Chemical modification as a probe of the topography and reactivity of horse-spleen apoferritin.

In apoferritin, but not in ferritin, 1.0 +/- 0.1 cysteine residue per subunit can be modified. In ferritin 3.3 +/- 0.3 lysine residues and 7.1 +/- 0.7 carboxyl groups per subunit can be modified, whilst the corresponding values for apoferritin are 4.4 +/- 0.4 lysine residues and 11.0 +/- 0.4 carboxyl groups per subunit. Modification of lysine residues which maleic anhydride and carboxyl groups with glycineamide in apoferritin which has been dissociated and denatured in guanidine hydrochloride leads to the introduction of 9.1 +/- 0.5 maleyl groups per subunit and 22.0 +/- 0.9 glycineamide residues per subunit. Whereas unmodified apoferritin subunit can be reassociated from guanidine hydrochloride to apoferritin monomer, the ability of maleylated apoferritin to reassociate is impaired. Apoferritin in which all the carboxyl groups have been blocked with glycineamide cannot be reassociated to apoferritin and exists in solution as stable subunits. The modification of one cysteine residue per subunit, of 3 or 4 lysine residues per subunit or of 7 carboxyl groups per subunit has no effect on the catalytic activity of apoferritin. In contrast the modification of 11 carboxyl groups per subunit completely abolishes the catalytic properties of the protein. We conclude that one or more carboxyl groups are essential for the catalytic activity of horse spleen apoferritin.     

The incorporation of iron into chicken apoferritin in the presence of ceruloplasmin

After assaying the appropriate conditions for the experiments, the oxidation of iron with incorporation into chicken apoferritin was studied in the presence of ceruloplasmin, analysing the roles of iron, apoferritin and ceruloplasmin. The results show that the process is hastened by both apoferritin and ceruloplasmin. The dependence of the rate with respect to iron, apoferritin and ceruloplasmin concentrations was in general linear in the studied range. However, for low concentrations of iron or apoferritin the behaviour deviated from the linearity, suggesting that significant changes can happen in the mechanism of iron incorporation into apoferritin when the ratio of iron to apoferritin varies, which is in accordance with previous works. Finally, some differences found in the influence of the species on the process, with respect to an earlier report, open the possibility of differences in the affinity for iron between avian and mammalian apoferritins.     

Fabrication of nickel and chromium nanoparticles using the protein cage of apoferritin

The iron storage protein, apoferritin, has a cavity in which iron is oxidized and stored as a hydrated oxide core. The size of the core is about 7 nm in diameter and is regulated by the cavity size. The cavity can be utilized as a nanoreactor to grow inorganic crystals. We incubated apoferritin in nickel or chromium salt solutions to fabricate hydroxide nanoparticles in the cavity. By using a solution containing dissolved carbon dioxide and by precisely controlling the pH, we succeeded in fabricating nickel and chromium cores. During the hydroxylation process of nickel ions a large portion of the apoferritin precipitated through bulk precipitation of nickel hydroxide. Bulk precipitation was suppressed by adding ammonium ions. However, even in the presence of ammonium ions the core did not form using a degassed solution. We concluded that carbonate ions were indispensable for core formation and that the ammonium ions prevented precipitation in the bulk solution. The optimized condition for nickel core formation was 0.3 mg/mL horse spleen apoferritin and 5 mM ammonium nickel sulfate in water containing dissolved carbon dioxide. The pH was maintained at 8.65 using two buffer solutions: 150 mM HEPES (pH 7.5) and 195 mM CAPSO (pH 9.5) with 20 mM ammonium at 23 degrees C. The pH had not changed after 48 h. After 24 h of incubation, all apoferritins remained in the supernatant and all of them had cores. Recombinant L-ferritin showed less precipitation even above a pH of 8.65. A chromium core was formed under the following conditions: 0.1 mg/mL apoferritin, 1 mM ammonium chromium sulfate, 100 mM HEPES (pH 7.5) with a solution containing dissolved carbon dioxide. About 80% of the supernatant apoferritin (0.07 mg/mL) formed a core. In nickel and chromium core formation, carbonate ions would play an important role in accelerating the hydroxylation in the apoferritin cavity compared to the bulk solution outside.     

