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PubMed2000

Interactions and aggregation of apoferritin molecules in solution: effects of added electrolytes.

We have studied the structure of the protein species and the protein-protein interactions in solutions containing two apoferritin molecular forms, monomers and dimers, in the presence of Na(+) and Cd(2+) ions. We used chromatographic, and static and dynamic light scattering techniques, and atomic force microscopy (AFM). Size-exclusion chromatography was used to isolate these two protein fractions. The sizes and shapes of the monomers and dimers were determined by dynamic light scattering and AFM. Although the monomer is an apparent sphere with a diameter corresponding to previous x-ray crystallography determinations, the dimer shape corresponds to two, bound monomer spheres. Static light scattering was applied to characterize the interactions between solute molecules of monomers and dimers in terms of the second osmotic virial coefficients. The results for the monomers indicate that Na(+) ions cause strong intermolecular repulsion even at concentrations higher than 0.15 M, contrary to the predictions of the commonly applied Derjaguin-Landau-Verwey-Overbeek theory. We argue that the reason for such behavior is hydration force due to the formation of a water shell around the protein molecules with the help of the sodium ions. The addition of even small amounts of Cd(2+) changes the repulsive interactions to attractive but does not lead to oligomer formation, at least at the protein concentrations used. Thus, the two ions provide examples of strong specificity of their interactions with the protein molecules. In solutions of the apoferritin dimer, the molecules attract even in the presence of Na(+) only, indicating a change in the surface of the apoferritin molecule. In view of the strong repulsion between the monomers, this indicates that the dimers and higher oligomers form only after partial denaturation of some of the apoferritin monomers. These observations suggest that aggregation and self-assembly of protein molecules or molecular subunits may be driven by forces other than those responsible for crystallization and other phase transitions in the protein solution.

Apoferritins↗

PubMed1997

Two-dimensional crystals of apoferritin.

A simple 2D crystallization method using unfolded protein film as a supporting film of crystals was described, which allows modification of protein surfaces by injecting chemical reagents into the subphase after the crystal formation. As an example, glutaraldehyde was used to cross-link adjacent proteins and then stabilize protein crystals. The second layer of other proteins can also be formed on the apoferritin array using cross-linkers. The array of apoferritin is not only beneficial for electron crystallography but also for practical applications. For example, apoferritin produces a mineral core with a size which can be adjusted by the size to the cavity (i.e. 6 nm). Fabrication of such a small size of well defined fine particles is currently not easy using physical or chemical procedures. Using apoferritin, however, it is easy to produce uniform fine particles. If the core is designed to add interesting properties such as magnetism it is possible to make the highest class of magnetic film with ferritin 2D crystals. Basic researches toward practical applications of 2D protein crystal is now under way in various fields. The well defined size and function of protein molecules will benefit to many applications. The function and crystalline order can be designed by site-directed mutagenesis with the development of protein engineering.

Animals↗

PubMed1987

Iron(III) clusters bound to horse spleen apoferritin: an X-ray absorption and Mössbauer spectroscopy study that shows that iron nuclei can form on the protein.

Ferritin is a complex of a hollow, spherical protein and a hydrous, ferric oxide core of less than or equal to 4500 iron atoms inside the apoprotein coat; the apoprotein has multiple (ca. 12) binding sites for monoatomic metal ions, e.g., Fe(II), V(IV), Tb(III), that may be important in the initiation of iron core formation. In an earlier study we observed that the oxidation of Fe(II) vacated some, but not all, of the metal-binding sites, suggesting migration of some Fe during oxidation, possibly to form nucleation clusters; some Fe(III) remained bound to the protein. Preliminary extended X-ray absorbance fine structure (EXAFS) analysis of the same Fe(III)-apoferritin complex showed an environment distinct from ferritin cores, but the data did not allow a test of the Fe cluster hypothesis. In this paper, with improved EXAFS data and with Mössbauer data on the same complex formed with 57Fe, we clearly show that the Fe(III) in the distinctive environment is polynuclear (Fe atoms with Fe-Fe = 3.5 A and TB = 7 K). Moreover, the arrangement of atoms is such that Fe(III) atoms appear to have both carboxylate-like ligands, presumably from apoferritin, and oxo bridges to the other iron atoms. Thus the protein provides sites not only for initiation but also for nucleation of the iron core. Sites commodious enough and with sufficient conserved carboxylate ligands to accommodate such a nucleus occur inside the protein coat at the subunit dimer interfaces. Such Fe(III)-apoferritin nucleation complexes can be used to study the properties of the several members of the apoferritin family.

Animals↗

PubMed2002

Two pathways of iron uptake in bovine spleen apoferritin dependent on iron concentration.

