Fe-binding Proteins Research Articles

The determination of zinc, copper and iron oxidation state in invasive ductal carcinoma of breast tissue and normal surrounding tissue using XANES

X-ray absorption near-edge structure (XANES) spectroscopy was used to examine the oxidation state of Zn, Fe and Cu in 22 normal and 23 tumour regions spread over 30 formalin-fixed, paraffin-embedded tissue samples of human primary invasive breast cancer. A micro-mapping analysis of the metal distribution in the tissue was performed prior to the XANES analysis to identify and localise the metals in the tumour and normal tissue regions. The aim of this study was to identify the oxidation state of Zn, Fe and Cu in normal and tumour tissues of the breast, in order to correlate the oxidation state of these elements with the carcinogenesis process. The position of the Zn K-edge in normal and tumour tissues suggests that Zn exists in a bounded form. The shape of the Cu K-edge XANES spectra and the first derivative spectra of normal and tumour tissues shows that a significant portion of the total copper is present as Cu (I). Nevertheless, the position of the edges in the normal and tumour tissue spectra does not exclude the presence of Cu (II). The shape and position of both normal and tumour regions of the tissue suggest that they contain mixtures of Fe (II) and Fe (III) ions with a significant fraction being Fe (III). However, normal tissue regions were found to have a higher fraction of Fe (II) compared to the tumour tissues. In order to estimate the best target for therapy, more information is required about the relative abundance of Zn, Fe and Cu binding proteins, their oxidation state and their localisation at the subcellular level. Copyright © 2010 John Wiley & Sons, Ltd.

Gallium Disrupts Iron Uptake by Intracellular and ExtracellularFrancisellaStrains and Exhibits Therapeutic Efficacy in a Murine Pulmonary Infection Model

Francisella tularensis requires iron (Fe) for growth, but the biologic sources of Fe for this organism are largely unknown. We found that Francisella sp. growing in broth culture or within human macrophages can acquire Fe from the two major host Fe-binding proteins, lactoferrin (Lf) and transferrin (Tf). Fe acquisition is a potential target for novel therapies. Gallium (Ga) is a transition metal that interferes with cellular Fe metabolism by competing with Fe for uptake/utilization. Growth of either F. tularensis live vaccine strain (LVS) or Francisella novicida was inhibited by >or=2 microM Ga chelated to Tf or Lf, with GaLf being somewhat more potent. Francisella spp. express two Fe-containing antioxidant enzymes, catalase (KatG) and Fe cofactored superoxide dismutase (FeSOD). Growth of LVS with 10 muM GaTf or GaLf led to a dramatic decrease in bacterial catalase activity and in FeSOD activity that was associated with an increased susceptibility to H(2)O(2). Ga also protected mice from intranasal challenge with F. novicida. Whereas 100% of the F. novicida-infected mice died by day 9, 75% of the mice receiving Ga continued to survive to at least day 15. Thus, a single intranasal dose of Ga followed by daily intraperitoneal Ga at a dose tolerated by the animals resulted in prolonged survival. These data support the potential utility of Ga as a therapy for F. tularensis infection of the lung.

Oxidative damage in human epithelial alveolar cells exposed in vitro to oil fly ash transition metals

