Ferritin Aggregates Research Articles

Differential Degradation of Ferritin H- and L-Chains: Accumulation of L-Chain-Rich Ferritin in Lens Epithelial Cells

The storage of iron by ferritin is determined by tissue-specific composition of its 24 subunits, which are designated as either heavy (H) or light (L). For a better understanding of how lens epithelial cells regulate their ferritin subunit makeup, the degradation pattern of each subunit type was analyzed. In addition, age-related changes in ferritin concentration and subunit makeup were determined. Ferritin turnover in primary cultures of canine lens epithelial cells was determined by metabolic labeling with [(35)S]-methionine. Transient transfection with vectors containing coding sequences for either H- or L-chains were used to modify ferritin subunit makeup. Ferritin concentration was measured by ELISA. Immunodetection and fluorescence immunocytochemistry were used to study age-related changes in ferritin chain concentration. Inhibition of the proteasomal protein degradation pathway by clastolactacystin-beta-lactone had no effect on ferritin degradation, whereas inhibition of lysosomal degradation markedly increased ferritin levels, confirming that this system is involved in ferritin turnover. H-chain ferritin degraded at a faster rate than the L-chain. L-chain-rich ferritin in L-chain-transfected cells formed inclusion bodies that were localized to the cytosol. Similar inclusion bodies were found in older lens cells kept in cell culture for more than 8 days. Steady degradation of H-chain ferritin contributed to the maintenance of a constant level of this chain within the lens epithelial cells. In contrast, slower turnover of the L-chain resulted in accumulation of L-chain-enriched ferritin associated with cytoplasmic inclusion bodies. These L-chain-containing inclusion bodies were found in the cytosol of cells overexpressing L-ferritin chain and in nontransfected cells maintained in culture for 8 to 35 days. Overexpression of the L-chain has been associated with the formation of premature cataracts in humans with hereditary hyperferritinemia cataract syndrome. The formation of inclusion bodies in older lens epithelial cells, as demonstrated in the current investigation, is intriguing and could point to possible involvement of cytoplasmic L-chain-enriched ferritin aggregates in the formation of age-related cataract.

Image analysis of bioparticles accumulation and diamagnetic alignment in high-gradient magnetic field

Magnetic properties of biological particles are measured in high-gradient magnetic separation (HGMS) analysis, revealing the concentrating process of nucleoprotein particles, ferritin, red blood cells, and eggs. A magnetic force acting on micrometer and submicrometer biological particles having diamagnetic or paramagnetic susceptibility with respect to the solution causes their movement and accumulation in gradient magnetic fields dependent on the values of the magnetic moments. The methods developed enable us to obtain the magnetic moments values of single particles and their assembly directly from magnetic separation and image analyses without assuming the detection of sizes. Our precision methods for the measurement of the capture traveling (magnetic diffusion) time and the accumulation (magnetic sedimentation) radius in HGMS show that it is really possible to determine the weak dia- or paramagnetic shifts of magnetic susceptibility up to 0.7x10(-10) (SI units). HGMS analysis of the concentrating process of nucleoprotein granules (microcells, DNA granules, or nucleosome core particles) with polarization microscopy reveals phase transitions for DNA in granules, and separation accumulation of particles enables the determination of the diamagnetic susceptibility and anisotropy properties. Magnetic concentration effects always occur in living systems because micrometer-located gradient magnetic fields inside an organism are strong enough to cause drifts of cellular complexes and organelles of micrometer and submicrometer sizes. We report the appearance of superparamagnetic contamination inside developing shrimp eggs. In the developing shrimps eggs, ferritin aggregates are observed under weak gradient magnetic fields and diaparaferromagnetic changes are detected. A significant interruption of egg development is revealed in such fields.

Adult‐onset generalized dystonia due to a mutation in the neuroferritinopathy gene

Neuroferritinopathy is a recently recognized autosomal dominant disorder that results in abnormal aggregates of iron and ferritin in the brain due to a mutation in the ferritin light chain gene on chromosome 19q13.3. We present the clinical details of a patient with adult-onset generalized dystonia associated with this mutation. Neuroferritinopathy appears to be a rare disorder; hence, there is a need to report new cases to further our understanding of the clinical phenotype, diagnostic challenges, the course of the condition and imaging characteristics.

