Ferritins Research Articles

A Hollow NaGdF4/AFn Nanosystem Based on “Relay Race” Release for Therapy

To develop a multifunctional nanomaterial for dual-mode imaging and synergetic chemotherapy, curcumin (CUR) was physically entrapped into hollow upconversion NaGdF4 nanomaterial, then apoferritin (AFn) loaded with doxorubicin (DOX) was attached to the NaGdF4 surface. Subsequent modification with the targeting reagent folic acid (FA) led to generation of the CUR/NaGdF4 -DOX/AFn-FA conjugate for cancer treatment. X-ray diffraction, scanning (SEM) and transmission electron microscopy (TEM), and Fourier transform infrared (FTIR) spectroscopy demonstrated the successful preparation of hexagonal-phase NaGdF4 and NaGdF4 -AFn-FA. Moreover, no toxicity was observed for NaGdF4 -AFn-FA. In vitro and in vivo experiments demonstrated that the two drugs are sequentially released from the nanocomposites. This two-drug system showed strong growth inhibitory effects on MCF-7 cells. Upconversion luminescence imaging and magnetic resonance (MR) imaging of NaGdF4 -AFn-FA were carried out. The results of this study show that NaGdF4 -AFn-FA can be used for targeted anticancer drug delivery as well as imaging, a novel multi-pronged theranostic system for tumor treatment.

Commentary on "Ferritins: Furnishing proteins with iron"

Commentary on "Ferritins: Furnishing proteins with iron"

Research progress of clinical values of ferritin

A growing number of clinical roles have been attributed to ferritin in the aspects of diagnosis, monitoring and treatment. Low level of serum ferritin has high specificity to diagnosis of iron deficiency anemia. Low level of serum ferritin may be involved in the pathogenesis of nervous system diseases in children and senior patients. Hyperferritinemia has uniquely value in the diagnosis and monitoring of chronic kidney disease, autoimmune disease and hemophagocytic lymphohistiocytosis. Serum ferritin level is positive correlation with the degree of liver fibrosis and metabolism syndrome. Ferritin is not only the biomarker of malignant diseases, but also the excellent nanoparticle carrier for treatment of malignant diseases. In this review, we summarize the clinical value of ferritin for a comprehensive understanding of the role of ferritin in clinical practice. Key words: Ferritins; Disease; Institutional practice

Construction of magnetic-carbon-quantum-dots-probe-labeled apoferritin nanocages for bioimaging and targeted therapy.

Carbon dots (CDs) are one of the most highlighted carbon-based materials for biological applications, such as optical imaging nanoprobes, which are used for labeling cells in cancer treatment mainly due to their biocompatibility and unique optical properties. In this study, gadolinium (Gd)-complex-containing CDs were obtained through a one-step microwave method to develop multimodal nanoprobes integrating the advantages of optical and magnetic imaging. The obtained Gd-CDs exhibited highly fluorescent properties with excellent water solubility and biological compatibility. Natural apoferritin (AFn) nanocages, an excellent drug delivery carrier, are hollow in structure, with their pH-dependent, unfolding–refolding process at pH 2.0 and 7.4. The chemotherapeutic drug doxorubicin (DOX) can be highly effective and encapsulated into AFn cavity. A widely used tumor-targeting molecule, folic acid (FA), functionalized the surface of AFn to obtain an active tumor targeting effect on MCF-7 cells and malignant tumors in mice models. In this study, an AFn nanocarrier encapsulating high concentration of DOX labeled with magnetic and fluorescent Gd-CDs probe was developed. Gd-CDs exhibited a unique green photoluminescence and almost no toxicity compared with free GdCl3. Furthermore, Gd-doped CDs significantly increased the circulation time and decreased the toxicity of Gd3+ in in vitro and in vivo magnetic resonance imaging, which demonstrated that the AFn nanocages labeled with Gd-CD compounds could serve as an excellent T1 contrast agent for magnetic resonance imaging. The self-assembling multifunctional Gd-CDs/AFn (DOX)/FA nanoparticles have a great potential for cancer theranostic applications.

