Iron-binding Ability Research Articles

Antioxidant and free radical scavenging activity of iron chelators

Inside the human body, reactive derivatives of oxygen, known as reactive oxygen species (ROS) such as the superoxide radical (O2), hydroxyl radical (OH) and hydrogen peroxide (H2O2), are constantly generated. The ROS easily cause oxidative damage to various biomolecules such as proteins, lipids and DNA leading to various disease conditions. Iron chelators function as antioxidants by scavenging ROS and also reduce the amount of available iron thereby decreasing the quantity of OH generated by Fenton reactions. In this study, the antioxidant activity of the iron chelators: caffeic acid (CA), 2,3-dihydroxybenzoic acid (DHBA), desferroxamine B (FOB) and benzohydroxamic acid (BHA) were determined using five different in vitro antioxidant assays. The antioxidant assays used were: iron binding ability, reducing ability using the potassium ferricyanide reduction method, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, H2O2 scavenging activity and OH scavenging activity. The standard used for the iron binding ability was Na2EDTA whereas vitamin C was used as a standard for the remaining assays. The iron chelators showed a concentration dependent increase in their radical scavenging activities as well as their reducing ability. At the concentration of 1mM, FOB had the highest iron binding ability of 93.7% whereas DHBA had the lowest iron binding ability of 5.0% compared to the standard Na2EDTA which had 94.8%. The iron chelators, with the exception of BHA, showed good reducing ability than vitamin C. Caffeic acid showed significant DPPH, hydrogen peroxide and hydroxyl radical scavenging activities of 84.7%, 99.8% and 14.5%, respectively. All the iron chelators were observed to show significant activities in all five antioxidant assays.

Bordetella pertussis FbpA binds both unchelated iron and iron siderophore complexes.

Bordetella pertussis is the causative agent of whooping cough. This pathogenic bacterium can obtain the essential nutrient iron using its native alcaligin siderophore and by utilizing xeno-siderophores such as desferrioxamine B, ferrichrome, and enterobactin. Previous genome-wide expression profiling identified an iron repressible B. pertussis gene encoding a periplasmic protein (FbpABp). A previously reported crystal structure shows significant similarity between FbpABp and previously characterized bacterial iron binding proteins, and established its iron-binding ability. Bordetella growth studies determined that FbpABp was required for utilization of not only unchelated iron, but also utilization of iron bound to both native and xeno-siderophores. In this in vitro solution study, we quantified the binding of unchelated ferric iron to FbpABp in the presence of various anions and importantly, we demonstrated that FbpABp binds all the ferric siderophores tested (native and xeno) with μM affinity. In silico modeling augmented solution data. FbpABp was incapable of iron removal from ferric xeno-siderophores in vitro. However, when FbpABp was reacted with native ferric-alcaligin, it elicited a pronounced change in the iron coordination environment, which may signify an early step in FbpABp-mediated iron removal from the native siderophore. To our knowledge, this is the first time the periplasmic component of an iron uptake system has been shown to bind iron directly as Fe3+ and indirectly as a ferric siderophore complex.

Lipoxygenase inhibitory activity of alkyl protocatechuates

Alkyl 3,4-dihydroxybenzoates (protocatechuates) inhibited linoleic acid peroxidation catalyzed by soybean lipoxygenase-1 (EC 1.13.11.12, Type 1). Their inhibitory activities displayed a parabolic function of their lipophilicity and maximized with alkyl chain lengths of between C11 and C14. Tetradecanyl protocatechuate exhibited the most potent inhibition with an IC50 of 0.05μM, followed by dodecyl (lauryl) protocatechuate with an IC50 of 0.06μM. However, their parent compound, protocatechuic acid, did not show this inhibitory activity up to 200μM, indicating that the alkyl chain length is significantly related to the inhibition activity. The allosteric (or cooperative) inhibition of soybean lipoxygenase-1 of longer alkyl protocatechuates is reversible but in combination with their iron binding ability to disrupt the active site competitively and to interact with the hydrophobic portion surrounding near the active site (sequential action). In the case of dodecyl protocatechuate, the enzyme quickly binds this protocatechuate and then its dodecyl group undergoes a slow interaction with the hydrophobic domain in close proximity to the active site in the enzyme. The inhibition kinetics analyzed by Lineweaver–Burk plots indicates that octyl protocatechuate is a competitive inhibitor and the inhibition constant (Ki) was obtained as 0.23μM but dodecyl protocatechuate is a slow binding inhibitor.

