Free Hemin Research Articles

Involvement of Methemoglobin (MetHb) Formation and Hemin Loss in the Pro-oxidant Activity of Fish Hemoglobins

The capacity of Atlantic pollock ( Pollachius pollachius ), seabass ( Dicentrarchus labrax ), and horse mackerel ( Trachurus trachurus ) hemoglobin (Hb) to promote lipid oxidation has been evaluated in liposomes and washed minced horse mackerel muscle. The pro-oxidant ability of fish Hbs was related with their vulnerability to suffer oxidation to metHb and release hemin either in spontaneous situation or in the presence of two representative lipid oxidation products, linolein hydroperoxides and trans-2-pentenal. The results indicated similar effectiveness to promote lipid oxidation in liposomes and washed fish muscle: pollock Hb > horse mackerel Hb > seabass Hb. Pollock Hb showed a more elevated autoxidation rate and spontaneous hemin loss and also faster oxidation to metHb in the presence of hydroperoxides and trans-2-pentenal. The autoxidation and spontaneous hemin loss rates were intermediate for horse mackerel Hb, whereas seabass Hb exhibited the highest stability. The isoelectrofocusing (IEF) pattern of pollock Hb revealed the presence of isoforms with elevated anionic character, which are known to have poor oxygen affinity at the pH values found in fish muscle (pH 7.0-5.5). In agreement with the IEF patterns, pollock Hb was less oxygenated at pH 6.8, and seabass Hb exhibited more oxygenation than did horse mackerel. MetHb forms were significantly more effective in catalyzing lipid oxidation than the corresponding reduced fish Hbs and free hemin. The present investigation highlights a direct correlation between the pro-oxidant capacity of fish Hbs and their susceptibility to undergo metHb formation and hemin loss and also suggests a potential role of lipid oxidation byproducts in activating the pro-oxidative action of hemoglobin.

Hemin Augments Growth and Hemoglobinization of Erythroid Precursors Derived from Patients with Diamond-Blackfan Anemia (DBA).

DBA is a congenital form of pure red cell anemia characterized by a macrocytic anemia, reticulocytopenia, and a block in erythroid differentiation at the proerythroblast stage, often in association with physical anomalies and growth retardation. About 25% of the patients carry mutations in genes that encode for proteins (RPS19, RPS24 and RPS17) that bind to the 40S subunit of the ribosome. The resultant defect in ribosomal biogenesis has been proposed to impair the initiation of globin translation, leading to mismatch between intracellular levels of heme and globin chains. It has been hypothesized that the transient excess of intracellular free heme resulting from the delay in globin synthesis exerts direct toxicity to erythroid precursors and plays a major role in pathogenesis of DBA through apoptosis of proerythroblasts (Keel et al., Science319;825,2008).Free hemin, however, is not necessarily toxic to developing erythroid precursors. Exogenously supplied hemin is readily taken up by erythroid cells in culture and its iron is incorporated into hemoglobin or stored in ferritin (Fibach et al., J Cell Physiol130;460,1987). Following addition of succinylacetone, a potent inhibitor of heme synthesis, exogenously supplied hemin can replace intracellularly synthesized heme and be incorporated into de novo formed hemoglobin (Fibach et al., Blood85;2967,1995). Hemin supplementation to semi-solid cultures promotes the growth of normal erythroid precursors (e.g., Lu and Broxmeyer, Exp Hematol11;721,1983). We showed in a two-phase liquid culture that exogenous hemin promotes normal erythropoiesis by accelerating the proliferation and hemoglobinization of erythroid precursors in the presence or absence of transferrin (Fibach et al., Blood85;2967,1995). This effect was particularly prominent during the early stages of hemoglobinization, when iron-uptake and heme synthesis are rate-limiting. In the present study we show that surplus hemin (10 - 50 mM) supplemented to cultures at early stage of erythroid development is well tolerated. Although the generation of reactive oxygen species (measured by staining with dichlorofluorescein diacetate) was modestly (50 ± 15%, N=4) increased, it was not associated with increased apoptosis, as measured by binding of annexin V, nor necrosis as measured by propidium iodide staining.Having demonstrated the growth and differentiation promoting potential of exogenous hemin on normal erythroid precursors and lack of overt toxicity, we studied the effect of exogenous heme in cultures of erythroid cells derived from six patients with DBA. We show that hemin, added as heme chloride or heme arginate, circumvented the primary defect and significantly stimulated (4 - 20-fold, p<0.001) ) the growth of DBA erythroid cells and their hemoglobinization.In conclusion, our results show thatexogenous hemin is taken up by developing erythroid cells and can supplement or substitute endogenously synthesized heme;excess heme stimulates free radical generation moderately but does not cause apoptosis or necrosis;addition of hemin to cultured erythroid precursors derived from normal donors stimulates their growth and hemoglobinization, and in DBA, in contrast to the recently proposed scheme, heme can actually restore the growth and differentiation potential of the DBA-erythroid precursors.The beneficial effect of hemin on DBA erythroid precursors may be related to its effect on translation initiation factors, such as eIF-2 , and suggests a therapeutic potential.

