Binding Specificity of the Porphyromonas gingivalis Heme and Hemoglobin Receptor HmuR, Gingipain K, and Gingipain R1 for Heme, Porphyrins, and Metalloporphyrins

Previous genetic and biochemical studies have confirmed that hemoglobin and hemin utilization in Porphyromonas gingivalis is mediated by the outer membrane hemoglobin and heme receptor HmuR, as well as gingipain K (Kgp), a lysine-specific cysteine protease, and gingipain R1 (HRgpA), one of two arginine-specific cysteine proteases. In this study we report on the binding specificity of the recombinant P. gingivalis HmuR protein and native gingipains for hemoglobin, hemin, various porphyrins, and metalloporphyrins as assessed by spectrophotometric assays, by affinity chromatography, and by enzyme-linked immunosorbent assay. Protoporphyrin, mesoporphyrin, deuteroporphyrin, hematoporphyrin, and some of their iron, copper, and zinc derivatives were examined to evaluate the role of both the central metal ion and the peripheral substituents on binding to recombinant HmuR and soluble gingipains. Scatchard analysis of hemin binding to Escherichia coli cells expressing recombinant membrane-associated six-His-tagged HmuR yielded a linear plot with a binding affinity of 2.4 x 10(-5) M. Recombinant E. coli cells bound the iron, copper, and zinc derivatives of protoporphyrin IX (PPIX) with similar affinities, and approximately four times more tightly than PPIX itself, which suggests that the active site of HmuR contains a histidine that binds the metal ion in the porphyrin ring. Furthermore, we found that recombinant HmuR prefers the ethyl and vinyl side chains of the PPIX molecule to either the larger hydroxyethyl or smaller hydrogen side chains. Kgp and HRgpA were demonstrated to bind various porphyrins and metalloporphyrins with affinities similar to those for hemin, indicating that the binding of Kgp and HRgpA to these porphyrins does not require a metal within the porphyrin ring. We did not detect the binding of RgpB, the arginine-specific cysteine protease that lacks a C-terminal hemagglutinin domain, to hemoglobin, porphyrins, or metalloporphyrins. Kgp and HRgpA, but not RgpB, were demonstrated to bind directly to soluble recombinant six-His-tagged HmuR. Several possible mechanisms for the cooperation between outer membrane receptor HmuR and proteases Kgp and HRgpA in hemin and hemoglobin binding and utilization are discussed.     

Site-selective binding of Zn(II) to metallo-β-lactamase L1 from Stenotrophomonas maltophilia

Extended X-ray absorption fine structure studies of the metallo-β-lactamase L1 from Stenotrophomonas maltophilia containing 1 and 2 equiv of Zn(II) and containing 2 equiv of Zn(II) plus hydrolyzed nitrocefin are presented. The data indicate that the first, catalytically dominant metal ion is bound by L1 at the consensus Zn₁ site. The data further suggest that binding of the first metal helps preorganize the ligands for binding of the second metal ion. The di-Zn enzyme displays a well-defined metal–metal interaction at 3.42 Å. Reaction with the β-lactam antibiotic nitrocefin results in a product-bound species, in which the ring-opened lactam rotates in the active site to present the S1 sulfur atom of nitrocefin to one of the metal ions for coordination. The product bridges the two metal ions, with a concomitant lengthening of the Zn–Zn interaction to 3.62 Å.     

Heme-mediated binding of α-casein to ferritin: evidence for preferential α-casein binding to ferrous iron

Bovine milk α-casein was identified as a ferritin-binding protein, and ferritin is known to be a heme-binding protein. In this study, we found that the binding of α-casein to bovine spleen ferritin in vitro was blocked by hemin, but not by iron-free hemin (protoporphyrin IX) or zinc-protoporphyrin IX, suggesting that the presence of iron in heme play a key role in this interaction. Indeed, the binding of α-casein to ferritin and biotinylated hemin was inhibited by adding excess ferrous ammonium sulfate (FAS). To further elucidate the binding mechanism of α-casein to biotinylated hemin, Ferrozine and nitrilotriacetic acid (NTA) were used as ferrous and ferric iron chelators, respectively. FAS-mediated inhibition of α-casein to biotinylated hemin was neutralized with Ferrozine, but not NTA, while FAS- as well as ferric chloride-mediated inhibition in their interaction was neutralized by NTA. The following ions also inhibited α-casein-biotinylated hemin binding in order of potency of inhibition: FAS (Fe²⁺) ≪ ferric chloride (Fe³⁺) < copper sulfate (Cu²⁺) < zinc sulfate (Zn²⁺) < manganese chloride (Mn²⁺) < calcium chloride (Ca²⁺) < magnesium sulfate (Mg²⁺). These results suggests that the binding of α-casein to ferritin is heme-mediated through direct binding of α-casein to iron in the heme on the surface of ferritin molecule, and that α-casein preferentially binds Fe²⁺ compared with any other metal ions, including Fe³⁺.     