Detection of serum ferritin in patients with idiopathic inflammatory myopathies and its clinical significance

Objective To determine serum ferritin levels in patients with idiopathic inflammatory myopathies(IIM), including dermatomyositis(DM), clinical amyopathic dermatomyositis(CADM)and polymyositis(PM), and to evaluate their association with disease activity, especially with interstitial lung disease(ILD). Methods Clinical data and laboratory results were collected from 120 patients with IIM. A double-site enzyme immunoassay was conducted to measure serum ferritin levels in 120 patients with IIM and 63 healthy human controls. Statistical analysis was carried out to assess the relationship of serum ferritin levels with inflammatory biomarkers, autoantibodies, serum muscle enzymes, and severity of ILD in patients with IIM. Results Serum ferritin levels were elevated in 36 out of the 120 patients, but normal in the other 84 patients. Compared with patients with normal serum ferritin levels, those with elevated serum ferritin levels showed increased levels of C-reactive protein(14.1 ± 6.5 vs. 3.6 ± 1.7 mg/L, P < 0.01), aspartate aminotransferase(111.8 ± 44.6 vs. 46.0 ± 9.0 U/L, P< 0.01), lactate dehydrogenase(388.6 ± 81.5 vs. 260.7 ± 29.1 U/L, P< 0.01), as well as erythrocyte sedimentation rate(34.8 ± 8.2 vs. 15.4 ± 2.7 mm/h, P< 0.01). However, no significant difference was observed in the level of creatine kinase between patients with elevated and normal serum ferritin levels. Moreover, serum ferritin levels were significantly higher in both IIM patients complicated with acute/subacute interstitial pneumonia(A/SIP, 650.5 ± 268.5 ng/ml)and those with chronic interstitial pneumonia(CIP, 489.9 ± 157.3 ng/ml)than in those without ILD(155.7 ± 90.8 ng/ml)and those with imageological changes but no clinical symptoms of ILD(193.3 ± 62.1 ng/ml)(all P < 0.01). Moreover, compared with patients with normal serum ferritin levels, those with elevated serum ferritin levels showed a significant increase in the incidence of CIP(52.8% vs. 25.9%, P < 0.01), A/SIP(22.2% vs. 3.5%, P < 0.01), as well as appearance of ground-glass opacity(67.6% vs. 43.4%, P < 0.05)and honeycomb-like changes(14.7% vs. 2.6%, P < 0.05)on high-resolution CT scans. Conclusion Serum ferritin levels are highly correlated with erythrocyte sedimentation rate, C-reactive protein, aspartate aminotransferase, lactate dehydrogenase, ILD and high-resolution CT findings, and may be of great clinical significance for the evaluation of disease activity, especially the severity of ILD. Key words: Myositis; Dermatomyositis; Ferritins; Lung diseases, interstitial; Idiopathic interstitial pneumonias

Site-Directed Conjugation of Antibodies to Apoferritin Nanocarrier for Targeted Drug Delivery to Prostate Cancer Cells.

Herein, we describe a novel approach for targeting of ubiquitous protein apoferritin (APO)-encapsulating doxorubicin (DOX) to prostate cancer using antibodies against prostate-specific membrane antigen (PSMA). The conjugation of anti-PSMA antibodies and APO was carried out using HWRGWVC heptapeptide, providing their site-directed orientation. The prostate-cancer-targeted and nontargeted nanocarriers were tested using LNCaP and HUVEC cell lines. A total of 90% of LNCaP cells died after treatment with DOX (0.25 μM) or DOX in nontargeted and prostate-cancer-targeted APO, proving that the encapsulated DOX toxicity for LNCaP cells remained the same. Free DOX showed higher toxicity for nonmalignant cells, whereas the toxicity was lower after treatment with the same dosage of APO-encapsulated DOX (APODOX) and even more in prostate-cancer-targeted APODOX. Hemolytic assay revealed exceptional hemocompatibility of the entire nanocarrier. The APO encapsulation mechanism ensures applicability using a wide variety of chemotherapeutic drugs, and the presented surface modification enables targeting to various tumors.

