoxidation than Hepes. A n Eadie-Scatchard plot (v/[s] versus v) of the iron uptake rate in Hepes wa s a curved rather than a straight line, suggesting that there are two iron uptake pathways. On the other hand, the Eadie-Scatchard plots of Tris and o f Hepes after the addition of phosphate showed a straight line. Phosphate a ccelerated the iron uptake rate. The iron loading kinetics of apoferritin i n Hepes was dependent on apoferritin concentration. The K-m value obtained from iron uptake kinetics was 4.5 muM, corresponding to the physiological i ron concentration. These results demonstrate that iron loading of apoferrit in was accomplished at physiological iron concentrations, which is essentia l for iron uptake, via two uptake pathways of dependent on iron concentrati on.