Ultrastructural silver enhancement of Prussian blue-reactive iron in hematopoietic and intestinal cells

Prussian blue has been widely used to localize iron in a variety of tissues at the light and electron microscopic level. In the present study, thin sections of human marrow and blood cells and rat duodenal cells were exposed to silver proteinate (SP) after staining en bloc with acid ferrocyanide (AF), with and without prior iron saturation using iron nitrilotriacetate (FeNTA). Silver deposition was observed over Prussian blue-reactive sites and significantly enhanced sites of minimal AF and FeNTA-AF staining. AF-SP stain deposits were present in the cytoplasmic matrix, granules, and occasionally on the surfaces of macrophages, monocytes, and erythroblasts. FeNTA-AF-SP stained additional cytoplasmic and surface sites in erythroblasts and stained neutrophil granules intensely. Duodenal epithelium from iron-loaded rats demonstrated strong AF-SP staining of ferric iron in microvilli, apical cytoplasmic matrix, and lateral membranes. Similar preparations from iron-replete rats stained sparsely; however, intense AF-SP staining was observed after iron saturation with FeNTA. SP similarly enhanced luminal ferrous iron deposits stained with acid ferricyanide in rats given intraluminal ferrous iron. AF-SP stain deposits were removed by exposure of thin sections to NH4OH, KCN, or HNO3 but were not affected by prior exposure to HIO4 or NaBH4, consistent with a silver cyanide or complex stain precipitate rather than reduced silver or silver ferriferrocyanide. SP enhancement of Prussian blue allows identification of reactive sites not readily visualized with AF or FeNTA-AF alone, and offers the potential for differentiating AF staining from other deposits or organelles of comparable density.     

Structure of frataxin iron cores: an X-ray absorption spectroscopic study

X-ray absorption spectroscopy at the iron K-edge indicates that the iron cores of human and yeast frataxin polymers assembled in vitro are identical to each other and are similar but not identical to ferritin cores. Both frataxin polymers contain ferrihydrite, a biomineral composed of ferric oxide/hydroxide octahedra. The ferrihydrite in frataxin is less ordered than iron cores of horse spleen ferritin, having fewer face-sharing Fe-Fe interactions but similar double corner-sharing interactions. The extended X-ray absorption fine structure (EXAFS) analysis agrees with previous electron microscopy data showing that frataxin cores are composed of very small ferrihydrite crystallites.     

Magnetic resonance cell-tracking studies: spectrophotometry-based method for the quantification of cellular iron content after loading with superparamagnetic iron oxide nanoparticles

The purpose of this article is to present a user-friendly tool for quantifying the iron content of superparamagnetic labeled cells before cell tracking by magnetic resonance imaging (MRI). Iron quantification was evaluated by using Prussian blue staining and spectrophotometry. White blood cells were labeled with superparamagnetic iron oxide (SPIO) nanoparticles. Labeling was confirmed by light microscopy. Subsequently, the cells were embedded in a phantom and scanned on a 3 T magnetic resonance tomography (MRT) whole-body system. Mean peak wavelengths λ(peak) was determined at A(720 nm) (range 719-722 nm). Linearity was proven for the measuring range 0.5 to 10 μg Fe/mL (r = .9958; p = 2.2 × 10(-12)). The limit of detection was 0.01 μg Fe/mL (0.1785 mM), and the limit of quantification was 0.04 μg Fe/mL (0.714 mM). Accuracy was demonstrated by comparison with atomic absorption spectrometry. Precision and robustness were also proven. On T(2)-weighted images, signal intensity varied according to the iron concentration of SPIO-labeled cells. Absorption spectrophotometry is both a highly sensitive and user-friendly technique that is feasible for quantifying the iron content of magnetically labeled cells. The presented data suggest that spectrophotometry is a promising tool for promoting the implementation of magnetic resonance-based cell tracking in routine clinical applications (from bench to bedside).     