Ratiometric Time-Gated Luminescence Probe for Nitric Oxide Based on an Apoferritin-Assembled Lanthanide Complex-Rhodamine Luminescence Resonance Energy Transfer System

Using apoferritin (AFt) as a carrier, a novel ratiometric luminescence probe based on luminescence resonance energy transfer (LRET) between a Tb(3+) complex (PTTA-Tb(3+)) and a rhodamine derivative (Rh-NO), PTTA-Tb(3+)@AFt-Rh-NO, has been designed and prepared for the specific recognition and time-gated luminescence detection of nitric oxide (NO) in living samples. In this LRET probe, PTTA-Tb(3+) encapsulated in the core of AFt is the energy donor, and Rh-NO, a NO-responsive rhodamine derivative, bound on the surface of AFt is the energy acceptor. The probe only emits strong Tb(3+) luminescence because the emission of rhodamine is switched off in the absence of NO. Upon reaction with NO, accompanied by the turn-on of rhodamine emission, the LRET from Tb(3+) complex to rhodamine occurs, which results in the remarkable increase and decrease of the long-lived emissions of rhodamine and PTTA-Tb(3+), respectively. After the reaction, the intensity ratio of rhodamine emission to Tb(3+) emission, I565/I539, is ∼24.5-fold increased, and the dose-dependent enhancement of I565/I539 shows a good linearity in a wide concentration range of NO. This unique luminescence response allowed PTTA-Tb(3+)@AFt-Rh-NO to be conveniently used as a ratiometric probe for the time-gated luminescence detection of NO with I565/I539 as a signal. Taking advantages of high specificity and sensitivity of the probe as well as its good water-solubility, biocompatibility, and cell membrane permeability, PTTA-Tb(3+)@AFt-Rh-NO was successfully used for the luminescent imaging of NO in living cells and Daphnia magna. The results demonstrated the efficacy of the probe and highlighted it's advantages for the ratiometric time-gated luminescence bioimaging application.

Dragon fruit-like biocage as an iron trapping nanoplatform for high efficiency targeted cancer multimodality imaging

Natural biopolymer based multifunctional nanomaterials are perfect candidates for multimodality imaging and therapeutic applications. Conventional methods of building multimodal imaging probe require either cross-linking manners to increase its in vivo stability or attach a target module to realize targeted imaging. In this study, the intrinsic photoacoustic signals and the native strong chelating properties with metal ions of melanin nanoparticle (MNP), and transferrin receptor 1 (TfR1) targeting ability of apoferritin (APF) was employed to construct an efficient nanoplatform (AMF) without tedious assembling process. Smart APF shell significantly increased metal ions loading (molar ratio of 1:800, APF/Fe3+) and therefore improved magnetic resonance imaging (MRI) sensitivity. Moreover, synergistic use of Fe3+ and APF contributed to high photoacounstic imaging (PAI) sensitivity. AMF showed excellent bio-stability and presented good in vivo multimodality imaging (PET/MRI/PAI) properties (good tumor uptake, high specificity and high tumor contrast) in HT29 tumor because of its targeting property combined with the enhanced permeability and retention (EPR) effect, making it promising in theranostics and translational nanomedicine.

