Kinetics Of Iron Release Research Articles

The model pH-controlled delivery system based on gelatin-tannin hydrogels containing ferrous ascorbate: iron release in vitro

Hydrogels have become an essential class among all biomaterials. The specialized biomaterials are highly valued in the field of biomedical applications. One of the problems in wound management is local microelement deficiency associated with extensive wound lesions. The significant lack of elemental iron in the human body leads to serious consequences and prolongs treatment. The synthesis of gelatin-tannin hydrogels with ion delivery function is proposed in this study. The ability to release ions in low acid solution is a sphere of great interest. The pH drop in the wound cavity is usually associated with the contamination of some bacterial cultures. pH-controlled delivery of iron in buffer solutions (рН = 5.5/6.4/7.4) was considered for these hydrogels. The kinetics of iron release was determined by visible spectroscopy. Theoretical models were applied to describe the process of ion delivery. The structure of materials was examined by IR-spectroscopy and demonstrated the incorporation of ferrous ascorbate into hydrogel matrix. Thermal analysis was used to point out the key differences in thermal behavior by isoconversional methods (Flynn-Wall-Ozawa/Kissinger-Akahira-Sunose). The mechanical properties of the materials have been studied. The effect of iron ascorbate on polymer network parameters was discussed. The current study demonstrated the possibility of obtaining gelatin-tannin hydrogels for pH-dependent iron delivery. That provides future perspectives to expand the set of releasing microelements for biomedical applications.

Iron Mobilization from Ferritin in Yeast Cell Lysate and Physiological Implications.

Most in vitro iron mobilization studies from ferritin have been performed in aqueous buffered solutions using a variety of reducing substances. The kinetics of iron mobilization from ferritin in a medium that resembles the complex milieu of cells could dramatically differ from those in aqueous solutions, and to our knowledge, no such studies have been performed. Here, we have studied the kinetics of iron release from ferritin in fresh yeast cell lysates and examined the effect of cellular metabolites on this process. Our results show that iron release from ferritin in buffer is extremely slow compared to cell lysate under identical experimental conditions, suggesting that certain cellular metabolites present in yeast cell lysate facilitate the reductive release of ferric iron from the ferritin core. Using filtration membranes with different molecular weight cut-offs (3, 10, 30, 50, and 100 kDa), we demonstrate that a cellular component >50 kDa is implicated in the reductive release of iron. When the cell lysate was washed three times with buffer, or when NADPH was omitted from the solution, a dramatic decrease in iron mobilization rates was observed. The addition of physiological concentrations of free flavins, such as FMN, FAD, and riboflavin showed about a two-fold increase in the amount of released iron. Notably, all iron release kinetics occurred while the solution oxygen level was still high. Altogether, our results indicate that in addition to ferritin proteolysis, there exists an auxiliary iron reductive mechanism that involves long-range electron transfer reactions facilitated by the ferritin shell. The physiological implications of such iron reductive mechanisms are discussed.

Effect of chaotropes on the kinetics of iron release from ferritin by flavin nucleotides

BackgroundFerritins are ubiquitous multi-subunit iron storage and detoxification proteins that play a critical role in iron homeostasis. Ferrous ions that enter the protein's shell through hydrophilic channels are rapidly oxidized at dinuclear centers on the H-subunit before transfer to the protein's cavity for storage. The mechanisms of iron loading have been extensively studied, but little is known about iron mobilization. Fe(III) reduction can occur via rapid reduction by suitable reducing agents followed by chelation of Fe(II) ions or via direct and slow Fe(III) chelation. Here, the iron release kinetics from ferritin by FMNH2 in the presence of various chaotropic agents are studied and their in-vivo physiological significance discussed. MethodsThe iron release kinetics from horse and human ferritins by FMNH2 were monitored at 522nm where the Fe(II)–bipyridine complex absorbs. The experiments were performed in the presence of different concentrations of three chaotropic agents, urea, guanidine HCl, and triton. Results and conclusionsUnder our experimental conditions, iron reductive mobilization by the non-enzymatic FMN/NAD(P)H system is limited by the concentration of FMNH2 and is independent on the type or amount of chaotropes present. Diffusion of FMNH2 through the ferritin pores is an unlikely mechanism for ferritin iron reduction. An iron mobilization mechanism involving rapid electron transfer through the protein shell is discussed. General significanceCaution must be exercised when interpreting the kinetics of iron mobilization from ferritin using the FMN/NAD(P)H system. The kinetics are highly dependent on the amount of dissolved oxygen and the concentration of reagents used.