Iron incorporation by bovine spleen apoferritin either with ferrous ammonium sulfate in different buffers or with ferrous ammonium sulfate and phosphate was studied. Iron uptake and iron autoxidation were recorded spectrophotomerically. The buffers [4-(2-hydroxyethyl)-1-piperazinyl]ethanesulphonic acid (Hepes) and tris(hydroxymethyl)aminoethane (Tris) exhibited pH-dependent iron autoxidation, with Tris showing less iron autoxidation than Hepes. An Eadie-Scatchard plot (v/[s] versus v) of the iron uptake rate in Hepes was a curved rather than a straight line, suggesting that there are two iron uptake pathways. On the other hand, the Eadie-Scatchard plots of Tris and of Hepes after the addition of phosphate showed a straight line. Phosphate accelerated the iron uptake rate. The iron loading kinetics of apoferritin in Hepes was dependent on apoferritin concentration. The Km value obtained from iron uptake kinetics was 4.5 microM, corresponding to the physiological iron concentration. These results demonstrate that iron loading of apoferritin was accomplished at physiological iron concentrations, which is essential for iron uptake, via two uptake pathways of dependent on iron concentration.

Animals↗

PubMed2002

Apoferritin attenuates experimental allergic encephalomyelitis in SJL mice.

Ferritin has been shown to attenuate iron-catalyzed oxidative damage in several experimental conditions. Since oxidative damage has been implicated in the pathogenesis of multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE), an animal model of MS, we tested the hypothesis that ferritin would act to attenuate disease. The experimental design was to increase plasma ferritin levels during the active stage of EAE by giving systemic injections of apoferritin and then compare disease activity between these mice and EAE mice administered vehicle. Additional mice received systemic injections of iron, which induces ferritin synthesis, in order to test the effects of exogenous iron on the disease course. Although plasma levels of ferritin were found to be elevated in both apoferritin and iron-injected EAE mice, only apoferritin treatment resulted in a reduction in disease activity compared to EAE mice given vehicle. The suppressive effects of apoferritin administration suggest that the increase in endogenous ferritin levels that have been previously observed in the cerebrospinal fluid of chronic progressive MS patients with active disease might be functioning to limit the severity and spread of tissue damage.

Animals↗

PubMed1979

Ferritin- and apoferritin-induced immune complex glomerulonephritis in mice.

In order to study the effects of the protein moiety independent of the protein-iron complex in the development of ferritin-induced glomerulonephritis, we compared the effects of ferritin, equimolar amounts of apoferritin, and equimolar amounts of iron dextran in Swiss albino mice. The results were compared to both saline-injected and non-injected controls. Ferritin resulted in a glomerulonephritis associated with predominantly mesangial deposition of immune complexes. Tubulo-interstitial changes occurred as well. Iron dextran resulted in similar but less severe tubulo-interstitial changes and evoked no glomerular alterations. Apoferritin resulted in an immune complex glomerulonephritis usually associated with membranous deposits. No tubular or interstitial changes occurred. Proteinuria developed in animals receiving apoferritin. Since the protein-iron complex caused tubular and interstitial damage, apoferritin may provide a more suitable model of immune-complex-mediated glomerulonephritis.

Animals↗

PubMed1987

Assembly of apoferritin from horse spleen: comparison of the protein in its native and reassembled state.

The in vitro selfassembly of apoferritin after previous dissociation and unfolding in 7.2M guanidinium chloride, pH 3.5, yields up to 80% of a protein complex exhibiting the molecular mass of the native icositetramer of greater than or equal to 450 kDa. After removal of high molecular mass byproducts, the final reassembly product proves to be indistinguishable 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 transitions 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 preferential solvation of the protein in the presence of less than or equal to 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----hemosiderin transition.

Animals↗

PubMed1973

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.

Amino Acids↗

PubMed1983

Binding of terbium to apoferritin: a fluorescence study.

Luminescence measurements show that apoferritin binds three Tb(III) atoms per subunit in accordance with crystallographic evidence. Fe(II) competes with Tb(III) for at least some of the binding sites. This competition may be the molecular basis for the inhibition of iron incorporation into apoferritin brought about by Tb(III). Ca(II), which is generally replaced by Tb(III) in Ca(II) binding proteins, does not compete with the lanthanide for binding to apoferritin.

Animals↗

PubMed1990

Iron detoxifying activity of ferritin. Effects of H and L human apoferritins on lipid peroxidation in vitro.

Three recombinant human apoferritin variants were added to ferrous iron and the amount of lipid peroxidation produced by hydrogen peroxide was studied. The H-apoferritin had the strongest inhibitory effect on lipid peroxidation, probably due to its ferroxidase activity. The L-apoferritin inhibited lipid peroxidation slowly and only at neutral pH. The H-mutant 91, deleted of the last 22 C-terminal amino acids, and which is not able to form an iron core, had minimal effects on iron lipid peroxidation. It was concluded that both ferro-oxidase and iron mineralization activities are necessary for ferritin iron detoxifying action.