Heme-Mediated Production of Free Radicals via Preformed Lipid Hydroperoxide Fragmentation

Electron spin resonance (ESR) spectroscopy and the spin-trapping technique were used to investigate the capacity of several hemoglobin (Hb) forms of rainbow trout (oxyHb and metHb), free hemin (oxidized form of heme group), and hemin complexed with bovine serum albumin (BSA) to promote formation of free radicals via fragmentation of preformed lipid hydroperoxides. Cumene hydroperoxide (CumOOH) was used as a model for lipid hydroperoxide, and free radicals were monitored by stabilizing with the spin traps alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Two different types of free radicals, hydroxyl and carbon-centered radicals, were identified as a result of the interaction of the heme-containing systems and CumOOH. Carbon-centered radicals were found to be mainly heme-mediated because the addition of the iron-chelating agent EDTA did not affect the formation of POBN/carbon-centered adducts. Hemin alone was the best promoter for the production of POBN/carbon-centered radicals in the presence of low hydroperoxide concentrations (below equimolar condition over heme group), whereas hemin/BSA and oxyHb were more active in generating radicals at high hydroperoxide concentrations or after successive interactions with hydroperoxides. This finding can be explained by the coexistence of two different facts: (i) the interaction between hemin and lipid hydroperoxides seems to be more efficient in the case of free hemin compared to heme-protein complexes and (ii) a faster degradation of hemin is produced without the presence of a protein fraction, globin or albumin. The comparison of oxyHb and metHb also suggested that both Hb redox states have similar capacities to generate oxidative stress via cleavage of preformed lipid hydroperoxides.

A novel hemin-based organic phase artificial enzyme electrode and its application in different hydrophobicity organic solvents

Based on the newly discovered artificial enzyme formed by mixing hemin with supramolecular hydrogels via the self-assembly of amphiphilic oligopeptides, we prepared a novel organic phase artificial enzyme electrode by coating the artificial enzyme on an electrode which was then covered with sodium alginate for protection. Scanning electron micrograph showed that the supramolecular hydrogel kept its nanofibers structure on the electrode surface. Hemin dispersed in the supermolecular hydrogel as monomer greatly promotes its direct electrochemistry behavior in organic solvents. At the same time, this electrode exhibited higher electrocatalytic ability to tert-butyl hydroperoxide (TBHP) than free hemin modified electrode (free hemin mainly present as dimer). As low as 27μM TBHP could be detected with a linear range from 6.6×10−5 to 1.27×10−2M via amperometric method. The biosensor can reach 95% of the steady-state current in about 10±2s. More importantly, it can be applied in both hydrophilic and hydrophobic solvents without adding extra buffer or mediators to them that cannot be received by most traditional organic phase enzyme electrodes. This unique property greatly promotes the development of the organic phase enzyme electrodes by facilitating the detection of different kinds of substrates of the hemin-based artificial enzyme soluble in hydrophilic and hydrophobic solvents. The artificial enzyme electrode was successfully used to determine organic peroxides in body lotion samples.

