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Sample records for recombinant horseradish peroxidase

  1. Recombinant horseradish peroxidase variants for targeted cancer treatment.

    PubMed

    Bonifert, Günther; Folkes, Lisa; Gmeiner, Christoph; Dachs, Gabi; Spadiut, Oliver

    2016-06-01

    Cancer is a major cause of death. Common chemo- and radiation-therapies damage healthy tissue and cause painful side effects. The enzyme horseradish peroxidase (HRP) has been shown to activate the plant hormone indole-3-acetic acid (IAA) to a powerful anticancer agent in in vitro studies, but gene directed enzyme prodrug therapy (GDEPT) studies showed ambivalent results. Thus, HRP/IAA in antibody directed enzyme prodrug therapy (ADEPT) was investigated as an alternative. However, this approach has not been intensively studied, since the enzyme preparation from plant describes an undefined mixture of isoenzymes with a heterogenic glycosylation pattern incompatible with the human system. Here, we describe the recombinant production of the two HRP isoenzymes C1A and A2A in a Pichia pastoris benchmark strain and a glyco-engineered strain with a knockout of the α-1,6-mannosyltransferase (OCH1) responsible for hypermannosylation. We biochemically characterized the enzyme variants, tested them with IAA and applied them on cancer cells. In the absence of H2 O2 , HRP C1A turned out to be highly active with IAA, independent of its surface glycosylation. Subsequent in vitro cytotoxicity studies with human T24 bladder carcinoma and MDA-MB-231 breast carcinoma cells underlined the applicability of recombinant HRP C1A with reduced surface glycoslyation for targeted cancer treatment. Summarizing, this is the first study describing the successful use of recombinantly produced HRP for targeted cancer treatment. Our findings might pave the way for an increased use of the powerful isoenzyme HRP C1A in cancer research in the future. PMID:26990592

  2. Formation and properties of dimeric recombinant horseradish peroxidase in a system of reversed micelles.

    PubMed Central

    Gazaryan, I G; Klyachko, N L; Dulkis, Y K; Ouporov, I V; Levashov, A V

    1997-01-01

    Wild-type recombinant horseradish peroxidase purified and refolded from Escherichia coli inclusion bodies has been studied in the system of bis(2-ethylhexyl)sulphosuccinate sodium salt (Aerosol OT)-reversed micelles in octane. In contrast with native horseradish peroxidase the wild-type recombinant enzyme forms dimeric structures as judged by sedimentation analysis. Peroxidase substrates affect the equilibrium between monomeric and dimeric enzyme forms. The dependence of the catalytic activity of recombinant peroxidase on the degree of hydration of the surfactant exhibits two maxima with pyrogallol, o-phenylene- diamine, guaiacol and o-dianisidine, with different ratios of activities for the first and second maxima. The differences in activities of monomeric and dimeric forms of the recombinant horseradish peroxidase provide evidence for active-site screening in dimeric forms. This has been used to model a dimeric structure of recombinant horseradish peroxidase with the screened entrance to the active site. In the model structure obtained, three of eight glycosylation sites were screened. This might explain the absence of dimeric structures in native enzyme peroxidase. The system of reversed micelles provides, for the first time, evidence for the formation of dimeric structures by recombinant plant peroxidase with an altered substrate specificity compared with the native enzyme. Thus one can assume that haem-containing peroxidases in general are able to form dimeric structures. PMID:9371726

  3. Oxidation of 4-bromophenol by the recombinant fused protein cellulose-binding domain-horseradish peroxidase immobilized on cellulose.

    PubMed

    Levy, Ilan; Ward, Gary; Hadar, Yitzhak; Shoseyov, Oded; Dosoretz, Carlos G

    2003-04-20

    A fused protein consisting of cellulose-binding domain (CBD) and horseradish peroxidase (HRP) was constructed and expressed in Escherichia coli. Refolded recombinant CBD-HRP (95% recovery yield) was bound to microcrystalline cellulose and applied for the oxidation of a model toxic phenol, 4-bromophenol (BP). Oxidation of BP by CBD-HRP resulted in the formation of dimers to pentamers as evidenced by mass spectrometry analysis. When immobilized, the vast majority of the oxidation products adsorbed to the cellulose matrix. CBD-HRP (0.75 pyrogallol units) bound to 0.1 g cellulose was packed in a column, connected to an HPLC pump and monitoring system, and column performance and capacity were studied under various operating conditions. When performance was studied as a function of BP loading rate at a constant H(2)O(2) loading rate of 1500 nmol/min, V(app) (max) and K(m) (app) were calculated to be 5.29 +/- 0.46 micromol mL min and 644.9 +/- 114.3 microM, respectively. Immobilized CBD-HRP exhibited enhanced stability to H(2)O(2) and oxidized considerably more BP than free CBD-HRP. Inclusion of gelatin, which suppresses product-dependent inactivation, further increased the amount of BP oxidation. These findings may have potential impact in terms of enzyme supply in high-rate treatment of wastewater contaminated with toxic phenols, since the susceptibility of peroxidases to both H(2)O(2) - and product-dependent inactivation demands continuous supply of fresh enzyme. PMID:12584764

  4. Bioconjugation of antibodies to horseradish peroxidase (hrp)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The bioconjugation of an antibody to an enzymatic reporter such as horseradish peroxidase (HRP) affords an effective mechanism by which immunoassay detection of a target antigen can be achieved. The use of heterobifunctional cross—linkers to covalently link antibodies to HRP provides a simple and c...

  5. Peroxidase gene discovery from the horseradish transcriptome

    PubMed Central

    2014-01-01

    Background Horseradish peroxidases (HRPs) from Armoracia rusticana have long been utilized as reporters in various diagnostic assays and histochemical stainings. Regardless of their increasing importance in the field of life sciences and suggested uses in medical applications, chemical synthesis and other industrial applications, the HRP isoenzymes, their substrate specificities and enzymatic properties are poorly characterized. Due to lacking sequence information of natural isoenzymes and the low levels of HRP expression in heterologous hosts, commercially available HRP is still extracted as a mixture of isoenzymes from the roots of A. rusticana. Results In this study, a normalized, size-selected A. rusticana transcriptome library was sequenced using 454 Titanium technology. The resulting reads were assembled into 14871 isotigs with an average length of 1133 bp. Sequence databases, ORF finding and ORF characterization were utilized to identify peroxidase genes from the 14871 isotigs generated by de novo assembly. The sequences were manually reviewed and verified with Sanger sequencing of PCR amplified genomic fragments, resulting in the discovery of 28 secretory peroxidases, 23 of them previously unknown. A total of 22 isoenzymes including allelic variants were successfully expressed in Pichia pastoris and showed peroxidase activity with at least one of the substrates tested, thus enabling their development into commercial pure isoenzymes. Conclusions This study demonstrates that transcriptome sequencing combined with sequence motif search is a powerful concept for the discovery and quick supply of new enzymes and isoenzymes from any plant or other eukaryotic organisms. Identification and manual verification of the sequences of 28 HRP isoenzymes do not only contribute a set of peroxidases for industrial, biological and biomedical applications, but also provide valuable information on the reliability of the approach in identifying and characterizing a large group

  6. Horseradish peroxidase catalyzed hydroxylations: mechanistic studies.

    PubMed

    Dordick, J S; Klibanov, A M; Marletta, M A

    1986-05-20

    The hydroxylation of phenol to hydroquinone and catechol in the presence of dihydroxyfumaric acid and oxygen catalyzed by horseradish peroxidase was studied under conditions where the product yield was high and the side reactions were minimal. The reaction is partially uncoupled with a molar ratio of dihydroxyfumaric acid consumed to hydroxylated products of 12:1. Hydrogen peroxide does not participate in the reaction as evidenced by the lack of effect of catalase and by the direct addition of hydrogen peroxide. Conversely, superoxide and hydroxyl radicals are involved as their scavengers are potent inhibitors. Experiments were all consistent with the involvement of compound III (oxygenated ferrous complex) of peroxidase in the reaction. Compound III is stable in the presence of phenol alone but decomposes rapidly in the presence of both phenol and dihydroxyfumaric acid with the concomitant formation of product. Therefore, phenol and dihydroxyfumaric acid must be present with compound III in order for the hydroxylation reaction to occur. A mechanism consistent with the experimental results is proposed. PMID:3718931

  7. Horseradish peroxidase catalyzed nitric oxide formation from hydroxyurea.

    PubMed

    Huang, Jinming; Sommers, Erin M; Kim-Shapiro, Daniel B; King, S Bruce

    2002-04-01

    Hydroxyurea represents an approved treatment for sickle cell anemia and a number of cancers. Chemiluminescence and electron paramagnetic resonance spectroscopic studies show horseradish peroxidase catalyzes the formation of nitric oxide from hydroxyurea in the presence of hydrogen peroxide. Gas chromatographic headspace analysis and infrared spectroscopy also reveal the production of nitrous oxide in this reaction, which provides evidence for nitroxyl, the one-electron reduced form of nitric oxide. These reactions also generate carbon dioxide, ammonia, nitrite, and nitrate. None of these products form within 1 h in the absence of hydrogen peroxide or horseradish peroxidase. Electron paramagnetic resonance spectroscopy and trapping studies show the intermediacy of a nitroxide radical and a C-nitroso species during this reaction. Absorption spectroscopy indicates that both compounds I and II of horseradish peroxidase act as one-electron oxidants of hydroxyurea. Nitroxyl, generated from Angeli's salt, reacts with ferric horseradish peroxidase to produce a ferrous horseradish peroxidase-nitric oxide complex. Electron paramagnetic resonance experiments with a nitric oxide specific trap reveal that horseradish peroxidase is capable of oxidizing nitroxyl to nitric oxide. A mechanistic model that includes the observed nitroxide radical and C-nitroso compound intermediates has been forwarded to explain the observed product distribution. These studies suggest that direct nitric oxide producing reactions of hydroxyurea and peroxidases may contribute to the overall pharmacological properties of this drug. PMID:11916434

  8. Production and purification of the multifunctional enzyme horseradish peroxidase

    PubMed Central

    Spadiut, Oliver; Herwig, Christoph

    2014-01-01

    The oxidoreductase horseradish peroxidase (HRP) is used in numerous industrial and medical applications. In this review, we briefly describe this well-studied enzyme and focus on its promising use in targeted cancer treatment. In combination with a plant hormone, HRP can be used in specific enzyme–prodrug therapies. Despite this outstanding application, HRP has not found its way as a biopharmaceutical into targeted cancer therapy yet. The reasons therefore lie in the present low-yield production and cumbersome purification of this enzyme from its natural source. However, surface glycosylation renders the recombinant production of HRP difficult. Here, we compare different production hosts for HRP and summarize currently used production and purification strategies for this enzyme. We further present our own strategy of glycoengineering this powerful enzyme to allow recombinant high-yield production in Pichia pastoris and subsequent simple downstream processing. PMID:24683473

  9. Immobilization of horseradish peroxidase onto kaolin.

    PubMed

    Šekuljica, Nataša Ž; Prlainović, Nevena Ž; Jovanović, Jelena R; Stefanović, Andrea B; Djokić, Veljko R; Mijin, Dušan Ž; Knežević-Jugović, Zorica D

    2016-03-01

    Kaolin showed as a very perspective carrier for the enzyme immobilization and it was used for the adsorption of horseradish peroxidase (HRP). The effects of the enzyme concentration and pH on the immobilization efficiency were studied in the reaction with pyrogallol and anthraquinone dye C.I. Acid Violet 109 (AV 109). In addition, Fourier transform infrared spectroscopy, scanning electron microscopy and analysis by Brunauer-Emmett-Teller were performed for kaolin, thermally activated kaolin and the immobilized enzyme. It has been shown that 0.1 IU of HRP-kaolin decolorized 87 % of dye solution, under the optimal conditions (pH 5.0, temperature 24 °C, dye concentration 40 mg/L and 0.2 mM of H2O2) within 40 min. The immobilized HRP decolorization follows the Ping Pong Bi-Bi mechanism with dead-end inhibition by the dye. The biocatalyst retained 35 ± 0.9 % of the initial activity after seven cycles of reuse in the decolorization reaction of AV 109 under optimal conditions in a batch reactor. The obtained kinetic parameters and reusability study confirmed improvement in performances of k-HRP compared to free, indicating that k-HRP has a great potential for environmental purposes. PMID:26747440

  10. Structural studies by proton-NMR spectroscopy of plant horseradish peroxidase C, the wild-type recombinant protein from Escherichia coli and two protein variants, Phe41----Val and Arg38----Lys.

    PubMed

    Veitch, N C; Williams, R J; Bray, R C; Burke, J F; Sanders, S A; Thorneley, R N; Smith, A T

    1992-07-15

    Wild-type recombinant horseradish peroxidase isoenzyme C and two protein variants, Phe41----Val and Arg38----Lys, have been characterised using both one- and two-dimensional NMR spectroscopy. Proton NMR spectra recorded in both resting and cyanide-ligated states of the proteins were compared with those of the corresponding plant peroxidase. The latter contains 18% carbohydrate in eight N-linked oligosaccharide side chains whereas the recombinant proteins are expressed in nonglycosylated form. The spectra of the plant enzyme and refolded recombinant protein are essentially identical with the exception of carbohydrate-linked resonances in the former, indicating that their solution structures are highly similar. This comparison also identifies classes of carbohydrate resonances in the plant enzyme which provides new information on the local environment and mobility of the oligosaccharide side chains. Comparison of the spectra of the cyanide-ligated states of the two variants and those of plant horseradish peroxidase C indicated that there were significant differences with respect to haem and haem-linked resonances. These could not be rationalised simply on the basis of the local perturbation expected from a single-site substitution. The two substitutions made to residues on the distal side of the haem apparently influenced the degree of imidazolate character of the proximal His170 imidazole ring thus perturbing the magnetic environment of the haem group. Inspection of the spectra of the Phe41----Val variant also showed that the resonances of a phenylalanine residue in the haem pocket had been incorrectly assigned to Phe41 in a previous study. A new assignment, based on additional information from two-dimensional nuclear Overhauser enhancement spectroscopy, was made to Phe152. The assignments made for the Phe41----Val variant were also used as a basis to investigate the structure of the complex formed with the aromatic donor molecule, benzhydroxamic acid. PMID:1633807

  11. Horseradish peroxidase: a modern view of a classic enzyme.

    PubMed

    Veitch, Nigel C

    2004-02-01

    Horseradish peroxidase is an important heme-containing enzyme that has been studied for more than a century. In recent years new information has become available on the three-dimensional structure of the enzyme and its catalytic intermediates, mechanisms of catalysis and the function of specific amino acid residues. Site-directed mutagenesis and directed evolution techniques are now used routinely to investigate the structure and function of horseradish peroxidase and offer the opportunity to develop engineered enzymes for practical applications in natural product and fine chemicals synthesis, medical diagnostics and bioremediation. A combination of horseradish peroxidase and indole-3-acetic acid or its derivatives is currently being evaluated as an agent for use in targeted cancer therapies. Physiological roles traditionally associated with the enzyme that include indole-3-acetic acid metabolism, cross-linking of biological polymers and lignification are becoming better understood at the molecular level, but the involvement of specific horseradish peroxidase isoenzymes in these processes is not yet clearly defined. Progress in this area should result from the identification of the entire peroxidase gene family of Arabidopsis thaliana, which has now been completed. PMID:14751298