Among particulate matter emissions from combustion processes, oil fly ash (OFA) displays a marked oxidative and inflammogenic reactivity, due to the high content of bioavailable transition metals. In the present study, we evaluated the biological effects of an OFA water solution, composed of the transition metals Fe (57.5%), V (32.4%), and Ni (10.1%), in human epithelial alveolar cells (A549 line). The fluorimetric analysis by 2',7'-dichlorofluorescein showed a significant, dose- and time-dependent induction of intracellular reactive oxygen species (ROS) triggered by OFA metal components at subtoxic doses. The metal chelator deferoxamine and the radical scavenger dimethylsulfoxide attenuated the metal-induced generation of ROS. Confocal microscopy observations strengthened these findings and showed an intense cytoplasmic fluorescence with perinuclear thickenings in A549 cells, in the absence of morphological damage. Metal-induced generation of ROS was significantly correlated with a dose- and time-dependent DNA damage, as assessed by single cell gel electrophoresis (comet assay). Catalase was able to decrease dramatically DNA damage. Fluorimetric analyses by diphenyl-1-pyrenylphosphine showed a parallelism between generation of ROS and formation of lipid peroxides. The results obtained in the experiments evaluating the effects of individual metal solutions did not show any significant difference in DNA damage between Fe(III) and V(IV), but highlighted the higher capability of V(IV) to increase ROS in the cytoplasmic compartment. The different behavior of these two elements, confirmed by the weak Fe-induced lipid peroxidation, may be ascribed to the presence of Fe-binding proteins, such as ferritin, in the cytoplasm. Finally, Ni(II) had negligible effects on ROS production. On the whole, the results obtained in this study show the strong capability of transition metals adsorbed to OFA to cause widespread damage to biological macromolecules, and suggest potential health effects resulting from exposure to power plant emissions in industrialized sites.

Dietary inulin affects the expression of intestinal enterocyte iron transporters, receptors and storage protein and alters the microbiota in the pig intestine

Inulin, a linear beta fructan, is present in a variety of plants including chicory root and wheat. It exhibits prebiotic properties and has been shown to enhance mineral absorption and increase beneficial bacteria in the colon. The aim of the present study was to assess the effect of dietary inulin on the gene expression of selected intestinal Fe transporters and binding proteins. Anaemic piglets at age 5 weeks were allocated to a standard maize-soya diet (control) or the same diet supplemented with inulin at a level of 4 %. After 6 weeks, the animals were killed and caecum contents and sections of the duodenum and colon were removed. Segments of the genes encoding for the pig divalent metal transporter 1 (DMT1) and duodenal cytochrome-b reductase (Dcytb) were isolated and sequenced. Semi-quantitative RT-PCR analyses were performed to evaluate the expression of DMT1, Dcytb, ferroportin, ferritin, transferrin receptor (TfR) and mucin genes. DMT1, Dcytb, ferroportin, ferritin and TfR mRNA levels in duodenal samples were significantly higher in the inulin group (P < or = 0.05) compared with the control. In colon, DMT1, TfR and ferritin mRNA levels significantly increased in the inulin group. Additionally, the caecal content microflora was examined using 16S rDNA targeted probes from bacterial DNA. The Lactobacillus and Bifidobacterium populations were significantly increased in the inulin group (P < or = 0.05) compared with the control group. These results indicate that dietary inulin might trigger an up regulation of genes encoding for Fe transporters in the enterocyte. The specific mechanism for this effect remains to be elucidated.

Modern proteomes contain putative imprints of ancient shifts in trace metal geochemistry

Because of the rise in atmospheric oxygen 2.3 billion years ago (Gya) and the subsequent changes in oceanic redox state over the last 2.3-1 Gya, trace metal bioavailability in marine environments has changed dramatically. Although theorized to have influenced the biological usage of metals leaving discernable genomic signals, a thorough and quantitative test of this hypothesis has been lacking. Using structural bioinformatics and whole-genome sequences, the Fe-, Zn-, Mn-, and Co-binding metallomes of 23 Archaea, 233 Bacteria, and 57 Eukarya were constructed. These metallomes reveal that the overall abundances of these metal-binding structures scale to proteome size as power laws with a unique set of slopes for each Superkingdom of Life. The differences in the power describing the abundances of Fe-, Mn-, Zn-, and Co-binding proteins in the proteomes of Prokaryotes and Eukaryotes are similar to the theorized changes in the abundances of these metals after the oxygenation of oceanic deep waters. This phenomenon suggests that Prokarya and Eukarya evolved in anoxic and oxic environments, respectively, a hypothesis further supported by structures and functions of Fe-binding proteins in each Superkingdom. Also observed is a proliferation in the diversity of Zn-binding protein structures involved in protein-DNA and protein-protein interactions within Eukarya, an event unlikely to occur in either an anoxic or euxinic environment where Zn concentrations would be vanishingly low. We hypothesize that these conserved trends are proteomic imprints of changes in trace metal bioavailability in the ancient ocean that highlight a major evolutionary shift in biological trace metal usage.