Pathway for Heme Uptake from Human Methemoglobin by the Iron-regulated Surface Determinants System of Staphylococcus aureus

The iron-regulated surface proteins IsdA, IsdB, and IsdC and transporter IsdDEF of Staphylococcus aureus are involved in heme acquisition. To establish an experimental model of heme acquisition by this system, we have investigated hemin transfer between the various couples of human methemoglobin (metHb), IsdA, IsdB, IsdC, and IsdE by spectroscopic and kinetic analyses. The efficiencies of hemin transfer from hemin-containing donors (holo-protein) to different hemin-free acceptors (apo-protein) were examined, and the rates of the transfer reactions were compared with that of indirect loss of hemin from the relevant donor to H64Y/V68F apomyoglobin. The efficiencies, spectral changes, and kinetics of the transfer reactions demonstrate that: 1) metHb directly transfers hemin to apo-IsdB, but not to apo-IsdA, apo-IsdC, and apo-IsdE; 2) holo-IsdB directly transfers hemin to apo-IsdA and apo-IsdC, but not to apo-IsdE; 3) apo-IsdE directly acquires hemin from holo-IsdC, but not from holo-IsdB and holo-IsdA; and 4) IsdB and IsdC enhance hemin transfer from metHb to apo-IsdC and from holo-IsdB to apo-IsdE, respectively. Taken together with our recent finding that holo-IsdA directly transfers its hemin to apo-IsdC, these results provide direct experimental evidence for a model in which IsdB acquires hemin from metHb and transfers it directly or through IsdA to IsdC. Hemin is then relayed to IsdE, the lipoprotein component of the IsdDEF transporter.

A novel mimetic peroxidase catalyst by using magnetite-containing silica nanoparticles as carriers

A method for coating magnetite and mimetic enzyme (hemin) with amorphous silica to form a novel mimetic peroxidase (magnetite-hemin/SiO2) has been developed by combining reverse microemulsion and the modified Stober method. The magnetic silica nanoparticle supported hemin has a long-term stability toward temper- ature and good reusability. They can be easily separated from the reaction solution by using an external magnetic field and reused directly for next round of reaction. The peroxidase activity of the magnetite-hemin/SiO2 was studied based on its catalytic effect on the reaction of p- hydroxyphenylacetic acid and H2O2. The results indicated that the catalytic activity of the new mimetic enzyme catalyst is higher than that of the free hemin. The possibility of its application was proven by the determination of H2O2, with the detection limits of 7.3 nmol L �1 H2O2.

Direct Hemin Transfer from IsdA to IsdC in the Iron-regulated Surface Determinant (Isd) Heme Acquisition System of Staphylococcus aureus

The iron-regulated surface determinants (Isd) of Staphylococcus aureus, including surface proteins IsdA, IsdB, IsdC, and IsdH and ATP-binding cassette transporter IsdDEF, constitute the machinery for acquiring heme as a preferred iron source. Here we report hemin transfer from hemin-containing IsdA (holo-IsdA) to hemin-free IsdC (apo-IsdC). The reaction has an equilibrium constant of 10 +/- 5 at 22 degrees C in favor of holo-IsdC formation. During the reaction, holo-IsdA binds to apo-IsdC and then transfers the cofactor to apo-IsdC with a rate constant of 54.3 +/- 1.8 s(-1) at 25 degrees C. The transfer rate is >70,000 times greater than the rate of simple hemin dissociation from holo-IsdA into solvent (k transfer = 54.3 s(-1) versus k -hemin = 0.00076 s(-1)). The standard free energy change, Delta G 0, is -27 kJ/mol for the formation of the holo-IsdA-apo-IsdC complex. IsdC has a higher affinity for hemin than IsdA. These results indicate that the IsdA-to-IsdC hemin transfer is through the activated holo-IsdA-apo-IsdC complex and is driven by the higher affinity of apo-IsdC for the cofactor. These findings demonstrate for the first time in the Isd system that heme transfer is rapid, direct, and affinity-driven from IsdA to IsdC. These results also provide the first example of heme transfer from one surface protein to another surface protein in Gram-positive bacteria and, perhaps most importantly, indicate that the mechanism of activated heme transfer, which we previously demonstrated between the streptococcal proteins Shp and HtsA, may apply in general to all bacterial heme transport systems.