  12. Glyco-variant library of the versatile enzyme horseradish peroxidase

    PubMed Central

    Capone, Simona; Pletzenauer, Robert; Maresch, Daniel; Metzger, Karl; Altmann, Friedrich; Herwig, Christoph; Spadiut, Oliver

    2014-01-01

    When the glycosylated plant enzyme horseradish peroxidase (HRP) is conjugated to specific antibodies, it presents a powerful tool for medical applications. The isolation and purification of this enzyme from plant is difficult and only gives low yields. However, HRP recombinantly produced in the yeast Pichia pastoris experiences hyperglycosylation, which impedes the use of this enzyme in medicine. Enzymatic and chemical deglycosylation are cost intensive and cumbersome and hitherto existing P. pastoris strain engineering approaches with the goal to avoid hyperglycosylation only resulted in physiologically impaired yeast strains not useful for protein production processes. Thus, the last resort to obtain less glycosylated recombinant HRP from P. pastoris is to engineer the enzyme itself. In the present study, we mutated all the eight N-glycosylation sites of HRP C1A. After determination of the most suitable mutation at each N-glycosylation site, we physiologically characterized the respective P. pastoris strains in the bioreactor and purified the produced HRP C1A glyco-variants. The biochemical characterization of the enzyme variants revealed great differences in catalytic activity and stability and allowed the combination of the most promising mutations to potentially give an unglycosylated, active HRP C1A variant useful for medical applications. Interestingly, site-directed mutagenesis proved to be a valuable strategy not only to reduce the overall glycan content of the recombinant enzyme but also to improve catalytic activity and stability. In the present study, we performed an integrated bioprocess covering strain generation, bioreactor cultivations, downstream processing and product characterization and present the biochemical data of the HRP glyco-library. PMID:24859724

  13. The Reaction of Coumarins with Horseradish Peroxidase 1

    PubMed Central

    Miller, Richard W.; Sirois, J.-Claude; Morita, Hirokazu

    1975-01-01

    The peroxidase catalyzed oxidation of indole-3-acetate is inhibited by naturally occurring coumarins such as scopoletin. This inhibition is due to the preferential reactivity of the coumarins with the peroxidase compounds I, II, and III. In view of the possible growth regulatory role of coumarins in plants, the mechanism of oxidation of scopoletin by horse-radish peroxidase has been investigated. Peroxidase catalyzed coumarin oxidation requires either an electron donor and molecular oxygen or hydrogen peroxide. If peroxide is present, the reaction is mediated by peroxidase compound II which reacts rapidly and stoichiometrically with scopoletin. Different oxidation products are formed, depending on whether IAA or hydrogen peroxide promotes the reaction. A scopoletin-free radical intermediate has been isolated from the peroxide reaction mixture but was not detected in the peroxide-free system. When indole-3-acetate is the electron donor, reduced peroxidase combines with molecular oxygen to give peroxidase compound III. Added scopoletin is cooxidized with indole-3-acetate. Compared to the scopoletin peroxidation, this reaction is slower and yields fewer coumarin oxidation products. The differences observed between the two scopoletin oxidation pathways reflect: (a) the competition between indole-3-acetate and scopoletin for peroxidase compounds; (b) the lower reactivity of scopoletin with peroxidase compound III compared with peroxidase compound II. The peroxide-promoted reaction is eliminated by catalase, while the indole-3-acetate initiated oxidation is not affected by excess quantities of either catalase or superoxidase dismutase. PMID:16659024

  14. Detoxification of pesticides aqueous solution using horseradish peroxidase.

    PubMed

    El-Said, Saad Mohamed

    2013-03-15

    There are pesticide residues in agriculture wastewater and that compounds must be removed before discharge of wastewater in native waters. Thus the aim of this study was to remove toxic pesticide in waste water by the addition of horseradish peroxidase enzyme. The process of pesticide (methyl-parathion (O,O-Diethyl- O-4-nitro-phenylthiophosphate), atrazine (1-chloro-3-ethylamino-5-isopropylamino-2,4,6-triazine) and triazophos (O,O-diethyl O-1-phenyl-1H-1,2,4- triazol-3-yl phosphorothioate) removal from synthetic wastewater using horseradish peroxidase and hydrogen peroxide has been analyzed. The technical feasibility of the process was studied using 0.001-3.0 mM synthetic pesticides solutions. Experiments were carried out at different time, HRP and H2O2 dose and pH to determine the optimum removing conditions. The removal of the three pesticides increases with an increase in HRP and hydrogen peroxide dose. The optimum HRP dose is 2.0 U L(-1) and 10 mM for H2O2. The contact needed to reach equilibrium was found to be 360 min. Maximum removal was achieved up to 74% at pH 8. Also, Chemical Oxygen Demand (COD) of the effluent reduced at the end of 6 h from 2111-221 mg L(-1) (at pH 8). Tests based upon horseradish peroxidase, at optimized parameters, show the reduction of toxicity to non-toxic levels. PMID:24498792

  15. Removal of chlorophenols from wastewater by immobilized horseradish peroxidase

    SciTech Connect

    Tatsumi, Kenji; Wada, Shinji; Ichikawa, Hiroyasu

    1996-07-05

    Immobilization of horseradish peroxidase on magnetite and removal of chlorophenols using immobilized enzyme were investigated. Immobilization by physical adsorption on magnetite was much more effective than that by the crosslinking method, and the enzyme was found to be immobilized at 100% of retained activity. In addition, it was discovered that horseradish peroxidase was selectively adsorbed on magnetite, and the immobilization resulted in a 20-fold purification rate for crude enzyme. When immobilized peroxidase was used to treat a solution containing various chlorophenols, p-chlorophenol, 2,4-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol, and pentachlorophenol, each chlorophenol was almost 100% removed, and also the removal of total organic carbon (TOC) and adsorbable organic halogen (AOX) reached more than 90%, respectively. However, in the case of soluble peroxidase, complete removal of each chlorophenol could not be attained, and in particular, the removal of 2,4,5-trichlorophenol was the lowest, with a removal rate of only 36%.

  16. FTIR study of horseradish peroxidase in reverse micelles.

    PubMed

    Chen, J; Xia, C; Niu, J; Li, S

    2001-04-20

    Fourier transform infrared (FTIR) method was used to study the secondary structures of horseradish peroxidase (HRP) in aqueous solution and in reverse micelles for the first time. Results indicated that the structure of HRP in sodium bis(2-ethylhexy)sulfosuccinate (AOT) reverse micelles was close to that in aqueous solution. In cetyltrimethylammonium bromide (CTAB) and sodium dodecylfate (SDS) reverse micelles the position of some bands changed. Results indicated that the secondary structure had a close relationship with the surfactant species of the reverse micelles. Among the three types of reverse micelles, the system of AOT reverse micelles was probably the most beneficial reaction media to HRP. PMID:11302746

  17. Preparation of horseradish peroxidase hydrazide and its use in immunoassay.

    PubMed

    Shrivastav, Tulsidas G

    2003-01-01

    Preparation of horseradish peroxidase (HRP) hydrazide that is HRP linked to adipic acid dihydrazide (HRP-ADH) and its use in enzyme immunoassay (EIA) is described. In this new strategy, horseradish peroxidase was conjugated to adipic acid dihydrazide using a carbodiimide coupling method. The resulting HRP-ADH was then coupled to cortisol-21-hemisuccinate (Cortisol-21-HS) to prepare enzyme conjugate. The prepared cortisol-21-HS coupled ADH-HRP (Cortisol-21-HS-ADH-HRP) enzyme conjugate was used for the development of an enzyme linked immunosorbent assay (ELISA) for direct estimation of cortisol. To the cortisol antibody coated microtiter wells, standard or serum samples (50 microL), along with cortisol-21-HS-ADH-HRP enzyme conjugate (100 microL) were incubated for 1 h at 37 degrees C. Bound enzyme activity was measured by using tetramethyl benzidine/hydrogen peroxide (TMB/H2O2) as substrate. The sensitivity of the assay was 0.05 microg/dL and the analytical recovery ranged from 92.9 to 101.7%. PMID:12953974

  18. Relative Binding Affinities of Monolignols to Horseradish Peroxidase.

    PubMed

    Sangha, Amandeep K; Petridis, Loukas; Cheng, Xiaolin; Smith, Jeremy C

    2016-08-11

    Monolignol binding to the peroxidase active site is the first step in lignin polymerization in plant cell walls. Using molecular dynamics, docking, and free energy perturbation calculations, we investigate the binding of monolignols to horseradish peroxidase C. Our results suggest that p-coumaryl alcohol has the strongest binding affinity followed by sinapyl and coniferyl alcohol. Stacking interactions between the monolignol aromatic rings and nearby phenylalanine residues play an important role in determining the calculated relative binding affinities. p-Coumaryl and coniferyl alcohols bind in a pose productive for reaction in which a direct H-bond is formed between the phenolic -OH group and a water molecule (W2) that may facilitate proton transfer during oxidation. In contrast, in the case of sinapyl alcohol there is no such direct interaction, the phenolic -OH group instead interacting with Pro139. Since proton and electron transfer is the rate-limiting step in monolignol oxidation by peroxidase, the binding pose (and thus the formation of near attack conformation) appears to play a more important role than the overall binding affinity in determining the oxidation rate. PMID:27447548

  19. Effects of microwaves (900 MHz) on peroxidase systems: A comparison between lactoperoxidase and horseradish peroxidase.

    PubMed

    Barteri, Mario; De Carolis, Roberta; Marinelli, Fiorenzo; Tomassetti, Goliardo; Montemiglio, Linda Celeste

    2016-01-01

    This work shows the effects of exposure to an electromagnetic field at 900 MHz on the catalytic activity of the enzymes lactoperoxidase (LPO) and horseradish peroxidase (HRP). Experimental evidence that irradiation causes conformational changes of the active sites and influences the formation and stability of the intermediate free radicals is documented by measurements of enzyme kinetics, circular dichroism spectroscopy (CD) and cyclic voltammetry. PMID:25577980

  20. Adsorption and inactivation behavior of horseradish peroxidase on various substrates.

    PubMed

    Di Risio, Sabina; Yan, Ning

    2010-09-01

    To produce bioactive papers, i.e. papers incorporating biomolecules that are useful for analyte detection, adequate immobilization strategies should be devised. In this article, the physical immobilization behavior and activity of the enzyme horseradish peroxidase (HRP) on various papermaking substrates were studied. The papermaking substrates included amorphous and crystalline cellulose, calcium carbonate, styrene butadiene latex, polystyrene, and both negatively charged rayon and rayon with a positively charged layer. It was found that HRP adsorption improves as the hydrophobicity of the substrate increases; however, excessive hydrophobicity produces enzyme deactivation. HRP-calcium carbonate binding was weak and the enzyme loading was scant. These results provided a possible explanation for the poor analytical signals observed in pigment-coated papers when used as bioactive paper supports. Electrostatic effects played a minor role in HRP adsorption behavior. PMID:20570116

  1. Horseradish Peroxidase Inactivation: Heme Destruction and Influence of Polyethylene Glycol

    PubMed Central

    Mao, Liang; Luo, Siqiang; Huang, Qingguo; Lu, Junhe

    2013-01-01

    Horseradish peroxidase (HRP) mediates efficient conversion of many phenolic contaminants and thus has potential applications for pollution control. Such potentially important applications suffer however from the fact that the enzyme becomes quickly inactivated during phenol oxidation and polymerization. The work here provides the first experimental data of heme consumption and iron releases to support the hypothesis that HRP is inactivated by heme destruction. Product of heme destruction is identified using liquid chromatography with mass spectrometry. The heme macrocycle destruction involving deprivation of the heme iron and oxidation of the 4-vinyl group in heme occurs as a result of the reaction. We also demonstrated that heme consumption and iron releases resulting from HRP destruction are largely reduced in the presence of polyethylene glycol (PEG), providing the first evidence to indicate that heme destruction is effectively suppressed by co-dissolved PEG. These findings advance a better understanding of the mechanisms of HRP inactivation. PMID:24185130

  2. Polymerization reactivity of sulfomethylated alkali lignin modified with horseradish peroxidase.

    PubMed

    Yang, Dongjie; Wu, Xiaolei; Qiu, Xueqing; Chang, Yaqi; Lou, Hongming

    2014-03-01

    Alkali lignin (AL) was employed as raw materials in the present study. Sulfomethylation was conducted to improve the solubility of AL, while sulfomethylated alkali lignin (SAL) was further polymerized by horseradish peroxidase (HRP). HRP modification caused a significant increase in molecular weight of SAL which was over 20 times. It was also found to increase the amount of sulfonic and carboxyl groups while decrease the amount of phenolic and methoxyl groups in SAL. The adsorption quantity of self-assembled SAL film was improved after HRP modification. Sulfonation and HRP modification were mutually promoted. The polymerization reactivity of SAL in HRP modification was increased with its sulfonation degree. Meanwhile, HRP modification facilitated SAL's radical-sulfonation reaction. PMID:24534439

  3. New pteridine substrates for dihydropteridine reductase and horseradish peroxidase.

    PubMed Central

    Armarego, W L; Ohnishi, A; Taguchi, H

    1986-01-01

    The oxidation of 4,5-diaminopyrimidin-6(1H)-one, 5,6,7,8-tetrahydropteridin-4(3H)-one, its 6-methyl and cis-6,7-dimethyl derivatives, and 6-methyl- and cis-6-7-dimethyl-5,6,7,8-tetrahydropterins, by horseradish peroxidase/H2O2 is enzymic and follows Michaelis-Menten kinetics, and its Km and kcat. values were determined. This oxidation of 5,6,7,8-tetrahydropterins produces quinonoid dihydropterins of established structure, and they are known to be specific substrates for dihydropteridine reductase. By analogy the peroxidase/H2O2 oxidation of the 5,6,7,8-tetrahydropteridin-4(3H)-ones should produce similar quinonoid dihydro species. The quinonoid species derived from 5,6,7,8-tetrahydropteridin-4(3H)-one and its 6-methyl and cis-6,7-dimethyl derivatives are shown to be viable substrates for human brain dihydropteridine reductase, and apparent Km and Vmax. values are reported. PMID:3718470

  4. Horseradish peroxidase and chitosan: activation, immobilization and comparative results.

    PubMed

    Mohamed, Saleh A; Al-Malki, Abdulrahman L; Kumosani, Taha A; El-Shishtawy, Reda M