Influence of site-directed modifications on the formation of iron cores in ferritin

The structure and crystal chemical properties of iron cores of reconstituted recombinant human ferritins and their site-directed variants have been studied by transmission electron microscopy and electron diffraction. The kinetics of Fe uptake have been compared spectrophotometrically. Recombinant L and H-chain ferritins, and recombinant H-chain variants incorporating modifications in the threefold (Asp131----His or Glu134----Ala) and fourfold (Leu169----Arg) channels, at the partially buried ferroxidase sites (Glu62,His65----Lys,Gly), a putative nucleation site on the inner surface (Glu61,Glu64,Glu67----Ala), and both the ferroxidase and nucleation sites (Glu62,His65----Lys,Gly and Glu61,Glu64,Glu67----Ala), were investigated. An additional H-chain variant, incorporating substitution of the last ten C-terminal residues for those of the L-chain protein, was also studied. Most of the proteins assimilated iron to give discrete electron-dense cores of the Fe(III) hydrated oxide, ferrihydrite (Fe2O3.nH2O). No differences were observed for variants modified in the three- or fourfold channels compared with the unmodified H-chain ferritin. The recombinant L-chain ferritin and H-chain variant depleted of the ferroxidase site, however, showed markedly reduced uptake kinetics and comprised cores of increased diameter and regularity. Depletion of the inner surface Glu residues, whilst maintaining the ferroxidase site, resulted in a partially reduced rate of Fe uptake and iron cores of wider particle size distribution. Modification of both ferroxidase and inner surface Glu residues resulted in complete inhibition of iron uptake and deposition. No cores were observed by electron microscopy although negative staining showed that the protein shell was intact. The general requirement of an appropriate spatial charge density across the cavity surface rather than specific amino acid residues could explain how, in spite of an almost complete lack of identity between the amino acid sequences of bacterioferritin and mammalian ferritins, ferrihydrite is deposited within the cavity of both proteins under similar reconstitution conditions.     

Rate of iron transfer through the horse spleen ferritin shell determined by the rate of formation of Prussian Blue and Fe-desferrioxamine within the ferritin cavity

Iron (2+ and 3+) is believed to transfer through the three-fold channels in the ferritin shell during iron deposition and release in animal ferritins. However, the rate of iron transit in and out through these channels has not been reported. The recent synthesis of [Fe(CN)6]3-, Prussian Blue (PB) and desferrioxamine (DES) all trapped within the horse spleen ferritin (HoSF) interior makes these measurements feasible. We report the rate of Fe2+ penetrating into the ferritin interior by adding external Fe2+ to [Fe(CN)6]3- encapsulated in the HoSF interior and measuring the rate of formation of the resulting encapsulated PB. The rate at which Fe2+ reacts with [Fe(CN)6]3- in the HoSF interior is much slower than the formation of free PB in solution and is proceeded by a lag period. We assume this lag period and the difference in rate represent the transfer of Fe2+ through the HoSF protein shell. The calculated diffusion coefficient, D approximately 5.8x10(-20) m2/s corresponds to the measured lag time of 10-20 s before PB forms within the HoSF interior. The activation energy for Fe2+ transfer from the outside solution through the protein shell was determined to be 52.9 kJ/mol by conducting the reactions at 10 approximately 40 degrees C. The reaction of Fe3+ with encapsulated [Fe(CN)6]4- also readily forms PB in the HoSF interior, but the rate is faster than the corresponding Fe2+ reaction. The rate for Fe3+ transfer through the ferritin shell was confirmed by measuring the rate of the formation of Fe-DES inside HoSF and an activation energy of 58.4 kJ/mol was determined. An attempt was made to determine the rate of iron (2+ and 3+) transit out from the ferritin interior by adding excess bipyridine or DES to PB trapped within the HoSF interior. However, the reactions are slow and occur at almost identical rates for free and HoSF-encapsulated PB, indicating that the transfer of iron from the interior through the protein shell is faster than the rate-limiting step of PB dissociation. The method described in this work presents a novel way of determining the rate of transfer of iron and possibly other small molecules through the ferritin shell.     