A Direct Electrode-Driven Immobilized Catalase for Biocatalysis of Peroxides

The direct electrochemistry of catalase (Cat) immobilized in agarose hydrogel film on glassy carbon (GC) electrode was investigated. A pair of well-defined and quasi-reversible redox peaks both in aqueous buffer and ethanol-aqueous mixture was obtained. The formal potential (E°') indicates the reactions origin from the direct electron transfer between the CatFe(III)/(II) couple and the electrode surface. The E°' is linearly dependent on solution pH (redox Bohr effect), indicating the electron transfer of the CatFe(III)/(II) redox couple accompanied with the transfer of a proton. The electron transfer can be facilitated in ethanol-aqueous mixture for the reason of partial dehydration of the distal heme pocket. The water pools in agarose hydrogel play an important role in preventing either the change of conformation or the inactivity of Cat from polar organic solvent. Cat retains its original conformation in the agarose film that was confirmed by UV-Vis spectra and electrochemical behaviors in comparison to the controlled experiments using free hemin. The catalytic reactions to peroxides (hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, 2-butanone peroxide) using Cat-agarose modified electrode were also explored both in aqueous buffer and ethanol-aqueous mixture. The method could be application to the preparation of the hydrogel based third generation biosensors or bioreactors in organic media.

Bis-methionyl Coordination in the Crystal Structure of the Heme-binding Domain of the Streptococcal Cell Surface Protein Shp

Surface proteins Shr, Shp, and the ATP-binding cassette (ABC) transporter HtsABC are believed to make up the machinery for heme uptake in Streptococcus pyogenes. Shp transfers its heme to HtsA, the lipoprotein component of HtsABC, providing the only experimentally demonstrated example of direct heme transfer from a surface protein to an ABC transporter in Gram-positive bacteria. To understand the structural basis of heme transfer in this system, the heme-binding domain of Shp (Shp 180) was crystallized, and its structure determined to a resolution of 2.1 Å. Shp 180 exhibits an immunoglobulin-like β-sandwich fold that has been recently found in other pathogenic bacterial cell surface heme-binding proteins, suggesting that the mechanisms of heme acquisition are conserved. Shp shows minimal amino acid sequence identity to these heme-binding proteins and the structure of Shp 180 reveals a unique heme–iron coordination with the axial ligands being two methionine residues from the same Shp molecule. A negative electrostatic surface of protein structure surrounding the heme pocket may serve as a docking interface for heme transfer from the more basic outer cell wall heme receptor protein Shr. The crystal structure of Shp 180 reveals two exogenous, weakly bound hemins, which form a large interface between the two Shp 180 molecules in the asymmetric unit. These “extra” hemins form a stacked pair with a structure similar to that observed previously for free hemin dimers in aqueous solution. The propionates of the protein-bound heme coordinate to the iron atoms of the exogenous hemin dimer, contributing to the stability of the protein interface. Gel filtration and analytical ultracentrifugation studies indicate that both full-length Shp and Shp 180 are monomeric in dilute aqueous solution.

Spectroelectrochemical Study of the Hemin - Glutathione Interaction in the Absence and Presence of Surfactants

The interaction of hemin with reduced glutathione (GSH) was studied using cyclic and linear sweep voltammetry with stationary and rotating disc electrodes and UV-VIS spectroelectrochemical experiments. A 1:1 global stoichiometry and similar values for the hemin - GSH binding constant were obtained in aqueous media in the presence of sodium dodecyl sulfate (SDS) with those in dimethylsulphoxide (DMSO). A comparison between the family of curves obtained during the electrochemical reduction of hemin with that recorded at the hemin - GSH interaction points to different absorption bands, leading to the idea of a complexation of the FeIII by GSH, rather than a Fe III → Fe II reduction. The biochemical relevance of the hemin-glutathione interaction is related to the presence of GSH in normal erythrocytes in high concentrations (1 - 4 mM), being involved as an agent in the trapping of free hemin from the cell cytosol.