    2013-09-01

    Recently, horseradish peroxidase (HRP) was immobilized on activated wool and we envisioned that the use of chitosan would be interesting instead of wool owing to its simple chemical structure, abundant nature and biodegradability. In this work, HRP was immobilized on chitosan crosslinked with cyanuric chloride. FT-IR spectroscopy and scanning electron microscopy were used to characterize immobilized HRP. The number of ten reuses of immobilized HRP has been detected. The pH was shifted from 5.5 for soluble HRP to 5.0 for immobilized enzyme. The soluble HRP had an optimum temperature of 30 °C, which was shifted to 35 °C for immobilized enzyme. The soluble HRP and immobilized HRP were thermal stable up to 35 and 45 °C, respectively. The apparent kinetic constant values (K(m)) of soluble HRP and chitosan-HRP were 35 mM and 40 mM for guaiacol and 2.73 mM and 5.7 mM for H2O2, respectively. Immobilization of HRP partially protected them from metal ions compared to soluble enzyme. The chitosan-HRP was remarkably more stable against urea, Triton X-100 and organic solvents. Chitosan-HRP exhibited large number of reuses and more resistance to harmful compounds compared with wool-HRP. On the basis of results obtained in the present study, chitosan-HRP could be employed in bioremediation application. PMID:23769933

  5. Immobilization of horseradish peroxidase on modified chitosan beads.

    PubMed

    Monier, M; Ayad, D M; Wei, Y; Sarhan, A A

    2010-04-01

    A method has been developed to immobilize horseradish peroxidase (HRP) on modified chitosan beads by means of graft copolymerization of polyethylacrylate in presence of potassium persulphate and Mohr's salt redox initiator. The activity of free and immobilized HRP was studied. FTIR spectroscopy and scanning electron microscopy were used to characterize HRP immobilization. The efficiency of the immobilization was investigated by examining the relative enzymatic activity of free enzyme before and after the HRP immobilization. The obtained values were found to reach 98.4%. The results show that the optimum temperature of immobilized HRP was 45 degrees C, which was identical to that of free enzyme, and the immobilized HRP exhibited a higher relative activity than that of free HRP over 45 degrees C. The optimal pH for immobilized HRP was 10, which was higher than that of the free HRP (pH 9.0), and the immobilization resulted in stabilization of enzyme over a broader pH range. The apparent kinetic constant value (K(m)) of immobilized HRP was 3.784 mmol ml(-1), which was higher than that of free HRP. On the other hand, the activity of immobilized HRP decreased slowly against time when compared to that of the free HRP, and could retain 65.8% residual activity after 6 consecutive cycles. PMID:20060854

  6. A genetically engineered fusion protein with horseradish peroxidase as a marker enzyme for use in competitive immunoassays.

    PubMed

    Grigorenko, V; Andreeva, I; Börchers, T; Spener, F; Egorov, A

    2001-03-15

    Horseradish peroxidase is one of the most widely used marker enzymes in immunoassays. Several disadvantages are encountered upon chemical conjugation of peroxidase with antibodies or antigens, as are low reproducibility and undefined stoichiometry. We here describe for the first time the production of a recombinant fusion of a protein analyte with horseradish peroxidase in Escherichia coli, employing refolding of inclusion bodies and reconstitution with heme. The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and defined structure. As a protein analyte, the human heart fatty acid binding protein (H-FABP) was chosen, which is a new and sensitive marker for acute myocardial infarction. The recombinant conjugate was fully active [650 U/mg with 2,2-azino-bis(3-ethyl-thiazoline-6-sulfonate) as substrate] and obtained in a yield of 12 mg/L of E. coli culture, which is better than that for recombinant peroxidase alone. The competitive immunoassay that was developed with the recombinant conjugate requires fewer incubation steps than the traditional sandwich ELISA format. It permitted the detection of H-FABP directly in plasma in the range of 10-1500 ng/mL which is the relevant range for clinical decision-making. PMID:11305642

  7. The horseradish peroxidase technique for cell lineage studies.

    PubMed

    Peralta, M; Denaro, F J

    2003-12-01

    The identification of cell lineage for a given cell type of a particular tissue is an important step in understanding how this process contributes to histogenesis. The importance in understanding cell lineage has relevance for both theoretical and practical reasons. For example, delineating and identifying histogenic principals is required to advance stem cell research and tissue regeneration. To document cell lineage in a given experimental preparation, a number of techniques have been developed. This typically requires the injection of a tracer compound into a founder cell. As this cell produces progeny, the tracer is passed on to the daughter cells. By detecting the tracer in the progeny or daughter cells the investigator can determine which cells originated from the cell that was originally injected with the tracer. By using such an approach it is possible to trace the developmental path from the first cells of the embryo to the specialized cells making the tissue of the adult. A number of tracer compounds have been used with good results in lineage tracing. One of these tracer compounds is horseradish peroxidase (HRP). Several variations of the technique are available depending on what species are studied or what histological requirements are made by the study. A basic technique that can be adapted to individual needs is presented. Included in this protocol on lineage tracing are the procedures for injection, fixation, and the microscope evaluation of labelled cells in the Helobdella triseralis embryo. A brief discussion of the technique will note its advantages and disadvantages. Examples of labelled cell preparations are given to illustrate the technique. PMID:14984012

  8. The enzyme horseradish peroxidase is less compressible at higher pressures.

    PubMed Central

    Smeller, László; Fidy, Judit

    2002-01-01

    Fluorescence line-narrowing (FLN) spectroscopy at 10 K was used to study the effect of high pressure through the prosthetic group in horseradish peroxidase (HRP), which was Mg-mesoporphyrin (MgMP) replacing the heme of the enzyme. The same measurement was performed on MgMP in a solid-state amorphous organic matrix, dimethyl sulfoxide (DMSO). Series of FLN spectra were registered to determine the (0, 0) band shape through the inhomogeneous distribution function (IDF). In the range of 0-2 GPa a red-shift of the IDF was determined, and yielded the isothermal compressibility of MgMP-HRP as 0.066 GPa(-1), which is significantly smaller than that found earlier as 0.106 GPa(-1) by fine-tuning the pressure in the range up to 1.1 MPa. The vibrational frequencies also shifted with pressure increase, as expected. The compressibility in the DMSO matrix was smaller, 0.042 GPa(-1), both when the pressure was applied at room temperature before cooling to 10 K, or at 10 K. At 200 K or above, the bimodal (0, 0) band shape in DMSO showed a population conversion under pressure that was not observed at or below 150 K. A significant atomic rearrangement was estimated from the volume change, 3.3 +/- 0.7 cm(3)/mol upon conversion. The compressibility in proteins and in amorphous solids seems not to significantly depend on the temperature and in the protein it decreases toward higher pressures. PMID:11751329

  9. Electrochemical aptasensor based on the dual-amplification of G-quadruplex horseradish peroxidase-mimicking DNAzyme and blocking reagent-horseradish peroxidase.

    PubMed

    Yuan, Yali; Gou, Xuxu; Yuan, Ruo; Chai, Yaqin; Zhuo, Ying; Mao, Li; Gan, Xianxue

    2011-06-15

    A simple electrochemical aptasensor for sensitive detection of thrombin was fabricated with G-quadruplex horseradish peroxidase-mimicking DNAzyme (hemin/G-quadruplex system) and blocking reagent-horseradish peroxidase as dual signal-amplification scheme. Gold nanoparticles (nano-Au) were firstly electrodeposited onto single wall nanotube (SWNT)-graphene modified electrode surface for the immobilization of electrochemical probe of nickel hexacyanoferrates nanoparticles (NiHCFNPs). Subsequently, another nano-Au layer was electrodeposited for further immobilization of thrombin aptamer (TBA), which later formed hemin/G-quadruplex system with hemin. Horseradish peroxidases (HRP) then served as blocking reagent to block possible remaining active sites and avoided the non-specific adsorption. In the presence of thrombin, the TBA binded to thrombin and the hemin released from the hemin/G-quadruplex electrocatalytic structure, increasing steric hindrance of the aptasensor and decomposing hemin/G-quadruplex electrocatalytic structure, which finally decreased the electrocatalytic efficiency of aptasensor toward H(2)O(2) in the presence of NiHCFNPs with a decreased electrochemical signal. On the basis of the synergistic amplifying action, a detection limit as low as 2 pM for thrombin was obtained. PMID:21536422

  10. Horseradish peroxidase-catalyzed polymerization of cardanol in the presence of redox mediators.

    PubMed

    Won, Keehoon; Kim, Yong Hwan; An, Eun Suk; Lee, Yeon Soo; Song, Bong Keun

    2004-01-01

    Horseradish peroxidase-catalyzed polymerization of cardanol in aqueous organic solvent was investigated in the presence of a redox mediator. Cardanol is a phenol derivative from a renewable resource mainly having a C15 unsaturated hydrocarbon chain with mostly 1-3 double bonds at a meta position. Unlike soybean peroxidase (SBP), it has been shown that horseradish peroxidase (HRP) is not able to perform oxidative polymerization of phenol derivatives having a bulky meta substituent such as cardanol. For the first time, redox mediators have been applied to enable horseradish peroxidase to polymerize cardanol. Veratryl alcohol, N-ethyl phenothiazine, and phenothiazine-10-propionic acid were tested as a mediator. It is surprising that the horseradish peroxidase-catalyzed polymerization of cardanol took place in the presence of N-ethyl phenothiazine or phenothiazine-10-propionic acid. However, veratryl alcohol showed no effect. FT-IR and GPC analysis of the product revealed that the structure and properties of polycardanol formed by HRP with a mediator were similar to those by SBP. This is the first work to apply a redox mediator to enzyme-catalyzed oxidative polymerization. Our new finding that oxidative polymerization of a poor substrate, which the enzyme is not active with, can take place in the presence of an appropriate mediator will present more opportunities for the application of enzyme-catalyzed polymerization. PMID:14715000

  11. Evaluation of seven cosubstrates in the quantification of horseradish peroxidase enzyme by square wave voltammetry.

    PubMed

    Kergaravat, Silvina V; Pividori, Maria Isabel; Hernandez, Silvia R

    2012-01-15

    The electrochemical detection for horseradish peroxidase-cosubstrate-H(2)O(2) systems was optimized. o-Phenilendiamine, phenol, hydroquinone, pyrocatechol, p-chlorophenol, p-aminophenol and 3,3'-5,5'-tetramethylbenzidine were evaluated as cosubstrates of horseradish peroxidase (HRP) enzyme. Therefore, the reaction time, the addition sequence of the substrates, the cosubstrate:H(2)O(2) ratio and the electrochemical techniques were elected by one-factor optimization assays while the buffer pH, the enzymatic activity and cosubstrate and H(2)O(2) concentrations for each system were selected simultaneously by response surface methodology. Then, the calibration curves for seven horseradish peroxidase-cosubstrate-H(2)O(2) systems were built and the analytic parameters were analyzed. o-Phenilendiamine was selected as the best cosubstrate for the HRP enzyme. For this system the reaction time of 60s, the phosphate buffer pH 6.0, and the concentrations of 2.5×10(-4)molL(-1) o-phenilendiamine and of 1.25×10(-4)molL(-1) H(2)O(2) were chosen as the optimal conditions. In these conditions, the calibration curve of horseradish peroxidase by square wave voltammetry showed a linearity range from 9.5×10(-11) to 1.9×10(-8)molL(-1) and the limit of detection of 3.8×10(-11)molL(-1) with RSD% of 0.03% (n=3). PMID:22265528

  12. Primary product of the horseradish peroxidase-catalyzed oxidation of pentachlorophenol

    SciTech Connect

    Kazunga, C.; Aitken, M.D.; Gold, A.

    1999-05-01

    Peroxidases are a class of enzymes that catalyze the oxidation of various phenolic substrates by hydrogen peroxide. They are common enzymes in soil and are also available commercially, so that they have been proposed as agents of phenolic pollutant transformation both in the environment and in engineered systems. Previous research on the peroxidase-catalyzed oxidation of pentachlorophenol (PCP) has suggested that tetrachloro-p-benzoquinone (chloranil) is the principal product and that a considerable fraction of the PCP added to reaction mixtures appears to be resistant to oxidation. In experiments employing alternative methods of product separation and analysis, the authors found that both of these observations are artifacts of extraction and analytical methods used in previous studies. The major product of the horseradish peroxidase-catalyzed oxidation of pentachlorophenol from pH 4--7 was 2,3,4,5,6-pentachloro-4-pentachlorophenoxy-2,5-cyclohexadienone (PPCHD), which is formed by the coupling of two pentachlorophenoxyl radicals.

  13. Differential activity and structure of highly similar peroxidases. Spectroscopic, crystallographic, and enzymatic analyses of lignifying Arabidopsis thaliana peroxidase A2 and horseradish peroxidase A2.

    PubMed

    Nielsen, K L; Indiani, C; Henriksen, A; Feis, A; Becucci, M; Gajhede, M; Smulevich, G; Welinder, K G

    2001-09-18

    Anionic Arabidopsis thaliana peroxidase ATP A2 was expressed in Escherichia coli and used as a model for the 95% identical commercially available horseradish peroxidase HRP A2. The crystal structure of ATP A2 at 1.45 A resolution at 100 K showed a water molecule only 2.1 A from heme iron [Ostergaard, L., et al. (2000) Plant Mol. Biol. 44, 231-243], whereas spectroscopic studies of HRP A2 in solution at room temperature [Feis, A., et al. (1998) J. Raman Spectrosc. 29, 933-938] showed five-coordinated heme iron, which is common in peroxidases. Presented here, the X-ray crystallographic, single-crystal, and solution resonance Raman studies at room temperature confirmed that the sixth coordination position of heme iron of ATP A2 is essentially vacant. Furthermore, electronic absorption and resonance Raman spectroscopy showed that the heme environments of recombinant ATP A2 and glycosylated plant HRP A2 are indistinguishable at neutral and alkaline pH, from room temperature to 12 K, and are highly flexible compared with other plant peroxidases. Ostergaard et al. (2000) also demonstrated that ATP A2 expression and lignin formation coincide in Arabidopsis tissues, and docking of lignin precursors into the substrate binding site of ATP A2 predicted that coniferyl and p-coumaryl alcohols were good substrates. In contrast, the additional methoxy group of the sinapyl moiety gave rise to steric hindrance, not only in A2 type peroxidases but also in all peroxidases. We confirm these predictions for ATP A2, HRP A2, and HRP C. The specific activity of ATP A2 was lower than that of HRP A2 (pH 4-8), although a steady-state study at pH 5 demonstrated very little difference in their rate constants for reaction with H2O2 (k1 = 1.0 microM(-1) x s(-1). The oxidation of coniferyl alcohol, ferulic, p-coumaric, and sinapic acids by HRP A2, and ATP A2, however, gave modest but significantly different k3 rate constants of 8.7 +/- 0.3, 4.0 +/- 0.2, 0.70 +/- 0.03, and 0.04 +/- 0.2 microM(-1) x

  14. Toxicity of textile dyes and their degradation by the enzyme horseradish peroxidase (HRP).

    PubMed

    Ulson de Souza, Selene Maria Arruda Guelli; Forgiarini, Eliane; Ulson de Souza, Antônio Augusto

    2007-08-25

    The enzyme peroxidase is known for its capacity to remove phenolic compounds and aromatic amines from aqueous solutions and also to decolorize textile effluents. This study evaluates the potential of the enzyme horseradish peroxidase (HRP) in the decolorization of textile dyes and effluents. Some factors such as pH and the amount of H(2)O(2) and the enzyme were evaluated in order to determine the optimum conditions for the enzyme performance. For the dyes tested, the results indicated that the decolorization of the dye Remazol Turquoise Blue G 133% was approximately 59%, and 94% for the Lanaset Blue 2R; for the textile effluent, the decolorization was 52%. The tests for toxicity towards Daphnia magna showed that there was a reduction in toxicity after the enzymatic treatment. However, the toxicity of the textile effluent showed no change towards Artemia salina after the enzyme treatment. This study verifies the viability of the use of the enzyme horseradish peroxidase in the biodegradation of textile dyes. PMID:17628340

  15. 3,3',5,5'-Tetramethylbenzidine Oxidation on Paper Devices for Horseradish Peroxidase-based Assays.