The presence of heat-labile factors interfering with binding analysis of fibrinogen with ferritin in horse plasma

Background

Horse fibrinogen has been identified as a plasma specific ferritin-binding protein. There are two ways in the binding of ferritin-binding protein with ferritin: one is direct binding and the other is indirect binding which is heme-mediated. The aim of this study was to analyze the binding between horse fibrinogen and ferritin.

Findings

Although fibrinogen in horse plasma did not show the binding to ferritin coated on the plate wells, after following heat-treatment (60°C, 30 min) of horse plasma, plasma fibrinogen as well as purified horse fibrinogen bound to plates coated with horse spleen ferritin, but not with its apoferritin which lost heme as well as iron after the treatment of reducing reagent. Binding of purified or plasma fibrinogen to ferritin was inhibited by hemin and Sn-protoporphyrin IX (Sn-PPIX), but not by PPIX or Zn-PPIX.

Conclusions

Heat-treatment of horse plasma enabled plasma fibrinogen to bind to plate well coated with holo-ferritin. From the binding analysis of fibrinogen and ferritin, it is suggested that horse fibrinogen recognized iron or tin in complexed with the heme- or the hemin-ring, and also suggest that some fibrinogens circulate in the form of a complex with ferritin and/or heat-labile factors which inhibit the binding of fibrinogen with ferritin.

     

Iron-dependent binding of bovine milk α-casein with holo-lactoferrin, but not holo-transferrin

Bovine milk α-casein has been identified as an iron- and heme-binding protein. However, the physiological role of its iron-binding remains to be elucidated in more detail. α-Casein was immobilized on CNBr-activated Sepharose 4B beads, and the α-casein agarose beads efficiently bound hemin as well as ferrous ammonium sulfate (Fe(2+)) as compared with control beads. Additionally, α-casein-beads bound bovine holo-lactoferrin (Lf), but not holo-transferrin. Lf caused the release of Fe(2+) which had bound to the α-casein-agarose beads beforehand. These results suggest that bovine α-casein iron-dependently binds holo-bovine Lf more strongly than Fe(2+), and that strong binding between them may play a physiological role in regulating iron homeostasis in the bovine mammary gland.     

Physiological implications of mammalian ferritin-binding proteins interacting with circulating ferritin and a new aspect of ferritin- and zinc-binding proteins

Serum ferritin levels are relatively low (<1 µg/ml) and serum ferritin generally disappears rapidly from the circulation (t _1/2 < 10 min). There are various mammalian ferritin-binding proteins (FBPs) in the blood. Ferritin is cleared by direct uptake by ferritin receptors and by indirect receptor-mediated uptake of FBP complexed with ferritin. Mammalian ferritin binds both heme and iron, and binding occurs through two mechanisms: direct binding with ferritin to H-kininogen and anti-ferritin autoantibody, and indirect heme-mediated binding of fibrinogen and apolipoprotein B to ferritin. Anti-ferritin autoantibody and fibrinogen are proposed to be common mammalian FBPs, as is α₂-macroglobulin. FBP-ferritin binding may affect blood coagulation and influence iron metabolism, oxidative condition, angiogenesis, inflammatory condition and immune response. Aside from apolipoprotein B, FBPs bind zinc ion to form antioxidant and anti-inflammatory agents. The possible simultaneous uptake of zinc ion with FBP-ferritin complex is likely to attenuate iron- and/or heme-mediated oxidative damage and inflammatory response.     

Ferritin—A general metal detoxicant

Binding of nonferrous metal ions to ferritin was compared to that of the phosphate-free or phosphate containing synthetic iron cores. The Scatchard plots for the synthetic cores reveal a high affinity site for Cd, Zn, Be, and Al, with K_D in the range 10^?5–10^?7 M. Preloading the cores with phosphate increased the number of metal ions bound without altering the K_D. The metal ions with smaller ionic radii (Be, Al) were bound in larger numbers than those with larger ionic radii (Cd, Zn). Ferritin isolated from soybean (Glycina max), horse spleen, and rat liver bound the metal ions in amounts larger than predicted from their iron core. Whereas the iron cores and their nonferrous metal ion complexes were insoluble, those in the protein shell remained in solution. Thus apoferritin precipitated with lower concentrations of aluminum than did holoferritin. Also, Al bound to apoferritin reduced the rate of iron loading into the protein.     