Hereditary causes of disturbed iron homeostasis in the central nervous system.

Iron is essential for oxidation-reduction catalysis and bioenergetics; however, unless appropriately shielded, this metal plays a crucial role in the formation of toxic oxygen radicals that can attack all biological molecules. Organisms are equipped with specific proteins designed for iron acquisition, export and transport, and storage, as well as with sophisticated mechanisms that maintain the intracellular labile iron pool at an appropriate level. Despite these homeostatic mechanisms, organisms often face the threat of either iron deficiency or iron overload. This review describes several hereditary iron-overloading conditions that are confined to the brain. Recently, a mutation in the L-subunit of ferritin has been described that causes the formation of aberrant L-ferritin with an altered C-terminus. Individuals with this mutation in one allele of L-ferritin have abnormal aggregates of ferritin and iron in the brain, primarily in the globus pallidus. Patients with this dominantly inherited late-onset disease present with symptoms of extrapyramidal dysfunction. Mice with a targeted disruption of a gene for iron regulatory protein 2 (IRP2), a translational repressor of ferritin, misregulate iron metabolism in the intestinal mucosa and the central nervous system. Significant amounts of ferritin and iron accumulate in white matter tracts and nuclei, and adult IRP2-deficient mice develop a movement disorder consisting of ataxia, bradykinesia, and tremor. Mutations in the frataxin gene are responsible for Friedreich's ataxia, the most common of the inherited ataxias. Frataxin appears to regulate mitochondrial iron-sulfur cluster formation, and the neurologic and cardiac manifestations of Friedreich's ataxia are due to iron-mediated mitochondrial toxicity. Patients with Hallervorden-Spatz syndrome, an autosomal recessive, progressive neurodegenerative disorder, have mutations in a novel pantothenate kinase gene (PANK2). The cardinal feature of this extrapyramidal disease is pathologic iron accumulation in the globus pallidus. The defect in PANK2 is predicted to cause the accumulation of cysteine, which binds iron and causes oxidative stress in the iron-rich globus pallidus. Finally, aceruloplasminemia is an autosomal recessive disorder of iron metabolism caused by loss-of-function mutations in ceruloplasmin gene that leads to misregulation of both systemic and central nervous system iron trafficking. Affected individuals suffer from extrapyramidal signs, cerebellar ataxia, progressive neurodegeneration of retina, and diabetes mellitus. Excessive iron depositions are found in the brain, liver, pancreas, and other parenchymal cells, but plasma iron concentrations are decreased. These conditions are not common, but awareness about them is important for differential diagnosis of various neurodegenerative disorders.

On the magnetic ordering of the iron storage proteins in tissues

Abstract Small angle neutron scattering data are indicative of ferritin aggregation in tissues (Rg of ca. 21–25 nm) in contrast to the isolated purified ferritin protein (Rg of ca. 5 nm). Dependence of the shape and position of the ferritin electron spin resonance spectra in tissues upon sample orientation relative steady state magnetic field points to the magnetic susceptibility anisotropy of ferritin aggregates. The magnetic ordering of ferritin results in dramatic increase in magnetic susceptibility of the ferritin iron in tissues compared to the crude and purified ferritin extracts.

Neuroferritinopathy: A Window on the Role of Iron in Neurodegeneration

ABSTRACT Neuroferritinopathy is a recently recognised genetic disease resulting in a dominantly inherited movement disorder. The condition was mapped by linkage analysis to chromosome 19q13.3 and found to be due to a single adenine insertion in the ferritin light chain ( FTL) gene at position 460–461 which is predicted to alter the C terminus of the FTL polypeptide. Clinical features of neuroferritinopathy are highly variable, with chorea, dystonia, and Parkinsonian features predominating in different affected individuals. The most consistent feature is a dystonic dysarthria. Symptoms and abnormal physical signs appear to be restricted to the nervous system and onset is typically in the fourth to sixth decades. Low serum ferritin also characterises this condition. Brain MR imaging of affected patients demonstrates iron deposition in the basal ganglia, progressing over years to cystic degeneration, and brain histochemistry shows abnormal aggregates of ferritin and iron. Now that the molecular basis of the condition is known, therapeutic interventions to reduce or reverse brain iron deposition are being evaluated. This rare disease provides evidence of a central role for iron metabolism in neurodegenerative disorders.