    PubMed

    Busa, Lori Shayne Alamo; Komatsu, Takeshi; Mohammadi, Saeed; Maeki, Masatoshi; Ishida, Akihiko; Tani, Hirofumi; Tokeshi, Manabu

    2016-01-01

    We report on the colorimetric oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by hydrogen peroxide using horseradish peroxidase on photolithography-fabricated (P-PAD) and wax-printed (W-PAD) paper-based analytical devices. Fabricating PADs via photolithography exposes the hydrophilic areas to polymers (photoresists) and solvents, not only reducing the hydrophilicity, but also affecting the TMB-H2O2 assay system with an unavoidable incomplete elimination of photoresist during fabrication. Detection signals are then observed in the presence of photoresist residues on the P-PAD, even at a blank HRP concentration. PMID:27506705

  16. A simple device for injection of small calibrated amounts of horseradish peroxidase into the cerebral cortex.

    PubMed

    Bullier, J; Henry, G H; Baker, W J

    1980-02-01

    A simple device for injecting horseradish peroxidase into the cerebral cortex is described. It consists of a syringe connected by tubing to a hydraulic adaptor which provides a sealed connection to a disposable glass micropipette. Replaceable micropipettes may be prepared by drawing capillary glass under heat to a fine diameter tip that provides easy penetration into the cortex without dimpling the surface grey matter or damaging the underlying white matter. The hydraulic seal achieved by the adaptor allows volumes of the order of 0.05 microliter to be readily injected into nervous tissue. PMID:7035757

  17. Well-Defined Macromolecules Using Horseradish Peroxidase as a RAFT Initiase.

    PubMed

    Danielson, Alex P; Bailey-Van Kuren, Dylan; Lucius, Melissa E; Makaroff, Katherine; Williams, Cameron; Page, Richard C; Berberich, Jason A; Konkolewicz, Dominik

    2016-02-01

    Enzymatic catalysis and control over macromolecular architectures from reversible addition-fragmentation chain transfer polymerization (RAFT) are combined to give a new method of making polymers. Horseradish peroxidase (HRP) is used to catalytically generate radicals using hydrogen peroxide and acetylacetone as a mediator. RAFT is used to control the polymer structure. HRP catalyzed RAFT polymerization gives acrylate and acrylamide polymers with relatively narrow molecular weight distributions. The polymerization is rapid, typically exceeding 90% monomer conversion in 30 min. Complex macromolecular architectures including a block copolymer and a protein-polymer conjugate are synthesized using HRP to catalytically initiate RAFT polymerization. PMID:26748786

  18. Conformational relaxation of a low-temperature protein as probed by photochemical hole burning. Horseradish peroxidase.

    PubMed Central

    Zollfrank, J; Friedrich, J; Vanderkooi, J M; Fidy, J

    1991-01-01

    For the first time, conformational relaxation processes have been measured in a small protein, mesoporphyrin-horseradish peroxidase via their influence on spectral diffusion broadening of holes burnt in the fluorescence excitation spectrum of free base mesoporphyrin. Holes were burnt in three 0----0 bands of different tautomeric forms of the chromophore at 1.5 and 4 K, and the spectral diffusion broadening was measured in temperature cycling experiments between 4 and 30 K. The inhomogeneous linewidth for the tautomeric 0----0 bands was estimated to be 60-70 cm-1; the hole width was found narrow, being in the order of 350 MHz (10(-2) cm-1) at 1.5 K what allowed for an extremely sensitive detection of the conformational changes. Though proteins have many features in common with glasses, the spectral diffusion broadening of photochemical holes under temperature cycling conditions in mesoporphyrin horseradish peroxidase has a very different pattern as a function of temperature. Up to 12 K, the linewidth did not significantly change, then around 14 K; a steplike broadening was observed for all three tautomers, although to a different extent. The total magnitude of line broadening up to 30 K was large and also different for the tautomers. We argue that the difference between the behavior of this protein and that of glassy matrices originate from finite size effects; the protein may be characterized by a small number of TLS, and their distribution may bear discrete features. PMID:2009354

  19. Kinetic model for removal of phenol by horseradish peroxidase with PEG

    SciTech Connect

    Wu, Y.; Taylor, K.E.; Biswas, N.; Bewtra, J.K.

    1999-05-01

    A kinetic model has been developed to simulate horseradish peroxidase-catalyzed polymerization of phenol in the presence of polyethylene glycol based on the following kinetics. The phenol conversion expression is a second-order Michaelis-Menten equation with respect to the concentrations of phenol and hydrogen peroxide. The enzyme inactivation is attributed to the polymer and hydrogen peroxide simultaneously. The inactivation by polymer is an apparently second-order reaction, first-order in each of enzyme and phenol, whereas the inactivation by peroxide is also an apparently second-order reaction, first-order in each of enzyme and hydrogen peroxide. The rates of consumption of hydrogen peroxide and polyethylene glycol are directly proportional to the rate of conversion of phenol. Experimental data show that the model output can predict the phenol removal and activity depletion realistically under a variety of reaction conditions. The model has been verified by predicting some independent experimental results on reaction stoichiometry, horseradish peroxidase dose effect, and semibatch operation with respect to hydrogen peroxide.

  20. Inactivation of aminated horseradish peroxidase by interaction with S-Sepharose.

    PubMed

    Allen, M P; Choo, S H; Li, T M; Parrish, R F

    1991-02-01

    Horseradish peroxidase which had been aminated by periodate oxidation and reductive amination was purified by cation-exchange chromatography on S-Sepharose. Instead of the expected single peak of aminated enzyme, two distinct peaks of protein were eluted from the column. Evaluation of the protein in each of the two distributions showed that peak number 1 had spectral properties and specific activity similar to those of native enzyme. Distribution number 2 had a threefold reduction in the extinction in the Soret region at 404 nm and was completely devoid of enzymatic activity. This inactivation was caused by a specific interaction between the aminated peroxidase and the S-Sepharose matrix, resulting in a displacement of the heme prosthetic group out of its native orientation. The inactivation of the aminated peroxidase was found to be dependent on time, pH, and the support matrix itself. These results indicate that the S-Sepharose and Mono-S resins are not interchangeable, despite the chemical similarities of the two resins. PMID:2035845

  1. Combining Protein and Strain Engineering for the Production of Glyco-Engineered Horseradish Peroxidase C1A in Pichia pastoris

    PubMed Central

    Capone, Simona; Ćorajević, Lejla; Bonifert, Günther; Murth, Patrick; Maresch, Daniel; Altmann, Friedrich; Herwig, Christoph; Spadiut, Oliver

    2015-01-01

    Horseradish peroxidase (HRP), conjugated to antibodies and lectins, is widely used in medical diagnostics. Since recombinant production of the enzyme is difficult, HRP isolated from plant is used for these applications. Production in the yeast Pichia pastoris (P. pastoris), the most promising recombinant production platform to date, causes hyperglycosylation of HRP, which in turn complicates conjugation to antibodies and lectins. In this study we combined protein and strain engineering to obtain an active and stable HRP variant with reduced surface glycosylation. We combined four mutations, each being beneficial for either catalytic activity or thermal stability, and expressed this enzyme variant as well as the unmutated wildtype enzyme in both a P. pastoris benchmark strain and a strain where the native α-1,6-mannosyltransferase (OCH1) was knocked out. Considering productivity in the bioreactor as well as enzyme activity and thermal stability, the mutated HRP variant produced in the P. pastoris benchmark strain turned out to be interesting for medical diagnostics. This variant shows considerable catalytic activity and thermal stability and is less glycosylated, which might allow more controlled and efficient conjugation to antibodies and lectins. PMID:26404235

  2. Effect of architecture on the activity of glucose oxidase/horseradish peroxidase/carbon nanoparticle conjugates.

    PubMed

    Ciaurriz, Paula; Bravo, Ernesto; Hamad-Schifferli, Kimberly

    2014-01-15

    We investigate the activity of glucose oxidase (GOx) together with horseradish peroxidase (HRP) on carbon nanoparticles (CNPs). Because GOx activity relies on HRP, we probe how the arrangement of the enzymes on the CNPs affects enzymatic behavior. Colorimetric assays to probe activity found that the coupling strategy affects activity of the bienzyme-nanoparticle complex. GOx is more prone than HRP to denaturation on the CNP surface, where its activity is compromised, while HRP activity is enhanced when interfaced to the CNP. Thus, arrangements where HRP is directly on the surface of the CNP and GOx is not are more favorable for overall activity. Coverage also influenced activity of the bienzyme complex, but performing the conjugation in the presence of glucose did not improve GOx activity. These results show that the architecture of the assembly is an important factor in optimization of nanoparticle-protein interfaces. PMID:24231087

  3. Carbon Based Electrodes Modified with Horseradish Peroxidase Immobilized in Conducting Polymers for Acetaminophen Analysis

    PubMed Central

    Tertis, Mihaela; Florea, Anca; Sandulescu, Robert; Cristea, Cecilia

    2013-01-01

    The development and optimization of new biosensors with horseradish peroxidase immobilized in carbon nanotubes-polyethyleneimine or polypyrrole nanocomposite film at the surface of two types of transducer is described. The amperometric detection of acetaminophen was carried out at −0.2 V versus Ag/AgCl using carbon based-screen printed electrodes (SPEs) and glassy carbon electrodes (GCEs) as transducers. The electroanalytical parameters of the biosensors are highly dependent on their configuration and on the dimensions of the carbon nanotubes. The best limit of detection obtained for acetaminophen was 1.36 ± 0.013 μM and the linear range 9.99–79.01 μM for the HRP-SWCNT/PEI in GCE configuration. The biosensors were successfully applied for the detection of acetaminophen in several drug formulations. PMID:23580052

  4. Compound I in horseradish peroxidase enzyme: Magnetic state assessment by quadratric configuration interaction calculations

    NASA Astrophysics Data System (ADS)

    Zazza, Costantino; Sanna, Nico; Tatoli, Simone; Aschi, Massimiliano; Palma, Amedeo

    Quadratic configuration interaction procedure with single and double electronic excitations (QCISD) has been used, for the first time, to calculate the electronic structure of the Compound I (CpdI), which represents a key intermediate in the catalytic cycle of Horseradish Peroxidase (HRP) enzyme. The QCISD method is applied to lowest quasi-isoenergetic doublet and quartet spin multiplicity and results compared with density functional theory (DFT/B3LYP) data. This investigation shows that, at present, QCISD is more accurate than DFT-based approach in discriminating between the two lowest magnetic states of CpdI complex in HRP enzyme. Such a result opens the possibility of theoretically addressing the reaction mechanism leading to CpdI complex in HRP using a correlated wavefunction based approach.

  5. Catalytic and Inhibitory Kinetic Behavior of Horseradish Peroxidase on the Electrode Surface

    PubMed Central

    Huang, Jitao; Huang, Wei; Wang, Titi

    2012-01-01

    Enzymatic biosensors are often used to detect trace levels of some specific substance. An alternative methodology is applied for enzymatic assays, in which the electrocatalytic kinetic behavior of enzymes is monitored by measuring the faradaic current for a variety of substrate and inhibitor concentrations. Here we examine a steady-state and pre-steady-state reduction of H2O2 on the horseradish peroxidase electrode. The results indicate the substrate-concentration dependence of the steady-state current strictly obeys Michaelis-Menten kinetics rules; in other cases there is ambiguity, whereby he inhibitor-concentration dependence of the steady-state current has a discontinuity under moderate concentration conditions. For pre-steady-state phases, both catalysis and inhibition show an abrupt change of the output current. These anomalous phenomena are universal and there might be an underlying biochemical or electrochemical rationale. PMID:23202175

  6. Enzymatic growth of quantum dots: applications to probe glucose oxidase and horseradish peroxidase and sense glucose.

    PubMed

    Saa, Laura; Pavlov, Valeri

    2012-11-19

    Three innovative assays are developed for the detection of enzymatic activities of glucose oxidase (GOx) and horseradish peroxidase (HRP) by the generation of CdS quantum dots (QDs) in situ using non-conventional enzymatic reactions. In the first assay, GOx catalyzes the oxidation of 1-thio-β-D-glucose to give 1-thio-β-D-gluconic acid. The latter is spontaneously hydrolyzed to β-D-gluconic acid and H2 S, which in the presence of cadmium nitrate yields fluorescent CdS nanoparticles. In the second assay HRP catalyzes the oxidation of sodium thiosulfate with hydrogen peroxide generating H2 S and consequently CdS QDs. The combination of GOx with HRP, allowed quantification of glucose in plasma by following growth of fluorescent QDs. PMID:22887879

  7. Electrochemical properties of seamless three-dimensional carbon nanotubes-grown graphene modified with horseradish peroxidase.