Catalytical oxidation of styrene by molecularly imprinted polymer with phenylacetic acid as template and hemin as co-monomer

This letter used the molecular imprinting technology to build up the microenvironment around co-monomer hemin to mimic the cytochrome P450 catalyzing the epoxidation of styrene. The results showed that the conversion rates of products were obviously enhanced by molecularly imprinted polymers, compared to free hemin solution, using three kinds of oxidants. The used axial ligand in polymers synthesis also improved the total conversion rates.

Noncovalent porphyrin-functionalized single-walled carbon nanotubes: solubilization and spectral behaviors

We describe the solubilization/dispersion of as-produced and purified single-walled carbon nanotubes (raw-SWNTs and p-SWNTs) with protoporphyrin IX derivatives and tetraphenylporphyrin iron(III) chloride, the spectral behavior of the octaalkylporphyrins-solubilized shortened-SWNTs (s-SWNTs), and the electrochemistry of a p-SWNTs/FePP cast film on a glassy carbon electrode. Transmission electron and atomic force microscopies, as well as UV-visible-near IR spectroscopy, revealed that the protoporphyrin IX derivatives individually dissolved the p-SWNTs in polar solvents under mild conditions of sonication using a bath-type sonicator, followed by centrifugation at 1000 g. The raw-SWNTs were more easily dissolved than the p-SWNTs with protoporphyrin IX zinc(II) ( ZnPP ), whereas the amount of the solubilized/dispersed p-SWNTs did not depend on the concentration of the solubilizer and sonication time. The absorption and fluorescence spectral measurements of the octaalkylporphyrins in dimethylformamide containing 1 vol% tetrahydrofuran with various amounts of the s-SWNTs showed that the absorption maxima of the octaalkylporphyrins decreased with an increase in the concentration of the s-SWNTs without wavelength shift and the fluorescence of the porphyrins was quenched by the addition of the s-SWNTs. These spectral behaviors are direct evidence for the interaction between the nanotube sidewall and the porphyrins in the solutions. The cyclic voltammograms of the p-SWNTs/hemin ( FePP ) and free hemin ( FePP ) suggest that the nanotubes act as a conduction passage for electrons between hemin ( FePP ) and the glassy carbon electrode.

A Supramolecular‐Hydrogel‐Encapsulated Hemin as an Artificial Enzyme to Mimic Peroxidase

Faking it: The use of a supramolecular hydrogel as the structural component of artificial enzymes provides a new and useful approach to the development of biomimetic catalysts. In toluene, hemin chloride encapsulated in such a hydrogel achieves about 60 % nascent catalytic activity of horseradish peroxidase. Additionally, the activity of hemin in the hydrogel is 387.1 times greater than that of free hemin.

Theoretical study of “deep” fragmentation of hemin ion with successive loss of methyl and vinyl groups

The electronic and geometric structures, energy stability, normal mode frequencies, and spin density distribution have been calculated by the density functional theory B3LYP method with the Gen = 6-31+G*(Fe) + 6-31G(C,H,N,O), 6-31G*, and 6-311++G** basis sets for the deep fragmentation products of the free hemin ion with successive removal of methyl and vinyl groups in the electronic states with different multiplicities. The computation results are compared with the available experimental data and previous computation results for the fragmentation products of the isolated heme molecule and hemin ion with removal of carboxymethyl groups. The trends in the behavior of these properties are analyzed as a function of multiplicity, external charge, and the number of peripheral substituents at the porphyrin core.