Iron Release from the Siderophore Pyoverdine in Pseudomonas aeruginosa Involves Three New Actors: FpvC, FpvG, and FpvH.

Siderophores are iron chelators produced by bacteria to access iron, an essential nutriment. Pyoverdine (PVDI), the major siderophore produced by Pseudomonas aeruginosa PAO1, consists of a fluorescent chromophore linked to an octapeptide. The ferric form of PVDI is transported from the extracellular environment into the periplasm by the outer membrane transporter, FpvA. Iron is then released from the siderophore in the periplasm by a mechanism that does not involve chemical modification of the chelator but an iron reduction step. Here, we followed the kinetics of iron release from PVDI, in vitro and in living cells, by monitoring its fluorescence (as apo PVDI is fluorescent, whereas PVDI-Fe(III) is not). Deletion of the inner membrane proteins fpvG (PA2403) and fpvH (PA2404) affected 55Fe uptake via PVDI and completely abolished PVDI-Fe dissociation, indicating that these two proteins are involved in iron acquisition via this siderophore. PVDI-Fe dissociation studies, using an in vitro assay, showed that iron release from this siderophore requires the presence of an iron reducer (DTT) and an iron chelator (ferrozine). In this assay, DTT could be replaced by the inner membrane protein, FpvG, and ferrozine by the periplasmic protein, FpvC, suggesting that FpvG acts as a reductase and FpvC as an Fe2+ chelator in the process of PVDI-Fe dissociation in the periplasm of P. aeruginosa cells. This mechanism of iron release from PVDI is atypical among Gram-negative bacteria but seems to be conserved among Pseudomonads.

Iron release from ferritin by flavin nucleotides

BackgroundExtensive in-vitro studies have focused on elucidating the mechanism of iron uptake and mineral core formation in ferritin. However, despite a plethora of studies attempting to characterize iron release under different experimental conditions, the in-vivo mobilization of iron from ferritin remains poorly understood.Several iron-reductive mobilization pathways have been proposed including, among others, flavin mononucleotides, ascorbate, glutathione, dithionite, and polyphenols. Here, we investigate the kinetics of iron release from ferritin by reduced flavin nucleotide, FMNH2, and discuss the physiological significance of this process in-vivo. MethodsIron release from horse spleen ferritin and recombinant human heteropolymer ferritin was followed by the change in optical density of the Fe(II)–bipyridine complex using a Cary 50 Bio UV–Vis spectrophotometer. Oxygen consumption curves were followed on a MI 730 Clark oxygen microelectrode. ResultsThe reductive mobilization of iron from ferritin by the nonenzymatic FMN/NAD(P)H system is extremely slow in the presence of oxygen and might involve superoxide radicals, but not FMNH2. Under anaerobic conditions, a very rapid phase of iron mobilization by FMNH2 was observed. ConclusionsUnder normoxic conditions, FMNH2 alone might not be a physiologically significant contributor to iron release from ferritin. General significanceThere is no consensus on which iron release pathway is predominantly responsible for iron mobilization from ferritin under cellular conditions. While reduced flavin mononucleotide (FMNH2) is one likely candidate for in-vivo ferritin iron removal, its significance is confounded by the rapid oxidation of the latter by molecular oxygen.

An ABC Transporter with Two Periplasmic Binding Proteins Involved in Iron Acquisition in Pseudomonas aeruginosa

