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PubMed1991

Initial iron oxidation in horse spleen apoferritin. Characterization of a mixed-valence iron(II)-iron(III) complex.

In ferritin, iron is stored by oxidative deposition of the ferrous ion to form a hydrous ferric oxide mineral core. Two intermediates, formed during the initial stages of iron accumulation in apoferritin, have been observed previously in our laboratory and have been identified as a mononuclear Fe3(+)-protein complex and a mixed-valence Fe2(+)-Fe3(+)-protein complex. The physical characteristics of the mixed-valence Fe2(+)-Fe3+ complex and its relationship to the mononuclear Fe3+ complex in horse spleen apoferritin samples to which 0-240 iron atoms were added was examined by EPR spectroscopy. The results indicate that the mononuclear complex is not a precursor to the formation of the mixed-valence complex. Competitive binding studies with Cd2+, Zn2+, Tb3+, and UO2+(2) suggest that the mixed-valence complex is formed on the interior of the protein in the vicinity of the 2-fold axis of the subunit dimer. The mixed-valence complex could be generated by the partial oxidation of Fe2+ in apoferritin containing 120 Fe2+ or by the addition of up to 120 Fe2+ to ferritin already containing 18 Fe3+/protein molecule. The fact that the complex is generated during early Fe2+ oxidation suggests that it may be a key intermediate during the initial oxidative deposition of iron in the protein. The unusual EPR powder lineshape at 9.3 GHz of the mixed-valence complex was simulated with a rhombic g-tensor (gx = 1.95, gy = 1.88, gz = 1.77) and large linewidths and g-strain parameters. The presence of significant g-strain in the complex probably accounts for the failure to observe an EPR signal at 35 GHz and likely reflect considerable flexibility in the structure of the metal site. The temperature dependence of the EPR intensity in the range 8-38 K was modeled successfully by an effective spin Hamiltonian including exchange coupling (-2JS1.S2) and zero-field terms, from which an antiferromagnetic coupling of J = -4.0 +/- 0.5 cm-1 was obtained. This low value for J may reflect the presence of a mu-oxo bridge(s) in the dimer.

Animals↗
PubMed1987

Time-resolved fluorescence of apoferritin and its subunits.

The decay of the intrinsic fluorescence of the apoferritin polymer and its subunits has been studied by pulse and phase shift techniques. Both techniques show that the fluorescence decay of all the samples tested cannot be described by a single exponential function. The fluorescence decay data of the apoferritin subunits obtained with either technique can be fitted satisfactorily with a function resulting from the sum of two exponential components. However, the polymer data obtained with the high resolution phase shift technique operated either by synchrotron radiation or by a mode-locked argon ion laser can be fitted better using a bimodal gaussian continuous distribution of lifetime components. The molecular basis for this distribution of lifetime values may lie in the heterogeneity of the tryptophan environment generated by the assembly of the subunits into the polymer. The binding of the first 100 irons to apoferritin quenches the intrinsic fluorescence without affecting the lifetimes in a proportional way. This finding may be taken as an indication that the quenching of the tryptophan fluorescence induced by the binding of iron has both static and dynamic components.

Apoferritins↗
PubMed1983

Subunit dimers in sheep spleen apoferritin. The effect on iron storage.