The preparation of apo-Cu,Zn superoxide dismutase by ion-exchange chromatography on iminodiacetic acid-sepharose

The superoxide dismutases (EC 1.15.1.1) are a family of enzymes that catalyze the dismutation of superoxide radical anion to dioxygen and hydrogen peroxide. The active site contains a critical metal ion such as manganese, iron, or copper. The copper-containing protein also has one zinc ion bound per subunit. The standard method used to remove the metal ions from Cu,Zn superoxide dismutase has been to exhaustively dialyze the protein against chelating agents at low pH. We have developed a new method where the protein is bound to ion-exchange medium based on iminodiacetic acid immobilized on Sepharose. The bound protein is treated with a buffer containing edta at pH 3.5 to remove metal ions; the buffer is then exchanged for acetate buffer to remove edta, after which the protein is eluted by a salt gradient. An advantage of this method is that a single chromatography step is sufficient to produce apo protein. Results are shown for both human and bovine dimeric Cu,Zn superoxide dismutase and the monomeric Escherichia coli Cu,Zn superoxide dismutase. In every case, the metals were removed efficiently.     

Metal dependent hydrolysis of β‐casein by sIgA antibodies from human milk

We present the first evidence that electrophoretically and immunologically homogeneous sIgAs purified from milk of healthy human mothers by chromatography on Protein A‐Sepharose and FPLC gel filtration contain intrinsically bound metal ions (Ca > Mg ≥ Al > Fe ≈ Zn ≥ Ni ≥ Cu ≥ Mn), the removal of which by a dialysis against ethylenediamine tetraacetic acid (EDTA) leads to a significant decrease in the β‐casein‐hydrolyzing activity of these antibodies (Abs). An affinity chromatography of total sIgAs on benzamidine‐Sepharose interacting with canonical serine proteases separates a small metalloprotease sIgA fraction (6.8 ± 2.4%) from the main part of these Abs with a serine protease‐like β‐casein‐hydrolyzing activity. The relative activity of this metalloprotease sIgA fraction containing intrinsically bound metal ions increases ～1.2–1.9‐fold after addition of external metal ions (Mg²⁺ > Fe²⁺ > Cu²⁺ ≥ Ca²⁺ ≥ Mn²⁺) but decreases by 85 ± 7% after the removal of the intrinsically bound metals. The metalloprotease sIgA fraction free of intrinsic metal ions demonstrates a high β‐casein‐hydrolyzing activity in the presence of individual external metal ions (Fe²⁺ > Ca²⁺ > Co²⁺ ≥ Ni²⁺) and especially several combinations of metals: Co²⁺ + Ca²⁺ < Mg²⁺ + Ca²⁺ < Ca²⁺ + Zn²⁺ < Fe²⁺ + Zn²⁺ < Fe²⁺ + Co²⁺ < Fe²⁺ + Ca²⁺. The patterns of hydrolysis of a 22‐mer oligopeptide corresponding to one of sIgA‐dependent specific cleavage sites in β‐casein depend significantly on the metal used. Metal‐dependent sIgAs demonstrate an extreme diversity in their affinity for casein‐Sepharose and chelating Sepharose, and interact with Sepharoses bearing immobilized monoclonal mouse IgGs against λ‐ and κ‐type light chains of human Abs. Possible ways of the production of metalloprotease abzymes (Abz) by human immune system are discussed. Copyright © 2010 John Wiley & Sons, Ltd.     

