Ferritin reconstitution

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)... 

The microenvironment inside the protein coat of ferritin has recently been modeled by encapsulating ferrous ion inside phosphatidylcholine vesicles and studying the oxidation of iron as the pH is raised. The efficacy of such a model is indicated by the observation of relatively stable mixtures of Fe(II)/Fe(III) inside the vesicles, as have also been observed in ferritin reconstituted experimentally from protein coats and ferrous ion. - " ... 

Table 12-1 Reductive Fe release from recombinant ferritins reconstituted to constant mineral size (480 Fe/molecule) the effect of proline substitution for the highly conserved leucine 134.

Why mammalian ferritin cores contain ferrihydrite-like structures rather than some other mineral phase is less easy to understand, and presumably reflects the way in which the biomineral is built up within the interior of the protein shell together with the geometry of the presumed nucleation sites. The phosphate content in the intracellular milieu can readily be invoked to explain the amorphous nature of the iron core of bacterioferritins and plants. Indeed, when the iron cores of bacterioferritins are reconstituted in the absence of phosphate, they are found to be more highly ordered than their native counterparts, and give electron diffraction lines typical of the ferrihydrite structure. Recently it has been reported that the 12 subunit ferritin-like Dps protein (Figure 19.6), discussed in Chapter 8, forms a ferrihydrite-like mineral core, which would seem to imply that deposition of ferric oxyhydroxides within a hollow protein cavity (albeit smaller) leads to the production of this particular mineral form (Su et al., 2005 Kauko et al., 2006). 

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

Since the (Fen05(0H)6> unit is stable, it has been speculated(8b,17b) that it might also be present in the ferritin core. Since the majority of phosphate in ferritin is adventitious, surface bound and the metallic core can be reconstituted in the absence of phosphate groups with no change in the X-ray powder diffraction pattem(l), replacement of bridging phosphate by bridging carboxylate groups should not influence the three dimensional structure of the core. Calculations show that -409 Fell nnits could fill the apoferritin inner cavity. Further details can be found in reference 17. 

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). 

Efficacy is appreciated clinically and has two distinct components. Initially there is reversal in the impaired effort tolerance that parallels regeneration of the haemoglobin levels. This is followed by a longer period when cognitive function gradually improves after stores are reconstituted. Oral iron typically needs to be given between 3 and 6 months and certainly until both percentage saturation of transferrin and serum, or preferably red cell, ferritin are normal. It is furthermore prudent that treated indi-... 

Ferritin has also been compared with iron-dextran by the EXAFS technique.1108 The apoferritin controls the deposition of the core. Reconstitution of ferritin under a range of conditions always gives the same structure, which is not the case in the absence.of apoferritin. There are metal-binding sites on the protein shell. There is evidence for the binding of iron to apoferritin, probably by carboxyl groups, but there is little detailed information on these sites.1098 On the other hand, other metal ions inhibit the formation of ferritin and may do this by binding at or close to the iron sites. Of most significance appear to be results on Tb3+, Zn2+ and V02+,... 

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 etalP were able to form crystalline cores in bacterioferritins, and Rohrer formed cores of iron-... 

Ferritin induced nanoparticle synthesis was adapted from a number of different synthetic strategies reliant upon the physical nature of ferritin. For instance, ferritin can readily exist in two stable forms (native ferritin with an intact iron oxide core or apo-ferritin lacking a mineral core) owing to the enhanced structural integrity of the protein shell. As a result, two general reaction schemes were adopted. The first route utilized the iron oxide core of native or reconstituted ferritin as a precursor to different mineral phases and types of iron nanoparticles, while the second invokes mineralization within the empty cavity of apo-ferritin. In the latter approach, the native protein must be demetallated by reductive dissolution with thioglycolic acid to yield apo-ferritin. Ultimately, apo-ferritin provides a widely applicable means to the synthesis of various nanoparticle compositions under many conditions. 

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. 

In vitro a crystalline iron core can be laid down in apoferritin by the addition of an oxidant, such as O2, to an aqueous solution of a ferrous salt and apoferritin (32, 132, 140). The reconstituted core of horse ferritin prepared in the absence of phosphate and with O2 as oxidant is very similar to the native core in terms of its size and Mossbauer properties (85). Electron microscopy, however, reveals that it is less well ordered. Reconstitution in the presence of phosphate leads to smaller cores. Reconstituted A. vinelandii cores in the absence of phosphate were more ordered than were the native cores, and clearly contained ferrihydrite particles and, in some cases, crystal domains (85). Thus the nature of the core is not determined solely by the protein coat the conditions of core formation are also important. This is also indicated by Mossbauer spectroscopy studies of P. aeruginosa cells grown under conditions different than those employed for the large-scale pu-... 

The ease with which the core of ferritin and bacfer can be reconstituted with Fe and an oxidant has led to work with Fe and other metals. Fe, added as a citrate, oxalate, or nitrilotriacetate complex, to horse holoferritin does enter the core, but only a small amount of Fe is taken up (139). No Fe + was taken up by apoferritin. This work emphasizes the requirement for core formation to occur by the oxidation of Fe +, a subject we discuss in the following section. 

Given the poor uptake of Fe + by ferritin, it is not surprising that is not taken up in large amounts. Both we (F. H. A. Kadir and G. R. Moore, unpublished observations) and Dedman et al. (36) have found that apoferritin and holoferritin will only take up at most 40 Al atoms per ferritin molecule after prolonged incubation with aluminum citrate. However, in their more extensive study, Dedman et al. found that 120 Al atoms per ferritin molecule were bound to the iron core when the exposure to aluminum citrate occurred during its reconstitution. This is comparable to the 164 aluminum atoms bound to holoferritin reported by Sczekan and Joshi (122). 

In contrast to ferritin, very little work has been done on the reconstitution of BFR cores, other than the experiments mentioned above that showed that, in the absence of phosphate, crystalline ferrihydrite formed inside the protein shell. The intermediate stages in this process are unknown, but the sigmoid iron uptake behavior (25) suggests there could be a similar succession of events oxidation and nucleation on the protein shell followed by direct oxidation on the core. The influence of the heme, if any, on BFR iron core formation also awaits investigation. As mentioned above, the presence of the iron core influences the heme redox potential, but it is not known whether the presence of heme influences the redox potential of the nonheme iron. 

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... 

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