Ferritin with high and low iron content, 2000 and 790 iron atoms/molecule, was isolated from the spleens of copper-poisoned and control lambs, respectively. Differences in the iron content in vivo were reflected in the properties of the apoferritin protein shells, since the apoprotein from the low iron ferritin took up iron relatively more slowly (0.52 +/- 0.09) and released it more rapidly (1.68 +/- 0.06) in vitro. Although the two types of apoferritin were indistinguishable in terms of surface charge (pI range 4.98-5.43) and in consisting of both heavy and light subunits, the subunit interactions differed markedly; 40-50% of the subunits of low iron ferritin were in dimers stable to reduction and carboxylmethylation, 4% mercaptoethanol, 8% sodium dodecyl sulfate, and 100 degrees C for 30 min, 70% formic acid, and 30% methanol. Subunit dimers were also observed in liver ferritin from mouse and neonatal pig and were enriched in a low iron fraction of horse spleen ferritin. Based on cyanogen bromide fragmentation and NH2-terminal analysis, the natural and chemically cross-linked subunit dimers had two peptides in common; natural subunit dimers also appeared to have a second region cross-linked, suggesting the possibility of both intra- and intersubunit links in the natural dimers. In sheep spleen ferritin, both heavy and light subunits appeared to participate in subunit dimerization. Natural subunit dimers were enriched in low iron ferritin fractions of all ferritin preparations tested (linear correlation = 0.94) and can explain, at least in part, the previously observed effects of iron core size on the apoferritin shell. Whether the subunit cross-links represent part of the subunit assembly process subsequently cleaved by iron (or copper) or whether the cross-links form after iron core formation in vivo has yet to determined. In either case, it is clear that such post-translational variations can affect iron uptake and release and emphasize the importance of the protein shell in determining the iron storage properties of ferritin.

Amino Acids↗
PubMed1980

Assembly of intra- and interspecies hybrid apoferritins.

An intraspecies hybrid apoferritin was assembled by mixing subunits of horse heart ferritin, which consists mainly of H-type subunits, and horse spleen ferritin, in which L-type subunits predominate. Interspecies hybrid apoferritins were reconstituted from subunits of human liver-horse spleen ferritins and from rat liver-horse spleen ferritins. All the hybrid ferritins migrated as single zones with electrophoretic mobilities intermediate between those of the parent ferritins. Isoelectric focusing data and immunological patterns were consistent with the view that the reassembled apoferritins were composite molecules that contained subunits from each of the interacting forms. Reconstitution occurred in a random manner, as there was no apparent preference for assembly of homologous subunits. These results suggest that intersubunit interaction domains and recognition mechanisms that dictate formation of the highly specific quaternary structure assumed by this protein are common for different species of ferritins.

Animals↗
PubMed1993

[Monoclonal antibodies to human spleen ferritin. I. Preparation and study of the interaction with isoferritin and apoferritin].

Three monoclonal antibodies to human spleen ferritin were produced and their interaction with soluble and immobilized ferritins studied. An immunoassay was developed to monitor the interaction of soluble biotinylated ferritin and the antibody with subsequent separation of the soluble complexes on streptavidin-cellulose. Analysis of immunoreactivities of a series of isoferritins (human liver, spleen, heart and equine spleen ferritins) revealed that all the three monoclonal antibodies bound to only human L-type ferritins (spleen and liver ferritins), suggesting a high species- and tissue specificity of these antibodies. The monoclonal antibodies were specifically directed against conformation-dependent antigenic determinants as could be evidenced from the lack of their binding to the subunits of the dissociated ferritin. The affinity of the monoclonal antibody for the ferritin deprived of iron (apoferritin) was higher than that for native ferritin due to the greater conformational flexibility of the apoferritin molecule. The latter property may underly a complete loss of immunoreactivity by the apoprotein adsorbed on the polystyrene surface as a result of conformational changes induced in the apoferritin molecule by adsorption. These findings provide additional support for recognition by all of the three antibodies of conformational antigenic epitopes in the ferritin molecule.

Antibodies, Monoclonal↗
PubMed2001

A Model for Hydration Interactions between Apoferritin Molecules in Solution.

We have studied how non-DLVO forces between molecules of the globular protein apoferritin in solution affect its osmotic second virial coefficient. A model explaining the effects of the solution ionic strength and pH on the interprotein interaction is developed, to give a physical interpretation of recently published experimental findings showing that the second virial coefficient of the protein apoferritin, supported by acetate buffer, goes through a minimum as a function of ionic strength. At low ionic strengths, the apoferritin second virial coefficient initially decreases with increasing sodium ion concentration, as DLVO theory predicts. However, non-DLVO hydration forces due to overlapping of the Stern layers of the protein molecules increase the second virial coefficient with further increase of sodium ion concentration, again as found experimentally at higher ionic strengths. The non-DLVO effect arises from ionic exchange between hydrogen and sodium ions at the protein surface. An adsorption shell of hydrated sodium ions forms around the protein molecules with increasing buffer concentration. Copyright 2001 Academic Press.