Overexpression and Characterization of an Iron Storage and DNA-Binding Dps Protein from Trichodesmium erythraeum

Although the role of iron in marine productivity has received a great deal of attention, no iron storage protein has been isolated from a marine microorganism previously. We describe an Fe-binding protein belonging to the Dps family (DNA binding protein from starved cells) in the N(2)-fixing marine cyanobacterium Trichodesmium erythraeum. A dps gene encoding a protein with significant levels of identity to members of the Dps family was identified in the genome of T. erythraeum. This gene codes for a putative Dps(T. erythraeurm) protein (Dps(tery)) with 69% primary amino acid sequence similarity to Synechococcus DpsA. We expressed and purified Dps(tery), and we found that Dps(tery), like other Dps proteins, is able to bind Fe and DNA and protect DNA from degradation by DNase. We also found that Dps(tery) binds phosphate, like other ferritin family proteins. Fe K near-edge X-ray absorption of Dps(tery) indicated that it has an iron core that resembles that of horse spleen ferritin.

RNase E-based ribonucleoprotein complexes: mechanical basis of mRNA destabilization mediated by bacterial noncoding RNAs

Hfq-binding antisense small RNAs of Escherichia coli, SgrS and RyhB, mediate the destabilization of target mRNAs in an RNase E-dependent manner. SgrS, whose expression is induced in response to phosphosugar stress, act on the ptsG mRNA encoding a major glucose transporter, while RyhB, whose expression is induced in response to Fe depletion, acts on several mRNAs encoding Fe-binding proteins. In this report, we addressed the question of how SgrS and RyhB RNAs cooperate with RNase E to destabilize the target mRNAs. We demonstrate that Hfq along with SgrS and RyhB copurified with RNase E but not with truncated RNase E. In addition, we show that RNase E but not other degradosome components copurified with Hfq. Taken together, we conclude that RNase E forms variable ribonucleoprotein complexes with Hfq/small RNAs through its C-terminal scaffold region. These complexes, distinct from the RNA degradosome, may act as specialized RNA decay machines that initiate the degradation of mRNAs targeted by each small RNA. The present finding has uncovered the mechanical basis of mRNA destabilization mediated by bacterial small RNAs. The formation of ribonucleoprotein complexes containing RNases could be a general way by which small RNAs destabilize target mRNAs in both prokaryotes and eukaryotes.

Heme oxygenase-1: redox regulation and role in the hepatic response to oxidative stress.

Heme oxygenase (HO) catalyzes the oxidative cleavage of the alpha-mesocarbon of Fe-protoporphyrin-IX yielding equimolar amounts of biliverdin-IXalpha, free divalent iron, and carbon monoxide (CO). Among the three isoenzymes cloned to date, only HO-1 can be induced by a variety of seemingly disparate stimuli, most of which are linked by their ability to provoke oxidative stress. Although constitutive expression of HO-1 in the liver is restricted to Kupffer cells, the gene is inducible in nonparenchymal as well as in parenchymal liver cells. HO-1 induction potentially confers protection against oxidative stress in a variety of experimental models, such as liver ischemia/reperfusion secondary to transplantation or hemorrhage/resuscitation. Induction of HO-1 may protect the cell against oxidative injury by (a) controlling intracellular levels of "free" heme (a prooxidant), (b) producing biliverdin (an antioxidant), (c) improving nutritive perfusion via CO release, and (d) fostering the synthesis of the Fe-binding protein ferritin. Although protective effects of up-regulation of the HO pathway--presumably through production of bile pigments and CO--have been reported for a variety of cells and tissues, including the liver, evidence suggests that the protective action might be restricted to a rather narrow threshold of overexpression. High levels of HO-1 may even sensitize the cell to oxidative stress, e.g., through release of reactive iron. Transcriptional activation of the HO-1 gene is an integral part of the cellular response to oxidative stress, but its induction seems to be neither exclusively cytoprotective nor exclusively cytotoxic.