Pyoverdine I is the main siderophore secreted byPseudomonas aeruginosa PAO1 to obtain access to iron. After extracellular iron chelation, pyoverdine-Fe uptake into the bacteria involves a specific outer-membrane transporter, FpvA. Iron is then released in the periplasm by a mechanism involving no siderophore modification but probably iron reduction. The proteins involved in this dissociation step are currently unknown. The pyoverdine locus contains the fpvCDEF operon, which contains four genes. These genes encode an ABC transporter of unknown function with the distinguishing characteristic of encompassing two periplasmic binding proteins, FpvC and FpvF, associated with the ATPase, FpvE, and the permease, FpvD. Deletion of these four genes partially inhibited cytoplasmic uptake of (55)Fe in the presence of pyoverdine and markedly slowed down the in vivo kinetics of iron release from the siderophore. This transporter is therefore involved in iron acquisition by pyoverdine in P. aeruginosa. Sequence alignments clearly showed that FpvC and FpvF belong to two different subgroups of periplasmic binding proteins. FpvC appears to be a metal-binding protein, whereas FpvF has homology with ferrisiderophore binding proteins. In vivo cross-linking assays and incubation of purified FpvC and FpvF proteins showed formation of complexes between both proteins. These complexes were able to bind in vitro PVDI-Fe, PVDI-Ga, or apo PVDI. This is the first example of an ABC transporter involved in iron acquisition via siderophores, with two periplasmic binding proteins interacting with the ferrisiderophore. The possible roles of FpvCDEF in iron uptake by the PVDI pathway are discussed.

Biochemical and structural characterization of recombinant human serum transferrin from rice (Oryza sativa L.)

The Fe3+ binding protein human serum transferrin (hTF) is well known for its role in cellular iron delivery via the transferrin receptor (TFR). A new application is the use of hTF as a therapy and targeted drug delivery system for a number of diseases. Recently, production of hTF in plants has been reported; such systems provide a relatively inexpensive, animal-free (eliminating potential contamination by animal pathogens) method to produce large amounts of recombinant proteins for such biopharmaceutical applications. Specifically, the production of Optiferrin™ (hTF produced in rice, Oryza sativa, from InVitria) has been shown to yield large amounts of functional protein for use in culture medium for cellular iron delivery to promote growth. In the present work we describe further purification (by gel filtration) and characterization of hTF produced in rice (purified Optiferrin™) to determine its suitability in biopharmaceutical applications. The spectral, mass spectrometric, urea gel and kinetic analysis shows that purified Optiferrin™ is similar to recombinant nonglycosylated N-His tagged hTF expressed by baby hamster kidney cells and/or serum derived glycosylated hTF. Additionally, in a competitive immunoassay, iron-loaded Optiferrin™ is equivalent to iron-loaded N-His hTF in its ability to bind to the soluble portion of the TFR immobilized in an assay plate. As an essential requirement for any functional hTF, both lobes of purified Optiferrin™ bind Fe3+ tightly yet reversibly. Although previously shown to be capable of delivering Fe3+ to cells, the kinetics of iron release from iron-loaded Optiferrin™/sTFR and iron-loaded N-His hTF/sTFR complexes differ somewhat. We conclude that the purified Optiferrin™ might be suitable for consideration in biopharmaceutical applications.

Protonation and Anion Binding Control the Kinetics of Iron Release from Human Transferrin

Iron release in vitro from human serum diferric transferrin (hFe(2)Tf) in acidic media (4.2 ≤ pH ≤ 5.4) in the presence of nonsynergistic anions occurs in at least five kinetic steps. Step 1 (most rapid) involves proton assisted release of carbonate from the protein. In subsequent steps, iron release from both the N- and C-terminal lobes is controlled by slow proton transfers and anion binding. In step 2, the N-terminal lobe takes up one proton with kinetic linkage to the binding of one anion. In step 3, iron release from the anion-linked N-terminal lobe is controlled by slow uptake of two protons with rate-constant, k(2N), of 2.6(6) × 10(7), 6.1(6) × 10(7), and 9(1) × 10(7) M(-2) s(-1) in the presence of Cl(-), NO(3)(-), and SO(4)(2-), respectively. In step 4, the C-terminal lobe takes up one proton with kinetic linkage to the binding of one anion. In step 5, iron release from the anion-linked C-terminal lobe is controlled by slow uptake of two protons with rate-constant, k(2C), of 8.4(2) × 10(4), 4.4(6) × 10(5), and 8.1(2) × 10(5) M(-2) s(-1) in the presence of Cl(-), NO(3)(-), and SO(4)(2-), respectively.