Rare causes of hereditary iron overload

Iron is a vitally important element in mammalian metabolism because of its unsurpassed versatility as a biologic catalyst. However, when not appropriately shielded or when present in excess, iron plays a key role in the formation of extremely toxic oxygen radicals, which ultimately cause peroxidative damage to vital cell structures. Organisms are equipped with specific proteins designed for iron acquisition, export, transport, and storage as well as with sophisticated mechanisms that maintain the intracellular labile iron pool at an appropriate level. These systems normally tightly control iron homeostasis but their failure can lead to iron deficiency or iron overload and their clinical consequences. This review describes several rare iron loading conditions caused by genetic defects in some of the proteins involved in iron metabolism. A dramatic decrease in the synthesis of the plasma iron transport protein, transferrin, leads to a massive accumulation of iron in nonhematopoietic tissues but virtually no iron is available for erythropoiesis. Humans and mice with hypotransferrinemia have a remarkably similar phenotype. Homozygous defects in a recently identified gene encoding transferrin receptor 2 lead to iron overload (hemochromatosis type 3) with symptoms similar to those seen in patients with HFE-associated hereditary hemochromatosis (hemochromatosis type 1). Transferrin receptor 2 is primarily expressed in the liver but it is unclear how mutant forms cause iron overload. Mutations in the gene encoding the iron exporter, ferroportin 1, cause iron overload characterized by iron accumulation in macrophages yet normal plasma iron levels. Plasma iron, together with dominant inheritance, discriminates iron overload due to ferroportin mutations (hemochromatosis type 4) from hemochromatosis type 1. Heme oxygenase 1 is essential for the catabolism of heme and in the recycling of hemoglobin iron in macrophages. Homozygous heme oxygenase 1 deletion in mice leads to a paradoxical accumulation of nonheme iron in macrophages, hepatocytes, and many other cells and is associated with low plasma iron levels, anemia, endothelial cell damage, and decreased resistance to oxidative stress. A similar phenotype occurred in a child with severe heme oxygenase 1 deficiency. Recently, a mutation in the L-subunit of ferritin has been described that causes the formation of aberrant L-ferritin with an altered C-terminus. Individuals with this mutation in one allele of L-ferritin have abnormal aggregates of ferritin and iron in the brain, primarily in the globus pallidus. Patients with this dominantly inherited late-onset disease present with symptoms of extrapyramidal dysfunction. Mice with a targeted disruption of a gene for iron regulatory protein 2 (IRP2), a translational repressor of ferritin, misregulate iron metabolism in the intestinal mucosa and the central nervous system. Significant amounts of ferritin and iron accumulate in white matter tracts and nuclei, and adult IRP2-deficient mice develop a movement disorder consisting of ataxia, bradykinesia, and tremor. Mutations in the frataxin gene are responsible for Friedreich ataxia, the most common of the inherited ataxias. Frataxin appears to regulate mitochondrial iron (or iron-sulfur cluster) export and the neurologic and cardiac manifestations of Friedreich ataxia are due to iron-mediated mitochondrial toxicity. Finally, patients with Hallervorden-Spatz syndrome, an autosomal recessive, progressive neurodegenerative disorder, have mutations in a novel pantothenate kinase gene (PANK2). The cardinal feature of this extrapyramidal disease is pathologic iron accumulation in the globus pallidus. The defect in PANK2 is predicted to cause the accumulation of cysteine, which binds iron and causes oxidative stress in the iron-rich globus pallidus. Semin Hematol 39:249-262. Copyright 2002, Elsevier Science (USA). All rights reserved.