Structure and composition of ferritin cores isolated from human spleen, limpet (Patella vulgata) hemolymph and bacterial (Pseudomonas aeruginosa) cells

Ferritin cores isolated from human spleen, limpet (Patella vulgata) hemolymph and bacterial (Pseudomonas aeruginosa) cells have been investigated by high resolution transmission electron microscopy, electron diffraction and chemical analysis. Hemosiderin particles isolated from thalassemic spleens also have been studied. The results show that there is a marked difference in structure and composition of the biomineral phases. Human ferritin and hemosiderin particles are single domain crystals of hydrated iron (III) oxide (ferrihydrite). Lattice fringes were low in contrast and often discontinuous within the central regions of the core. Heat treatment of human ferritins results in a 5 A shrinkage in particle size and an increase in the single crystalline nature of the core. In contrast, lattice images and electron diffraction of limpet and bacterial cores show no evidence of long-range crystallographic order. Chemical analysis indicates a high inorganic phosphate (Pi) (Fe/Pi = 1.71) content in bacterial ferritin compared with human ferritin (thalassemic) (Fe/Pi = 21.0). The high Pi content of bacterial ferritin suggests a hydrated amorphous iron (III) phosphate mineral core. Structural disorder within the limpet and bacterial cores may be associated with increased Pi content and increased oxidation in Fe(II), resulting in rapid mineral deposition. Growth of the iron (III) oxide cores in human ferritin is discussed on the basis of high resolution electron microscopy results.     

A sensitive post-DAB enhancement technique for demonstration of iron in the central nervous system

A technique is described for enhancing the reaction product of the staining reaction for iron in paraffin-embedded tissue from central nervous system (CNS). After amplification of the Prussian Blue staining reaction with 3,3-diaminobenzidine (DAB), the reaction product was further intensified using a stepwise treatment with silver methenamine, gold chloride and uranyl nitrate (post-DAB treatment). Following the Prussian Blue-DAB staining reaction, iron was seen only in glial cells and choroid plexus epithelial cells, whereas the post-DAB treatment revealed that neurons and endothelial cells of the brain capillaries were also positively stained. The post-DAB treatment resulted additionally in an increased intensity of the reaction product within choroid plexus epithelial cells compared to that obtained in sections subjected only to the Prussian Blue-DAB reaction. The reliability of the method was evaluated using liver sections as positive controls. Furthermore the higher sensitivity of the method was assessed using nitrocellulose filters containing serially diluted iron-saturated transferrin. The post-DAB method is simple and can easily be applied to formalin- or glutaraldehyde fixed, paraffin-embedded nervous and non-nervous tissue.     

Enhanced iron uptake of Saccharomyces cerevisiae by heterologous expression of a tadpole ferritin gene

We genetically engineered Saccharomyces cerevisiae to express ferritin, a ubiquitous iron storage protein, with the major heavy-chain subunit of tadpole ferritin. A 450-kDa ferritin complex can store up to 4,500 iron atoms in its central cavity. We cloned the tadpole ferritin heavy-chain gene (TFH) into the yeast shuttle vector YEp352 under the control of a hybrid alcohol dehydrogenase II and glyceraldehyde-3-phosphate dehydrogenase promoter. We confirmed transformation and expression by Northern blot analysis of the recombinant yeast, by Western blot analysis using an antibody against Escherichia coli-expressed TFH, and with Prussian blue staining that indicated that the yeast-expressed tadpole ferritin was assembled into a complex that could bind iron. The recombinant yeast was more iron tolerant in that 95% of transformed cells, but none of the recipient strain cells, could form colonies on plates containing 30 mM ferric citrate. The cell-associated concentration of iron was 500 microg per gram (dry cell weight) of the recombinant yeast but was 210 microg per gram (dry cell weight) in the wild type. These findings indicate that the iron-carrying capacity of yeast is improved by heterologous expression of tadpole ferritin and suggests that this approach may help relieve dietary iron deficiencies in domesticated animals by the use of the engineered yeast as a feed and food supplement.     

Reduction of Prussian Blue by the two iron-reducing microorganisms Geobacter metallireducens and Shewanella alga

Cyanide or cyanide-metal complexes are frequent contaminants of soil or aquifers at industrial sites, which can be released from such sites by outgassing or transport with the groundwater. They form very stable complexes with iron, which may occur in the subsurface as an insoluble blue mineral, the so-called Prussian Blue (Fe(4)[Fe(CN)(6)](3)). In this study, we show that the insoluble and colloidal Fe(III)-cyanide complex Prussian Blue can be reduced and utilized as electron acceptor by the dissimilatory iron-reducing bacteria Geobacter metallireducens and Shewanella alga strain BrY. The microbial reduction of the dark blue pigment Prussian Blue leads to the formation of a completely colourless solid mineral, presumably Prussian White (Fe(2)[Fe(CN)(6)]), which could be reoxidized through exposure to air, regaining the dark blue colour. In addition, the microorganisms were able to grow with Prussian Blue, using it as the sole electron acceptor. Geobacter metallireducens could also reduce Prussian Blue coatings on sand, which was sampled from a contaminated site.     