Transient state kinetic investigation of ferritin iron release

Increased iron concentration in tissues appears to be a factor in the genesis and development of inflammatory and degenerative diseases. By means of real-time small angle x-ray scattering measurements, we studied the kinetics of iron release from the ferritin inorganic core as a function of time and distance from the iron core centre. Accordingly, the iron release process follows a three step model: (i) a defect nucleation in the outer part of the mineral core, (ii) the diffusion of the reducing agent towards the inner part of the core, and (iii) the erosion of the core from the inner to the outer part.

Mass spectrometric characteristics and kinetics of iron release in visceral mass of Saccostrea cucullata

Ferritins with electrophoretic homogeneity were prepared from the visceral mass of Saccostrea cucullata in batch. The native PAGE approach showed similar electrophoretic mobility among pig pancreatic ferritin, liver ferritin of Dasyatis akajei, and visceral mass ferritin of Saccostrea cucullata. SDS-PAGE indicated that the Saccostrea cucullata visceral ferritin (SCVF) consisted of a single subunit type and had a molecular weight (MW) of approximately 20 kDa, suggesting that the protein shell in SCVF was composed of a single subunit. In addition, peptide mass fingerprinting and transmission electron microscopy were used to identify SCVF further, and to observe its molecular structure. We found that the molecular structure in SCVF was similar to that of most mammalian ferritins, which are composed of a protein shell and an iron core. The results of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry under the assistance of an acidic matrix, sinapic acid, also showed that SCVF was composed of a single subunit type and its subunit MW was calculated to be 19871.042 Da in the absence of heme. Kinetics analysis revealed that the complete process of iron release fitted the law of a first-order reaction, which is similar to that of most ferritins in mammals. Similar to bacterial ferritin, studies indicated that the shell consisted of a single subunit type and showed similar kinetics of iron release, suggesting that this subunit plays two important roles in iron release and storage, and that it shows different stability and intensity of interaction in carrying out its physiological functions in SCVF.

Functions of Double Subunits of a Type, Structure of Iron Core, and Kinetics of Iron Release from Membrane Ferritin of Human Placenta

Abstract Membrane ferritin of human placenta (HPMF) with electrophoresis purity was prepared in batch from human placental tissues. Several analysis methods were employed to study on the structure and functions of HPMF. The results of SDS-PAGE indicated that the protein shell of HPMF comprised different subunits, which belong to a similar type. The subunits were classified into two groups with the molecular weights of 15 and 20 kDa, which were named MF 15 and MF 20 subunits, respectively. In addition, relative amount of MF 15 subunit was about two folds more than that of M 20 subunit in SDS-PAGE gel. Both MF 15 and MF 20 subunits were identified by peptide mass fingerprinting (PMF) and showed high homology with L subunit of human ferritin. Accordingly, it was believed that HPMF was a novel ferritin of two subunits with different molecular weights belonging to a similar type. Using a matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), the subunit stability of HPMF was investigated, and five mass peak's corresponding ratio of mass to charge ( m / z ) such as 5071.25, 9962.51, 15131.55 (MF 15 ), 19936.40 (MF 20 ), and 20147.61 were detected, which indicated HPMF had five subunit formula of [MF 15 ] 3+ , [MF 20 ] 2+ , [MF 15 ] + , [MF 20 ] + and [MF 20 + Fe] + , respectively. Using inductively coupled plasma-mass spectrometry (ICP-MS) to analyze the elemental composition of HPMF, only 85 Fe 3+ and 15 inorganic phosphate (P i ) ions in the iron core of per molecule of HPMF were determined, which were significantly lower than that of most mammal ferritins, but higher than that of bacterial ferritin (BF). The kinetic studies showed that iron release from the complete HPMF followed the regulation of first-order reaction, and the time taken for the whole process of iron release from HPMF was about 750 min, which was slower than that from both horse spleen ferritin (HSF) and pig pancreatic ferritin (PPF), which were about 60 min. Accordingly, based on the difference of structure and function among HPMF, mammalian ferritins, plant ferritins, and BF, we proposed that M 15 and M 20 subunits play different roles in iron transporting from maternal blood to fetal blood.