Iron loading and large doses of intravenous ascorbic acid promote lipid peroxidation in whole serum in guinea pigs.

Large doses of ascorbic acid may mobilise Fe from Fe-binding proteins in vivo which in turn could catalyse lipid peroxidation, a process associated with degenerative diseases. This hypothesis was tested in vitro in the serum of Fe-loaded animals. Eighteen male guinea pigs weighing about 500 g on arrival were allocated to two groups of nine. Fe loading was induced in one group by two intraperitoneal injections of 200 mg iron dextran given on days 1 and 5. Blood (6 ml) was drawn from all animals on day 12 by cardiac puncture. Serum and LDL were separated. Serum was tested for loosely-bound Fe (bleomycin assay) and lipid peroxidation (thiobarbituric acid reactive substances (TBARS) assay) and LDL for susceptibility to in vitro oxidation (TBARS and conjugated diene assays). On day 12, another intraperitoneal injection of 200 mg iron dextran was given to the animals in the Fe-loaded group. On day 19, all animals were given 75 mg ascorbic acid by intraperitoneal injection. Blood (6 ml) was drawn 4 h later by cardiac puncture. Serum and LDL assays were repeated. Ascorbic acid increased loosely-bound Fe and in vitro oxidation in the serum from animals of the Fe-loaded group but not in the serum from animals of the control group. Susceptibility of LDL to in vitro oxidation increased after the ascorbic acid injection in the control group but there was no further increase in the Fe-loaded group. These data suggest that large doses of ascorbic acid promote Fe mobilisation and in vitro oxidation in the serum of Fe-loaded animals.

Molecular characterization of idiA and adjacent genes in the cyanobacteria Synechococcus sp. strains PCC 6301 and PCC 7942.

IdiA (iron-deficiency-induced protein A) is a protein expressed at highly elevated levels in Synechococcus sp. strains PCC 6301 and PCC 7942 under Fe- or Mn-limiting growth conditions. Besides being similar to two bacterial Fe-binding proteins, SfuA and FbpA, IdiA shows similarity to two ORFs (slr0513 and sir1295) of Synechocystis sp. PCC 6803. Northern blot analysis detected one transcript of about 1300 nt in RNA extracted from Synechococcus sp. PCC 6301 and PCC 7942 grown under Fe deficiency. The intensity of this transcript was considerably reduced in Fe-sufficient culture. It could be further shown that the regulation of IdiA expression is at the transcriptional level and that transcription and translation of IdiA are closely linked. Primer extension analysis indicated a single transcriptional start site 193 nt upstream of the first presumed translational start codon. Moreover, molecular characterization of the entire 5.8 kb chromosomal HindIII DNA fragment carrying the idiA gene from Synechococcus sp. PCC 6301 led to the identification of six long ORFs in addition to idiA. The two genes adjacent to idiA, and dpsA located 2018 nt downstream of idiA, were insertionally inactivated in Synechococcus sp. PCC 7942 and the corresponding mutants were partially characterized. These experiments provide evidence that the gene products of idiB, located immediately downstream of idiA, and of dpsA are involved in the activation of IdiA expression, since the absence of each of these two gene products prevents the greatly elevated expression of IdiA under nutrient deficiency.

Construction of recombinant chimeric human lactoferrin/bovine transferrins.