The incorporation of iron into apoferritin as mediated by ceruloplasmin

Ceruloplasmin, a copper ferroxidase, promotes the incorporation of Fe(III) into the iron storage protein, apoferritin. The product formed is identical to ferritin as judged by polyacrylamide electrophoresis and iron/protein measurements. Of several proteins examined, only apoferritin accumulates the Fe(III) produced by ceruloplasmin. When ceruloplasmin was replaced by tyrosinase, which we have shown to have ferroxidase activity, no iron incorporation into apoferritin was observed. It is proposed that Fe(III) is transferred directly and specifically to apoferritin. These data support a more specific role for ceruloplasmin in iron metabolism than has previously been proposed.     

The consequences of hydroxyl radical formation on the stoichiometry and kinetics of ferrous iron oxidation by human apoferritin

Despite previous detection of hydroxyl radical formation during iron deposition into ferritin, no reports exist in the literature concerning how it might affect ferritin function. In the present study, hydroxyl radical formation during Fe(II) oxidation by apoferritin was found to be contingent on the "ferroxidase" activity (i.e., H subunit composition) exhibited by apoferritin. Hydroxyl radical formation was found to affect both the stoichiometry and kinetics of Fe(II) oxidation by apoferritin. The stoichiometry of Fe(II) oxidation by apoferritin in an unbuffered solution of 50 mM NaCl, pH 7.0, was approximately 3.1 Fe(II)/O(2) at all iron-to-protein ratios tested. The addition of HEPES as an alternate reactant for the hydroxyl radical resulted in a stoichiometry of about 2 Fe(II)/O(2) at all iron-to-protein ratios. HEPES functioned to protect apoferritin from oxidative modification, for its omission from reaction mixtures containing Fe(II) and apoferritin resulted in alterations to the ferritin consistent with oxidative damage. The kinetic parameters for the reaction of recombinant human H apoferritin with Fe(II) in HEPES buffer (100 mM) were: K(m) = 60 microM, k(cat) = 10 s(-1), and k(cat)/K(m) = 1.7 x 10(5) M(-1) x (-1). Collectively, these results contradict the "crystal growth model" for iron deposition into ferritin and, while our data would seem to imply that the ferroxidase activity of ferritin is adequate in facilitating Fe(II) oxidation at all stages of iron deposition into ferritin, it is important to note that these data were obtained in vitro using nonphysiologic conditions. The possibility that these findings may have physiological significance is discussed.     

Apolipoprotein B binds ferritin by hemin-mediated binding: evidence of direct binding of apolipoprotein B and ferritin to hemin

Apolipoprotein B (apoB) is known to be a ferritin-binding protein. Here we show that apoB binds to ferritin through hemin-mediated binding. Human apoB bound to bovine spleen, horse spleen, and canine liver ferritins, but did not bind to bovine apoferritin, even after incorporation of iron into it. Incubation of apoferritin with hemin resulted in apoB binding with apoferritin at the same level as with holoferritin. In contrast, hemin inhibited binding of apoB to ferritin. Bovine spleen apoferritin bound biotinylated hemin, and hemin inhibited the binding between the apoferritin and biotinylated hemin, suggesting that ferritin binds hemin directly. ApoB and LDL containing apoB bound biotinylated hemin, and their bindings were also inhibited by hemin, but not protoporphyrin IX. These data demonstrate that binding of apoB to ferritin is mediated through ferritin’s binding to hemin, and also that apoB binds hemin directly.     