Identification of a Kinetically Significant Anion Binding (KISAB) Site in the N-Lobe of Human Serum Transferrin

Human serum transferrin (hTF) binds two ferric iron ions which are delivered to cells in a transferrin receptor (TFR) mediated process. Critical to the delivery of iron to cells is the binding of hTF to the TFR and the efficient release of iron orchestrated by the interaction. Within the endosome, iron release from hTF is also aided by lower pH, the presence of anions, and a chelator yet to be identified. We have recently shown that three of the four residues comprising a loop in the N-lobe (Pro142, Lys144, and Pro145) are critical to the high-affinity interaction of hTF with the TFR. In contrast, Arg143 in this loop does not participate in the binding isotherm. In the current study, the kinetics of iron release from alanine mutants of each of these four residues (placed into both diferric and monoferric N-lobe backgrounds) have been determined +/- the TFR. The R143A mutation greatly retards the rate of iron release from the N-lobe in the absence of the TFR but has considerably less of an effect in its presence. Our data definitively show that Arg143 serves as a kinetically significant anion binding (KISAB) site that is, by definition, sensitive to salt concentration and critical to the conformational change necessary to induce iron release from the N-lobe of hTF (in the absence of the TFR). This is the first identification of an authentic KISAB site in the N-lobe of hTF. The effect of the single R143A mutation on the kinetic profile of iron release provides a dramatic illustration of the dynamic nature of hTF.

The Ferrichrome Uptake Pathway inPseudomonas aeruginosaInvolves an Iron Release Mechanism with Acylation of the Siderophore and Recycling of the Modified Desferrichrome

The uptake of iron into Pseudomonas aeruginosa is mediated by two major siderophores produced by the bacterium, pyoverdine and pyochelin. The bacterium is also able of utilize several heterologous siderophores of bacterial or fungal origin. In this work, we have investigated the iron uptake in P. aeruginosa PAO1 by the heterologous ferrichrome siderophore. (55)Fe uptake assays showed that ferrichrome is transported across the outer membrane primarily (80%) by the FiuA receptor and to a lesser extent (20%) by a secondary transporter. Moreover, we demonstrate that like in the uptake of ferripyoverdine and ferripyochelin, the energy required for both pathways of ferrichrome uptake is provided by the inner membrane protein TonB1. Desferrichrome-(55)Fe uptake in P. aeruginosa was also dependent on the expression of the permease FiuB, suggesting that this protein is the inner membrane transporter of the ferrisiderophore. A biomimetic fluorescent analogue of ferrichrome, RL1194, was used in vivo to monitor the kinetics of iron release from ferrichrome in P. aeruginosa in real time. This dissociation involves acylation of ferrichrome and its biomimetic analogue RL1194 and recycling of both modified siderophores into the extracellular medium. FiuC, an N-acetyltransferase, is certainly involved in this mechanism of iron release, since its mutation abolished desferrichrome-(55)Fe uptake. The acetylated derivative reacts with iron in the extracellular medium and is able to be taken up again by the cells. All these observations are discussed in light of the current knowledge concerning ferrichrome uptake in P. aeruginosa and in Escherichia coli.

The Unique Kinetics of Iron Release from Transferrin: The Role of Receptor, Lobe–Lobe Interactions, and Salt at Endosomal pH

Transferrins are a family of bilobal iron-binding proteins that play the crucial role of binding ferric iron and keeping it in solution, thereby controlling the levels of this important metal. Human serum transferrin (hTF) carries one iron in each of two similar lobes. Understanding the detailed mechanism of iron release from each lobe of hTF during receptor-mediated endocytosis has been extremely challenging because of the active participation of the transferrin receptor (TFR), salt, a chelator, lobe–lobe interactions, and the low pH within the endosome. Our use of authentic monoferric hTF (unable to bind iron in one lobe) or diferric hTF (with iron locked in one lobe) provided distinct kinetic end points, allowing us to bypass many of the previous difficulties. The capture and unambiguous assignment of all kinetic events associated with iron release by stopped-flow spectrofluorimetry, in the presence and in the absence of the TFR, unequivocally establish the decisive role of the TFR in promoting efficient and balanced iron release from both lobes of hTF during one endocytic cycle. For the first time, the four microscopic rate constants required to accurately describe the kinetics of iron removal are reported for hTF with and without the TFR. Specifically, at pH 5.6, the TFR enhances the rate of iron release from the C-lobe (7-fold to 11-fold) and slows the rate of iron release from the N-lobe (6-fold to 15-fold), making them more equivalent and producing an increase in the net rate of iron removal from Fe2hTF. Calculated cooperativity factors, in addition to plots of time-dependent species distributions in the absence and in the presence of the TFR, clearly illustrate the differences. Accurate rate constants for the pH and salt-induced conformational changes in each lobe precisely delineate how delivery of iron within the physiologically relevant time frame of 2 min might be accomplished.