One of the contributing factors to bacterial pathogenesis is the ability to acquire iron (Fe) in the extremely Fe scarce environment of the host. The families Neisseriaceae and Pasteurellaceae, which include a number of human and veterinary pathogens, express outer membrane receptors that specifically bind and remove Fe from the hosts’ Fe binding proteins, transferrin (Tf) and lactoferrin (Lf)5. The bacterial Lf receptor was initially identified by affinity methods with immobilized human Lf (hLf) as the affinity matrix, using high salt and pH conditions to minimize nonspecific interactions11. These studies identified a single Fe-repressible outer membrane protein, lactoferrin binding protein (Lbp), in constrast to the two transferrin binding proteins, transferrin binding protein A (TbpA) and transferrin binding protein B (TbpB), that were isolated with a Tf affinity column11. Lbp shared several properties with TbpA and once the gene encoding Lbp was cloned9 it was apparent that the predicted protein sequence was homologous to TbpA.

19F NMR Studies of the Recombinant Human TransferrinN-Lobe and Three Single Point Mutants

19F NMR was utilitized to study human serum transferrin, which is a bilobal protein with a high-affinity metal-binding site in each lobe. The N-terminal lobe of the recombinant protein (residues 1–337; hTF/2N) and three single point mutants, H207E, W8Y and W128Y, were expressed in baby hamster kidney cells grown in media supplemented with 5-fluorotryptophan (5-F-Trp). The three tryptophan residues gave three well resolved 19F NMR resonances, which were assigned by site-directed mutagenesis of two of the three Trp residues to Tyr. It was found that conformational changes are induced by metal binding to hTF/2N and a site-directed mutant H207E which has a higher binding affinity for Fe(III). Shifts in the 19F NMR spectra indicated changes when proteins bound Fe(III) or Ga(III) along with a synergistic anion, e.g. oxalate or carbonate. The resonance for 5-F-Trp 264 did not change frequency during titration with either metal in hTF/2N or the H207E mutant. Two resonances corresponding to Trp 128 and Trp 8 showed high-field shifts upon metal binding. These studies have shown that the fluorine nucleus is sensitive to local conformational changes in the binding pocket when the synergistic anion is changed from carbonate to oxalate. In addition, the fluorine nucleus can identify areas of the protein which experience secondary structural change when ligand binds. The solution NMR studies showing dynamic changes are complementary to the crystal structures of this family of Fe(III) binding proteins. © 1997 John Wiley & Sons, Ltd.

Role of sialic acid residues in iron binding by human Lactoferrin - α

Lactoferrin (Lf - α) is a major constituent of the secondary granules of neutrophils. and is thought to be involved in bacteriostasis through high affinity binding of plasma Fe required for microbial growth. Lf - α is also the major Fe binding protein hi milk. Fe bound to Lf - α is available for metabolic use, but the mechanism of Fe release from Lf - α is unknown. Treatment of human Lf - α with neuraminidase to remove sialic acid residues reduced its ability to bind Fe and caused release of Fe already bound to the protein. Reduction in Fe binding capacity was dependent on tune of exposure to and concentration of neuraminidase, and was not due to Lf - α loss or degradation. Readdition of sialic acid to desialylated Lf - α using α - 2, 6 - sialyltransferase restored Fe binding. The results suggest that sialylation — desialylation reactions could play a role in physiologic binding and release of Fe from the very high affinity binding sites of Lf-α.

Regulation of iron absorption: proteins involved in duodenal mucosal uptake and transport.