The role of cysteine residues in the oxidation of ferritin

We have shown that ferritin is oxidized during iron loading using its own ferroxidase activity and that this oxidation results in its aggregation (Welch et al., Free Radic. Biol. Med. 31:999–1006; 2001). In this study we determined the role of cysteine residues in the oxidation of ferritin. Loading iron into recombinant human ferritin by its own ferroxidase activity decreased its conjugation by a cysteine specific spin label, indicating that cysteine residues were altered during iron loading. Using LC/MS, we demonstrated that tryptic peptides of ferritin that contained cysteine residues were susceptible to modification as a result of iron loading. To assess the role of cysteine residues in the oxidation of ferritin, we used site-directed mutagenesis to engineer variants of human ferritin H chain homomers where the cysteines were substituted with other amino acids. The cysteine at position 90, which is located at the end of the BC-loop, appeared to be critical for the formation of ferritin aggregates during iron loading. We also provide evidence that dityrosine moieties are formed during iron loading into ferritin by its own ferroxidase activity and that the dityrosine formation is dependent upon the oxidation of cysteine residues, especially cysteine 90. In conclusion, cysteine residues play an integral role in the oxidation of ferritin and are essential for the formation of ferritin aggregates.

A case of neuroferritinopathy

A new human disease akin to Huntington's disease and parkinsonism has been identified that is caused by the abnormal formation of ferritin aggregates in the basal ganglia of the brain. Curtis et al. (Nat. Genet. 28, 350–353) have now reported a mutation in the ferritin l-chain (19q13.3) that causes a dominant adult-onset neuropathology which they have coined as neuroferritinopathy. Symptoms of extrapyramidal dysfunction such as dystonia, spacticity and rigidity characterized this novel disease. In brain, ferritin (FT) functions as an intracellular store and detoxification site for iron. It is likely, the authors suggest, that the mutant l-chain discovered could increase its susceptibility to proteolysis and that the consequent release of highly toxic iron from putative storage proteins might induce oxidative stress and neuronal cell death. SS

Hollow-Fiber flow field-flow fractionation of synthetic polymers in organic solvents.

A modified polyacrylonitrile (PAN) hollow-fiber membrane from a commercial source has been applied as the separation channel in flow field-flow fractionation (FFF). With the PAN membrane fiber, the application range of flow FFF could be extended to synthetic polymers that are soluble in a variety of organic solvents. The PAN membrane was shown to be resistant to hydrophobic solvents, such as dichloromethane (DCM), tetrahydrofuran (THF), ethyl acetate, and methyl ethyl ketone (MEK), as was illustrated by the successful fractionation of different polymer standards in these solvents. The system performance was assessed using polystyrene (PS) standards with ethyl acetate as the solvent. For a 100 kDa PS standard, the average recoverywas 57%, but for standards with a molar mass of 400 kDa and higher, 100% recovery was obtained. A linear relationship between peak area and injected mass was found. The run-to-run and fiber-to-fiber repeatability was determined using 100- and 400 kDa PS standards. The repeatability appeared to be satisfactory, with relative standard deviations < 2% for the retention times and < 5% for the recoveries of the standards. Plate numbers for the 400 kDa standard on different fibers were in the order of 110. From measurements on the fractionation of ferritin aggregates, it is concluded that the instrumental band-broadening is negligible. For an accurate determination of diffusion coefficients and molecular sizes based on retention times, calibration of the channel with standards appeared to be necessary. However, it was shown that the FFF system could be coupled to a multiangle light scattering (MALS) detector, thus providing an alternative on-line method for calibration. Expressions for the maximum obtainable plate number per unit of time have been derived for a hollow-fiber flow FFF system. It is shown that an increase in the system performance can be expected from a scaling down of the fiber diameter.

Mutation in the gene encoding ferritin light polypeptide causes dominant adult-onset basal ganglia disease.