The Interaction of Nitric Oxide with Distinct Hemoglobins Differentially Amplifies Endothelial Heme Uptake and Heme Oxygenase-1 Expression

Heme is a strong inducer and substrate of the stress protein heme oxygenase-1 (HO-1), which produces carbon monoxide, iron, and bilirubin. We have reported recently that nitric oxide (NO) augments the incorporation of free hemin in endothelial cells, resulting in amplified HO-1 expression and production of bilirubin. Here, we extend our studies by showing that both NO+ and NO- donors interacted with reduced (HbA0) or oxidized (metHb) hemoglobin, as well as hemoglobin from sickle cell disease (HbS), to strongly magnify HO-1, with a pattern of induction dependent on the oxidation state of the hemoglobin used. A corresponding enhancement of endothelial heme uptake was observed following exposure of HbA0 or HbS to the NO donors, which also increased the uptake of free hemin. We postulated that this effect may be caused by formation of heme-nitrosyl (H-NO) complexes, and indeed endothelial cells exposed to preformed H-NO showed greater heme incorporation than free hemin. Furthermore, NO donors directly affected the permeability of membranes to free hemin. In conclusion, our data indicate a novel role for NO in the modulation of heme transport and HO-1 induction in endothelial cells, which may be relevant for hematological disorders characterized by disruption of the heme-NO equilibrium.

Phytol‐modified heme in mesoporous silica: Conjugates as models of hemoproteins

A ferriprotoporphyrin, hemin (Fe(3+)), modified with 3,7,11,15-tetramethyl-2-hexadecen-1-ol, phytol, was adsorbed in nano-spaces of about 4 nm in diameter in mesoporous silica (FSM; folded-sheet mesoporous material) forming a phytol-modified hemin (Fe(3+))-FSM nano-conjugate. The properties and the structure of the conjugate were studied by UV-visible light absorption, IR absorption spectroscopy, and a nitrogen adsorption isotherm. Although the hemin without phytol could not be adsorbed to the mesoporous silica, modification with phytol imparted preferential adsorption properties. The conjugate was not only stable but also had a peroxidase-like activity in a 0.1% hydrogen peroxide solution, while free hemin in the solution was easily destroyed. The hemin (Fe(3+)) in the FSM was reduced to heme (Fe(2+)) by hydrazine. The phytol-modified heme (Fe(2+))-FSM conjugate formed an O(2)-heme complex with a superoxide type structure, resembling oxyhemoglobin or oxymyoglobin, which has not been previously observed for free heme in solution. The addition of carbon monoxide or nitrogen monoxide to the phytol-modified heme (Fe(2+))-FSM conjugate caused the formation of CO- or NO-heme complex in the nano-spaces of the FSM. These properties are attributed not only to the Fe-complex but also to the cooperative functions of the heme with mesoporous silica, resembling properties of a natural heme-protein conjugate; hemoglobin or peroxidase. These results are an elegant example of biomimetic nano-technology.

Cooperativity and Pseudo-cooperativity in the Glutathione S-Transferase from Plasmodium falciparum

Binding and catalytic properties of glutathione S-transferase from Plasmodium falciparum (PfGST) have been studied by means of fluorescence, steady state and pre-steady state kinetic experiments, and docking simulations. This enzyme displays a peculiar reversible low-high affinity transition, never observed in other GSTs, which involves the G-site and shifts the apparent K(D) for glutathione (GSH) from 200 to 0.18 mM. The transition toward the high affinity conformation is triggered by the simultaneous binding of two GSH molecules to the dimeric enzyme, and it is manifested as an uncorrected homotropic behavior, termed "pseudo-cooperativity." The high affinity enzyme is able to activate GSH, lowering its pK(a) value from 9.0 to 7.0, a behavior similar to that found in all known GSTs. Using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, this enzyme reveals a potential optimized mechanism for the GSH conjugation but a low catalytic efficiency mainly due to a very low affinity for this co-substrate. Conversely, PfGST efficiently binds one molecule of hemin/monomer. The binding is highly cooperative (n(H) = 1.8) and occurs only when GSH is bound to the enzyme. The thiolate of GSH plays a crucial role in the intersubunit communication because no cooperativity is observed when S-methylglutathione replaces GSH. Docking simulations suggest that hemin binds to a pocket leaning into both the G-site and the H-site. The iron is coordinated by the amidic nitrogen of Asn-115, and the two carboxylate groups are in electrostatic interaction with the epsilon-amino group of Lys-15. Kinetic and structural data suggest that PfGST evolved by optimizing its binding property with the parasitotoxic hemin rather than its catalytic efficiency toward toxic electrophilic compounds.