Newly identified iron (Fe)-binding proteins isolated from both rat and human duodenal mucosa permit a better understanding of Fe absorption. Mucins bind Fe at acid pH to keep it soluble and available for absorption at the more alkaline pH of the duodenum; this explains the development of Fe deficiency in achlorhydric subjects. Integrin was identified on the surface of enterocytes in association with radioiron and is believed to facilitate the transfer of Fe through the microvillous membrane. Mobilferrin, a 56 kDa Fe-binding protein, was identified in enterocyte cytosol. It coprecipitates with integrin and appears in close association with integrin in the apical cytoplasm of absorptive cells. We postulate it accepts dietary Fe from integrin and acts as the shuttle protein from Fe in the cytoplasm. Since Fe in enterocytes remains in equilibrium with body stores, we postulate mucosal Fe uptake is regulated by the number of Fe-binding sites either occupied or unoccupied by Fe on mobilferrin. Fe repletion of enterocytes from body stores is probably accomplished via transferrin receptors on the basal membranes of enterocytes. Increased transfer of Fe from blood into absorptive enterocytes occurs in Fe-replete animals to inhibit mucosal uptake of dietary Fe. Little transfer of Fe from plasma to enterocytes occurs in Fe deficiency. Enhanced mucosal transfer into the body occurs with increased body need for Fe. The exact mechanism for mucosal transfer of Fe into the plasma has not been defined but may also be mediated by an integrin.(ABSTRACT TRUNCATED AT 250 WORDS)

A siderophore production mutant of Bordetella bronchiseptica cannot use lactoferrin as an iron source.

Bordetella bronchiseptica secreted a hydroxamate siderophore when grown in Fe-depleted medium. A Tn5lac insertion mutant of B. bronchiseptica, DBB22, did not produce this hydroxamate siderophore and was incapable of using lactoferrin as an Fe source. Our data suggest that B. bronchiseptica uses a siderophore for removal of Fe from lactoferrin and transferrin rather than relying upon a receptor for these host Fe-binding proteins.

Does the lens serve as a ‘sink’ for iron during ocular inflammation?

Twenty-four hours after induction of ocular inflammation by intravitreal injection of endotoxin (10 ng), the intraocular fluid (IOF, aqueous and vitreous humors) concentration of iron (Fe) increased. This was presumably due to entry of the plasma Fe-binding protein, transferrin, into the IOFs through disrupted blood ocular barriers. After 1 day of inflammation the Fe concentration in lenses from the inflamed eye was 60% greater than that measured in contralateral control lenses. By day 15, lens Fe concentration had returned to levels of the contralateral control lenses. There was a distinct relationship between the dose of endotoxin used and the amount of Fe accumulated by the lens. The Fe concentration in lenses from eyes injected with 100 ng endotoxin was 0·376±0·027 μg g −1 wet weight compared to 0·214±0·014 μg g −1 in lenses from eyes injected with 0·25 ng endotoxin. In a previous study, copper (Cu) concentration in the IOFs was elevated to the same extent as Fe in response to intravitreal injection of endotoxin. However, in the present study, lenticular Cu concentration was unaltered at the highest (100 ng) dose of endotoxin. Since the increase in lens uptake was selective for Fe, there may be a specific Fe uptake mechanism in this ocular tissue. The physiological reasons for and possible pathological consequences of such a process are discussed.

Transferrin as a muscle trophic factor.