Separation of Phosvitin from Egg Yolk without Using Organic Solvents.

The objective of this study was to develop a new method to separate phosvitin from egg yolk without using organic solvents. Phosvitin was extracted from yolk granules using 10% NaCl or 10% (NH4)2SO4 (final concentration) and then treated with heat to precipitate the lipoproteins from the extracted solution. The optimal pH for the phosvitin extraction from yolk granules was determined, and the iron-binding ability of the extracted phosvitin (final product) was tested. Adding 10% (NH4)2SO4 disrupted the granules, and the subsequent thermal treatment at 90°C for 1 h precipitated low density and high density lipoproteins, which enabled separation of phosvitin by centrifugation. The phosvitin concentration in the extract was significantly higher when the pH of the solution was adjusted to pH ≥9. The purity and recovery rate of phosvitin at the end of the separation process were approximately 78% and 56%, respectively. The separated phosvitin was confirmed to have ferrous and ferric iron binding ability. The advantages of this new method compared with the traditional methods include no organic solvents and high-priced equipment are needed for the separation. Also, this method is more environment and consumer friendly than that of the traditional methods.

Imaging Interactions Between Transferrin‐containing Endosomes and Mitochondria

Iron transport occurs via iron binding to transferrin (Tfn), which is then taken up by the Tfn‐receptor (TFR) at the plasma membrane and delivered to endosomes, where upon acidification Tfn undergoes iron release. Iron is then transported to the mitochondria via a yet unknown mechanism; in the mitochondria, iron is incorporated into the heme groups of proteins involved in cellular respiration. The “kiss and run” hypothesis states that iron is transported into the mitochondria via a direct interaction between endosomes and mitochondria. To test the “kiss and run” hypothesis in epithelial cells, we will use time‐lapse live‐cell fluorescence TIRF microscopy to assay the colocalization of endosomes and mitochondria under various conditions that affect pH and Tfn‐iron binding capacity. Our goal is to develop a semi‐automated image analysis protocol based on the Imaris software to characterize putative “kiss and run” events. Our analysis shows that endosomal speed increases before the “kiss” as well as before the “run” of each “kiss and run” event. During the attachment period the endosome reduces its motility. We have developed an image analysis protocol that provides both qualitative and quantitative information on putative kiss and run events between endosomes and mitochondria, allowing us in the future to assay them functionally and ascertain their dependency on Tfn's iron binding ability as well as endocytic pH.

Understanding Magnetite Biomineralisation: The Effect of Short Amino Acid Sequences on the {100} and the {111} Surface

ABSTRACTMagnetite (Fe3O4) formation within Magnetospirillum magneticum strain AMB-1 occurs under the influence of the Mms6 protein. It is hypothesised that if key iron binding sites within the C-terminus of the Mms6 protein are substituted for alanine, the protein’s overall iron binding ability is diminished. In this study, an atomistic model of Mms6-driven magnetite formation was developed and the attachment of series amino acid repeats (alanine-alanine, alanine-glutamic acid & glutamic acid-glutamic acid) to the {100} & {111} magnetite surfaces were investigated. Our results suggest the substitution of glutamic acid for alanine residues significantly reduces iron binding affinity of the system, thus confirming the hypothesis. In addition, it is shown that the surface of preferable attachment is the {111} magnetite surface.

Microstructural design to reduce lipid oxidation in oil-inwater emulsions

The influence of emulsifier type (Tween 20 and sodium caseinate (CAS)) and oil phase volume fraction (5% and 30%) on emulsion oxidative stability was investigated. The primary and secondary lipid oxidation products of emulsions stored at 40°C were measured over 7 days. The results indicated that the oxidative stability of samples stabilised with CAS was significantly higher compared with emulsions stabilised with Tween 20. We propose that this is due to iron binding ability of CAS. Moreover, the impacts of Pickering emulsions (Silica particles) on lipid oxidation were studied and compared with Tween 20 stabilised emulsions. The results showed that silica particles could increase the oxidative stability of 20% sunflower oil-in-water emulsions by acting as a physical barrier between pro-oxidants located in continuous phase and hydroperxide at droplet interface.