Primary structure of rat liver apoferritin. The amino end

Rat liver apoferritin is known to have a blocked amino end. From a pronase digest of rat liver apoferritin we have isolated and purified by ion-exchange chromatography the blocked N-terminal tripeptide. Its sequence and the nature of the blocking group were shown to be Ser-Ser-Gln and an acetyl moiety, respectively. The N-terminal sequence of rat liver apoferritin is thus N-acetyl-Ser-Ser-Gln, which coincides with the N-terminal sequence of horse-spleen apoferritin, the only other apoferritin studied structurally at present.     

In vitro loading of apoferritin.

This study compared the effect of loading apoferritin either with ferrous ammonium sulfate in various buffers or with ceruloplasmin and chelated ferrous iron. It was shown that loading of apoferritin with ferrous ammonium sulfate was dependent on buffer and pH, and was directly related to the rate of iron autoxidation. The ceruloplasmin-dependent loading of apoferritin, however, was unaffected by these factors. Isoelectric focusing and amino acid analysis of the differently loaded ferritins showed that ferrous ammonium sulfate loading of apoferritin resulted in the depletion of the basic amino acids, lysine and histidine, probably as a result of protein oxidation. No significant differences in amino acid composition was noted for ceruloplasmin-loaded ferritin. Furthermore, ferritin loaded with ferrous ammonium sulfate released more iron than either native or ceruloplasmin-loaded ferritin when either paraquat or EDTA was used as an iron mobilizing agent. We suggest that the loading of apoferritin with ferrous ammonium sulfate occurred as a result of iron autoxidation and may result in oxidation of amino acids and loss of integrity of the protein, and that ceruloplasmin may act as a catalyst for the incorporation of iron into apoferritin in a manner more closely related to that occurring in vivo.     

Quaternary structure of apoferritin: the rotation function at 9 A resolution

The rotation function has been calculated for apoferritin using data at 9 Å resolution obtained from cubic crystals, space group F432, and compared with rotation functions of possible alternative model structures consisting of (a) 24 subunits at the vertices of a snub-cube (octahedral symmetry) and (b) 20 subunits at the vertices of a pentagonal dodecahedron (icosahedral symmetry). The apoferritin rotation function, like that of the 24 subunit model, had large peaks only on the crystallographic rotation axes. The 20 subunit model gave peaks on non-crystallographic axes, which were not observed with apoferritin. It is concluded that apoferritin molecules consist of 24 subunits arranged in 432 (octahedral) symmetry as suggested by the space group.     

pH-dependent structures of ferritin and apoferritin in solution: disassembly and reassembly

The pH-dependent structures of the ferritin shell (apoferritin, 24-mer) and the ferrihydrite core, under physiological conditions that permit enzymatic activity, were investigated by synchrotron small-angle X-ray scattering (SAXS). The solution structure of apoferritin was found to be nearly identical to the crystal structure. The shell thickness and hollow core volumes were estimated. The intact hollow spherical apoferritin was stable over a wide pH range, 3.40-10.0, and the ferrihydrite core was stable over the pH range 2.10-10.0. The apoferritin subunits underwent aggregation below pH 0.80, whereas the ferrihydrite cores aggregated below pH 2.10 as a result of the disassembly of the ferritin shell under the strongly acidic conditions. As the pH decreased from 3.40 to 0.80, apoferritin underwent stepwise disassembly by first forming a hollow sphere with two holes, then a headset-shaped structure, and, finally, rodlike oligomers. As the pH was increased from pH 1.96, the disassembled rodlike oligomers recovered only to the headset-shaped structure, and the disassembled headset-shaped intermediates recovered only to the hollow spherical structure with two hole defects. The apoferritin hole defects that formed during the disassembly process did not heal as the pH was increased to neutral or slightly basic conditions. The pH-induced apoferritin disassembly and reassembly processes were not fully reversible, although they were pseudoreversible over a limited pH range, between 10.0 and 2.66.     