Trapping Capacities, Stability and Interaction Intensity of Subunits from Bacterial Ferritin of Azotobacter Vinelandii

Bacterial ferritin with purity for analysis of mass spectrometry (MS) in Azotobacter vinelandii was prepared by column chromatography, electrophoresis, and RP-HPLC. Bacterial ferritin of Azotobacter vinelandii (AVBF) was further identified by both kinetics of iron release and peptide mass fingerprinting (PMF), respectively. Moreover, interaction intensity, stability, and polymer in AVBF subunits were revealed by both matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF MS) and electrophoresis. Bacterial ferritin of Azotobacter vinelandi can be used to trap the small organic molecules such as methylene blue (MB), and the trapping rate is approximately 15.0 ± 2.0 MB/AVBF. It is suggested that heme component located at the interface between subunits or monomers in AVBF participates this process for trapping MB. Bacterial ferritin of Azotobacter vinelandi and liver ferritin of shark (SLF) can directly release its unstable subunits for MS analysis under the conditions of 40%–50% concentration of both acetonitrile and acetone, respectively, but both the proteins cannot release its subunits for MS analysis under the condition of 20%–30% acetonitrile unless both proteins further absorb the laser from the mass spectrometer. It was well known that the interaction intensity among subunits in AVBF was lower than that of SLF. This intensity was strongly connected with the rates of iron release and its storage in ferritin.

Desorption/Ionization of Subunit and Kinetics of Iron Release Revealed with Matrix-assisted Laser Desorption Ionization-Time of Flight Mass spectrometry and Electronic Spectrum in Liver Ferritin of Aplysia

Liver ferritin of Aplysia (ALF) with purity of mass spectrometry (MS) was identified using peptide mass fingerprint. Both the lasers emitted from the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) and the matrix sinapic acid together decomposed ALF into molecular ion of the subunit for MS analysis, showing three molecular ions, such as single subunit with double [M + 2H] 2+ and single [M + H] + charges and dimeric subunit with single charge [2M + H] +. The ratio of mass to charge ( m/ z) in these ions were divided to be 9784.03, 19678.42 and 39387.80, respectively. The molecular weight of ALF was less than that of the ferritin in shark liver (SLF) to a certain extent. Under the condition of basic medium (pH 8.0), the experimental results used with electron spectral technology showed that ascorbic acid, as a half-order reaction, reduced the whole iron component within the ferritin core for releasing, which resulted in the release of iron by ALF as the first-order reaction rather than half-order reaction, showing two different rates of iron release. The results obtained in this article suggested that these novel phenomena to be tightly connected with the low iron content and the capacities of subunit regulation in ALF and with the evolution level in Aplysia.

Comparison of the kinetics of iron release from a marine (Trichodesmium erythraeum) Dps protein and mammalian ferritin in the presence and absence of ligands

The ferritin superfamily of iron storage proteins includes ferritin proper and Dps (DNA binding protein from starved cells) along with bacterioferritin. We examined the release of Fe from the Dps of Trichodesmium erythraeum (Dpstery) and compared it to the release of Fe from horse spleen ferritin (HoSF) under various conditions. Both desferrioxamine B (DFB), a Fe(III) chelator, and ascorbic acid were able to mobilize Fe from Dpstery at rates comparable to those observed for HoSF. The initial Fe release rate from both proteins increased linearly with the concentration of DFB, suggesting that the chelator binds to Fe in the protein. A small but significant rate obtained by extrapolation to zero concentration of DFB implies that Dpstery and HoSF might release Fe(III) spontaneously. A similar result was observed for HoSF in the presence of sulfoxine. In a different experiment, Fe(III) was transferred from holoferritin to apotransferrin across a dialysis membrane in the absence of chelator or reducing agent. The apparent spontaneous release of Fe from HoSF and Dpstery brings forth the hypothesis that the Fe core in Fe storage proteins might be continuously dissolving and re-precipitating in vivo, thus maintaining it in a highly reactive and bioavailable form.