ChemInform Abstract: Syntheses and Binding Studies of Oligonucleotides Containing N-Hydroxycarbamate Linkages: Potential DNA Cleaving Antisense Oligomers.

Abstract An efficient synthesis of N-hydroxycarbamate containing thymidine dimer 9 was accomplished and it was incorporated into automatic DNA syntheses to make thymidine 16mers T*-1, T*-2 and T*-3. Binding abilities of T*-1, T*-2 and T*-3 with poly (dA)-16mer were assayed by melting denaturation (Tm) studies of the corresponding duplexes. Iron binding ability of a thymidine oligomer with N-hydroxycarbamate linkages was studied by MALDI-MS. Nuclease stability assays showed that the modified oligonucleotides have enhanced resistance toward nuclease S1 (endonuclease) compared to natural oligonucleotides.

A Mammalian Siderophore Synthesized by an Enzyme with a Bacterial Homolog Involved in Enterobactin Production

Intracellular iron homeostasis is critical for survival and proliferation. Lipocalin 24p3 is an iron-trafficking protein that binds iron through association with a bacterial siderophore, such as enterobactin, or a postulated mammalian siderophore. Here, we show that the iron-binding moiety of the 24p3-associated mammalian siderophore is 2,5-dihydroxybenzoic acid (2,5-DHBA), which is similar to 2,3-DHBA, the iron-binding component of enterobactin. We find that the murine enzyme responsible for 2,5-DHBA synthesis, BDH2, is the homolog of bacterial EntA, which catalyzes 2,3-DHBA production during enterobactin biosynthesis. RNA interference-mediated knockdown of BDH2 results in siderophore depletion. Mammalian cells lacking the siderophore accumulate abnormally high amounts of cytoplasmic iron, resulting in elevated levels of reactive oxygen species, whereas the mitochondria are iron deficient. Siderophore-depleted mammalian cells and zebrafish embryos fail to synthesize heme, an iron-dependent mitochondrial process. Our results reveal features of intracellular iron homeostasis that are conserved from bacteria through humans.

Lactoferrin Protects Against Acetaminophen‐Induced Liver Injury in Mice

Acetaminophen‐induced liver injury (AILI) is a significant health problem and represents the most frequent cause of drug‐induced liver failure in the United States. The development and implementation of successful therapeutic intervention strategies have been demanding, due to significant limitations associated with the current treatment for AILI. Lactoferrin (Lac), a glycoprotein present in milk, has been demonstrated to possess a multitude of biological functions. Our study demonstrated a profound protective effect of Lac in a murine model of AILI, which was not dependent on its iron binding ability, inhibition of acetaminophen (APAP) metabolism, or a direct cytoprotective effect on hepatocytes. Instead, Lac treatment significantly attenuated APAP‐induced liver sinusoidal endothelial cell dysfunction and ameliorated hepatic microcirculation disorder. This protective effect of Lac appeared to be dependent on hepatic resident macrophages (Kupffer cells, KC). Collectively, our data indicated that Lac, through activation of KC, inhibited APAP‐induced liver sinusoidal endothelial cell damage and improved hepatic congestion, thereby protecting against AILI. These findings reveal the significant therapeutic potential of Lac during AILI and other types of liver diseases.