Selective adsorption of apoferritin on immobilized Fe(III): demonstration of Fe(III) binding sites

Immobilized metal ion affinity chromatography has been used to demonstrate and partially characterize Fe(III) binding sites on apoferritin. Binding of Fe(III) to these sites is influenced by pH, but not affected by high ionic strength. These results suggest that both ionic and coordinate covalent interactions are important in the formation of the Fe(III): apoferritin complex. This is, to our knowledge, the first demonstration of direct Fe(III) binding to apoferritin. Other immobilized metal ions, including Zn(II), Ni(II), Cu(II), Cr(III), Co(II), and Tb(III), displayed little or no adsorption of apoferritin. The analytical technique of immobilized metal ion affinity chromatography also shows great promise in the purification of apoferritin, ferritin, and other iron-binding proteins.     

The formation of ferritin from apoferritin. Catalytic action of apoferritin

The iron-storage protein ferritin consists of a protein shell and has an iron content of up to 4500 iron atoms as a microcrystalline ferric oxide hydrate. A study was made of the uptake of ferrous iron by apoferritin in the presence of an oxidizing agent at very low iron:protein ratios. At ratios of less than about 150 iron atoms per apoferritin molecule hyperbolic progress curves were obtained, whereas at higher ratios the curves became sigmoidal under the conditions used. A computer model, developed previously (Macara et al., 1972), was shown to account for this result. The experimental evidence indicates that apoferritin binds ferrous iron and catalyses the initial stage in the formation of the ferric oxide hydrate inside the protein shell. This stage involves the oxidation of sufficient iron within the protein molecule to form a stable nucleus on which the growth of the microcrystalline iron-core particles can proceed. A possible schematic mechanism for the action of apoferritin is suggested.     

The isolation and properties of porcine ferritin and apoferritin.

Porcine ferritin and apoferritin were purified to a greater degree of homogeneity than has been reported previously. Porcine ferritin was insoluble in the absence of a reducing agent, possessed a high content of iron, with an average $\text{Fe}\text{N}$ ratio of ~2.5, and contained almost no detectable endogenous apoferritin. The amino acid composition, ultraviolet-absorption spectrum, and ultraviolet-circular dichroism spectrum of porcine apoferritin are very similar to the respective parameters of equine apoferritin. The native and subunit molecular weights of porcine apoferritin are 503,000 and 20,000, respectively.     

Hydrogen ion interactions of horse spleen ferritin and apoferritin.

The interactions of horse spleen ferritin and its derivative apoferritin with H+ ions were studied by potentiometric and spectrophotometric titration; to aid in data analysis, heats of ionization over a limited pH range and amide content were also determined. Per apoferritin subunit, all tyrosine and cysteine side chains, two of the nine lysine side chains and at least three of the six histidine side chains were found not to titrate; a preliminary but self-consistent analysis of the titration data is proposed. The titration curve of ferritin was identical with that of apoferritin in the pH range 5.5 to 3. In addition, under the conditions used, the reactivities of ferritin histidines to bromoacetate and of ferritin lysines to formaldehyde were identical with those in apoferritin. Above pH 8, a time-dependent titration of the ferritin core occurs which prevents comparison of the titration curves of the two proteins in this region. However, in the pH regions 5.5 to 7.5, two extra groups per subunit titrate reversibly in ferritin relative to apoferritin. Moreover, although the isoionic points of ferritin and apoferritin are identical in water, the isoionic point of ferritin is 0.5 pH unit lower than that of apoferritin in 0.16 to 1 M KCl. The different effects of KCl and NaCl on the two proteins indicate the presence of cation binding sites in ferritin that are absent in apoferritin and possibly also the presence of anion binding sites in apoferritin that are occupied in ferritin by anions of the core. The difference between the isoionic points of the two proteins in KCl has been interpreted to indicate the presence of approximately 2 phosphate residues per ferritin subunit which serve as cation binding sites and which are negatively charged at the isoionic point in KCl. These phosphates may also represent the additional residues that titrate in ferritin between pH 5.5 and 7.5, or may interact with positively charged residues on the inner surface of the ferritin shell, or both.     