Mucosal iron binding proteins and the inhibition of iron absorption by endotoxin

The uptake of iron from a tied off jejunal segment into the body after the injection of a 59Fe labeled test dose was decreased after the administration of endotoxin by about 80% in both normal and iron deficient animals.--In the iron deficient group the distribution of 59Fe in the cytosol fraction of jejunal mucosa between transferrin and ferritin was determined chromatographically; the amount of 59Fe in the ferritin fraction increased remarkably after the endotoxin treatment and the ratio of both was changed in favor of ferritin.--It is hypothesized that the association of the diversion of iron to the mucosal ferritin with the decrease of the transport of iron into the blood caused by endotoxin might be the consequence of abnormal oxidations in the mucosa measured by others in liver tissue.     

Ferritin iron mineralization proceeds by different mechanisms in MOPS and imidazole buffers

The buffer used during horse spleen ferritin iron loading significantly influences the mineralization process and the quantity of iron deposited in ferritin. Ferritin iron loading in imidazole shows a rapid hyperbolic curve in contrast to iron loading in 3-(N-morpholino)propanesulfonic acid (MOPS), which displays a slower sigmoidal curve. Ferritin iron loading in an equimolar mixture of imidazole and MOPS produces an iron-loading curve that is intermediate between the imidazole and MOPS curves indicating that one buffer does not dominate the reaction mechanism. The UV-visible spectrum of the ferritin mineral has a higher absorbance from 250 to 450 nm when prepared in imidazole buffer than in MOPS buffer. These results suggest that different mineral phases form in ferritin by different loading mechanisms in imidazole and MOPS buffered reactions. Samples of 1500 Fe/ferritin were prepared in MOPS or imidazole buffer and were analyzed for crystallinity and using the electron diffraction capabilities of the electron microscope. The sample prepared in imidazole was significantly more crystalline than the sample prepared in MOPS. X-ray powder diffraction studies showed that small cores (~ 500 Fe/ferritin) prepared in MOPS or imidazole possess a 2-line ferrihydrite spectrum. As the core size increases the mineral phase begins to change from 2-line to 6-line ferrihydrite with the imidazole sample favoring the 6-line ferrihydrite phase. Taken together, these results suggest that the iron deposition mechanism in ferritin can be controlled by properties of the buffer with samples prepared in imidazole forming a larger, more ordered crystalline mineral than samples prepared in MOPS.     

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 M?ssbauer 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)     

Combined in situ zymography, immunofluorescence, and staining of iron oxide particles in paraffin-embedded, zinc-fixed tissue sections

Superparamagnetic iron oxide particles are used as potent contrast agents in magnetic resonance imaging. In histology, these particles are frequently visualized by Prussian blue iron staining of aldehyde-fixed, paraffin-embedded tissues. Recently, zinc salt-based fixative was shown to preserve enzyme activity in paraffin-embedded tissues. In this study, we demonstrate that zinc fixation allows combining in situ zymography with fluorescence immunohistochemistry (IHC) and iron staining for advanced biologic investigation of iron oxide particle accumulation. Very small iron oxide particles, developed for magnetic resonance angiography, were applied intravenously to BALB/c nude mice. After 3 hours, spleens were explanted and subjected to zinc fixation and paraffin embedding. Cut tissue sections were further processed to in situ zymography, IHC, and Prussian blue staining procedures. The combination of in situ zymography as well as IHC with subsequent Prussian blue iron staining on zinc-fixed paraffin-embedded tissues resulted in excellent histologic images of enzyme activity, protease distribution, and iron oxide particle accumulation. The combination of all three stains on a single section allowed direct comparison with only moderate degradation of fluorescein isothiocyanate-labeled substrate. This protocol is useful for investigating the biologic environment of accumulating iron oxide particles, with excellent preservation of morphology.     

Uptake and handling of iron from transferrin, lactoferrin and immune complexes by a macrophage cell line.