DsrR, a Novel IscA-Like Protein Lacking Iron- and Fe-S-Binding Functions, Involved in the Regulation of Sulfur Oxidation inAllochromatium vinosum

In the purple sulfur bacterium Allochromatium vinosum, the reverse-acting dissimilatory sulfite reductase (DsrAB) is the key enzyme responsible for the oxidation of intracellular sulfur globules. The genes dsrAB are the first and the gene dsrR is the penultimate of the 15 genes of the dsr operon in A. vinosum. Genes homologous to dsrR occur in a number of other environmentally important sulfur-oxidizing bacteria utilizing Dsr proteins. DsrR exhibits sequence similarities to A-type scaffolds, like IscA, that partake in the maturation of protein-bound iron-sulfur clusters. We used nuclear magnetic resonance (NMR) spectroscopy to solve the solution structure of DsrR and to show that the protein is indeed structurally highly similar to A-type scaffolds. However, DsrR does not retain the Fe-S- or the iron-binding ability of these proteins, which is due to the lack of all three highly conserved cysteine residues of IscA-like scaffolds. Taken together, these findings suggest a common function for DsrR and IscA-like proteins different from direct participation in iron-sulfur cluster maturation. An A. vinosum DeltadsrR deletion strain showed a significantly reduced sulfur oxidation rate that was fully restored upon complementation with dsrR in trans. Immunoblot analyses revealed a reduced level of DsrE and DsrL in the DeltadsrR strain. These proteins are absolutely essential for sulfur oxidation. Transcriptional and translational gene fusion experiments suggested the participation of DsrR in the posttranscriptional control of the dsr operon, similar to the alternative function of cyanobacterial IscA as part of the sense and/or response cascade set into action upon iron limitation.

Lactoferrin Protects Against Acetaminophen-Induced Liver Injury in Mice

Acetaminophen-induced liver injury (AILI) is a significant health problem and represents the most frequent cause of drug-induced liver failure in the United States. The development and implementation of successful therapeutic intervention strategies have been demanding, due to significant limitations associated with the current treatment for AILI. Lactoferrin (Lac), a glycoprotein present in milk, has been demonstrated to possess a multitude of biological functions. Our study demonstrated a profound protective effect of Lac in a murine model of AILI, which was not dependent on its iron-binding ability, inhibition of acetaminophen (APAP) metabolism, or a direct cytoprotective effect on hepatocytes. Instead, Lac treatment significantly attenuated APAP-induced liver sinusoidal endothelial cell dysfunction and ameliorated hepatic microcirculation disorder. This protective effect of Lac appeared to be dependent on hepatic resident macrophages (Kupffer cells [KCs]). Collectively, our data indicate that Lac, through activation of KCs, inhibited APAP-induced liver sinusoidal endothelial cell damage and improved hepatic congestion, thereby protecting against AILI. These findings reveal the significant therapeutic potential of Lac during AILI and other types of liver diseases.

Effect of Chelating Agents and Spice‐Derived Antioxidants on Myoglobin Oxidation in a Lipid‐Free Model System

This study compared myoglobin (Mb) oxidation in lipid-free model systems containing iron and Type I (radical quenching) or Type II (metal chelating) antioxidants. Oxidation was measured as loss of oxymyoglobin (MbO(2)) during 0 to 24 h holding at 22 degrees C. Sodium tripolyphosphate (STPP) demonstrated iron-binding ability at all concentrations tested (88% and 21% added iron bound at 1 and 0.05 mg/mL, respectively). Iron chelation was observed for phytic acid only at the highest concentration (9.5% bound at 1 mg/mL phytate). Neither Type I antioxidant (rosmarinate or eugenol) demonstrated any iron chelating ability (<0.5% bound). In presence of iron, Type I antioxidants had a significant (P < 0.05) prooxidant effect (54.7% retention of MbO(2) in control, 9.5% and 37.5% retention in rosmarinate and eugenol samples, respectively). The Type II antioxidants (STPP and phytate) were more effective inhibitors (P < 0.05) of Mb oxidation than Type I antioxidants, (68.7% and 61.1% for STPP and phytate, respectively). Type I antioxidants were capable of rapid reduction of ferric iron to the ferrous form, as measured by the ferrozine assay. This strong reducing ability accounted for the prooxidant effects of rosmarinic acid and eugenol, since ferrous iron is the form associated with generation of oxygen radicals, and subsequent Mb oxidation. Type II antioxidants chelated and thus prevented the oxidizing effect of added ferrous iron. Mb oxidation can proceed rapidly (within 15 min) in the presence of iron and the absence of lipid, especially if reducing compounds such as rosmarinic acid or eugenol are also present to maintain iron in an active ferrous form.