The changes of the serum ferritin,fibrinogen and D-dimer levels in patients with small vessel disease and its significance

Objective To explore the correlation of serum ferritin （SF）,fibrinogen（FIB） and D-dimer levels in patients with small vessel disease（SVD）.Methods 359 patients with SVD were selected,including 186 patients with lacunar infarction （ LI） and 173 patients with leukoaraiosis （ LA） .176 healthy controls were enrolled in this study as control group.The SF,FIB and D-dimer concentrations were measured by radioimmunoassay method and Clauss method respectively .Results The serum levels of SF , FIB and D-dimer in SVD patients were significantly higher than those in the controls [（171.82 ±156.28）μg/L vs （100.37 ±70.11）μg/L,（3.27 ±0.93） g/L vs （2.97 ± 0.88）g/L,（1.10 ±1.06）mg/L vs （0.83 ±0.55）mg/L]（all P〈0.05）.Compared with LI subgroup,the levels of SF,FIB and D-dimer were higher in LA subgroup [（185.22 ±108.55）μg/L vs （159.36 ±89.69）μg/L,（3.32 ± 0.86）g/L vs （3.23 ±0.99）g/L,（1.29 ±1.17）mg/L vs （1.03 ±0.80）mg/L],but the differences were not signifi-cant（all P〈0.05）.Multivariate regression analysis revealed that by comparison to the reference quartile ,the odds ra-tio （95%CI） for SVD patients in the higher quartile （SF>151.16μg/L,D-Dimer>1.02 mg/L） were 3.444（95%CI=2.097-5.158,P〈0.001） and 1.767（95%CI=1.114-2.803,P=0.016）,respectively after multivariable ad-justment.Conclusion The increased levels of SF ,FIB and D-dimer are closely related with SVD .FIB and D-dimer might come to be attractive approaches to prevent the progress of SVD . Key words: Small vessel disease; Ferritins; Fibrinogen; D-dimer

Direct evidence of spatially selective iron mineralization using an immobilized ferritin protein cage.

(Apo)ferritins are cage-shaped proteins which have recently received a great deal of attention because the inner cavity of the protein shell can be used as a size-restricted reaction field for the synthesis of nanomaterials. The biomineralization behavior and inorganic nanoparticle (NP) synthesis mechanism of (apo)ferritin in solution systems have been studied but the mineralization behavior of (apo)ferritin on the substrates has not yet been well studied. Here, we conducted quantitative and kinetic analyses of the mineralization behavior of immobilized (apo)ferritin on a polyelectrolyte multilayer (PEM) using quartz crystal microbalance (QCM), scanning electron microscopy (SEM), and X-ray photoelectron spectroscopy (XPS) techniques. We demonstrated that the (apo)ferritin immobilized on a substrate synthesizes a ferrihydrite core within the confines of the protein cage; similar to a solution dispersed system. In addition, we applied a ferritin/apoferritin blended monolayer to the study of iron mineralization and revealed that biomineralization in this system is spatially selective. It is important to understand the mineralization mechanisms for the synthesis of other functional NPs as this approach has potential for a broad range of magnetic, catalytic, and biomedical sensing applications.

Correlation between femoral head bone iron content, serum ferritin and bone mineral density of postmenopausal women with femoral neck fragility fracture

Objective To explore the correlation between femoral head bone iron content,serum ferritin (SF) and bone mineral density of postmenopausal women with femoral neck fragility fracture.Methods From June 2010 to March 2013,156 postmenopausal women with femoral neck fragility fractures aged from 56 to 92 years old,with an average age of 72.40±8.97 years,were divided into 5 groups based upon the stratification of 10-year age intervals (≤60,61-70,71-80,81-90 and ≥91).Blood samples were collected to determine iron metabolism and bone metabolic markers after overnight fasting.Bone specimens were obtained for detecting bone iron contents and performing iron stain respectively after hip arthroplasty surgery.BMD was measured by dual energy X-ray absorptiometry(DXA)at hip and lumbar spine from L1 to L4 ten days after surgery.Results There were significant differences in the values of bone iron content,SF,transferrin,total iron binding capacity,serum procollagen type Ⅰ N-terminal propeptide,β-carboxy terminal telopeptide of collagen type Ⅰ and BMD at hip and L1 to L4 in all groups.BMD at hip and L1 to L4 decreased while bone iron content and SF increased with age; the peaks of bone iron content and SF were both in 81-90 years group; the average of bone iron content and SF was 96.81 μg/g and 235.66 μg/L respectively.There were 100 patients (100/156,64.1％) whose SF was over 200 μg/L.Bone iron content combined with SF,age and BMI could be put into the regression models of BMD at various proximal femur sites (femoral neck R2=0.443,Wards triangle R2=0.397,greater trochanter R2=0.322 and total femur R2=0.379).After adjustment of age,weight,BMI and other factors,we found that bone iron content and SF were negatively correlated with BMD at proximal femur,but had no significant correlation with BMD at lumbar spine.Conclusion Iron accumulation is prevalent in postmenopausal women with hip fragility fractures.High bone iron content and SF are independent risk factors for low BMD at proximal femur.These results provide clinical evidences that iron accumulation is associated with postmenopausal osteoporosis. Key words: Osteoporosis, postmenopausal; Femur head; Ferritins; Bone density