Journal Article↗
PubMed1972

The formation of ferritin from apoferritin. Kinetics and mechanism of iron uptake.

Ferritin has a high capacity as an iron store, incorporating some 4500 iron atoms as a microcrystalline ferric oxide hydrate. Starting from apoferritin, or ferritin of low iron content, Fe(2+) and an oxidizing agent, the uptake of iron can be recorded spectrophotometrically. Progress curves were obtained and the reconstituted ferritin was shown by several physical methods to be similar to natural ferritin. The progress curves of iron uptake by apoferritin are sigmoidal; those for ferritins of low iron content are hyperbolic. The rate of iron uptake is dependent on the amount of iron already present in the molecule. The distribution of iron contents among reconstituted ferritin molecules is inhomogeneous. These findings are interpreted in terms of a crystal growth model. The surface area of the crystallites forming inside the protein increases until the molecule is half full, and then declines. This surface controls the rate at which new material is deposited. The experimental results can best be accounted for by a two-stage mechanism, an initial slow ;nucleation' stage, which is apparently zero order with respect to [Fe(2+)], followed by a more rapid ;growth' stage. The rate of Fe(2+) oxidation is increased in the presence of apoferritin as compared with controls. Ferritin can therefore be regarded as an enzyme to which the product remains firmly attached. The protein appears to increase the rate of ;nucleation'. The apparent zero order of this stage suggests the presence of binding sites on the protein, which are saturated with respect to Fe(2+). These sites are presumed also to be oxidation sites. The oxidation and subsequent formation of the ferric oxide hydrate may proceed according to one of three alternative models.

Apoproteins↗
PubMed1973

Subunit interactions in horse spleen apoferritin. Dissociation by extremes of pH.

1. The dissociation of horse spleen apoferritin as a function of pH was analysed by sedimentation-velocity techniques. The oligomer is stable in the range pH2.8-10.6. Between pH2.8 and 1.6 and 10.6 and 13.0 both oligomer and subunits can be detected. At pH values between 1.6 and 1.0 the subunit is the only species observed, although below pH1.0 aggregation of the subunits to a particle sedimenting much faster than the oligomer occurs. 2. When apoferritin is first dissociated into subunits at low pH values and then dialysed into buffers of pH1.5-5.0, the subunit reassociates to oligomer in the pH range 3.1-4.3. 3. U.v.-difference spectroscopy was used to study conformational changes occurring during the dissociation process. The difference spectrum in acid can be accounted for by the transfer of four to five tyrosine residues/subunit from the interior of the protein into the solvent. This process is reversed on reassociation, but shows the same hysteresis as found by sedimentation techniques. The difference spectrum in alkali is more complex, but is consistent with the deprotonation of tyrosine residues, which appear to have rather high pK values. 4. In addition to the involvement of tyrosine residues in the conformational change at low pH values, spectral evidence is presented that one tryptophan residue/subunit also changes its environment before dissociation and subsequent to reassociation. 5. Analysis of the dissociation and reassociation of apoferritin at low pH values suggests that this is a co-operative process involving protonation and deprotonation of at least two carboxyl functions of rather low intrinsic pK. The dissociation at alkaline pH values does not appear to be co-operative. 6. Of the five tyrosine residues/subunit only one can be nitrated with tetranitromethane. Guanidination of lysine residues results in the modification of seven out of a total of nine residues/subunit. Nine out of the ten arginine residues/subunit react with cyclohexanedione.

Amino Acids↗
PubMed2000

Molecular-level thermodynamic and kinetic parameters for the self-assembly of apoferritin molecules into crystals.