Molecular cloning and characterization of Taiwan macaque lactoferrin

Lactoferrin (LF) is an iron-binding glycoprotein that plays an important role in combating a wide range of pathogens and contributes innate protective defenses of mammals. We cloned and sequenced the full-length cDNA for LF from Taiwan macaque. The antimicrobial activity and iron-binding ability of the purified recombinant macaque LF (rmLF) were determined and compared with those of human LF (hLF). The complete mLF cDNA (GenBank: EU523857) encoded a 710-aa precursor with a 19-aa signal peptide. The nucleotide sequence of mLF showed the highest identity to the rhesus monkey LF (98%), whereas the putative amino acid sequence of mLF showed the highest identity to the hLF (90%). The rmLF and natural hLF showed almost equivalent antibacterial activities against Klebsiella pneumoniae and mLF presented a slightly lower activity against Listeria monocytogenes than natural hLF. In addition, the patterns of iron release from mLF and hLF were nearly identical.

Investigations into the uptake of copper, iron and selenium by a highly sulphated bacterial exopolysaccharide isolated from microbial mats

A bacterium isolated from microbial mats located on a polynesian atoll produced a high molecular weight (3,000 kDa) and highly sulphated exopolysaccharide. Previous studies showed that the chemical structure of this EPS consisted of neutral sugars, uronic acids, and high proportions of acetate and sulphate groups. The copper- and iron-binding ability of the purified pre-treated native EPS was investigated. Results showed that this EPS had a very high affinity for both copper (9.84 mmol g(-1) EPS) and ferrous iron (6.9 mmol g(-1) EPS). Amazingly, this EPS did not show any affinity for either ferric ions or selenium salts. This finding is one of the first steps in assessing the biotechnological potential of this polysaccharide.

Oxinobactin, a siderophore analogue to enterobactin involving 8-hydroxyquinoline subunits: Synthesis and iron binding ability

Oxinobactin, a siderophore analogue to enterobactin but possessing 8-hydroxyquinoline instead of catechol complexing subunits, has been synthesized starting from l-serine and 8-hydroxyquinoline. Comparative iron binding studies showed that oxinobactin is as effective as enterobactin for the complexation of Fe III at physiological pH but with improved complexing ability at acidic pH.

Lactoferrin: a review

This review discusses the biological properties of the glycoprotein lactoferrin. Lactoferrin has been identified in secretions from exocrine glands and in specific granules of neutrophils. After degranulation, neutrophils become the main source of lactoferrin in blood plasma. Lactoferrin possesses various biological functions, including roles in iron metabolism, cell proliferation and differentiation, and antibacterial, antiviral, and antiparasitic activity. Many of these functions do not appear to be connected with its iron binding ability. Of late, lactoferrin concentrations have been measured mostly in humans but also in some other species. However, the relationship between its concentration and physiological or pathological effects on body functions is not yet well characterised.

Enzymatic Hydrolysis of Heated Whey: Iron-Binding Ability of Peptides and Antigenic Protein Fractions