Clinical significance of urine ferritin in patients with cerebral infarction

Objective To find a indicator in urine to assist diagnosis of cerebral infarction, we investigated the changes of urine ferritin in patients with cerebral infarction. Methods Collected serum from 30 healthy volunteers and 53 patients with cerebral infarction(CI), with ratio of males to females 19∶11 vs. 30∶23, and average age was （56±11） vs. （63±11） respectively.Collected urine from 39 healthy volunteers and 33 patients with CI, with ratio of males to females 25∶14 vs. 20∶13, and average age was （57±10） vs. （64±11） respectively.Ferritin concentration in all samples was detected with the chemical luminescence method, and the urine ferritin/urine cretinin (uFer/uCr) ratio was calculated.Differences of serum ferritin and uFer/uCr between healthy volunteers and CI patients were analyzed with the statistical software under Wilcoxon test.Urinary proteins were extracted with absolute ethanol.The extracted ferritin was displayed with Western blot for light chains and heavy chains to investigate the difference between healthy volunteers and CI patients. Results uFer/uCr was higher than that from healthy volunteers（U=292.00，P<0.05）, and the median was (14.86 vs. 6.22) μg/g.Moreover, after extraction of urinary proteins with alcohol, both light chains and heavy chains of ferritin in urine from CI patients were much more obvious than that from healthy volunteers. Conclusion The uFer/uCr ratio could provide a new proof to assist the diagnosis of cerebral infarction.（Chin J Lab Med，2012,35：1146-1149） Key words: Brain infarction; Urinalysis; Ferritins

Preparation and evaluation of recombinant human ferritin light chain for quality control materials

Objective To construct a prokaryotic expression system of human ferritin L which could be overexpressed in E.coli, and then prepare quality control materials of rFL and evaluate the homogeneity, stability, precision and application value of the rFL.Methods RT-PCR was used to clone ferritin L gene with total RNA from peripheral blood.Ferritin L gene was inserted into plasmid pGM-T to construct subclone plasmid pGM-T/ferritin L, which was identified by sequencing.The recombinant plasmid pET-30a/ferritin L was transformed into E.coli BL21 (DE3) and efficiently expressed under IPTG induction.rFL was identified by SDS-PAGE, and its antigenicity was examined by WB.The concentration of rFL was measured by ACCESS immunoassay system.Fifteen control samples were randomly selected using random number table.The homogeneity and stability of rFL were evaluated by one-way analysis of variance (ANOVA) and a linear regression model, respectively.The uncertainties of homogeneity, stability and the precision were evaluated.Results A prokaryotic expression system of ferritin L was successfully constructed.Sequence analysis showed that the ferritin L gene inserted was identical with the one from GenBank(NM_000146.3).The PCR products were 527 bp long, in complete agreement with length of ferritin L gene.The molecular weight of rFL highly expressed in E.coli induced by IPTG was 19 000 Da.The WB analysis indicated that this rFL protein had good antigenicity.The concentration of rFL(1 000 times dilution) measured by ACCESS immunoassay system was (1115.84±38.38) ng/ml.Homogeneity evaluation of low-concentration QC sample and high-concentration QC sample of rFL showed that there were no statistical significances in the within-groups and between-groups (for high-concentration F=2.336, P>0.05 and for low-concentration F=0.730, P>0.05).A linear regression based on the stability test indicated that there was no statistically significant trend of instability in five and a half months (F=1.755, P>0.05). Precision analysis showed the within-run CV, the between-run CV, the between-day CV and the total CV of high-concentration QC sample were 2.06%, 2.12%, 0% and 2.96% respectively; the within-run CV, the between-run CV, the between-day CV and the total CV of low-concentration QC sample 2.03%, 2.08%, 0% and 2.90%, respectively.Conclusion This rFL for quality control materials with independent intellectual property rights could meet the clinical requirement of homogeneity, stability and precision, and could provide raw materials for preparation of mixed ferritin for quality control. Key words: Ferritins; Escherichia coli; Recombination,genetic; Gene expression; Quality control