Redox reaction of hemin-immobilized polyallylamine–polystyrene latex suspensions

A nearly mono-dispersed spherical electroactive particle, 1.16 μm in diameter, was synthesized by coating polystyrene particles successively with polyallylamine and hemin with the aim of investigating the redox reactivity of large particles. Each particle contains 2 × 10 7 hemin moieties. Its suspension in 2-propanol solution was stable without aggregation. The voltammograms showed a pair of redox waves due to the immobilized hemin. The cathodic current was controlled by diffusion of the particle toward the electrode. The amount of hemin participating in the electrode reaction was only a quarter of the amount of loaded hemin. Dioxygen in solution was reduced catalytically with the hemin-coated particles. The catalytic efficiency for the hemin-coated particles was higher than that for free hemin molecules, because dioxygen confined in particles as well as dissolved dioxygen can participate in the catalytic reaction. The catalytic reaction rate constant was evaluated on the assumption of a pseudo-first-order reaction to give 0.08 M −1 s −1, which was half the rate constant of hemin.

Silica-hemin composite nanoparticles as new biocatalyst to highly sensitive determination of glucose in human serum.

The preparation and utilization of a novel composite silica-hemin nanoparticles (CSHNs) as mimetic peroxidase are reported in this article. Experimental results showed that the composite nanoparticles had unique advantages over free hemin molecules in good stability and highly catalytic activity. By employing these nanoparticles as biocatalyst, we developed a new spectrofluorometric method for the determination of trace level glucose. The calibration graph for glucose was linear over the range 4.0 x 10(-7) mol/L - 7.0 x 10(-5) mol/L, with a detection limit of 1.0 x 10(-7) mol/L. The proposed method has been successfully applied to the determination of glucose in serum samples and offers the advantages of being rapid, stable, sensitive, and renewable.

Heme-iron increases levels of AP65-mediated adherence by Trichomonas vaginalis

Trichomonas vaginalis is a protozoan responsible for the number one, non-viral sexually transmitted disease. Surface proteins (AP65, AP51, AP33 and AP23) mediate adherence to vaginal epithelial cells (VECs). Iron increases growth of trichomonads and synthesis and surface placement of adhesins. We observed by immunofluorescence using monoclonal antibody (mAb) 12G4 the placement of AP65 on surfaces of trichomonads supplemented with hemoglobin or hemin as a source of iron. We, therefore, tested the hypothesis that heme-bound iron is an alternative source of iron important to trichomonal growth and regulation of expression of the adhesin genes. Here we show that the inhibition of parasite growth by the iron chelator 2,2-dipyridal is rescued by hemoglobin or hemin, but not protoporphyrin IX. Importantly, trichomonads grown in iron-limiting medium supplemented with free iron, hemoglobin and hemin had elevated levels of ap65 transcript that were 12.6-, 12.3- and 9.2-fold higher, respectively, than low-iron organisms, as determined by RT-PCR. Similarly, the amounts of AP65 were 8.9-, 11.2-, and 4.8-fold higher in parasites grown in free iron, hemoglobin and hemin, respectively, than organisms in low-iron medium. The heme-iron-regulated AP65 increased adherence of parasites to immortalized VECs. Not surprisingly, parasites pretreated with anti-AP65 serum IgG had decreased adherence compared to organisms incubated with prebleed serum IgG. These data illustrate that heme-bound iron is a source of iron similar to lactoferrin. This work extends our findings about the multiple sources of iron for regulating virulence genes of T. vaginalis.