Apoferritins↗

PubMed1993

Nucleation of the iron core occurs at the three-fold channels of horse spleen apoferritin: an EXAFS study on the native and chemically-modified protein.

Extended X-ray absorbance fine structure measurements have been carried out on the initial Fe(III)-apoferritin complex at a Fe/subunit ratio of 2 in native and modified horse spleen apoferritin. Analysis of the data indicates that in the native protein the iron forms a protein-bound polynuclear cluster (Fe-Fe distance 3.4 A) with a first coordination sphere constituted by 5-6 low-Z atoms, e.g., nitrogen atoms, carboxylate-like ligands or oxo bridges between the iron atoms. Modification of Cys-126, a residue localized on the outer surface of the hydrophilic three-fold channels, with p-chloromercuribenzoate (PMB) or phenylmercuric acetate (PMA) brings about distinctive differences. In particular, in the PMB-reacted protein the feature assigned to the iron-iron interaction disappears from the spectrum, whilst in the PMA-reacted protein the main differences with respect to the native protein are observed at the level of the first coordination sphere. These results confirm the formation of protein-Fe(III)-clusters and localize these sites at the hydrophilic three-fold channels of horse spleen apoferritin.

Animals↗

PubMed2006

Versatile apoferritin nanoparticle labels for assay of protein.

A versatile bioassay label based on marker-loaded apoferritin nanoparticles (MLANs) has been developed for sensitive protein detection. Dissociation and reconstitution characteristics at different pH as well as the special cavity structure of apoferritin provides a facile route to prepare nanoparticle labels and avoid the complicated and tedious synthesis process of conventional nanoparticle labels. The optical and electrochemical characteristics of the prepared nanoparticle labels are easily controlled by loading different optical or electrochemical markers. A fluorescence marker (fluorescein anion) and a redox marker [hexacyanoferrate(III)] were used as model markers to load into the cavity of apoferritin nanoparticles for microscopic fluorescence immunoassay and electrochemical immunoassay, respectively. Detection limits of 0.06 (0.39 pM) and 0.08 ng mL(-1) (0.52 pM) IgG were obtained with fluorescein MLAN and hexacyanoferrate MLANs, respectively. The new nanoparticle labels hold great promise for multiplex protein detection (in connection with nanoparticles loaded with different markers) and for enhancing the sensitivity of other bioassays.

Apoferritins↗

PubMed1987

On the mechanism of horse spleen apoferritin assembly: a sedimentation velocity and circular dichroism study.

The apoferritin shell is known to assemble spontaneously from its subunits obtained at acid pH upon neutralization. The reassembly of apoferritin from horse spleen has been followed by means of sedimentation velocity and circular dichroism experiments as a function of the pH and the nature of the assembly buffer in order to obtain information on the assembly pathway. In all the buffer systems tested the subunits sediment as a single peak of varying sedimentation and diffusion coefficients, and shell assembly starts at pH values around 3.5. In dilute glycine-acetate buffers the subunits are essentially dimeric up to this pH value. Therefore, the dimeric building blocks of the apoferritin shell that are apparent in the X-ray structure represent the first assembly intermediates. When the pH is increased to 4.0-4.3, the weight-average sedimentation velocity of the subunits increases to 3.6-4.7 S, respectively, and the subunit population becomes heterogeneous. Concomitantly, significant changes in the circular dichroism properties of the aromatic residues take place. On the basis of the X-ray structure, where aromatic residues appear to be located at or near the fourfold symmetry axes, these data suggest that assembly proceeds from dimers through tetramers and octamers. In the pH range 4.5-6.5 the reassembly process cannot be followed due to reversible precipitation of the subunits near their isoelectric point; at neutral pH values essentially quantitative reassembly is obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Identification of the iron entry channels in apoferritin. Chemical modification and spectroscopic studies.

The knowledge of the route through which iron can enter and leave the apoferritin shell is a prerequisite for the understanding of ferritin's function. The involvement of the hydrophilic 3-fold channels in the iron uptake process has been studied by taking advantage of the reactivity of specific residues that line such channels, i.e., glutamic acid-127 and aspartic acid-130, the major Cd(II) binding sites, and cysteine-126. 113Cd NMR experiments have provided direct evidence for the competition between Fe(II) and Cd(II) binding to major Cd(II) binding sites on the protein and or a higher affinity of Fe(II) for these sites, in line with the well-known inhibitory effect of Cd(II) on iron uptake. Further evidence for the use of the 3-fold channels in the iron entry process has been obtained by means of chemical modification of Cys-126 with different mercurials. In particular, the introduction of the additional carboxylate carried by p-(chloromercuri)benzoate near Asp-127 and Glu-130 increases the initial rate of iron uptake and affects the coordination geometry of the metal in the Fe(III)-apoferritin complex as indicated by optical absorption and EPR data. The assignment of these effects to the carboxylate moiety of p-(chloromercuri)benzoate is brought out by the observation that the introduction in the 3-fold channel of the benzene ring only by means of phenylmercuric acetate has no effect on the initial iron uptake kinetics and on the spectroscopic properties of the Fe(III)-apoferritin complex.