The murine macrophage-like cell line P388D1 has been used as a model to investigate whether iron acquired simultaneously from different sources (transferrin, lactoferrin, and ovotransferrin-anti-ovotransferrin immune complexes) is handled in the same way. P388D1 cells bound both lactoferrin and transferrin, but over a 6 h incubation period only the latter actually donated iron to the cells. When the cells were incubated with [55Fe]transferrin and [59Fe]ovotransferrin-anti-ovotransferrin immune complexes iron was acquired from both sources. However, there was a difference in the intracellular distribution of the two isotopes, proportionally more 55Fe entering haem compounds and less entering ferritin. When the cells were precultured in a low-iron serum-free medium almost no transferrin-iron was incorporated into ferritin, whereas the proportion of immune complex-derived iron incorporated into ferritin was unchanged. Lactoferrin enhanced the rate of cellular proliferation, as measured by [3H]thymidine incorporation, despite its inability to donate iron to the cells, suggesting a stimulatory effect independent of iron donation. In contrast immune complexes inhibited cell proliferation. These findings indicate that iron acquired from transferrin and iron acquired by scavenging mechanisms are handled differently, and suggest that more than one intracellular iron transit pool may exist.     

A comparison of an undecairon(III) complex with the ferritin iron core

The iron core of ferritin is comprised of up to 4,500 Fe(III) atoms as Fe2O3.nH2O, which is maintained in solution by a surrounding, spherical coat of protein. Organisms as diverse as bacteria and man use the ferritin iron-protein complex as a reservoir of stored iron for other essential proteins. To extend studies of the steps in polynuclear iron core formation, a recently characterized undecairon(III) oxo-hydroxo aggregate [Fe11 complex] (Gorun et al., J. Am. Chem. Soc. 109, 3337 [1987]) was examined by x-ray absorption spectroscopy as a model for an intermediate. The results, which are comparable to the previous x-ray diffraction studies, show near neighbors (Fe-O) at 1.90 A that are distinct from those in ferritin and a longer distance of 2.02 A. However, contributions from neighbors (Fe-C) known to exist at ca. 2.7 A were obscured by a highly ordered Fe-Fe interaction and were not detectable in the Fe11 complex in contrast to a previously characterized Fe(III) cluster bound to the protein coat. Of the two Fe-Fe interactions detectable in the Fe11 complex, the shortest, at 3.0 A is particularly interesting, occurring at the same distance as a full shell (CN = 6) in ferritin, but having fewer Fe neighbors (CN = 2-3) characteristic of an intermediate in core formation. The incomplete Fe-Fe shell is much more ordered than in ferritin, suggesting that the disorder in ferritin cores may be associated with the later steps of the core growth. Differences between the Fe11 complex and the full core of ferritin indicate the possibility of intermediates in ferritin iron formation that might be like Fe11.     

Ferrocyanide staining of transferrin and ferritin-conjugated antibody to transferrin.

To evaluate the ultrastructural distribution of transferrin on the surface of L1210 ascites tumor cells, we used ferrocyanide to stain ferric iron (Prussian blue reaction) in transferrin, as well as in ferritin conjugated to antibody that was immunologically attached to the transferrin. Small deposits averaging 5 nm in diameter identified transferrin iron, whereas large cuboidal deposits averaging 50 nm in diameter stained ferritin conjugated-antibody that was bound to both transferrin and apotransferrin on the cell surface. The ability of transferrin to deliver iron to ascites tumor cells was confirmed by kinetic studies of transferrin labeled with 59Fe and 125I. These preliminary results are consistent with release of transferrin iron at the cell surface and demonstrate additional uses for ferrocyanide in ultrastructural cytochemical techniques.     

Internal iron biomineralization in Imperata cylindrica, a perennial grass: chemical composition, speciation and plant localization

* The analysis of metal distribution in Imperata cylindrica, a perennial grass isolated from the banks of Tinto River (Iberian Pyritic Belt), an extreme acidic environment with high content in metals, has shown a remarkable accumulation of iron. This property has been used to study iron speciation and its distribution among different tissues and structures of the plant. * Mossbauer (MS) and X-ray diffraction (XRD) were used to determine the iron species, scanning electron microscopy (SEM) to locate iron biominerals among plant tissue structures, and energy-dispersive X-ray microanalysis (EDAX), X-ray fluorescence (TXRF) and inductively coupled plasma emission spectroscopy (ICP-MS) to confirm their elemental composition. * The MS spectral analysis indicated that iron accumulated in this plant mainly as jarosite and ferritin. The presence of jarosite was confirmed by XRD and the distribution of both minerals in structures of different tissues was ascertained by SEM-EDAX analysis. * The convergent results obtained by complementary techniques suggest a complex iron management system in I. cylindrica, probably as a consequence of the environmental conditions of its habitat.     