Ferritin as a photocatalyst and scaffold for gold nanoparticle synthesis

The ferrihydrite mineral core of ferritin is a semi-conductor capable of catalyzing oxidation/reduction reactions. This report shows that ferritin can photoreduce AuCl4 − to form gold nanoparticles (AuNPs). An important goal was to identify innocent reaction conditions that prevented formation of AuNPs unless the sample was illuminated in the presence of ferritin. TRIS buffer satisfied this requirement and produced AuNPs with spherical morphology with diameters of 5.7 ± 1.6 nm and a surface plasmon resonance (SPR) peak at 530 nm. Size-exclusion chromatography of the AuNP–ferritin reaction mixture produced two fractions containing both ferritin and AuNPs. TEM analysis of the fraction close to where native ferritin normally elutes showed that AuNPs form inside ferritin. The other peak eluted at a volume indicating a particle size much larger than ferritin. TEM analysis revealed AuNPs adjacent to ferritin molecules suggesting that a dimeric ferritin–AuNP species forms. We propose that the ferritin protein shell acts as a nucleation site for AuNP formation leading to the AuNP-ferritin dimeric species. Ferrihydrite nanoparticles (~10 nm diameter) were unable to produce soluble AuNPs under identical conditions unless apo ferritin was present indicating that the ferritin protein shell was essential for stabilizing AuNPs in aqueous solution.

Characterization and cellular uptake of platinum anticancer drugs encapsulated in apoferritin

Clinical application of platinum-based anticancer drugs is largely limited by severe general toxicity and drug resistance. Drug delivery systems with tumor-targeting potential are highly desired for improving the efficacy and applicability of these drugs. This study describes an alternative strategy for the delivery of platinum drugs (cisplatin, carboplatin and oxaliplatin) by encapsulating each of them in the cavity of apoferritin (AFt). The encapsulation was achieved through manipulating the pH-dependent unfolding-refolding process of AFt at pH 2.0 and 7.4, respectively, in saturated drug solution. UV-vis spectrometry, circular dichroism spectrometry, dynamic light scattering, and inductively coupled plasma mass spectrometry were used to characterize the AFt-drug complexes. The loading capacity of AFt varies with respective drugs and the structural integrity of the protein shell remains intact after encapsulation. In vitro assays on the rat pheochromocytoma cell line (PC12) show that AFt-cisplatin inhibits the cells in a slow but sustaining mode and the cellular uptake of platinum is enhanced by AFt. AFt-carboplatin and AFt-oxaliplatin complexes only exhibit a marginal cytotoxicity towards this cell line under similar concentrations.

