Assembly of Apoferritin from Horse Spleen: Comparison of the Protein in its Native and Reassembled State

Abstract

The in vitro selfassembly of apoferritin after previous dissociation and unfolding in 7.2 M guanidinium chloride, pH 3.5, yields up to 80% of a protein complex exhibiting the molecular mass of the native icositetramer of .gtoreq. 450 kDa. After removal of high molecular mass byproducts, the final reassembly product proves to be indistinguisable from native apoferritin with respect to its functional and conformational properties. These refer to the intrinsic fluorescence and to the far and near UV circular dichroism. The unfolding trnasitions of the native and reassembled protein in aqueous guanidinium chloride or at acid pH coincide within the range of error. The reassembled protein is also able to catalyze the oxidation of Fe(II). Higher polymers of the apoferritin complex represent most of the residual 20% of the reconstituted protein. They are stabilized by non-covalent (preferentially hydrophobic) interactions, and may be disassembled to the icositetramer by preferrential solvation of the protein in the presence of .ltoreq. 50% (v/v) ethylene glycol. The change in fluorescence emission accompanying polymerization reflects altered surface properties of the apoferritin subunits compatible with those reported for the ferritin.fwdarw.hemosiderin transition.