We describe here a previously unknown, dominantly inherited, late-onset basal ganglia disease, variably presenting with extrapyramidal features similar to those of Huntington's disease (HD) or parkinsonism. We mapped the disorder, by linkage analysis, to 19q13.3, which contains the gene for ferritin light polypeptide (FTL). We found an adenine insertion at position 460-461 that is predicted to alter carboxy-terminal residues of the gene product. Brain histochemistry disclosed abnormal aggregates of ferritin and iron. Low serum ferritin levels also characterized patients. Ferritin, the main iron storage protein, is composed of 24 subunits of two types (heavy, H and light, L) which form a soluble, hollow sphere. Brain iron deposition increases normally with age, especially in the basal ganglia, and is a suspected causative factor in several neurodegenerative diseases in which it correlates with visible pathology, possibly by its involvement in toxic free-radical reactions. We found the same mutation in five apparently unrelated subjects with similar extrapyramidal symptoms. An abnormality in ferritin strongly indicates a primary function for iron in the pathogenesis of this new disease, for which we propose the name 'neuroferritinopathy'.

Loop mutations affect ferritin solubility causing non-native aggregation of subunits or precipitation of fully assembled polymers

As a consequence of elevated expression rates, the intracellular aggregation of polypeptide chains is commonly observed in E. coli. Although wild-type human ferritin, a polymeric iron storage protein, accumulates in the soluble form at high level in the bacterial cytoplasmic fraction, some amino acid substitutions in an exposed loop direct the synthesis of a highly insoluble product. We found that two mechanisms can lead to the aggregation of ferritin. While some mutations prevent ferritin polymerisation, others cause the precipitation of molecules in the assembled state.

SCANNING TUNNELING MICROSCOPY OF FERRITIN NANOSTRUCTURES

In molecular electronics, components and circuits are made of biological molecules.1 It is important to study how the molecules arrange themselves on a substrate and to arrange them artificially. The ferritin molecule is a good candidate for making molecular devices. Previously, transmission electron microscopy was used to image only the metallic core of the ferritin molecule. We use scanning tunneling microscopy to study the behavior of ferritin deposited on a surface by imaging its protein shell. We have observed different kinds of ferritin aggregates as well as single molecules under different deposition conditions. For the first time ordered self-assemble of ferritin aggregates was observed. The aggregates were also found to be arranged in artificially made patterns. We suggest a possible mechanism of the formation of the observed structures.

Morphine enhances deposition of ferritin-antiferritin complexes in the glomerular mesangium.

Since increased mesangial accumulation of matrix has been considered to be an important event in the development of focal glomerulosclerosis, we investigated whether morphine, an active metabolite of heroin, can modulate mesangial accumulation of immune complexes. Control or morphine-dependent rats were administered intraperitoneal ferritin (8 mg/100 g body weight) daily for 6 weeks. Body weight, blood pressure, serum creatinine, 24-hour urinary protein and creatinine excretion rates were measured at 3-week intervals. Rats were sacrificed at the end of 6 weeks and kidney tissue was studied by light, immunofluorescence and electron microscopy. Serum creatinine levels and urinary protein excretion rates were not different between control and morphine-dependent rats. All morphine-dependent rats developed hematuria, whereas only 1 control rat developed hematuria. Light microscopy revealed no proliferation of mesangial cells and only a minimal increase in the mesangial matrix. Electron-microscopic studies showed deposition of immune complexes in the mesangial region. Mesangial cells showed aggregation of ferritin in lysosomes. Immunofluorescence studies revealed the presence of IgG staining predominantly in the mesangial region. The majority (60%) of morphine-dependent rats showed a diffuse mesangial deposition of IgG when compared to control rats (83%) who showed only focal deposition. These results indicate that morphine enhances deposition of immune complexes in the mesangium. Morphine-induced matrix but may also change its quality. This may play a pathogenic role in the development of glomerular lesions in patients who abuse opiates.

