Kinetic analysis of bovine spleen apoferritin and recombinant H and L chain homopolymers: Iron uptake suggests early stage H chain ferroxidase activity and second stage L chain cooperation

Abstract

Ferritin utilizes ferroxidase activity to incorporate iron. Iron uptake kinetics of bovine spleen apoferritin (H: L = 1 : 1.1) were compared with those of recombinant H chain ferritin and L chain ferritin homopolymers. H chain ferritin homopolymer showed an iron uptake rate identical to bovine spleen apoferritin (0.19 and 0.21 mmol/min/micromol of protein, respectively), and both showed iron concentration-dependent uptake. In contrast, the L chain homopolymer, which lacks ferroxidase, did not incorporate iron and showed the same level of iron autoxidation in the absence of ferritin. Bovine spleen apoferritin was shown to have two iron concentration-dependent uptake pathways over a range of 0.02-0.25 mM ferrous ammonium sulfate (FAS) by an Eadie-Scatchard plot (v/[FAS] versus v), whereas the H chain ferritin homopolymer was found to have only one pathway. Of the two Km values found in bovine spleen apoferritin, the lower mean Km value was 9.0 microM, while that of the H chain homopolymer was 11.0 microM. H chain ferritin homopolymer reached a saturating iron uptake rate at 0.1 mM FAS, while bovine spleen apoferritin incorporated more iron even at 0.25 mM FAS. These results suggest that the intrinsic ferroxidase of ferritin plays a significant role in iron uptake, and the L chain cooperates with the H chain to increase iron uptake.

Binding of Mammalian and Avian Ferritins with Biotinylated Hemin: Demonstration of Preferential Binding of the H Subunit to Heme

“…The commercial ferritin was almost entirely composed of L subunits, and the L/H subunit ratios of the purified horse spleen, canine liver, and bovine spleen ferritins were 4.0, 2.3 and 1.1, respectively, these results are in agreement with those in previous reports [17,18,30]. Apoferritins were prepared from each of the above ferritins using a reducing reagent as described previously [18]. Biotinylated hemin was used to examine the binding of holoferritin and its apoferritin with heme as described previously [9,27].…”

“…The expressed and purified H and L subunit homopolymers contained little iron (0.4 ng/µg of protein), as previously reported [18]. The expressed bovine H subunit homopolymer showed significantly higher binding activity to biotinylated hemin than the L subunit homopolymer expressed by the same expression system (Fig.…”

“…However, it remains to be demonstrated whether or not the H subunit binds heme. An H subunit gene deletion is lethal to the early mouse embryo because of a lack of ferroxidase essential to incorporate iron [7,18], and the H subunit is an ancestral protein because the L subunit is not found in bird ferritin [4,28]. The hemebinding activity of the H subunit is evolutionally significant.…”

“…Ferritin and apoferritin preparation: Horse spleen ferritin monomers were purified from commercial horse spleen ferritin as described previously [20]. Horse spleen, bovine spleen, and canine liver ferritin monomers were purified from pieces of frozen horse spleen, bovine spleen and canine liver, respectively, as described previously [13,17,18]. Chicken liver ferritin monomers were also purified from pieces of frozen pooled livers according to the method described previously [13] with minor modifications.…”

“…Commercial horse spleen, horse spleen ferritin, bovine spleen and canine liver apoferritins were prepared by dialysis of their holoferritins with 100 mM thioglycolic acid in 100 mM acetate buffer (pH 5.5) followed by phosphate-buffered saline (PBS, 150 mM NaCl, 20 mM sodium phosphate, pH 7.2). Recombinant bovine H-and L-subunit homopolymers were obtained by expression of bovine ferritin H-and Lsubunit cDNAs using a baculovirus expression system with Spodoptera frugiperda as described previously [17,18].…”