Agglomeration of proteins in acoustically levitated droplets

An ultrasonic trap (acoustic levitator) was used as an analytical tool to allow container-free handling of proteins in small sample volumes. This trap was combined for the first time with synchrotron small-angle X-ray scattering (SAXS) for structure analysis of biological macromolecules in a solution. The microfocus beamline at BESSY was used as a source of intense X-ray radiation. Apoferritin (APO) was used as a model protein, and its aggregation behavior in a levitator was followed from a diluted solution to the solid state. Different stages of APO agglomeration were observed without solid container walls, which may influence aggregation behavior and produce a parasitic scattering background. Starting with a volume of 5 microL we analyzed the concentration dependence of APO structure factors in the range from 5 to 1,200 mg/mL (solid protein). The solution was stirred automatically due to convection inside the droplet caused by the ultrasonic field. SAXS data recording of APO was performed in time intervals of 60 s during an aggregation experiment of 30 to 60 min.

The effects of temperature and radiation on holo and apo ferritin crystals

The effects of temperature and radiation on holo and apo ferritin crystals

Effect of microheterogeneity on horse spleen apoferritin crystallization

Apoferritin (APO) is an interesting model protein for crystal growth studies, as an alternative to the widely used hen egg white lysozyme. The effect of naturally occurring oligomers on the crystallization of isolated, microhomogeneous APO monomers (24 subunits, M r=440 000) was investigated. SDS PAGE analysis and immunoblotting showed that commercial APO was free of foreign proteins (>99.9% w/w). The quaternary structure of APO oligomers that form prior to the addition of precipitant was analyzed in native 4–15% T (1–2% C) gradient PAGE. Optical densitometry of these gels showed that oligomers (>24 subunit monomer) constituted approximately 45% w/w of the total APO. The primary oligomeric contaminants were dimers (48 subunits) with 35% w/w, and several bands constituting trimers (∼72 subunits) with 10% w/w. Directly determined physical molecular weights ( M w) and conformational data for oligomers obtained by analytical gel filtration fast protein-liquid-chromatography separations utilizing UV and multi-angle laser light scattering detectors (GF-FPLC-MALLS) confirmed and expanded the native PAGE results. This technique allowed the discovery of large oligomers ( M w=5 000 000 and 80 000 000) present in concentrations <1% w/w. Semi-preparative GF-FPLC was used to quantitatively reduce oligomer contamination to 5% w/w, and to produce 0.25 g of microhomogeneous monomers from 0.5 g APO. Crystallization from microhomogeneous monomer solutions yielded large crystals 0.5–1.0 mm in size. These crystals yielded an X-ray diffraction resolution of 1.8 Å. Reconstitutive experiments in which isolated oligomers were added to monomer preparations showed that dimers perturb the growth habit and reduce the crystal growth, without significantly affecting the nucleation. On trimer addition, the nucleation was increased and the crystal growth decreased. Addition of cadmium sulfate precipitant to unpurified APO did not affect the nature or quantity of the oligomers. These effects of oligomers on crystallization underline microheterogeneity as a critical factor in protein crystallization.

Isoferritins from tumorous and non-tumorous human liver.

Human ferritins purified from normal liver (LF) and from hepatocellular carcinoma (HF) showed different biochemical and immunological characteristics. Subunit preparations from the isoferritins showed differences in isoelectric focusing patterns, although both HF and LF were found to be composed of H and L subunits with apparently the same molecular weights. It is postulated that heterogeneities between the two isoferritins are not merely due to different proportions of H and L subunits, but also result from the existence of distinct species of the subunits bearing different surface electric charges.

Correlations between iron content and isoferritin profiles of normal and malignant tissues.

Isoelectric focusing profiles of ferritins isolated from livers of anemic rats and 3'-methyl-4-dimethylaminoazobenzene-induced hepatomas were more acidic than their normal counterparts, and exhibited patterns similar to those of heart ferritin. Iron administration induced a shift to more basic components for the normal and tumor ferritins. The isoferritin patterns reflected the subunit populations of the protein, as the more acidic components had greater proportions of H-type subunits and iron induced an increase in L subunits with concomitant increases in more basic isoferritins. For both normal and malignant tissues there was a direct correlation between iron content of the tissue and subunit composition. The apparently unique isoferritin patterns in the tumor tissue may, therefore, be ascribed primarily to the lower iron contents.