Stability of two-dimensional crystalline aggregates of a protein studied by molecular dynamics

Two-dimensional protein (ferritin) aggregates with a square lattice symmetry, which were formed within a thin liquid layer on a mercury surface, were studied by molecular dynamics (MD) simulation. For the simulation, the ferritin molecule was modeled by an assembly of 49 spheres, and the intermolecular interactions were given by simple formulae. During the simulation, molecules were confined within a layer, which corresponds to the thin liquid layer. An annealing MD simulation was done starting from a random molecular configuration within the layer, and aggregates with the square lattice symmetry were also obtained. To study the stability of aggregates, dissociation processes of the aggregates were analyzed using MD simulations at room temperature. Interactions between the nearest-neighbor molecules were regarded as bonds. Mean bond energies and correlation coefficients between the bond energies were calculated from the MD trajectories. A decay profile according to the dissociation was obtained, yielding a dissociation rate constant. Buried bonds were stronger than peripheral bonds. The larger the aggregate size, the stronger the bond for each of the buried and peripheral bonds. A simple theoretical account, which is applicable to a general bonded network, was introduced to analyze the dynamics of the aggregates. © 1994 by John Wiley & Sons, Inc.

Light-scattering investigations of nucleation processes and kinetics of crystallization in macromolecular systems.

Quasi-elastic light scattering (QELS) was used to investigate quantitatively the mechanisms of nucleation, postnucleation growth, and dissolution in ensembles of both crystalline and amorphous aggregates of satellite tobacco mosaic virus (STMV), ferritin, apoferritin and pumpkin seed globulin. At low supersaturation conditions, as described previously for small molecule crystallization, the metastable region was obtained. Under these conditions aggregation took place, but crystallization did not proceed and critical nuclei did not form over a long period of time. The critical solution supersaturation necessary to obtain crystals, sigma = ln(c/s) where c and s are concentration and solubility of protein, varied from approximately 0.1 for pumpkin seed globulin to approximately 0.9 for STMV. For higher supersaturation conditions when aggregation processes leading to formation of crystals are not established immediately but after a certain induction period, the supersaturation-dependent critical nuclear size, R(c), for different macromolecular systems was estimated from time-dependent size-distribution analyses to be in the range of approximately 10(3) for proteins such as pumpkin globulin to approximately 10 for virus particles. From the same data, the molar interfacial free energy was deduced to be 3.3-9.2 kJ mol(-1). These are believed to be among the first estimates for macromolecular crystals. Under conditions of moderate supersaturation where induction periods preceded the appearance of critical nuclei, the potential barriers for formation were estimated to be in the range 8.3-50 kJ mol(-1). Growth and dissolution kinetics for pumpkin seed globulin were investigated. These experiments allowed determination of protein solubility versus solution temperature, protein and precipitant concentrations. Aggregation patterns which lead to crystal formation are distinctly different to those which produce an amorphous precipitate. The results provide additional evidence that QELS can be used to find general criteria that allow one to discriminate between conditions for a given protein system leading to crystalline or amorphous states at early stages of the aggregation process.

Transport of siderotic Kupffer cells to the lung and bronchial excretion of iron in experimental iron-overload.

In 3,5,5-trimethylhexanoylferrocene-induced iron overload of rats, three different types of iron-loaded macrophages and derivatives thereof were found in the lungs. On the basis of their localization and of their pattern of iron load it was possible to distinguish: (1) Resident macrophages, showing an alveolar localization and a moderate iron content represented by lysosomal ferritin and haemosiderin. (2) Liver-derived macrophages and giant cells, as well as fragments of them. They showed an exclusive localization in capillaries and alveolar septa, and high concentrations of free ferritin molecules in addition to polymorphous ferritin- and haemosiderin-containing siderosomes. (3) Monocyte-derived intravascular pulmonary macrophages. Initially, they contained iron only as lysosomal aggregates of ferritin and haemosiderin, as a result of phagocytosis of liver-derived macrophageal cell fragments. Later in iron overload, they also showed free ferritin molecules in the cytosol and fused intrapulmonarily to giant cells. The resident as well as the liver-derived siderotic pulmonary macrophages provide a way for iron excretion through the airways.