Animals↗

PubMed1976

The monomers and oligomers of ferritin and apoferritin: association and dissociation.

We have reinvestigated the association and dissociation of ferritin and apoferritin in phosphate buffer (pH 7.2, I = 0.05). When oligomer-enriched solutions of horse spleen ferritin were mixed with more concentrated, but unenriched solutions of horse spleen apoferritin, there was dissociation of the ferritin oligomers, as determined by polyacrylamide gel electrophoresis and from iron/protein ratios. Some evidence was also obtained for association of monomers in the mixture of ferritin and apoferritin after pelleting and redissolution of pellets in minimal volumes of the phosphate buffer. Monomer-enriched, biosynthetically labeled rat liver ferritin was pelleted, redissolved in minimal volumes of phosphate buffer, and separated by polyacrylamide gel electrophoresis; the fractions were isolated and counted. The results revealed that an association of monomers of the rat liver ferritin had taken place which doubled the concentration of dimers. However, our results also indicate that association by concentration was limited to a fraction of monomers.

Animals↗

PubMed1992

Uranium-loaded apoferritin with antibodies attached: molecular design for uranium neutron-capture therapy.

A method is described to deliver 235U to tumors; the isotope would then be fissioned by incident neutrons, producing localized lethal radiation sufficient for therapy. Apoferritin was loaded with an average of approximately 800 238U atoms per molecule. Stability of the loaded apoferritin in solution was improved, so that only 8% loss of uranium occurred after 8 days at pH 7. Fab' antibody fragments were covalently attached to the uranium-loaded apoferritin, and the immunoreactivity of the conjugate was 92% of that for antibody alone. Such bio-uranium constructions should provide significant advantages over boronated antibodies to meet the requirements for clinical neutron-capture therapy.

Apoferritins↗

PubMed1976

Synthesis of apoferritin in mouse peritoneal macrophages. Characterization of 20 S particles.

Apoferritin particles were found in mouse peritoneal macrophages cultured in vitro. They were found as 20S particles in the "ribosomal fraction" of macrophages labeled with L-[14C]glutamic acid. Possibilities that they were breakdown products of ribosomes or of other well-known contaminants of the ribosomal fraction were excluded because they did not incorporate [5-3H]uridine. They were resistant to RNase and were relatively resistant to detergent. The antibody against horse spleen apoferritin precipitated about 70% of the particles in the 20S region, judging by measurement of radioactivity. On in vitro incubation with Fe2+ and suitable oxidizing agents the sedimentation coefficient of 80% of the 20S particles changed to about 60S, which corresponds to that of ferritin. SDS-polyacrylamide gel electrophoresis revealed the presence of subunit structures with the same molecular size as that of mouse liver apoferritin. Under the electron microscope, the particles appeared spherical with a relatively uniform diameter of about 130 A.

Animals↗

PubMed2006

Magnetic resonance visualization of tumor angiogenesis by targeting neural cell adhesion molecules with the highly sensitive gadolinium-loaded apoferritin probe.

Tumor vessel imaging could be useful in identifying angiogenic blood vessels as well as being a potential predictive marker of antiangiogenic treatment response. We recently reported the expression of the neural cell adhesion molecule (NCAM) in the immature and tumor endothelial cell (TEC) lining vessels of human carcinomas. Exploiting an in vivo model of human tumor angiogenesis obtained by implantation of TEC in Matrigel in severe combined immunodeficiency mice, we aimed to image angiogenesis by detecting the expression of NCAM with magnetic resonance imaging. The imaging procedure consisted of (a) targeting NCAMs with a biotinylated derivative of C3d peptide that is known to have high affinity for these epitopes and (b) delivery of a streptavidin/gadolinium (Gd)-loaded apoferritin 1:1 adduct at the biotinylated target sites. The remarkable relaxation enhancement ability of the Gd-loaded apoferritin system allowed the visualization of TEC both in vitro and in vivo when organized in microvessels connected to the mouse vasculature. Gd-loaded apoferritin displayed good in vivo stability and tolerability. The procedure reported herein may be easily extended to the magnetic resonance visualization of other epitopes suitably targeted by proper biotinylated vectors.

Animals↗