ABCG2 Transports and Transfers Heme to Albumin through Its Large Extracellular Loop

ABCG2 is an ATP-binding cassette (ABC) transporter preferentially expressed by immature human hematopoietic progenitors. Due to its role in drug resistance, its expression has been correlated with a protection role against protoporhyrin IX (PPIX) accumulation in stem cells under hypoxic conditions. We show here that zinc mesoporphyrin, a validated fluorescent heme analog, is transported by ABCG2. We also show that the ABCG2 large extracellular loop ECL3 constitutes a porphyrin-binding domain, which strongly interacts with heme, hemin, PPIX, ZnPPIX, CoPPIX, and much less efficiently with pheophorbide a, but not with vitamin B12. K_d values are in the range 0.5–3.5 μm, with heme displaying the highest affinity. Nonporphyrin substrates of ABCG2, such as mitoxantrone, doxo/daunorubicin, and riboflavin, do not bind to ECL3. Single-point mutations H583A and C603A inside ECL3 prevent the binding of hemin but hardly affect that of iron-free PPIX. The extracellular location of ECL3 downstream from the transport sites suggests that, after membrane translocation, hemin is transferred to ECL3, which is strategically positioned to release the bound porphyrin to extracellular partners. We show here that human serum albumin could be one of these possible partners as it removes hemin bound to ECL3 and interacts with ABCG2, with a K_d of about 3 μm.     

Kinetics of conformational changes induced by the binding of various metal ions to bovine alpha-lactalbumin.

The kinetic effects of the binding of various metal ions (Ca(2+), Cd(2+), Co(2+), Mg(2+), Mn(2+), Sr(2+) and Zn(2+)) to apo bovine alpha-lactalbumin has been monitored by means of stopped-flow fluorescence spectroscopy. Our results show that the measured rate constant for the binding of metal ions to the Ca(2+)-site increases with increasing binding constant. This is, however, not the case for metal ions binding to the Zn(2+)-site. The binding experiments performed at different temperatures allowed us to calculate the activation energy for the transition from the metal-free to the metal-loaded state of the protein. These values do not depend on the nature of the metal ion but are correlated with the type of binding site. As a result, we were able to demonstrate that Mg(2+), a metal ion which was thought to bind to the Ca(2+)-site, shows the same binding characteristics as Co(2+) and Zn(2+) and therefore most likely interacts with the residues belonging to the Zn(2+)-binding site.     

Effect of Zinc Sulfate Fortificant on Iron Absorption from Low Extraction Wheat Flour Co-Fortified with Ferrous Sulfate

The co-fortification of wheat flour with iron (Fe) and zinc (Zn) is a strategy used to prevent these deficiencies in the population. Given that Zn could interact negatively with Fe, the objective was to assess the effect of Zn on Fe absorption from bread prepared with wheat flour fortified with Fe and graded levels of Zn fortificant. Twelve women aged 30–43 years, with contraception and a negative pregnancy test, participated in the study. They received on four different days, after an overnight fast, 100 g of bread made with wheat flour (70 % extraction) fortified with 30 mg Fe/kg as ferrous sulfate (A) or prepared with the same Fe-fortified flour but with graded levels of Zn, as zinc sulfate: 30 mg/kg (B), 60 mg/kg (C), or 90 mg/kg (D). Fe radioisotopes (⁵⁹Fe and ⁵⁵Fe) of high specific activity were used as tracers and Fe absorption iron was measured by the incorporation of radioactive Fe into erythrocytes. Results: The geometric mean and range of ±1 SD of Fe absorption were: A?=?19.8 % (10.5–37.2 %), B?=?18.5 % (10.2–33.4 %), C?=?17.7 % (7.7–38.7 %), and D?=?11.2 % (6.2–20.3 %), respectively; ANOVA for repeated measures F?=?5.14, p?<?0.01 (Scheffè’s post hoc test: A vs D and B vs D, p?<?0.05). We can conclude that Fe is well absorbed from low extraction flour fortified with 30 mg/kg of Fe, as ferrous sulfate, and up to 60 mg/kg of Zn, as Zn sulfate. A statistically significant reduction of Fe absorption was observed at a Zn fortification level of 90 mg Zn/kg.     

Fibrous polymer-grafted chitosan/clay composite beads as a carrier for immobilization of papain and its usability for mercury elimination

Papain, which is an industrially important enzyme, has been immobilized on fibrous polymer-modified composite beads, namely poly(methacrylic acid)-grafted chitosan/clay. Characterization studies have been done using FTIR and SEM analysis. Operating parameters such as pH and initial concentration of papain have been varied to obtain the finest papain immobilized polymer-modified composite beads. The immobilization capacity of composite beads has been determined as 34.47 ± 1.18 (n = 3) mg/g. The proteolytic activity of immobilized papain was operated using bovine serum albumin (BSA) and maximum velocity (V _max) and Michaelis–Menten constant (K_m) values of the free and immobilized enzymes were determined using Lineweaver–Burk and Eadie–Hofstee equations. Usability of papain immobilized polymer-modified composite beads as adsorbents for the elimination of mercury was investigated. The maximum removal capacity of PIPMC beads has been found to be 4.88 ± 0.21 mg Hg/g when the initial metal concentration and weight of polymer-modified composite beads were 50 mg/L and 0.04 g at pH 7, respectively. Mercury removal performance of the papain immobilized polymer-modified composite beads was investigated in conjunction with Cu (II), Zn (II) and Cd (II) ions. The mercury adsorption capacity of papain immobilized polymer-modified composite beads was a slight reduction from 1.15 to 0.89 mg/g in presence of multiple metal salts.     