    PubMed

    Komori, Kikuo; Terse-Thakoor, Trupti; Mulchandani, Ashok

    2016-10-01

    Horseradish peroxidase (HRP) was immobilized through sodium dodecyl sulfate (SDS) on the surface of a seamless three-dimensional hybrid of carbon nanotubes grown at the graphene surface (HRP-SDS/CNTs/G) and its electrochemical properties were investigated. Compared with graphene alone electrode modified with HRP via SDS (HRP-SDS/G electrode), the surface coverage of electroactive HRP at the CNTs/G electrode surface was approximately 2-fold greater because of CNTs grown at the graphene surface. Based on the increase in the surface coverage of electroactive HRP, the sensitivity to H2O2 at the HRP-SDS/CNTs/G electrode was higher than that at the HRP-SDS/G electrode. The kinetics of the direct electron transfer from the CNTs/G electrode to compound I and II of modified HRP was also analyzed. PMID:27224430

  8. Horseradish peroxidase-encapsulated chitosan nanoparticles for enzyme-prodrug cancer therapy.

    PubMed

    Cao, Xiaodan; Chen, Chao; Yu, Haijun; Wang, Ping

    2015-01-01

    Among various enzyme-based therapies, enzyme-prodrug therapy (EPT) promises minimized side effects in that it activates non-toxic prodrugs locally where the enzymes are placed. The success of such an approach requires high enzyme stability against both structural denaturation and potential immunogenicity. This work examines the efficiency of nanoparticles for enzyme protection in EPT applications. Specifically, horseradish peroxidase (HRP)-encapsulated chitosan nanoparticles (HRP-CSNP) were constructed and examined with respect to stability enhancement. HRP-CSNP retained enzyme activity and had improved stability at 37 °C in the presence of a denaturant, urea. The nanoparticles effectively bound to the surface of human breast cancer cell Bcap37 and led to over 80 % cell death when applied with a prodrug indole-3-acetic acid. PMID:25257586

  9. Mechanistic analysis of ultrasound assisted enzymatic desulfurization of liquid fuels using horseradish peroxidase.

    PubMed

    Bhasarkar, Jaykumar; Borah, Arup Jyoti; Goswami, Pranab; Moholkar, Vijayanand S

    2015-11-01

    This study has attempted to gain physical insight into ultrasound-assisted enzymatic desulfurization using system comprising horseradish peroxidase enzyme and dibenzothiophene (DBT). Desulfurization pathway (comprising DBT-sulfoxide and DBT-sulfone as intermediates and 4-methoxy benzoic acid as final product) has been established with GC-MS analysis. Intrinsic fluorescence and circular dichroism spectra of ultrasound-treated enzyme reveal conformational changes in secondary structure (reduction in α-helix and β-conformations and increase in random coil content) leading to enhancement in activity. Concurrent analysis of desulfurization profiles, Arrhenius and thermodynamic parameters, and simulations of cavitation bubble dynamics reveal that strong micro-convection generated by sonication enhances enzyme activity and desulfurization kinetics. Parallel oxidation of DBT by radicals generated from transient cavitation gives further boost to desulfurization kinetics. However, random motion of enzyme molecules induced by shock waves reduces frequency factor and limits the ultrasonic enhancement of enzymatic desulfurization. PMID:26231128

  10. Horseradish peroxidase-driven fluorescent labeling of nanotubes with quantum dots.

    PubMed

    Didenko, Vladimir V; Baskin, David S

    2006-03-01

    We describe the first enzyme-driven technique for fluorescent labeling of single-walled carbon nanotubes (SWNTs). The labeling was performed via enzymatic biotinylation of nanotubes in the tyramide-horseradish peroxidase (HRP) reaction. Both direct and indirect fuorescent labeling of SWNTs was achieved using either biotinyl tyramide or fluorescently tagged tyramides. Biotinylated SWNTs later reacted with streptavidin-conjugated fluorophores. Linking semiconductor nanocrystals, quantum dots (Q-dots), to the surface of nanotubes resulted in their fluorescent visualization, whereas conventional fluorophores bound to SWNTs directly or through biotin-streptavidin linkage, were completely quenched. Enzymatic biotinylation permits fluorescent visualization of carbon nanotubes, which could be useful for a number of biomedical applications. In addition, other organic molecules such as proteins, antibodies, or DNA can be conjugated to biotinylated SWNTs using this approach. PMID:16568818

  11. Horseradish-peroxidase-catalyzed polymerization of amphiphilic tyrosine derivatives in micelles

    NASA Astrophysics Data System (ADS)

    Sarma, Rupmoni; Alva, Shridhara; Marx, Kenneth A.; Akkara, Joseph A.; Kaplan, David L.; Tripathy, Sukant K.

    1998-04-01

    There has been much interest in enzyme catalyzed organic synthesis because it allows the design and synthesis of new materials via chemically mild reaction schemes. This study reports on the horseradish peroxidase catalyzed polymerization of the amphiphilic, C10 alkyl monomer derivative of d and l isomers of tyrosine in micellar solutions. The methodology has been developed to improve the solubility and hence processability of these phenolic polymers. The technique involves the formation of emulsions or micelles of the amphiphilic tyrosines in aqueous medium through manipulation of the solution pH and subsequent enzymatic polymerization. The solution pH, concentrations of the tyrosine derivatives, hydrogen peroxide and the enzyme have been optimized for maximum conversion. The physico- chemical properties of the resulting polymers have been studied by various spectroscopic techniques. Limited stereo- specificity of the reaction has been demonstrated by kinetic methods. Thin films of these polymeric materials have been fabricated using the Langmuir-Blodgett film technique.

  12. Deuterium NMR study of structural and dynamic properties of horseradish peroxidase

    SciTech Connect

    La Mar, G.N.; Thanabal, V.; Johnson, R.D.; Smith, K.M.; Parish, D.W.

    1989-04-05

    High field deuterium NMR spectra have been recorded for various horseradish peroxidase complexes reconstituted with hemins possessing specific 2H labels. The line width of the 2H NMR signals of deuteroheme reconstituted-horseradish peroxidase (HRP) and its cyano complex for the immobilized skeletal 2-2H and 4-2H labels yield the overall protein rotational correlation time (22 ms at 55 degrees C), which is consistent with expectations based on molecular weight. Meso-2H4 labels yield broad (1.3 kHz) signals just upfield from the diamagnetic protein envelope for HRP, and in the central portion of the protein envelope for the CN- ligated resting state HRP. Meso-2H4-labeled mesohemin-reconstituted HRP exhibits a similar signal but shifted further upfield by approximately 10 ppm. The net upfield meso-H hyperfine shifts confirm a five-coordinate structure for resting state HRP. 2Ha resonances for essentially rotationally immobile vinyl groups were detected in both resting state HRP and CN- ligated resting state HRP. Heme methyl-2H-labeling yields relatively narrow lines (approximately 80 Hz) indicative of effective averaging of the quadrupolar relaxation by rapid methyl rotation. Thus the 2H line width of rapidly rotating methyls in hemoproteins can be used effectively to determine the overall protein tumbling rate. Preliminary 2H experiments in meso-2H4-labeled compound I do not support large pi spin density at these positions on the porphyrin cation radical, and argue for a a1u rather than a a2u orbital ground state.

  13. Decolorization of anthraquinonic dyes from textile effluent using horseradish peroxidase: optimization and kinetic study.

    PubMed

    Šekuljica, Nataša Ž; Prlainović, Nevena Ž; Stefanović, Andrea B; Žuža, Milena G; Čičkarić, Dragana Z; Mijin, Dušan Ž; Knežević-Jugović, Zorica D

    2015-01-01

    Two anthraquinonic dyes, C.I. Acid Blue 225 and C.I. Acid Violet 109, were used as models to explore the feasibility of using the horseradish peroxidase enzyme (HRP) in the practical decolorization of anthraquinonic dyes in wastewater. The influence of process parameters such as enzyme concentration, hydrogen peroxide concentration, temperature, dye concentration, and pH was examined. The pH and temperature activity profiles were similar for decolorization of both dyes. Under the optimal conditions, 94.7% of C.I. Acid Violet 109 from aqueous solution was decolorized (treatment time 15 min, enzyme concentration 0.15 IU/mL, hydrogen peroxide concentration 0.4 mM, dye concentration 30 mg/L, pH 4, and temperature 24°C) and 89.36% of C.I. Acid Blue 225 (32 min, enzyme concentration 0.15 IU/mL, hydrogen peroxide concentration 0.04 mM, dye concentration 30 mg/L, pH 5, and temperature 24°C). The mechanism of both reactions has been proven to follow the two substrate ping-pong mechanism with substrate inhibition, revealing the formation of a nonproductive or dead-end complex between dye and HRP or between H2O2 and the oxidized form of the enzyme. Both chemical oxygen demand and total organic carbon values showed that there was a reduction in toxicity after the enzymatic treatment. This study verifies the viability of use of horseradish peroxidase for the wastewaters treatment of similar anthraquinonic dyes. PMID:25685837

  14. Decolorization of Anthraquinonic Dyes from Textile Effluent Using Horseradish Peroxidase: Optimization and Kinetic Study

    PubMed Central

    Šekuljica, Nataša Ž.; Prlainović, Nevena Ž.; Stefanović, Andrea B.; Žuža, Milena G.; Čičkarić, Dragana Z.; Mijin, Dušan Ž.; Knežević-Jugović, Zorica D.

    2015-01-01

    Two anthraquinonic dyes, C.I. Acid Blue 225 and C.I. Acid Violet 109, were used as models to explore the feasibility of using the horseradish peroxidase enzyme (HRP) in the practical decolorization of anthraquinonic dyes in wastewater. The influence of process parameters such as enzyme concentration, hydrogen peroxide concentration, temperature, dye concentration, and pH was examined. The pH and temperature activity profiles were similar for decolorization of both dyes. Under the optimal conditions, 94.7% of C.I. Acid Violet 109 from aqueous solution was decolorized (treatment time 15 min, enzyme concentration 0.15 IU/mL, hydrogen peroxide concentration 0.4 mM, dye concentration 30 mg/L, pH 4, and temperature 24°C) and 89.36% of C.I. Acid Blue 225 (32 min, enzyme concentration 0.15 IU/mL, hydrogen peroxide concentration 0.04 mM, dye concentration 30 mg/L, pH 5, and temperature 24°C). The mechanism of both reactions has been proven to follow the two substrate ping-pong mechanism with substrate inhibition, revealing the formation of a nonproductive or dead-end complex between dye and HRP or between H2O2 and the oxidized form of the enzyme. Both chemical oxygen demand and total organic carbon values showed that there was a reduction in toxicity after the enzymatic treatment. This study verifies the viability of use of horseradish peroxidase for the wastewaters treatment of similar anthraquinonic dyes. PMID:25685837

  15. Size-dependent tuning of horseradish peroxidase bioreactivity by gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Wu, Haohao; Liu, Yi; Li, Meng; Chong, Yu; Zeng, Mingyong; Lo, Y. Martin; Yin, Jun-Jie

    2015-02-01

    Molecules with diverse biological functions, such as heme peroxidases, can be useful tools for identifying potential biological effects of gold nanoparticles (AuNPs) at the molecular level. Here, using UV-Vis, circular dichroism, dynamic light scattering, and electron spin resonance spectroscopy, we report tuning of horseradish peroxidase (HRP) bioactivity by reactant-free AuNPs with diameters of 5, 10, 15, 30 and 60 nm (Au-5 nm, Au-10 nm, Au-15 nm, Au-30 nm and Au-60 nm). HRP conjugation to AuNPs was observed with only Au-5 nm and Au-10 nm prominently increasing the α-helicity of the enzyme to extents inversely related to their size. Au-5 nm inhibited both HRP peroxidase activity toward 3,3',5,5'-tetramethylbenzidine and HRP compound I/II reactivity toward 5,5-dimethyl-1-pyrroline N-oxide. Au-5 nm enhanced the HRP peroxidase activity toward ascorbic acid and the HRP compound I/II reactivity toward redox-active residues in the HRP protein moiety. Further, Au-5 nm also decreased the catalase- and oxidase-like activities of HRP. Au-10 nm showed similar, but weaker effects, while Au-15 nm, Au-30 nm and Au-60 nm had no effect. Results suggest that AuNPs can size-dependently enhance or inhibit HRP bioreactivity toward substrates with different redox potentials via a mechanism involving extension of the HRP substrate access channel and decline in the redox potentials of HRP catalytic intermediates.Molecules with diverse biological functions, such as heme peroxidases, can be useful tools for identifying potential biological effects of gold nanoparticles (AuNPs) at the molecular level. Here, using UV-Vis, circular dichroism, dynamic light scattering, and electron spin resonance spectroscopy, we report tuning of horseradish peroxidase (HRP) bioactivity by reactant-free AuNPs with diameters of 5, 10, 15, 30 and 60 nm (Au-5 nm, Au-10 nm, Au-15 nm, Au-30 nm and Au-60 nm). HRP conjugation to AuNPs was observed with only Au-5 nm and Au-10 nm prominently increasing the

  16. The Molecular Mechanism of the Catalase-like Activity in Horseradish Peroxidase.

    PubMed

    Campomanes, Pablo; Rothlisberger, Ursula; Alfonso-Prieto, Mercedes; Rovira, Carme

    2015-09-01

    Horseradish peroxidase (HRP) is one of the most relevant peroxidase enzymes, used extensively in immunochemistry and biocatalysis applications. Unlike the closely related catalase enzymes, it exhibits a low activity to disproportionate hydrogen peroxide (H2O2). The origin of this disparity remains unknown due to the lack of atomistic information on the catalase-like reaction in HRP. Using QM(DFT)/MM metadynamics simulations, we uncover the mechanism for reduction of the HRP Compound I intermediate by H2O2 at atomic detail. The reaction begins with a hydrogen atom transfer, forming a peroxyl radical and a Compound II-like species. Reorientation of the peroxyl radical in the active site, concomitant with the transfer of the second hydrogen atom, is the rate-limiting step, with a computed free energy barrier (18.7 kcal/mol, ∼ 6 kcal/mol higher than the one obtained for catalase) in good agreement with experiments. Our simulations reveal the crucial role played by the distal pocket residues in accommodating H2O2, enabling formation of a Compound II-like intermediate, similar to catalases. However, out of the two pathways for Compound II reduction found in catalases, only one is operative in HRP. Moreover, the hydrogen bond network in the distal side of HRP compensates less efficiently than in catalases for the energetic cost required to reorient the peroxyl radical at the rate-determining step. The distal Arg and a water molecule in the "wet" active site of HRP have a substantial impact on the reaction barrier, compared to the "dry" active site in catalase. Therefore, the lower catalase-like efficiency of heme peroxidases compared to catalases can be directly attributed to the different distal pocket architecture, providing hints to engineer peroxidases with a higher rate of H2O2 disproportionation. PMID:26274391

  17. Immobilization of horseradish peroxidase on β-cyclodextrin-capped silver nanoparticles: Its future aspects in biosensor application.