Computer simulation of surface-induced aggregation of ferritin

Models are presented describing the transient mass-transport limited adsorption and cluster growth of ferritin at a solid surface. Computer simulations are carried out on a hexagonal lattice using a computer model that can be characterized as a two-dimensional stochastic cellular automaton allowing different rules regarding association, lateral interaction and dissociation to be incorporated in the model. The fractal dimensions of individual clusters were extracted from simulated aggregates and for similar rules found to be consistent with literature values on reversible diffusion-limited aggregation in two dimensions. The distribution of clusters versus free surface were shown to be affected by neighbor-dependent association probability. Low fractal dimension clusters were generated by a combination of strong lateral cohesion and neighbor-dependent dissociation to the bulk. By comparing computer simulated aggregation to experimental electron micrographs of adsorbed ferritin layers it is suggested that neighbor-dependent association, neighbor-dependent dissociation and lateral interactions are important factors in the complex dynamics of adsorbed protein layers.

Characterization and accumulation of ferritin in hepatocyte nuclei of mice with iron overload

After a single subcutaneous dose of iron-dextran (600 mg of iron/kg), iron overload developed in C57BL/10ScSn mice. At 4, 24 and 78 wk liver nonheme iron concentrations were 67-, 42- and 21-fold higher than controls, respectively. Much of the iron was in macrophages, but hepatocytes were also strongly positive for Perls' stainable iron. One feature was the development of iron-positive nuclear inclusions in hepatocytes. After a delay of at least 8 wk when no stainable iron was evident, a maximum of 37% of periportal hepatocytes contained inclusions by 24 wk. Although this proportion remained constant for the remainder of the study, the size of the inclusions (which were not membrane-limited) increased to greater than 3 microns in diameter, occupying greater than 25% of the nuclear volume. The presence of iron in the inclusions was confirmed by energy dispersive x-ray microanalysis. Immunocytochemical studies showed that the iron was present as aggregates of ferritin. Quantitation of nonaggregated ferritin molecules by image analyses after electron microscopy demonstrated that within 4 wk ferritin levels in cytoplasm and nucleoplasm had greatly increased but that there was a concentration gradient of approximately one order of magnitude across the nuclear envelope. These findings are consistent with the hypothesis that in iron-loaded mouse hepatocytes there is a slow passage of ferritin-molecules through the nuclear pores; the gradient is maintained by the continual aggregation of ferritin within the nucleus. Intranuclear ferritin may provide a source of iron for catalyzing hydroxyl radical formation in nuclei during some toxic, carcinogenic and aging processes.

Surface-induced aggregation of ferritin

The adsorption of ferritin from a water solution to a hydrophobic methylised quartz surface was studied by transmission electron microscopy, allowing direct examination of the iron core of the molecule without further preparation. The initial adsorption was seen to result in small clusters of molecules, the number of sites/cm(2) being concentration dependent. The adsorption process continued via cluster growth. The rate of adsorption increased and the process became mass transport limited. The clusters formed initially had low fractal dimensions (D approximately 1.0) and a coordination number, cn of 2.6-2.8, which increased with time. These clusters were abruptly restructured at a coordination number of 3.5, and the apparent rate of adsorption decreased during the reorganisation of the adsorbed layer. Finally, an equilibrium level was reached which was stable for at least 24 h. The distribution of ferritin molecules at equilibrium was in clusters with a fractal dimension of D = 1.14 +/- 0.16 and D= 1.33 +/- 0.08, respectively, for ferritin concentrations in the bulk of 10 and 100 microg/ml. Rinsing of adsorbed ferritin layers with buffered salt solution resulted in a rapid transient condensation of the clusters, but the net dissociation of protein was slow with the rate of dissociation being proportional to the logarithm of time. The condensed clusters were slowly restructured to linear polymers of ferritin molecules with a coordination number of 1.9 after 24 h of rinsing. The dissociation of protein molecules continued slowly for more than 3 days of rinsing. The results of the present study indicate that the rate of protein adsorption and desorption is strongly related to the supramolecular structure of the adsorbed protein film. Dense clusters of protein are not stable and this phenomenon may explain the formation of a dynamic equilibrium in spite of the fact that protein adsorption to a solid phase may appear to be practically irreversible.