Morphological demonstration of non-heme iron and iron-binding proteins in placentas from cases of congenital nephrotic syndrome of Finnish type

Two placentas (gestational age 36 and 39 weeks respectively), obtained from Congenital Nephrotic Syndrome of Finnish type (CNF) cases, have been studied by histochemistry in relation to the presence of non-heme iron and iron-binding proteins (ferritin, transferrin and lactoferrin). Ten control placentas of gestational age ranging from 35 to 40 weeks have been also examined. Employing the Prussian Blue ferrocyanide method, an intense positive reaction of the trophoblastic basament membrane (TBM) was observed only in cases of CNF. By immunoperoxidase techniques, ferritin was demonstrated in the villous stroma whereas lactoferrin was identified only in the cytoplasm of polymorphonuclear neutrophils within fetal stem vessels; a clear positivity for transferrin was encountered on the apical site of the syncytial trophoblast. All these immunohistochemical findings were similar in CNF cases and in control placentas. Ultrastructural observations of terminal villi revealed the presence of osmiophilic granulo-filamentous hemosiderin-like bodies in TBM of placentas from CNF. Although the role of iron-binding proteins in the iron transport through the placenta seems to be unaltered in CNF, the presence of non-heme iron in TBM could represent a histochemical approach for placental diagnosis in this type of nephrotic syndrome.     

Separation of cellular iron containing compounds by electrophoresis

High resolution separation of metalloproteins and other iron compounds based on native gel electrophoresis followed by⁵⁹Fe autoradiography is described. Lysates of mouse spleen erythroid cells metabolically labeled with⁵⁹Fe-transferrin were separated on 3–20% polyacrylamide gradient gels in the presence of Triton X100 and detected by autoradiography. In addition to ferritin and hemoglobin, several compounds characterized by their binding of iron under different conditions were described. Iron chelatable by desferrioxamine migrated in the region where several high-molecular weight compounds were detected by silver staining. The technique is nondissociative, allowing identification of iron compounds with the use of specific antibodies. Cellular iron transport and the action of iron chelators on specific cellular targets can be investigated in many small biological samples in parallel.     

Ferritin iron kinetics and protein turnover in K562 cells

The binding, incorporation, and release of iron by ferritin were investigated in K562 cells using both pulse-chase and long term decay studies with 59Fe-transferrin as the labeled iron source. After a 20-min pulse of labeled transferrin, 60% of the 59Fe was bound by ferritin with the proportion increasing to 70% by 4 h. This initial binding was reduced to 35% when the cells were exposed to the chelator desferrioxamine (5 mM) for an additional 30 min. By 4 h the association of 59Fe with ferritin was unaffected by the presence of the chelator, and levels of 59Fe-ferritin were identical to those in control cells (70%). Between 4-10h there was a parallel decline in 59Fe-ferritin in both control and desferrioxamine-treated cells. When incoming iron was bound by ferritin it was, therefore, initially chelatable but with time progressed to a further, nonchelatable compartment. In turnover studies where ferritin was preloaded with 59Fe by overnight incubation, 50% of the label was released from the protein by 18 h, contrasting with a t 1/2 for cellular iron release of approximately 70 h. The half-time of 59Fe release from ferritin was accelerated to 11 h by the presence of desferrioxamine. The half-time for ferritin protein turnover determined by [35S]methionine labeling was approximately 12 h in the presence or absence of the chelator. Thus, when the reassociation of iron with ferritin was prevented by the exogenous chelator there was a concordant decay of both protein and iron moieties. The direct involvement of lysosomes in this turnover was demonstrated by the use of the inhibitors leupeptin and methylamine which stabilized both 59Fe (t 1/2 = 24 h) and 35S (t 1/2 = 25.6 h) labels. We conclude that in this cell type the predominant mechanism by which iron is released from ferritin is through the constitutive degradation of the protein by lysosomes.