    PubMed

    Karim, Zoheb; Khan, Mohd Jahir; Maskat, Mohamad Yusof; Adnan, Rohana

    2016-05-18

    This study aimed to work out a simple and high-yield procedure for the immobilization of horseradish peroxidase on silver nanoparticle. Ultraviolet-visible (UV-vis) and Fourier-transform infrared spectroscopy and transmission electron microscopy were used to characterize silver nanoparticles. Horseradish peroxidase was immobilized on β-cyclodextrin-capped silver nanoparticles via glutaraldehyde cross-linking. Single-cell gel electrophoresis (Comet assay) was also performed to confirm the genotoxicity of silver nanoparticles. To decrease toxicity, silver nanoparticles were capped with β-cyclodextrin. A comparative stability study of soluble and immobilized enzyme preparations was investigated against pH, temperature, and chaotropic agent, urea. The results showed that the cross-linked peroxidase was significantly more stable as compared to the soluble counterpart. The immobilized enzyme exhibited stable enzyme activities after repeated uses. PMID:25830286

  18. Silica nanoparticles coencapsulating gadolinium oxide and horseradish peroxidase for imaging and therapeutic applications

    PubMed Central

    Gupta, Nikesh; Shrivastava, Anju; Sharma, Rakesh K

    2012-01-01

    Mesoporous silica nanoparticles coencapsulating gadolinium oxide and horseradish peroxidase (HRP) have been synthesized in the aqueous core of sodium bis-(2-ethylhexyl) sulfosuccinate (AOT)–hexane–water reverse micelle. The average diameter of these silica particles is around 25 nm and the particles are spherical and highly monodispersed as depicted using transmission electron microscopy. The entrapment efficiency of HRP was found to be as high as 95%. Practically, the entrapped enzyme shows zero leachability up to 90 days. The enzyme entrapped in these silica nanoparticles follows Michaelis–Menten kinetics. Peroxidase entrapped in silica nanoparticles shows higher stability towards temperature and pH change as compared to free enzymes. The gadolinium oxide-doped silica nanoparticles are paramagnetic as observed from the nuclear magnetic resonance line-broadening effect on the proton spectrum of the surrounding water molecule. The entrapped enzyme, HRP, has been used to convert a benign prodrug, indole-3-acetic acid (IAA), to a toxic oxidized product and its toxic effect has been tested on cancerous cell lines through thiazolyl blue tetrazolium blue (MTT) assay. In vitro studies on different cancerous cell lines show that the enzyme has been entrapped and retains its activity inside the silica nanoparticles. IAA alone has no cytotoxic effect and it becomes active only after oxidative decarboxylation by HRP. PMID:23233799

  19. Silica nanoparticles coencapsulating gadolinium oxide and horseradish peroxidase for imaging and therapeutic applications.

    PubMed

    Gupta, Nikesh; Shrivastava, Anju; Sharma, Rakesh K

    2012-01-01

    Mesoporous silica nanoparticles coencapsulating gadolinium oxide and horseradish peroxidase (HRP) have been synthesized in the aqueous core of sodium bis-(2-ethylhexyl) sulfosuccinate (AOT)-hexane-water reverse micelle. The average diameter of these silica particles is around 25 nm and the particles are spherical and highly monodispersed as depicted using transmission electron microscopy. The entrapment efficiency of HRP was found to be as high as 95%. Practically, the entrapped enzyme shows zero leachability up to 90 days. The enzyme entrapped in these silica nanoparticles follows Michaelis-Menten kinetics. Peroxidase entrapped in silica nanoparticles shows higher stability towards temperature and pH change as compared to free enzymes. The gadolinium oxide-doped silica nanoparticles are paramagnetic as observed from the nuclear magnetic resonance line-broadening effect on the proton spectrum of the surrounding water molecule. The entrapped enzyme, HRP, has been used to convert a benign prodrug, indole-3-acetic acid (IAA), to a toxic oxidized product and its toxic effect has been tested on cancerous cell lines through thiazolyl blue tetrazolium blue (MTT) assay. In vitro studies on different cancerous cell lines show that the enzyme has been entrapped and retains its activity inside the silica nanoparticles. IAA alone has no cytotoxic effect and it becomes active only after oxidative decarboxylation by HRP. PMID:23233799

  20. Enzymatic etching of gold nanorods by horseradish peroxidase and application to blood glucose detection

    NASA Astrophysics Data System (ADS)

    Saa, Laura; Coronado-Puchau, Marc; Pavlov, Valeri; Liz-Marzán, Luis M.

    2014-06-01

    Gold nanorods (AuNRs) have become some of the most used nanostructures for biosensing and imaging applications due to their plasmon-related optical response, which is highly sensitive toward minute changes in the AuNR aspect ratio. In this context, H2O2 has been used to trigger the chemical etching of AuNRs, thereby inducing a decrease of their aspect ratio. However, special conditions and relatively high concentrations of H2O2 are usually required, preventing the applicability of the system for biodetection purposes. To overcome this limitation we have introduced a biocatalytic species, the enzyme horseradish peroxidase (HRP) that is able to induce a gradual oxidation of AuNRs in the presence of trace concentrations of H2O2. Interestingly, the presence of halide ions has also been found to be essential for this process. As a consequence, other enzymatic reactions, such as those catalyzed by glucose oxidase, can be easily coupled to HRP activity, allowing the detection of different amounts of glucose. On the basis of these findings, we developed a highly sensitive and simple colorimetric assay that can be read out by the naked eye and allows the detection of physiological glucose concentrations in human serum.Gold nanorods (AuNRs) have become some of the most used nanostructures for biosensing and imaging applications due to their plasmon-related optical response, which is highly sensitive toward minute changes in the AuNR aspect ratio. In this context, H2O2 has been used to trigger the chemical etching of AuNRs, thereby inducing a decrease of their aspect ratio. However, special conditions and relatively high concentrations of H2O2 are usually required, preventing the applicability of the system for biodetection purposes. To overcome this limitation we have introduced a biocatalytic species, the enzyme horseradish peroxidase (HRP) that is able to induce a gradual oxidation of AuNRs in the presence of trace concentrations of H2O2. Interestingly, the presence of

  1. Oxidative metabolism of the anti-cancer agent mitoxantrone by horseradish, lacto-and lignin peroxidase.

    PubMed

    Brück, Thomas B; Brück, Dieter W

    2011-02-01

    Mitoxantrone (MH(2)X), an anthraquinone-type anti-cancer agent used clinically in the treatment of human malignancies, is oxidatively activated by the peroxidase/H(2)O(2) enzyme system. In contrast to the enzymatic mechanisms of drug oxidation, the chemical transformations of MH(2)X are not well described. In this study, MH(2)X metabolites, produced by the horseradish, lacto- or lignin peroxidase (respectively HRP, LPO and LIP)/H(2)O(2) system, were investigated by steady-state spectrokinetic and HPLC-MS methods. At an equimolar mitoxantrone/H(2)O(2) ratio, the efficacy of the enzyme-catalyzed oxidation of mitoxantrone decreased in the following order: LPO > HRP > LIP, which accorded with the decreasing size of the substrate access channel in the enzyme panel examined. In all cases, the central drug oxidation product was the redox-active cyclic metabolite, hexahydronaphtho-[2,3-f]-quinoxaline-7,12-dione (MH(2)), previously identified in the urine of mitoxantrone-treated patients. As the reaction progressed, data gathered in this study suggests that further oxidation of the MH(2) side-chains occurred, yielding the mono- and dicarboxylic acid derivatives respectively. Based on the available data a further MH(2) derivative is proposed, in which the amino-alkyl side-chain(s) are cyclised. With increasing H(2)O(2) concentrations, these novel MH(2) derivatives were oxidised to additional metabolites, whose spectral properties and MS data indicated a stepwise destruction of the MH(2) chromophore due to an oxidative cleavage of the 9,10-anthracenedione moiety. The novel metabolites extend the known sequence of peroxidase-induced mitoxantrone metabolism, and may contribute to the cytotoxic effects of the drug in vivo. Based on the structural features of the proposed MH(2) oxidation products we elaborate on various biochemical mechanisms, which extend the understanding of mitoxantrone's pharmaceutical action and its clinical effectiveness with a particular focus on

  2. Catalytic properties of horseradish peroxidase reconstituted with the 8-(hydroxymethyl)- and 8-formylheme derivatives.

    PubMed

    Harris, R Z; Liddell, P A; Smith, K M; Ortiz de Montellano, P R

    1993-04-13

    Recent studies suggest that 8-(thiomethyl)- and 8-formylheme modifications may be present in, respectively, lactoperoxidase and myeloperoxidase. To examine whether these heme modifications contribute to the unusual catalytic properties of the mammalian peroxidases, we have reconstituted apo-horseradish peroxidase (HRP) with 8-(hydroxymethyl)heme (8HM-HRP) and 8-formylheme (8F-HRP) and have characterized the reconstituted enzymes. Native HRP and 8HM-HRP have identical spectra in the ferric, compound I, and compound II states. In contrast, the Soret band of 8F-HRP is at 417 rather than 402 nm and that of its compound II species is at 436 rather than 416 nm. Compound I was observed as a transient species with 8F-HRP. The rate of formation of compound I was the same for native and 8HM-HRP, but the pseudo-first-order constant for decay of compound I was 0.021 s-1 for 8HM-HRP and 0.010 s-1 for native HRP. The rates of oxidation of guaiacol, iodide, and thioanisole are the same for native HRP and 8HM-HRP but are significantly slower for 8F-HRP. The stereospecificity of thioanisole oxidation is the same for native and 8HM-HRP, but differs for 8F-HRP. For guaiacol, which was studied in detail, Km = 2.3 mM and kcat = 33 s-1 for 8F-HRP versus Km = 1.8 mM and kcat = 104 s-1 for native HRP. 8HM-HRP oxidizes ethylhydrazine and azide to the ethyl and azidyl radicals, respectively, and is simultaneously inactivated. 8F-HRP is also slowly inactivated by ethylhydrazine and azide.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8385486

  3. Expressed Glycosylphosphatidylinositol-Anchored Horseradish Peroxidase Identifies Co-Clustering Molecules in Individual Lipid Raft Domains

    PubMed Central

    Miyagawa-Yamaguchi, Arisa; Kotani, Norihiro; Honke, Koichi

    2014-01-01

    Lipid rafts that are enriched in glycosylphosphatidylinositol (GPI)-anchored proteins serve as a platform for important biological events. To elucidate the molecular mechanisms of these events, identification of co-clustering molecules in individual raft domains is required. Here we describe an approach to this issue using the recently developed method termed enzyme-mediated activation of radical source (EMARS), by which molecules in the vicinity within 300 nm from horseradish peroxidase (HRP) set on the probed molecule are labeled. GPI-anchored HRP fusion proteins (HRP-GPIs), in which the GPI attachment signals derived from human decay accelerating factor and Thy-1 were separately connected to the C-terminus of HRP, were expressed in HeLa S3 cells, and the EMARS reaction was catalyzed by these expressed HRP-GPIs under a living condition. As a result, these different HRP-GPIs had differences in glycosylation and localization and formed distinct clusters. This novel approach distinguished molecular clusters associated with individual GPI-anchored proteins, suggesting that it can identify co-clustering molecules in individual raft domains. PMID:24671047

  4. Probing horseradish peroxidase catalyzed degradation of azo dye from tannery wastewater.

    PubMed

    Preethi, Sadhanandam; Anumary, Ayyappan; Ashokkumar, Meiyazhagan; Thanikaivelan, Palanisamy

    2013-01-01

    Biocatalysis based effluent treatment has outclassed the presently favored physico-chemical treatments due to nil sludge production and monetary savings. Azo dyes are commonly employed in the leather industry and pose a great threat to the environment. Here, we show the degradation of C. I. Acid blue 113 using horseradish peroxidase (HRP) assisted with H2O2 as a co-substrate. It was observed that 0.08 U HRP can degrade 3 mL of 30 mg/L dye up to 80% within 45 min with the assistance of 14 μL of H2O2 at pH 6.6 and 30°C. The feasibility of using the immobilized HRP for dye degradation was also examined and the results show up to 76% dye degradation under similar conditions to that of free HRP with the exception of longer contact time of 240 min. Recycling studies reveal that the immobilized HRP can be recycled up to 3 times for dye degradation. Kinetics drawn for the free HRP catalyzed reaction marked a lower K m and higher V max values, which denotes a proper and faster affinity of the enzyme towards the dye, when compared to the immobilized HRP. The applicability of HRP for treating the actual tannery dye-house wastewater was also demonstrated. PMID:23961406

  5. Investigation on binding of nitric oxide to horseradish peroxidase by absorption spectrometry

    NASA Astrophysics Data System (ADS)

    Qiang, Li; Zhu, Shuhua; Ma, Hongmei; Zhou, Jie

    2010-01-01

    Binding of nitric oxide to horseradish peroxidase (HRP) has been investigated by absorption spectrometry in 0.2 M anaerobic phosphate buffer solution (pH 7.4). Based on this binding equilibrium, a model equation for evaluating the binding constant of nitric oxide to HRP is developed and the binding constant is calculated to be (1.55 ± 0.06) × 10 4 M -1, indicating that HRP can form a stable complex with nitric oxide. The type of inhibition by nitric oxide is validated on the basis of studying initial reaction rates of HRP-catalyzed oxidation of guaiacol in the presence of hydrogen peroxide and nitric oxide. The inhibition mechanism is found to follow an apparent non-competitive inhibition by Lineweaver-Burk method. Based on this kinetic mechanism, the binding constant is also calculated to be (5.22 ± 0.06) × 10 4 M -1. The values of the binding constant determined by the two methods are almost identical. The non-competitive inhibition model is also applicable to studying the effect of nitric oxide on other metalloenzymes, which catalyze the two-substrate reaction with the "ping-pong" mechanism.

  6. Monitoring Synaptic Vesicle Protein Sorting with Enhanced Horseradish Peroxidase in the Electron Microscope.

    PubMed

    Schikorski, Thomas

    2016-01-01

    Protein sorting is the fundamental cellular process that creates and maintains cell organelles and subcellular structures. The synaptic vesicle (SV) is a unique cell organelle that contains a plethora of specific SV proteins and its protein composition is crucial for its function. Thus understanding the mechanisms that sort proteins to SVs and other cell organelles is central to neuroscience and cell biology.While in the past protein sorting was studied in the fluorescence and confocal microscope, we here present a protocol that reveals SV protein trafficking and sorting in the electron microscope (EM). The protocol exploits tagging SV proteins with a new genetically encoded label for EM: enhanced horseradish peroxidase (eHRP). eHRP gained its high sensitivity through direct evolution of its catalytic activity and is detectable in the EM and LM after expression in neurons and other mammalian cells. The protocol describes the use of eHRP, labeling of SVs in cultured hippocampal neurons, and analysis via serial section reconstruction. PMID:27515091

  7. Catalase-like activity of horseradish peroxidase: relationship to enzyme inactivation by H2O2.

    PubMed Central

    Hernández-Ruiz, J; Arnao, M B; Hiner, A N; García-Cánovas, F; Acosta, M

    2001-01-01

    H2O2 is the usual oxidizing substrate of horseradish peroxidase C (HRP-C). In the absence in the reaction medium of a one-electron donor substrate, H2O2 is able to act as both oxidizing and reducing substrate. However, under these conditions the enzyme also undergoes a progressive loss of activity. There are several pathways that maintain the activity of the enzyme by recovering the ferric form, one of which is the decomposition of H2O2 to molecular oxygen in a similar way to the action of catalase. This production of oxygen has been kinetically characterized with a Clark-type electrode coupled to an oxygraph. HRP-C exhibits a weak catalase-like activity, the initial reaction rate of which is hyperbolically dependent on the H2O2 concentration, with values for K(2) (affinity of the first intermediate, compound I, for H2O2) and k(3) (apparent rate constant controlling catalase activity) of 4.0 +/- 0.6 mM and 1.78 +/- 0.12 s(-1) respectively. Oxygen production by HRP-C is favoured at pH values greater than approx. 6.5; under similar conditions HRP-C is also much less sensitive to inactivation during incubations with H2O2. We therefore suggest that this pathway is a major protective mechanism of HRP-C against such inactivation. PMID:11171085