Muscle cells grow by proliferation and protein accumulation. During the initial stages of development the participation of nerves is not always required. Myoblasts and satellite cells proliferate, fusing to form myotubes which further differentiate to muscle fibers. Myotubes and muscle fibers grow by protein accumulation and fusion with other myogenic cells. Muscle fibers finally reach a quasi-steady state which is then maintained for a long period. The mechanism of maintenance is not well understood. However, it is clear that protein metabolism plays a paramount role. The role played by satellite cells in the maintenance of muscle fibers is not known. Growth and maintenance of muscle cells are under the influence of various tissues and substances. Among them are Tf and the motor nerve, the former being the main object of this review and essential for both DNA and protein synthesis. Two sources of Tf have been proposed, i.e., the motor nerve and the tissue fluid. The first proposal is that the nervous trophic influence on muscle cells is mediated by Tf which is released from the nerve terminals. In this model, the sole source of Tf which is donated to muscle cells should be the nerve, and Tf should not be provided for muscle fiber at sites other than the synaptic region; otherwise, denervation atrophy would not occur, since Tf provided from TfR located at another site would cancel the effect of denervation. The second proposal is that Tf is provided from tissue fluid. This implies that an adequate amount of Tf is transferred from serum to tissue fluid; in this case TfR may be distributed over the entire surface of the cells. The trophic effects of the motor neuron have been studied in vivo, but its effects of myoblast proliferation have not been determined. There are few experiments on its effects on myotubes. Most work has been made on muscle fibers, where innervation is absolutely required for their maintenance. Without it, muscle fibers atrophy, although they do not degenerate. In contrast, almost all the work on Tf has been performed in vitro. Its effects on myoblast proliferation and myotube growth and maintenance have been established; myotubes degenerate following Tf removal. But its effects on mature muscle fibers in vivo are not well understood. Muscle fibers possess TfR all over on their cell surface and contain a variety of Fe-binding proteins, such as myoglobin. It is entirely plausible that muscle fibers require an amount of Tf, and that this is provided by TfR scattered on the cell surface.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of cadmium on Fe+3-transferrin formation in the rat intestinal mucosa.

The effect of cadmium (Cd) on iron (Fe) metabolism has been an important subject for Cd toxicity, since anemia is usually observed in Itai-Itai patients who are exposed for long periods to Cd from the surrounding environment. It was previously accepted that Cd-induced anemia was not dependent on the route of administration. Thereafter, however, it was shown that oral Cd administration was essential for the development of anemia. Studies suggest that one of the possible sensitive sites of competition between Cd and Fe is in the gastrointestinal tract. Cd competes with Fe at one or more steps in the transport system and these metals undergo the same step(s) during their absorption. An hypothesis implies that these two metals possess a common carrier. Two Fe-binding proteins, ferritin and transferrin are well documented in the case of Fe deficiency but not in Cd exposed animals. Recently, the authors reported that the status of mucosal Fe-binding proteins in rats fed with Cd was similar to that in the Fe deficient rats. The present work was performed in an attempt to clarify the effect of in vivo and in vitro Cd on mucosal transferrin formation, which is one of two main Fe-binding proteins.

Transferrin, haematological indices and liver glycogen in field mice collected from a coal mining area and a control site in Poland

A polymorphism for serum transferrin in field mice from polluted and control sites was compared with body-morphological and haematological parameters, and with liver glycogen concentration. The distribution of genotypes in the polluted area was not panmictic, due to higher frequencies of the rare allele Trf c occurring mainly in males. In the polluted area, transferrin-c was accompanied by slowly migrating, Fe-binding protein fractions. Since the Trf c/Trf c homozygote possessed higher red blood cell parameters, it is suggested that these extra proteins have an Fe-transport function. The morpho-physiology of heterozygotes from the polluted area did not differ significantly from those from the control site. The animals from the polluted area possessed a high concentration of liver glycogen, indicative of poor environmental conditions. Also, a generally lower level of red blood cell parameters suggests that non-oxygenic processes dominate over oxygenic ones in the polluted area.

Identification of ferritin as a major high molecular weight zinc-binding protein in the tropical rock oyster,Saccostrea cuccullata

The metal-binding proteins of the body mass of the tropical rock oyster Saccostrea cuccullata from the iron-ore shipping terminals near Dampier, Western Australia, have been separated by gel chromatography and analyzed for Zn, Fe, and Cu. Appreciable amounts of Zn (40%) and Cu (25%) were eluted in association with a high molecular weight fraction (>60,000 daltons), the major component of which was identified as the heat stable Fe-binding protein, ferritin (approx. mol. wt. 550,000), which also contains 95% of the Fe detected. Ferritin thus accounts for significant amounts of protein-bound Zn in this species and most probably in other bivalves containing elevated tissue levels of Zn.