This study evaluated the influence of various enzymes on the hydrolysis of whey protein concentrate (WPC) to reduce its antigenic fractions and to quantify the peptides having iron-binding ability in its hydrolysates. Heated (for 10min at 100°C) WPC (2% protein solution) was incubated with 2% each of Alcalase, Flavourzyme, papain, and trypsin for 30, 60, 90, 120, 150, 180, and 240min at 50°C. The highest hydrolysis of WPC was observed after 240min of incubation with Alcalase (12.4%), followed by Flavourzyme (12.0%), trypsin (10.4%), and papain (8.53%). The nonprotein nitrogen contents of WPC hydrolysate followed the hydrolytic pattern of whey. The major antigenic fractions (β-lactoglobulin) in WPC were degraded within 60min of its incubation with Alcalase, Flavourzyme, or papain. Chromatograms of enzymatic hydrolysates of heated WPC also indicated complete degradation of β-lactoglobulin, α-lactalbumin, and BSA. The highest iron solubility was noticed in hydrolysates derived with Alcalase (95%), followed by those produced with trypsin (90%), papain (87%), and Flavourzyme (81%). Eluted fraction 1 (F-1) and fraction 2 (F-2) were the respective peaks for the 0.25 and 0.5 M NaCl chromatographic step gradient for analysis of hydrolysates. Iron-binding ability was noticeably higher in F-1 than in F-2 of all hydrolysates of WPC. The highest iron contents in F-1 were observed in WPC hydrolysates derived with Alcalase (0.2mg/kg), followed by hydrolysates derived with Flavourzyme (0.14mg/kg), trypsin (0.14mg/kg), and papain (0.08mg/kg). Iron concentrations in the F-2 fraction of all enzymatic hydrolysates of WPC were low and ranged from 0.03 to 0.05mg/kg. Fraction 1 may describe a new class of iron chelates based on the reaction of FeSO4·7H2O with a mixture of peptides obtained by the enzymatic hydrolysis of WPC. The chromatogram of Alcalase F-1 indicated numerous small peaks of shorter wavelengths, which probably indicated a variety of new peptides with greater ability to bind with iron. Alcalase F-1 had higher Ala (18.38%), Lys (17.97%), and Phe (16.58%) concentrations, whereas the presence of Pro, Gly, and Tyr was not detected. Alcalase was more effective than other enzymes at producing a hydrolysate for the separation of iron-binding peptides derived from WPC.

Separation of iron-binding protein from whey through enzymatic hydrolysis

Whey protein concentrate (WPC) was hydrolyzed by nine proteolytic enzymes to examine the effectiveness of the hydrolysates to bind iron. Degree of WPC hydrolysis was higher with pancreatin (13.91%), alcalase (13.60%), and flavourzyme (12.80%) compared with other enzymes (esperase, neutrase, papain, pepsin, protease and trypsin). Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high performance liquid chromatography analyses revealed maximum hydrolysis of α-LA and β-LG with alcalase. Molecular masses of peptides derived from alcalase hydrolysate were smaller than 6.5 kDa. Iron-binding by alcalase hydrolysate was the highest (97.6%) of all other hydrolysates. Using ion-exchange chromatography alcalase hydrolysate was eluted at a 0.25 m NaCl gradient concentration with higher iron-binding ability. This eluted fraction had higher Lys (18.09%), Ala (17.24%), and Phe (16.58%) contents. Alcalase showed noticeably better effectiveness than other enzymes to produce a hydrolysate for the separation of iron-binding peptides derived from WPC.

Molecular characterization of iron binding proteins from Glossina morsitans morsitans (Diptera: Glossinidae)

The regulation of iron is critical for maintaining homeostasis in the tsetse fly (Diptera: Glossinidae), in which both adult sexes are strict blood feeders. We have characterized the cDNAs for two putative iron-binding proteins (IBPs) involved in transport and storage; transferrin ( GmmTsf1) and ferritin from Glossina morsitans morsitans. GmmTsf1 transcripts are detected in the female fat body and in adult reproductive tissues, and only in the adult developmental stage in a bloodmeal independent manner. In contrast, the ferritin heavy chain ( GmmFer1HCH) and light chain ( GmmFer2LCH) transcripts are expressed ubiquitously, suggesting a more general role for these proteins in iron transport and storage. Protein domain predictions for each IBP suggest both the conservation and loss of several motifs present in their vertebrate homologues. In concert with many other described insect transferrins (Tfs), putative secreted GmmTsf1 maintains 3 of the 5 residues necessary for iron-binding in the N-terminal lobe, but exhibits a loss of this iron-binding ability in the C-terminal lobe as well as a loss of large sequence blocks. Both putative GmmFer1HCH and GmmFer2LCH proteins have signal peptides, similar to other insect ferritins. GmmFer2LCH has lost the 5′UTR iron-responsive element (IRE) and, thus, translation is no longer regulated by cellular iron levels. On the other hand, GmmFer1HCH maintains both the conserved ferroxidase center and the 5′UTR IRE; however, transcript variants suggest a more extensive regulatory mechanism for this subunit.