  8. Afferent neurons of the hypoglossal nerve of the rat as demonstrated by horseradish peroxidase tracing.

    PubMed

    Neuhuber, W; Mysicka, A

    1980-01-01

    Cell bodies of sensory neurons of the rat's hypoglossal nerve were demonstrated by the somatopetal horseradish peroxidase (HRP) transport technique. Labelled perikarya were found within the second and third cervical spinal ganglia and in the vagal sensory ganglia. After application of HRP to the cut peripheral trunk of the hypoglossal nerve about 200 labelled cell bodies were counted in each animal. The vast majority of the axons from cervical spinal ganglion cells reach the hypoglossal nerve via the descending ramus (N. descendens hypoglossi). However, there may exist an additional pathway, probably via the cervical sympathetic trunk. Application of HPR to the medial and lateral end branches led to a labelling of much fewer spinal ganglion cells while the number of labelled vegal sensory neurons remained unchanged. Thus, it is suggested that the majority of the cervical afferents of the hypoglossal nerve originates within the extrinsic tongue musculature and the geniohyoid muscle, whereas the vagal afferents may perhaps derive exclusively from the intrinsic muslces. Histograms of the mean diameters of labelled cell bodies show a predominance of very small perikarya. This contrasts with the diameter distribution of sensory perikarya labelled after HRP application to nerves supplying other skeletal muscles. It is therefore assumed that the afferent component of the hypoglossal nerve is composed mainly of small-calibre axons. PMID:7356184

  9. Immobilisation of horseradish peroxidase on EupergitC for the enzymatic elimination of phenol.

    PubMed

    Pramparo, L; Stüber, F; Font, J; Fortuny, A; Fabregat, A; Bengoa, C

    2010-05-15

    In this study, three different approaches for the covalent immobilisation of the horseradish peroxidase (HRP) onto epoxy-activated acrylic polymers (EupergitC) were explored for the first time, direct HRP binding to the polymers via their oxirane groups, HRP binding to the polymers via a spacer made from adipic dihydrazide, and HRP binding to hydrazido polymer surfaces through the enzyme carbohydrate moiety previously modified by periodate oxidation. The periodate-mediated covalent immobilisation of the HRP on hydrazido EupergitC was found to be the most effective method for the preparation of biocatalysts. In this case, a maximum value of the immobilised enzyme activity of 127 U/g(support) was found using an enzyme loading on the support of 35.2mg/g(support). The free and the immobilised HRP were used to study the elimination of phenol in two batch reactors. As expected, the activity of the immobilised enzyme was lower than the activity of the free enzyme. Around 85% of enzyme activity is lost during the immobilisation. However, the reaction using immobilised enzyme showed that it was possible to reach high degrees of phenol removal (around 50%) using about one hundredth of the enzyme used in the soluble form. PMID:20092944

  10. Improved covalent immobilization of horseradish peroxidase on macroporous glycidyl methacrylate-based copolymers.

    PubMed

    Prodanović, Olivera; Prokopijević, Miloš; Spasojević, Dragica; Stojanović, Zeljko; Radotić, Ksenija; Knežević-Jugović, Zorica D; Prodanović, Radivoje

    2012-11-01

    A macroporous copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate, poly(GMA-co-EGDMA), with various surface characteristics and mean pore size diameters ranging from 44 to 200 nm was synthesized, modified with 1,2-diaminoethane, and tested as a carrier for immobilization of horseradish peroxidase (HRP) by two covalent methods, glutaraldehyde and periodate. The highest specific activity of around 35 U g(-1) dry weight of carrier was achieved on poly(GMA-co-EGDMA) copolymers with mean pore diameters of 200 and 120 nm by the periodate method. A study of deactivation kinetics at 65 °C and in 80 % dioxane revealed that periodate immobilization also produced an appreciable stabilization of the biocatalyst, while stabilization factor depended strongly on the surface characteristics of the copolymers. HRP immobilized on copolymer with a mean pore diameter of 120 nm by periodate method showing not only the highest specific activity but also good stability was further characterized. It appeared that the immobilization resulted in the stabilization of enzyme over a broader pH range while the Michaelis constant value (K (m)) of the immobilized HRP was 10.8 mM, approximately 5.6 times higher than that of the free enzyme. After 6 cycles of repeated use in a batch reactor for pyrogallol oxidation, the immobilized HRP retained 45 % of its original activity. PMID:22941271

  11. Amperometric inhibitive biosensor based on horseradish peroxidase-nanoporous gold for sulfide determination.

    PubMed

    Sun, Huihui; Liu, Zhuang; Wu, Chao; Xu, Ping; Wang, Xia

    2016-01-01

    As a well-known toxic pollutant, sulfide is harmful to human health. In this study, a simple and sensitive amperometric inhibitive biosensor was developed for the determination of sulfide in the environment. By immobilizing nanoporous gold (NPG) on glassy carbon electrode (GCE), and encapsulating horseradish peroxidase (HRP) onto NPG, a HRP/NPG/GCE bioelectrode for sulfide detection was successfully constructed based on the inhibition of sulfide on HRP activity with o-Phenylenediamine (OPD) as a substrate. The resulted HRP/NPG/GCE bioelectrode achieved a wide linear range of 0.1-40 μM in sulfide detection with a high sensitivity of 1720 μA mM(-1) cm(-2) and a low detection limit of 0.027 μM. Additionally, the inhibition of sulfide on HRP is competitive inhibition with OPD as a substrate by Michaelis-Menten analysis. Notably, the recovery of HRP activity was quickly achieved by washing the HRP/NPG/GCE bioelectrode using differential pulse voltammetry (DPV) technique in deaerated PBS (50 mM, pH 7.0) for only 60 s. Furthermore, the real sample analysis of sulfide by the HRP/NPG/GCE bioelectrode was achieved. Based on above results, the HRP/NPG/GCE bioelectrode could be a better choice for the real determination of sulfide compared to inhibitive biosensors previously reported. PMID:27515253

  12. Amperometric inhibitive biosensor based on horseradish peroxidase-nanoporous gold for sulfide determination

    PubMed Central

    Sun, Huihui; Liu, Zhuang; Wu, Chao; Xu, Ping; Wang, Xia

    2016-01-01

    As a well-known toxic pollutant, sulfide is harmful to human health. In this study, a simple and sensitive amperometric inhibitive biosensor was developed for the determination of sulfide in the environment. By immobilizing nanoporous gold (NPG) on glassy carbon electrode (GCE), and encapsulating horseradish peroxidase (HRP) onto NPG, a HRP/NPG/GCE bioelectrode for sulfide detection was successfully constructed based on the inhibition of sulfide on HRP activity with o-Phenylenediamine (OPD) as a substrate. The resulted HRP/NPG/GCE bioelectrode achieved a wide linear range of 0.1–40 μM in sulfide detection with a high sensitivity of 1720 μA mM−1 cm−2 and a low detection limit of 0.027 μM. Additionally, the inhibition of sulfide on HRP is competitive inhibition with OPD as a substrate by Michaelis-Menten analysis. Notably, the recovery of HRP activity was quickly achieved by washing the HRP/NPG/GCE bioelectrode using differential pulse voltammetry (DPV) technique in deaerated PBS (50 mM, pH 7.0) for only 60 s. Furthermore, the real sample analysis of sulfide by the HRP/NPG/GCE bioelectrode was achieved. Based on above results, the HRP/NPG/GCE bioelectrode could be a better choice for the real determination of sulfide compared to inhibitive biosensors previously reported. PMID:27515253

  13. Biocatalysis in water-in-ionic liquid microemulsions: a case study with horseradish peroxidase.

    PubMed

    Moniruzzaman, M; Kamiya, N; Goto, M

    2009-01-20

    In this article we report the first results on the enzymatic activity of horseradish peroxidase (HRP) microencapsulated in water-in-ionic liquid (w/IL) microemulsions using pyrogallol as the substrate. Toward this goal, the system used in this study was composed of anionic surfactant AOT (sodium bis(2-ethyl-1-hexyl)sulfosuccinate)/hydrophobic IL [C(8)mim][Tf(2)N] (1-octyl-3-methyl imidazolium bis(trifluoromethylsulfonyl)amide)/water/1-hexanol. In this system, the catalytic activity of HRP was measured as a function of substrate concentrations, W(0) (molar ratio of water to surfactant), pH, and 1-hexanol content. The curve of the activity-W(0) profile was found to be hyperbolic for the new microemulsion. The apparent Michaelis-Menten kinetic parameters (k(cat) and K(m)) were estimated and compared to those obtained from a conventional microemulsion. Apparently, it was found that HRP-catalyzed oxidation of pyrogallol by hydrogen peroxide in IL microemulsuions is much more effective than in a conventional AOT/water/isooctane microemulsion. The stability of HRP solubilized in the newly developed w/IL microemulsions was examined, and it was found that HRP retained almost 70% of its initial activity after incubation at 28 degrees C for 30 h. PMID:19113810

  14. Chemically glycosylation improves the stability of an amperometric horseradish peroxidase biosensor

    PubMed Central

    Hernández-Cancel, Griselle; Suazo-Dávila, Damaris; Medina-Guzmán, Johnsue; Rosado-González, María; Díaz-Vázquez, Liz M.; Griebenow, Kai

    2014-01-01

    We constructed a biosensor by electrodeposition of gold nano-particles (AuNPs) on glassy carbon (GC) and subsequent formation of a 4-mercaptobenzoic acid self-assembled monolayer (SAM). The enzyme horseradish peroxidase (HRP) was then covalently immobilized onto the SAM. Two forms of HRP were employed: non-modified and chemically glycosylated with lactose. Circular dichroism (CD) spectra showed that chemical glycosylation did neither change the tertiary structure of HRP nor the heme environment. The highest sensitivity of the biosensor to hydroquinone was obtained for the biosensor with HRP-lactose 1 (414 nA μM−1 ) compared to 378 nA μM−1 for the one employing non-modified HRP. The chemically glycosylated form of the enzyme catalyzed the reduction of hydroquinone more rapidly than the native form of the enzyme. The sensor employing lactose-modified HRP also had a lower limit of detection (74 μM) than the HRP biosensor (83 μM). However, most importantly, chemically glycosylation improved the long-term stability of the biosensor, which retained 60% of its activity over a four-month storage period compared to only 10% for HRP. These results highlight improvements by an innovative stabilization method when compared to previously reported enzyme-based biosensors. PMID:25479876

  15. Enzymatic removal of paracetamol from aqueous phase: horseradish peroxidase immobilized on nanofibrous membranes.

    PubMed

    Xu, Ran; Si, Yifang; Li, Fengting; Zhang, Bingru

    2015-03-01

    Paracetamol is a widely used as an analgesic and an antipyretic that can easily accumulate in aquatic environments. This study aimed to enhance paracetamol removal efficiency from water by combining the biocatalytic activity of horseradish peroxidase (HRP) with the adsorption of nanofibrous membrane. Poly(vinyl alcohol)/poly(acrylic acid)/SiO2 electrospinning nanofibrous membrane was prepared with fiber diameters of 200 to 300 nm. The membrane was made insoluble by the thermal cross-linking process. HRP, which was previously activated by 1,1'-carbonyldiimidazole, was covalently immobilized on the surface of nanofibers. Immobilized HRP retained 79.4 % of the activity of free HRP. The physical, chemical, and biochemical properties of the immobilized HRP and its application in paracetamol removal were comprehensively investigated. Immobilized HRP showed better storage capability and higher tolerance to the changes in pH and temperature than free HRP. Paracetamol removal rate by immobilized HRP (83.5 %) was similar to that of free HRP (84.4 %), but immobilized HRP showed excellent reusability. The results signify that enzyme immobilized on nanofibers has great application potential in water treatment. PMID:25269844

  16. Sensitive electrochemical detection of horseradish peroxidase at disposable screen-printed carbon electrode

    SciTech Connect

    Lee, Ai Cheng; Liu, Guodong; Heng, Chew-Kiat; Tan, Swee-Nign; Lim, Tit-Meng; Lin, Yuehe

    2008-09-10

    A rapid, simple and sensitive electrochemical assay of horseradish peroxidase (HRP) performed on disposable screen-printed carbon electrode was developed. HRP activities were monitored by square-wave voltammetric (SWV) measuring the electroactive enzymatic product in the presence of o-aminophenol and hydrogen peroxide substrate solution. SWV analysis demonstrated a greater sensitivity and shorter analysis time than the widely used amperometric and differential-pulsed voltammetric methods. The voltammetric characteristics of substrate and enzymatic product as well as the parameters of SWV analysis were optimized. Under optimized conditions, a linear response for HRP from 0.003 - 0.1 U/mL and a detection limit of 0.002 U/mL (1.25×10-15 mol in 25 µL) were obtained with a good precision (RSD = 8%; n = 6). This rapid and sensitive HRP assay with microliters-assay volume could be readily integrated to portable devices and point-of-care (POC) diagnosis applications.

  17. Mechanisms for Covalent Immobilization of Horseradish Peroxidase on Ion-Beam-Treated Polyethylene

    PubMed Central

    Kondyurin, Alexey V.; Naseri, Pourandokht; Tilley, Jennifer M. R.; Nosworthy, Neil J.; Bilek, Marcela M. M.; McKenzie, David R.

    2012-01-01

    The surface of polyethylene was modified by plasma immersion ion implantation. Structure changes including carbonization and oxidation were observed. High surface energy of the modified polyethylene was attributed to the presence of free radicals on the surface. The surface energy decay with storage time after treatment was explained by a decay of the free radical concentration while the concentration of oxygen-containing groups increased with storage time. Horseradish peroxidase was covalently attached onto the modified surface by the reaction with free radicals. Appropriate blocking agents can block this reaction. All aminoacid residues can take part in the covalent attachment process, providing a universal mechanism of attachment for all proteins. The native conformation of attached protein is retained due to hydrophilic interactions in the interface region. The enzymatic activity of covalently attached protein remained high. The long-term activity of the modified layer to attach protein is explained by stabilisation of unpaired electrons in sp2 carbon structures. A high concentration of free radicals can give multiple covalent bonds to the protein molecule and destroy the native conformation and with it the catalytic activity. The universal mechanism of protein attachment to free radicals could be extended to various methods of radiation damage of polymers. PMID:24278665

  18. Effect of ozone and histamine on airway permeability to horseradish peroxidase in guinea pigs

    SciTech Connect

    Miller, P.D.; Gordon, T.; Warnick, M.; Amdur, M.O.