The self-assembly of apoferritin molecules into crystals is a suitable model for protein crystallization and aggregation; these processes underlie several biological and biomedical phenomena, as well as for protein and virus self-assembly. We use the atomic force microscope in situ, during the crystallization of apoferritin to visualize and quantify at the molecular level the processes responsible for crystal growth. To evaluate the governing thermodynamic parameters, we image the configuration of the incorporation sites, "kinks", on the surface of a growing crystal. We show that the kinks are due to thermal fluctuations of the molecules at the crystal-solution interface. This allows evaluation of the free energy of the intermolecular bond phi=3.0 k(B)T=7.3 kJ/mol. The crystallization free energy, extracted from the protein solubility, is -42 kJ/mol. Published determinations of the second virial coefficient and the protein solubility between 0 and 40 degrees C revealed that the enthalpy of crystallization is close to zero. Analyses based on these three values suggest that the main component in the crystallization driving force is the entropy gain of the water molecules bound to the protein molecules in solution and released upon crystallization. Furthermore, monitoring the incorporation of individual molecules in to the kinks, we determine the characteristic frequency of attachment of individual molecules at one set of conditions. This allows a correlation between the mesoscopic kinetic coefficient for growth and the molecular-level thermodynamic and kinetic parameters determined here. We found that step growth velocity, scaled by the molecular size, equals the product of the kink density and attachment frequency, i.e. the latter pair are the molecular-level parameters for self-assembly of the molecules into crystals.

Apoferritins↗
PubMed1991

Iron entry route in horse spleen apoferritin. Involvement of the three-fold channels as probed by selective reaction of cysteine-126 with the spin label 4-maleimido-tempo.

Apoferritin has been selectively labeled with a maleimide nitroxide derivative at Cys-126, located in the hydrophilic 3-fold channels. Titration of this derivative with Fe(II), which gives rise to the initial Fe(III)-apoferritin complex, produces, at low metal-to-protein ratios, a decrease of the intensity of the label EPR signal due to the occurrence of a magnetic dipolar interaction. A label-metal distance ranging between 8-12 A can be estimated from titrations performed with VO(IV), which is known to bind in the 3-fold channels, and likewise produces a decrease in the label EPR signal. The present findings indicate that iron binds in the hydrophilic channels in its higher oxidation state and that these channels represent the metal entry route at least at low metal-to-protein ratios.

Animals↗
PubMed1984

Spectroscopic studies on the binding of iron, terbium, and zinc by apoferritin.

Ultraviolet difference spectroscopy has been used to study Fe (III)-apoferritin complexes formed after addition of Fe (II) to apoferritin in air. At constant iron, the recorded spectra varied with time after Fe (II) addition and with the number of iron atoms/molecule (protein concentration). The results indicate that after production of an initial complex, rearrangement or migration of Fe (III) atoms occurs, with polynuclear species forming as end-product, probably by hydrolytic polymerization. The presence of Tb3+ or Zn2+ ions affected the Fe (III) spectra and their development in different ways. The combined data suggest that more than one site, or processes, are involved in ferritin iron-core formation and that some of the metal sites are clustered.

Animals↗
PubMed2006

Optical and EPR spectroscopic studies of demetallation of hemin by L-chain apoferritins.

Earlier crystallographic and spectroscopic studies had shown that horse spleen apoferritin was capable of removing the metal ion from hemin (Fe(III)-protoporphyrin IX) [G. Précigoux, J. Yariv, B. Gallois, A. Dautant, C. Courseille, B. Langlois d'Estaintot, Acta Cryst. D50 (1994) 739-743; R.R. Crichton, J.A. Soruco, F. Roland, M.A. Michaux, B. Gallois, G. Précigoux, J.-P. Mahy, D. Mansuy, Biochemistry 36 (1997) 15049-15054]. We have carried out a detailed re-analysis of this phenomenon using both horse spleen and recombinant horse L-chain apoferritins, by electron paramagnetic resonance spectroscopy (EPR) to unequivocally distinguish between heme and non-heme iron. On the basis of site-directed mutagenesis and chemical modification of carboxyl residues, our results show that the UV-visible difference spectroscopic method that was used to establish the mechanism of demetallation is not representative of hemin demetallation. EPR spectroscopy does establish, as in the initial crystallographic investigation, that hemin demetallation occurs, but it is much slower. The signal at g=4.3 corresponding to high spin non-heme-iron (III) increases while the signal at g=6 corresponding to heme-iron decreases. Demetallation by the mutant protein, while slower than the wild-type, still occurs, suggesting that the mechanism of demetallation does not only involve the cluster of four glutamate residues (Glu 53, 56, 57, 60), proposed in earlier studies. However, the mutant protein had lost its capacity to incorporate iron, as had the native protein in which the four Glu residues had been chemically modified. Interestingly, a signal at g=1.94 is also observed. This signal most likely corresponds to a mixed-valence Fe(II)-Fe(III) cluster suggesting that a redox reaction may also be involved in the mechanism of demetallation.