Kinetics of Iron Release from Ferric Binding Protein (FbpA): Mechanistic Implications in Bacterial Periplasm-to-Cytosol Fe3+ Transport

The ferric binding protein (FbpA) transports iron across the periplasmic space of certain Gram-negative bacteria and is an important component involved in iron acquisition by pathogenic Neisseria spp. (Neisseria gonorrheae and Neisseria meningitidis). Previous work has demonstrated that the synergistic anion, required for tight Fe(3+) sequestration by FbpA, also plays a key role in inserting Fe(3+) into the FbpA binding site. Here, we investigate the iron release process from various forms of holo-FbpA, Fe(3+)FbpA-X, during the course of a chelator competition reaction using EDTA and Tiron. Fe(3+)FbpA-X represents the protein assembly complex with different synergistic anions, X = PO(4)(3)(-) and NTA. Stepwise mechanisms of Fe(3+) release are proposed on the basis of kinetic profiles of these chelator competition reactions. Fe(3+)FbpA-PO(4) and Fe(3+)FbpA-NTA react differently with EDTA and Tiron during the Fe(3+)-exchange process. EDTA replaces PO(4)(3)(-) and NTA from the first coordination shell of Fe(3+) and acts as a synergistic anion to give a spectroscopically distinguishable intermediate, Fe(3+)FbpA-EDTA, prior to pulling Fe(3+) out of the protein. Tiron, on the other hand, does not act as a synergistic anion but is a more efficient competing chelator as it removes Fe(3+) from FbpA at rate much faster than EDTA. These results reaffirm the contribution of the synergistic anion to the FbpA iron transport process as the anion, in addition to playing a facilitative role in iron binding, appears to have a "gatekeeper" role, thereby modulating the Fe(3+) release process.

Iron release from transferrin, its C-lobe, and their complexes with transferrin receptor: presence of N-lobe accelerates release from C-lobe at endosomal pH.

Human transferrin, like other members of the transferrin class of iron-binding proteins, is a bilobal structure, the product of duplication and fusion of an ancestral gene during the course of biochemical evolution. Although the two lobes exhibit 45% sequence identity and identical ligand structures of their iron-binding sites (one in each lobe), they differ in their iron-binding properties and their responsiveness to complex formation with the transferrin receptor. A variety of interlobe interactions modulating these iron-binding functions has been described. We have now studied the kinetics of iron release to pyrophosphate from the isolated recombinant C-lobe and from that lobe in the intact protein, each free and bound to receptor. The striking finding is that the rates of iron release at the pH of the endosome to which transferrin is internalized by the iron-dependent cell are similar in the free proteins but 18 times faster from full-length monoferric transferrin selectively loaded with iron in the C-lobe than from isolated C-lobe when each is complexed to the receptor. The possibility that the faster release in the receptor complex of the full-length protein at endosomal pH contributes to the evolutionary advantage of the bilobal structure is considered.

Purification, electrophoretic behavior, and kinetics of iron release of liver ferritin of Dasyatis akajei.

From the liver of fish Dasyatis akajei, ferritin has been isolated by thermal denaturation and ammonium sulfate fractionation and then further purified by anion exchange chromatography and gel exclusion chromatography. The molecular weight of the liver ferritin of D. akajei (DALF) was measured to be 400 kDa by PAGE. Moreover, SDS-PAGE experimentation indicates that protein shell of DALF consists of the H and L subunits with molecular weight of 18 and 13 kDa, respectively. Using isoelectric focusing with pH ranging from 5.0 to 6.0, the ferritin purified by the PAGE exhibited three bands with different pI values in the gel slab. Diameters of the protein shell and iron core were also investigated by transmission electron microscope and determined to be 10-12 nm and 5-8 nm, respectively. A kinetic study of DALF reveals that the rate of self-regulation of the protein shell rather than the complex surface of the iron core plays an important role in forming a process for iron release with mixed orders.