Fractionation,mobility and multivariate statistical evaluation of metals in marine sediments of Cape Town Harbour,South Africa

Abstract

Distribution of possible chemical forms of Al, Si, Sn, Pb, Zn, Fe, Hg, Cd and Cu in marine sediments of Cape Town harbour was investigated using a modified Tessier’s sequential extraction procedure and ICP-MS and ICP-AES for heavy metals determination. The mean fractions for all metals at all locations were: 1.5–7196 mg kg^-1 for Si, 7.79–7266 mg kg^-1 for Al, 161-639 mg kg^-1 for Cu, 19–41978 mg kg^-1 for Fe, 2.83–5864 mg kg^-1 for Zn, 1.45–13.26 mg kg^-1for Cd, 9.87–223 mg kg^-1 for Sn, 11.98-979 mg kg^-1 for Pb and 0.13–5.93 mg kg^-1 for Hg. Si, Al and Zn were mostly associated with Fe–Mn oxides, whereas Sn and Hg were mainly bound to residual and organic matter. Pb existed mainly in the residual and iron/manganese oxide phases while Cd was evenly distributed in all the five phases. The loading plots of heavy metals bound to the various chemical forms, as well as Pearson correlation coefficients, enabled the determination binding relationship. Pb, Sn and Hg exhibited similar binding behaviour which indicated an anthropogenic point source from wastes from the ship maintenance workshop, and the presence of Sn in the organic phase can be identified with the use of anti-fouling paints at the harbour, whereas Al, Fe, Si, Cu and Zn would probably be of natural origin. Lastly Cd probably came from a diffuse pollution sources in the harbour due to its unique binding characteristic. The mobility of heavy metals varied depending on location and the heavy metal type. The mobility of metals followed the order: Si > Zn > Fe > Cu> Al> Cd> Pb > Sn > Hg. The high percentage of Cd and Pb in the bioavailable forms suggested the need to keep close surveillance on these metals because of their high toxicity.     

Mixture toxicity of copper and zinc to barley at low level effects can be described by the Biotic Ligand Model

Background and aims

The biotic ligand model (BLM) is a bioavailability model for metals based on the concept that toxicity depends on the concentration of metal bound to a biological binding site; the biotic ligand. Here, we evaluated the BLM to interpret and explain mixture toxicity of metals (Cu and Zn).

Methods

The mixture toxicity of Cu and Zn to barley (Hordeum vulgare L.) was tested with a 4 days root elongation test in resin buffered nutrient solutions. Toxicity of one toxicant was tested in presence or absence of a low effect level of the other toxicant or in a ray design with constant toxicant ratios. All treatments ran at three different Ca concentrations (0.3, 2.2 and 10?mM) to reveal ion interaction effects.

Results

The 50 % effect level (EC50) of one metal, expressed as the free ion in solution, significantly (p?<?0.05) increased by adding a low level effect of the other metal at low Ca. Such antagonistic interactions were smaller or became insignificant at higher Ca levels. The Cu EC10 was unaffected by Zn whereas the Zn EC10 increased by Cu at low Ca. These effects obeyed the BLM combined with the independent action model for toxicants.

Conclusions

The BLM model explains the observed interactions by accounting for competition between both metals free ions and Ca²⁺ at the Cu and Zn biotic ligands. The implications of these findings for Cu/Zn interactions in soil are discussed.     

Metal complexes of crosslinked chitosans: Correlations between metal ion complexation values and thermal properties

A series of heavy metal complexes of crosslinked chitosans were evaluated by thermogravimetric studies. The metal complexes with Cu, Cd and Hg ions exhibiting the highest complexing ability to chitosans (Hg 354–364, Cu 100–112, and Cd 121–160, in mg/g chitosan), had the lowest onset of degradation temperatures (range 194–210 °C) and the lowest final degradation temperatures (generally less than 294–304 °C for Hg, 296–338 °C for Cu, and 305–368 °C for Cd complexes). Mn ion, with the lowest binding to chitosans (Mn 5–7 mg/g), showed the reverse behavior, having onset (240–248 °C) and final degradation temperatures (range 300–368 °C). Zn (binding 74–87 mg/g) and Pb (binding 39–62 mg/g) ions have a binding ability intermediate to Cu/Cd/Hg and Mn extremes, and therefore the effects on onset and final degradation temperatures are intermediate to these values.     