Animals↗
PubMed1991

Iron binding to horse spleen apoferritin: a vanadyl ENDOR spin probe study.

The role of the protein shell in the formation of the hydrous ferric oxide core of ferritin is poorly understood. A VO2+ spin probe study was undertaken to characterize the initial complex of Fe2+ with horse spleen apoferritin (96% L-subunits). A competitive binding study of VO2+ and Fe2+ showed that the two metals compete 1:1 for binding at the same site or region of the protein. Curve fitting of the binding data showed that the affinity of VO2+ for the protein was 15 times that of Fe2+. Electron nuclear double resonance (ENDOR) measurements on the VO(2+)-apoferritin complex showed couplings from two nitrogen nuclei, tentatively ascribed to the N1 and N3 nitrogens of the imidazole ligand of histidine. The possibility that the observed nitrogen couplings are from two different ligands is not precluded by the data, however. A pair of exchangeable proton lines with a coupling of approximately 1 MHz is tentatively assigned to the NH proton of the coordinated nitrogen. A 30-40% reduction in the intensity of the 1H matrix ENDOR line upon D2O-H2O exchange indicates that the metal-binding site is accessible to solvent and, therefore, to molecular oxygen as well. The ENDOR data provide the first evidence for a principle iron(II)-binding site with nitrogen coordination in an L-subunit ferritin. The site may be important in Fe2+ oxidation during the beginning stages of core formation.

Animals↗
PubMed1991

Role of phosphate in initial iron deposition in apoferritin.

Ferritins from microorganisms to man are known to contain varying amounts of phosphate which has a pronounced effect on the structural and magnetic properties of their iron mineral cores. The present study was undertaken to gain insight into the role of phosphate in the early stages of iron accumulation by ferritin. The influence of phosphate on the initial deposition of iron in apoferritin (12 Fe/protein) was investigated by EPR, 57Fe Mössbauer spectroscopy, and equilibrium dialysis. The results indicate that phosphate has a significant influence on iron deposition. The presence of 1 mM phosphate during reconstitution of ferritin from apoferritin, Fe(II), and O2 accelerates the rate of oxidation of the iron 2-fold at pH 7.5. In the presence or absence of phosphate, the rate of oxidation at 0 degrees C follows simple first-order kinetics with respect to Fe(II) with half-lives of 1.5 +/- 0.3 or 2.8 +/- 0.2 min, respectively, consistent with a single pathway for iron oxidation when low levels of iron are added to the apoprotein. This pathway may involve a protein ferroxidase site where phosphate may bind iron(II), shifting its redox potential to a more negative value and thus facilitating its oxidation. Following oxidation, an intermediate mononuclear Fe(III)-protein complex is formed which exhibits a transient EPR signal at g' = 4.3. Phosphate accelerates the rate of decay of the signal by a factor of 3-4, producing EPR-silent oligonuclear or polynuclear Fe(III) clusters. In 0.5 mM Pi, the signal decays according to a single phase first-order process with a half-life near 1 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoferritins↗
PubMed1988

Self-assembly of apoferritin from horse spleen after reversible chemical modification with 2,3-dimethylmaleic anhydride.

