Ferritin reconstituted horse spleen

Fig. 14. Magnetization curves of reconstituted horse spleen ferritin. The solid lines are fits to expression (14). Reproduced with permission from Ref (35).

When pea seed apoferritin is reconstituted in vitro in the absence of phosphate, the reconstituted mineral core consists of crystalline ferrihydrite (Rohrer et ah,1990 Wade et ah, 1993 Waldo et ah, 1995). Conversely, horse spleen ferritin reconstituted in the presence of phosphate produces an amorphous core (Rohrer et ah,1990 St. Pierre et ah, 1996)... 

Formation of ferritin involves assemblage of the protein subunits to form the apo-ferritin shell which is then filled with the phosphated ferrihydrite core. The mechanism by which ferritin is filled and the iron core built up, has been investigated intensively in vitro. The experiments usually involved incubating apoferritin (from horse spleen) with Fe salts in the presence of an oxidant such as molecular oxygen. They showed that ferritin could be reconstituted from apoferritin and a source of Fe both the iron and the oxygen enter the protein shell, whereupon oxidation of Fe is catalysed by the interior surface of the protein shell (Macara et al., 1972). 

The storage and mobilization of surplus iron in eukaryotes and some prokaryotes are regulated by the iron storage protein, ferritin. Ferritin isolated from horse spleen consists of a hollow spherical shell of 24 symmetrically related protein subunits (—18 kDa per subunit) surrounding a core of inorganic hydrated iron(III) oxide 45). Phosphate may be associated with the surface of the iron oxide core in horse ferritin, but does not appear to be a critical factor for core formation in reconstituted ferritins (46). The diameter of the cavity set by the protein shell is of the order of 70-80 A, resulting in an upper limit of 4500 iron atoms (-30% wt/wt Fe) that can be stored within the molecule. 

Reconstitution experiments with apoferritins from animal and bacterial sources, whose native iron-loaded ferritins had crystalline and amorphous cores respectively, have been informative in showing that the core morphology is not determined by the protein shell. For example, Baaghil et al and Mann et al were able to form crystalline cores in bacterioferritins, and Rohrer et al formed cores of iron-phosphate in horse spleen ferritin with fewer short range Fe-Fe distances than in the native cores. Thus it appears that the composition of the medium within which the ferritin cores are laid down, particularly the phosphate content, influences strongly the nature of the core. This is consistent with the role of phosphate as an iron oxide chain terminator. 

In combination with the rapid freeze-quench methodology, EPR spectroscopy has been instrumental in the detection of reaction intermediates during the reconstitution of horse spleen ferritin from apoferritin, Fe , and O2 [486]. Within the first second after mixing of the reactants, a monomeric Fe -protein complex with a characteristic resonance at geff = 4.3, a mixed-valent Fe -Fe species with a peak 1-87 (which accumulates quantitatively from the monomeric Fe " species), and a radical species (gy = 2.042, = 2.0033) that may be associated with either... 

---