Characterization of horse spleen apoferritin reactive lysines by MALDI-TOF mass spectrometry combined with enzymatic digestion

Ferritins are a class of iron storage protein spheres found mainly in the liver and spleen, which have attracted many research interests due to their unique structural features and biological properties. Recently, ferritin and apoferritin (ferritin devoid of the iron core), have been employed as chemically addressable nanoscale building blocks for functional materials development. However, the reactive residues of apoferritin or ferritin have never been specified and it is still unclear about the chemoselectivity of apoferritin towards different kinds of bioconjugation reagents. In this work, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry combined with enzymatic digestion analysis was used to identify the reactive lysine residues of horse spleen apoferritin when conjugated with N-hydroxysuccinimide reagents. The result demonstrated that among all the lysine residues, K97, K83, K104, K67 and K143 are the reactive ones that can be addressed.     

The catalytic activity of horse spleen apoferritin. Preliminary kinetic studies and the effect of chemical modification

1. Horse spleen apoferritin catalyses the oxidation of Fe(2+) to Fe(3+) with molecular O(2) as electron acceptor under conditions where a number of other proteins have no such effect. The product is similar to ferritin by a number of criteria. 2. The progress curve is hyperbolic and the increase in initial velocity is linear with increasing apoferritin concentration. With respect to Fe(2+) the reaction follows Michaelis-Menten kinetics. The pH-dependence of the reaction was determined between pH4.3 and 6.0. 3. Modification of both tryptophan residues/apoferritin subunit with 2-nitrophenylsulphenyl chloride does not affect either k(cat.) or K(m) for the oxidation. Neither does the guanidination of seven out of nine lysine residues/subunit, the modification of nine out of ten arginine residues/subunit with cyclohexanedione, or the nitration of one out of five tyrosine residues/subunit with tetranitromethane. 4. The carboxymethylation of two out of three cysteine residues/subunit and of one out of six histidine residues/subunit can be achieved with iodoacetic acid. This carboxymethylated apoferritin is completely inactive in Fe(2+) oxidation. 5. Apoferritin does not take up Fe(3+). It appears from these results that Fe(2+) is the form in which iron is taken up by ferritin in a reaction where the protein acts as an enzyme which traps the product in the interior of the protein shell.     

Carbohydrate composition of horse spleen ferritin.

The carbohydrate composition of horse spleen ferritin was studied. 1 mol of the apoferritin, the protein moiety of ferritin, contains 25 mol of hexose, 3 mol of hexosamine and 10 mol of fucose. Same carbohydrate composition was detected in the apoferritin from iron rich ferritins. These results indicate that horse spleen ferritin is composed of non-identical subunits as regards its carbohydrate composition.     

Recognition of anesthetic barbiturates by a protein binding site: a high resolution structural analysis

Barbiturates potentiate GABA actions at the GABA_A receptor and act as central nervous system depressants that can induce effects ranging from sedation to general anesthesia. No structural information has been available about how barbiturates are recognized by their protein targets. For this reason, we tested whether these drugs were able to bind specifically to horse spleen apoferritin, a model protein that has previously been shown to bind many anesthetic agents with affinities that are closely correlated with anesthetic potency. Thiopental, pentobarbital, and phenobarbital were all found to bind to apoferritin with affinities ranging from 10–500 µM, approximately matching the concentrations required to produce anesthetic and GABAergic responses. X-ray crystal structures were determined for the complexes of apoferritin with thiopental and pentobarbital at resolutions of 1.9 and 2.0 Å, respectively. These structures reveal that the barbiturates bind to a cavity in the apoferritin shell that also binds haloalkanes, halogenated ethers, and propofol. Unlike these other general anesthetics, however, which rely entirely upon van der Waals interactions and the hydrophobic effect for recognition, the barbiturates are recognized in the apoferritin site using a mixture of both polar and nonpolar interactions. These results suggest that any protein binding site that is able to recognize and respond to the chemically and structurally diverse set of compounds used as general anesthetics is likely to include a versatile mixture of both polar and hydrophobic elements.     