X-ray structure of an unusual Ca2+ site and the roles of Zn2+ and Ca2+ in the assembly, stability, and storage of the insulin hexamer.

Metal ion binding to the insulin hexamer has been investigated by crystallographic analysis. Cadmium, lead, and metal-free hexamers have been refined to R values of 0.181, 0.172, and 0.172, against data of 1.9-, 2.5-, and 2.5-A resolution, respectively. These structures have been compared with each other and with the isomorphous two-zinc insulin. The structure of the metal-free hexamer shows that the His(B10) imidazole rings are arranged in a preformed site that binds a water molecule and is poised for Zn2+ coordination. The structure of the cadmium derivative shows that the binding of Cd2+ at the center of the hexamer is unusual. There are three symmetry-related sites located within 2.7 A of each other, and this position is evidently one-third occupied. It is also shown that the coordinating B13 glutamate side chains of this derivative have two partially occupied conformations. One of these conformations is two-thirds occupied and is very similar to that seen in two-zinc insulin. The other, one-third-occupied conformation, is seen to coordinate the one-third-occupied metal ion. The binding of Ca2+ to insulin is assumed to be essentially identical with that of Cd2+. Thus, we conclude that the Ca2+ binding site in the insulin hexamer is unlike that of any other known calcium binding protein. The crystal structures reported herein explain how binding of metal ions stabilizes the insulin hexamer. The role of metal ions in hexamer assembly and dissociation is discussed.     

Metal ion-induced conformational changes in Escherichia coli alkaline phosphatase.

Ultraviolet difference spectra are produced by the binding of divalent metal ions to metal-free alkaline phosphatase (EC 3.1.3.1). The interaction of the apoprotein with Zn2+, Mn2+, Co2+ and Cd2+, which induce the tight binding of one phosphate ion per dimer, give distinctly different ultraviolet spectra changes from Ni2+ and Hg2+ which do not induce phosphate binding. Spectrophotometric titrations at alkaline pH of various metallo-enzymes reveal a smaller number of ionizable tyrosines and a greater stability towards alkaline denaturation in the Zn2+- and Mn2+-enzymes than in the Ni2+-, Hg2+- and apoenzymes. The Zn2+- and Mn2+-enzymes have CD spectra in the region of the aromatic transitions that are different from the CD spectra of the Ni2+-, Hg2+- and apoenzymes. Modifications of arginines with 2,3-butanedione show that a smaller number of arginine residues are modified in the Zn2+-enzyme than in the Hg2+-enzyme. The presented data indicate that alkaline phosphatase from Escherichia coli must have a well-defined conformation in order to bind phosphate. Some metal ions (i.e. Zn2+, Co2+, Mn2+ and Cd2+), when interacting with the apoenzyme, alter the conformation of the protein molecule in such a way that it is able to interact with substrate molecules, while other metal ions (i.e. Ni2+ and Hg2+) are incapable of inducing the appropriate conformational change of the apoenzyme. These findings suggest an important structural function of the first two tightly bound metal ions in enzyme.     

Apolipoprotein B binds ferritin by hemin-mediated binding: evidence of direct binding of apolipoprotein B and ferritin to hemin

Apolipoprotein B (apoB) is known to be a ferritin-binding protein. Here we show that apoB binds to ferritin through hemin-mediated binding. Human apoB bound to bovine spleen, horse spleen, and canine liver ferritins, but did not bind to bovine apoferritin, even after incorporation of iron into it. Incubation of apoferritin with hemin resulted in apoB binding with apoferritin at the same level as with holoferritin. In contrast, hemin inhibited binding of apoB to ferritin. Bovine spleen apoferritin bound biotinylated hemin, and hemin inhibited the binding between the apoferritin and biotinylated hemin, suggesting that ferritin binds hemin directly. ApoB and LDL containing apoB bound biotinylated hemin, and their bindings were also inhibited by hemin, but not protoporphyrin IX. These data demonstrate that binding of apoB to ferritin is mediated through ferritin’s binding to hemin, and also that apoB binds hemin directly.