Apoferritin from horse spleen is composed of 24 subunits that undergo partial dissociation after chemical modification with 2,3-dimethylmaleic anhydride (DMMA), yielding dimeric, trimeric, and tetrameric intermediates, stable at pH 8.5 and 0 degrees C. Deacylation at neutral pH and elevated temperature provides a means to initiate reassembly by appropriate shifts of the solvent conditions. In order to monitor the pathway of self-assembly, starting from different intermediates of dissociation, dimers, trimers, and tetramers were isolated and investigated with respect to their capacity to accomplish reassociation. Intrinsic protein fluorescence, gel permeation chromatography, and analytical ultracentrifugation were applied to characterize the intermediate and final stages of association. The assembly of both the dimer and trimer yields greater than 85% of the native tetracosamer; the overall rate, starting from the dimer, exceeds the one starting from the trimer. Under comparable conditions, the tetramer exhibits only partial reassociation via the dimer and monomer; the corresponding dissociation reaction determines the observed slower rate. Significant assembly intermediates are "structured monomers", dimers, trimers, and dodecamers. Polymerization of the dimer via the tetramer, octamer, etc., does not occur on the pathway of assembly. The results confirm the assembly scheme proposed previously on the basis of cross-linking and spectroscopic experiments [Gerl, M., & Jaenicke, R. (1987) Eur. Biophys. J. 15, 103-109]. Comparison of structural models involving the different subunit interactions responsible for the sequential association supports the monomer----dimer----trimer----hexamer----dodecamer----tetracosamer mechanism of apoferritin self-assembly.

Animals↗
PubMed2000

Quasi-planar nucleus structure in apoferritin crystallization.

First-order phase transitions of matter, such as condensation and crystallization, proceed through the formation and subsequent growth of 'critical nuclei' of the new phase. The thermodynamics and kinetics of the formation of these critical nuclei depend on their structure, which is often assumed to be a compact, three-dimensional arrangement of the constituent molecules or atoms. Recent molecular dynamics simulations have predicted compact nucleus structures for matter made up of building blocks with a spherical interaction field, whereas strongly anisotropic, dipolar molecules may form nuclei consisting of single chains of molecules. Here we show, using direct atomic force microscopy observations, that the near-critical-size clusters formed during the crystallization of apoferritin, a quasi-spherical protein, and which are representative of the critical nucleus of this system, consist of planar arrays of one or two monomolecular layers that contain 5-10 rods of up to 7 molecules each. We find that these clusters contain between 20 and 50 molecules each, and that the arrangement of the constituent molecules is identical to that found in apoferritin crystals. We anticipate that similarly unexpected critical nucleus structures may be quite common, particularly with anisotropic molecules, suggesting that advanced nucleation theories should treat the critical nucleus structure as a variable.

Apoferritins↗
PubMed1995

Site-specific reactivities of cysteine residues in horse L-apoferritin.

Recombinant horse L-apoferritin and its mutants were used to compare the reactivities of two different cysteinyl residues with 7-fluoro-4-sulfamoyl-2,1,3-benzoxadiazole (ABD-F), p-chloromercuribenzoic acid (PCMB), N-(9-acridinyl)maleimide (NAM), and 4-maleimido-2,2,6,6-tetramethylpiperidine-N-oxyl (NEM-TEMPO). ABD-F selectively reacted with cysteine 52 (C52), which is located on the inner surface of the peptide shell of apoferritin. In contrast, PCMB reacted only with cysteine 130 (C130), which is located at the 3-fold channels of the shell. NAM and NEM-TEMPO reacted with both C52 and C130.

Amino Acid Sequence↗
PubMed1986

Cloning of the gene coding for human L apoferritin.

A recently reported cDNA clone coding for human promyelocytic L apoferritin, shows some differences with a liver L apoferritin cDNA. We have investigated if these differences are due to the expression of different genes or to an alternative transcription of an unique gene. In this paper we report data suggesting that a single gene is mainly expressed in several tissues examined. This gene has been cloned and characterized. Its sequence shows three introns: the exon sequence is identical to that of cDNA clone isolated from human liver. A minimum of five related pseudogenes have been also analysed. One of them is a processed pseudogene interrupted by an intron-like fragment.

Amino Acid Sequence↗