Uptake of iron by apoferritin from a ferric dihydrolipoate complex

A study was made on the uptake of iron by horse spleen apoferritin, by using as an iron source the same ferric dihydrolipoate complex which represents the major product in the anaerobic removal of ferritin-bound iron by dihydrolipoate at neutral pH. The ferric dihydrolipoate complex was chemically synthesized and used as an iron donor to apoferritin. Iron uptake was studied, at slightly alkaline pH and in anaerobic conditions, as a function of the concentration of both the iron donor and apoferritin. Isolation of ferritin from mixtures of ferric dihydrolipoate and apoferritin, and subsequent identification of the oxidation state of ferritin-bound iron, showed that the first metal atoms were taken up in the ferrous form and that this early step was accompanied by accumulation of ferric iron. Total iron uptake increased with the molar ratio of complex to apoprotein and ranged over 25-40% of the iron being supplied. The amount of ferrous iron found inside the protein did not exceed 50-60 mol iron/mol ferritin after a 48-h incubation. At this time, ferric iron represented a significant fraction of the iron found in the isolated ferritin. Analytical and spectroscopic data indicated that fractional rates and equilibria for disassembly of the ferric complex in the presence of apoferritin were independent of the concentration of the protein and of the complex itself.     

Mass spectrometry studies of demetallation of haemin by recombinant horse L chain apoferritin and its mutant (E 53,56,57,60 Q)

An essential difference between eukaryotic ferritins and bacterioferritins is that the latter contain naturally, in vivo haem as Fe-protoporphyrin IX. This haem is located in a hydrophobic pocket along the 2-fold symmetry axes and is liganded by two Met 52. However, in in vivo studies, a cofactor has been isolated in horse spleen apoferritin similar to protoporphyrin IX; in in vitro experiments, it has been shown that horse spleen apoferritin is able to interact with haem. Studies of haemin (Fe(III)-PPIX) incorporation into horse spleen apoferritin have been carried out, which show that the metal free porphyrin is found in a corresponding pocket to haem in bacterioferritins [Précigoux, G., Yariv, J., Gallois, B., Dautant, A., Courseille, C. and Langlois, d'Estaintot B. (1994) A crystallographic study of haem binding to ferritin. Acta Cryst. D 50, 739-743]. A mechanism of demetallation of haemin by L-chain apoferritin was proposed [Crichton, R.R., Soruco, J.A., Roland, F., Michaux, M.A., Gallois, B., Précigoux, G., Mahy, J.P. and Mansuy. (1997) Remarkable ability of horse spleen apoferritin to demetallate hemin and to metallate protoporphyrin IX as a function of pH. J. P. Biochem. 36, 49, 15049-15054]: this involved four Glu residues (53,56,57,60) situated at the entrance of the hydrophobic pocket and appeared to be favoured by acidic conditions. To verify this mechanism, we have mutated these four Glu to Gln and examined demetallation in both acidic and basic conditions. In this paper, we report the mass spectrometry studies of L-chain apoferritin and its mutant incubated with haemin and analysed after different times of incubation: 15 days, 2 months, 6 months, 9 months and 12 months. These studies show that the recombinant L-chain apoferritin and its mutant are able to demetallate haemin to give a hydroxyethyl protoporphyrin IX derivative in a dimeric form [Macieira, S., Martins, B. M. and Huber, R. (2003) Oxygen-dependent coproporphyrinogen IX oxidase from Escherichia coli: one-step purification and biochemical characterization. FEMS. Microbiology Letters 226, 31-37].