    1986-01-01

    Airway permeability was studied in groups of male guinea pigs at 2, 8, and 24 h after a 1-h exposure to 1 ppm ozone or at 2 h after a 1-h exposure to filtered air (control). Intratracheal administration of 2 mg horseradish peroxidase (HRP) was followed by blood sampling at 5-min intervals up to 30 min. The rate of appearance of HRP in plasma was significantly higher at 2 and 8 h after ozone exposure than that found in animals examined 2 h after air exposure or 24 h after ozone exposure. A dose of 0.12 mg/kg of subcutaneous histamine given after the 15 min blood sample significantly increased the already elevated permeability seen at 2 h post ozone, but had no effect on animals exposed to filtered air 2 h earlier or to ozone 24 h earlier. No difference was seen in the amount of subcutaneous radiolabeled histamine in the lungs of animals exposed 2 h earlier either to air or to ozone. These data indicate that a short-term exposure to ozone produced a reversible increase in respiratory epithelial permeability to HRP in guinea pigs. The potentiation of this increased permeability by histamine may be another manifestation of ozone-induced hyperreactivity.

  19. Investigations of Ferric Heme Cyanide Photodissociation in Myoglobin and Horseradish Peroxidase

    PubMed Central

    Zeng, Weiqiao; Sun, Yuhan; Benabbas, Abdelkrim; Champion, Paul M.

    2013-01-01

    The photodissociation of cyanide from ferric myoglobin (MbCN) and horseradish peroxidase (HRPCN) has been definitively observed. This has implications for the interpretation of ultrafast IR (Helbing et al. Biophys. J. 2004, 87, 1881–1891) and optical (Gruia et al. Biophys. J. 2008, 94, 2252–2268) studies that had previously suggested the Fe-CN bond was photostable in MbCN. The photolysis of ferric MbCN takes place with a quantum yield of ~75% and the resonance Raman spectrum of the photoproduct observed in steady-state experiments as a function of laser power and sample spinning rate is identical to that of ferric Mb (metMb). The data are quantitatively analyzed using a simple model where cyanide is photodissociated and, although geminate rebinding with a rate kBA ≈ (3.6 ps)−1 is the dominant process, some CN− exits from the distal heme pocket and is replaced by water. Using independently determined values for the CN− association rate, we find that the CN− escape rate from the ferric myoglobin pocket to the solution at 293 K is kout ≈ 1–2 × 107 s−1. This value is very similar to, but slightly larger than, the histidine gated escape rate of CO from Mb (1.1×107 s−1) under the same conditions. The analysis leads to an escape probability kout/(kout+kBA) ~ 10−4, which is unobservable in most time domain kinetic measurements. However, the photolysis is surprisingly easy to detect in Mb using cw resonance Raman measurements. This is due to the anomalously slow CN− bimolecular association rate (170 M−1s−1), which arises from the need for water to exchange at the ferric heme binding site of Mb. In contrast, ferric HRP does not have a heme bound water molecule and its CN− bimolecular association rate is larger by ~103 making the CN− photolysis more difficult to observe. PMID:23472676

  20. Horseradish peroxidase localization of sympathetic postganglionic and parasympathetic preganglionic neurons innervating the monkey heart.

    PubMed

    Chuang, King-Shun; Liu, Wan-Cherng; Liou, Nien-Hsien; Liu, Jiang-Chuan

    2004-06-30

    The localization of the sympathetic postganglionic and parasympathetic preganglionic neurons innervating the monkey heart were investigated through retrograde axonal transport with horseradish peroxidase (HRP). HRP (4 mg or 30 mg) was injected into the subepicardial and myocardial layers in four different cardiac regions. The animals were euthanized 84-96 hours later and fixed by paraformaldehyde perfusion via the left ventricle. The brain stem and the paravertebral sympathetic ganglia from the superior cervical, middle cervical, and stellate ganglia down to the T9 ganglia were removed and processed for HRP identification. Following injection of HRP into the apex of the heart, the sinoatrial nodal region, or the right ventricle, HRP-labeled sympathetic neurons were found exclusively in the right superior cervical ganglion (64.8%) or in the left superior cervical ganglion (35%). Fewer labeled cells were found in the right stellate ganglia. After HRP injection into the left ventricle, labeled sympathetic cells were found chiefly in the left superior cervical ganglion (51%) or in the right superior cervical ganglion (38.6%); a few labeled cells were seen in the stellate ganglion bilaterally and in the left middle cervical ganglion. Also, in response to administration of HRP into the anterior part of the apex, anterior middle part of the right ventricle, posterior upper part of the left ventricle, or sinoatrial nodal region, HRP-labeled parasympathetic neurons were found in the nucleus ambiguus on both the right (74.8%) and left (25.2%) sides. No HRP-labeled cells were found in the dorsal motor nucleus of the vagus on either side. PMID:15481792

  1. Effects of molecular confinement and crowding on horseradish peroxidase kinetics using a nanofluidic gradient mixer.

    PubMed

    Wichert, William R A; Han, Donghoon; Bohn, Paul W

    2016-03-01

    The effects of molecular confinement and crowding on enzyme kinetics were studied at length scales and under conditions similar to those found in biological cells. These experiments were carried out using a nanofluidic network of channels constituting a nanofluidic gradient mixer, providing the basis for measuring multiple experimental conditions simultaneously. The 100 nm × 40 μm nanochannels were wet etched directly into borosilicate glass, then annealed and characterized with fluorescein emission prior to kinetic measurements. The nanofluidic gradient mixer was then used to measure the kinetics of the conversion of the horseradish peroxidase (HRP)-catalyzed conversion of non-fluorescent Amplex Red (AR) to the fluorescent product resorufin in the presence of hydrogen peroxide (H2O2). The design of the gradient mixer allows reaction kinetics to be studied under multiple (five) unique solution compositions in a single experiment. To characterize the efficiency of the device the effects of confinement on HRP-catalyzed AR conversion kinetics were studied by varying the starting ratio of AR : H2O2. Equimolar concentrations of Amplex Red and H2O2 yielded the highest reaction rates followed by 2 : 1, 1 : 2, 5 : 1, and finally 1 : 5 [AR] : [H2O2]. Under all conditions, initial reaction velocities were decreased by excess H2O2. Crowding effects on kinetics were studied by increasing solution viscosity in the nanochannels in the range 1.0-1.6 cP with sucrose. Increasing the solution viscosities in these confined geometries decreases the initial reaction velocity at the highest concentration from 3.79 μM min(-1) at 1.00 cP to 0.192 μM min(-1) at 1.59 cP. Variations in reaction velocity are interpreted in the context of models for HRP catalysis and for molecular crowding. PMID:26792298

  2. Catalytic activity and thermal stability of horseradish peroxidase encapsulated in self-assembled organic nanotubes.

    PubMed

    Lu, Qin; Kim, Youngchan; Bassim, Nabil; Raman, Nisha; Collins, Greg E

    2016-03-21

    Horseradish peroxidase (HRP) was encapsulated in self-assembled lithocholic acid (LCA) based organic nanotubes and its catalytic activity before and after thermal treatment was measured for comparison with free HRP. The apparent kcat (kcat/Km) for nanotube encapsulated HRP remained almost the same before and after thermal treatment, reporting an average value of 3.7 ± 0.4 μM(-1) s(-1). The apparent kcat value for free HRP decreased from 14.8 ± 1.3 μM(-1) s(-1) for samples stored at 4 °C to 2.4 ± 0.1 μM(-1) s(-1) after thermal treatment for 8 h at 55 °C. The Michaelis-Menten constants, Km, determined for encapsulated HRP and free HRP were relatively unperturbed by storage conditions at 4 °C or thermally treated at 55 °C for varying time periods from 2-8 h, with encapsulated HRP having a slightly higher Km than free HRP (13.4 ± 0.9 μM versus 11.7 ± 0.4 μM). The amount of HRP encapsulated in LCA nanotubes increased dramatically when the mixture of HRP and LCA nanotubes was brought to an elevated temperature. Within 4 h of thermal treatment at 55 °C, the amount of HRP encapsulated by the LCA nanotubes was more than 4 times the amount of HRP encapsulated when equilibrated at 4 °C for 7 days. Molecular dynamics (MD) simulations show that the higher degree of exposure of hydrophobic residues in HRP at elevated temperatures enhances the hydrophobic interaction between HRP and the nanotube wall, resulting in the increased amount of HRP surface adsorption and, hence, the overall amount of encapsulation inside the nanotubes. PMID:26953357

  3. Immobilization of horseradish peroxidase on amidoximated acrylic polymer activated by cyanuric chloride.

    PubMed

    Mohamed, Saleh A; Al-Ghamdi, Saeed S; El-Shishtawy, Reda M

    2016-10-01

    Horseradish peroxidase (HRP) was immobilized on amidoximated acrylic fabric after being activated with cyanuric chloride. FT-IR spectroscopy and scanning electron microscopy were used to characterize fabrics. The maximum immobilization efficiency of HRP (70%) was detected at 4% cyanuric chloride and pH 7.0. The immobilized enzyme retained 45% of its initial activity after ten reuses. The immobilization of enzyme on the carrier is saturated after 6h of incubation time. The pH was shifted from 7.0 for soluble HRP to 7.5-8.0 for the immobilized enzyme. The soluble HRP and immobilized HRP had the same optimum activity at 40°C. The immobilized HRP is more thermal stable than soluble HRP. Substrate analogues were oxidized by immobilized HRP with higher efficiencies than those of soluble HRP. Km values of the soluble HRP and the immobilized HRP were 31 and 37mM for guiacol and 5.0 and 7.8mM for H2O2, respectively. The immobilized HRP had higher efficiency for removal of phenol than that of soluble HRP. The immobilized HRP had higher resistance toward heavy metal ions compared to the soluble enzyme. The immobilized HRP was more stable against high concentration of urea, Triton X-100 and isopropanol. The immobilized HRP exhibited high resistance to proteolysis by trypsin than soluble enzyme. In conclusion, the immobilized HRP could be used as a potential efficient catalyst for the removal of aromatic pollutants from wastewater. PMID:27264646

  4. Tyrosine Coupling Creates a Hyperbranched Multivalent Protein Polymer Using Horseradish Peroxidase via Bipolar Conjugation Points.

    PubMed

    Minamihata, Kosuke; Yamaguchi, Sou; Nakajima, Kei; Nagamune, Teruyuki

    2016-05-18

    Protein polymers of covalently cross-linked protein monomers are highly attractive biomaterials because each monomer unit possesses distinct protein functions. Protein polymers often show enhancement effects on the function by integrating a large number of molecules into one macromolecule. The cross-linking site of component proteins should be precisely controlled to avoid diminishing the protein function. However, preparing protein polymers that are cross-linked site-specifically with a high cross-linking degree is a challenge. Here, we demonstrate the preparation of a site-specifically cross-linked protein polymer that has a hyperbranched polymer-like structure with a high cross-linking degree. A horseradish peroxidase (HRP) reaction was used to achieve the protein polymerization through a peptide tag containing a tyrosine residue (Y-tag). Y-tag sequences were introduced to both N- and C-termini of a model protein, protein G. The dual Y-tagged protein G (Y-pG-Y) was treated with HRP to form a Y-pG-Y polymer possessing average and maximum cross-linking degree of approximately 70-mer and 150-mer, respectively. The Y-pG-Y polymer shows the highest cross-linking degree among the protein polymers reported, which are completely soluble in water and cross-linked via covalent bonding. The Y-pG-Y was cross-linked site-specifically at the Tyr residue in the Y-tag, retaining its function, and the Y-pG-Y polymer showed extremely strong avidity against immunoglobulin G. The reactivities of N- and C-terminal Y-tags were evaluated, and we revealed that the difference in the radical formation rate by HRP was the key for yielding highly cross-linked protein polymers. PMID:27093089

  5. Horseradish peroxidase-immobilized magnetic mesoporous silica nanoparticles as a potential candidate to eliminate intracellular reactive oxygen species

    NASA Astrophysics Data System (ADS)

    Shen, Yajing; Zhang, Ye; Zhang, Xiang; Zhou, Xiuhong; Teng, Xiyao; Yan, Manqing; Bi, Hong

    2015-02-01

    Horseradish peroxidase-immobilized magnetic mesoporous silica nanoparticles (MMSNs-HRP) have been synthesized by a NHS/EDC coupling between the amino groups of horseradish peroxidase (HRP) and the carboxyl groups on the MMSNs surface. It is found that the immobilized HRP on MMSNs still retain high activity and the MMSNs-HRP can eliminate the reactive oxygen species (ROS) in Chinese hamster ovary (CHO) cells induced by the addition of H2O2 aqueous solution. Further, the fluorescent MMSN-HRP-CD nanoparticles have been prepared by attaching biocompatible, fluorescent carbon dots (CDs) to MMSNs-HRP. We have also investigated the effect of an applied magnetic field on cellular uptake of MMSNs-HRP-CDs and found that the internalization of MMSNs-HRP-CDs by CHO cells could be enhanced within 2 hours under the magnetic field. This work provides us with a novel and efficient method to eliminate ROS in living cells by using HRP-immobilized nanoparticles.Horseradish peroxidase-immobilized magnetic mesoporous silica nanoparticles (MMSNs-HRP) have been synthesized by a NHS/EDC coupling between the amino groups of horseradish peroxidase (HRP) and the carboxyl groups on the MMSNs surface. It is found that the immobilized HRP on MMSNs still retain high activity and the MMSNs-HRP can eliminate the reactive oxygen species (ROS) in Chinese hamster ovary (CHO) cells induced by the addition of H2O2 aqueous solution. Further, the fluorescent MMSN-HRP-CD nanoparticles have been prepared by attaching biocompatible, fluorescent carbon dots (CDs) to MMSNs-HRP. We have also investigated the effect of an applied magnetic field on cellular uptake of MMSNs-HRP-CDs and found that the internalization of MMSNs-HRP-CDs by CHO cells could be enhanced within 2 hours under the magnetic field. This work provides us with a novel and efficient method to eliminate ROS in living cells by using HRP-immobilized nanoparticles. Electronic supplementary information (ESI) available: TEM image of CDs, BET XRD

  6. A fluorescence approach to the unfolding thermodynamics of horseradish peroxidase based on heme degradation by hydrogen peroxide

    NASA Astrophysics Data System (ADS)

    Ke, Zhigang; Ma, Shanshan; Li, Lamei; Huang, Qing

    2016-07-01

    Horseradish peroxidase (HRP) is a classical heme-containing protein which has been applied in many fields. The prosthetic group heme in HRP, especially in unfolded state, can react with hydrogen peroxide (H2O2) to produce a fluorescent product with the maximum emission wavelength at 450 nm. Utilizing this emission band as a fluorescence probe, the unfolding process of HRP in urea can be assessed quantitatively, and the calculated thermodynamic parameters are consistent with those determined by circular dichroism (CD) at 222 nm and steady-state tryptophan (Trp) fluorescence methods.

  7. Interaction of thiocyanate with horseradish peroxidase. 1H and 15N nuclear magnetic resonance studies.

    PubMed

    Modi, S; Behere, D V; Mitra, S

    1989-11-25

    Interaction of thiocyanate with horseradish peroxidase (HRP) was investigated by relaxation rate measurements (at 50.68 MHz) of the 15N resonance of thiocyanate nitrogen and by following the hyperfine shifted ring methyl proton resonances (at 500 MHz) of the heme group of SCN-.HRP solutions. At pH 4.0, the apparent dissociation constant (KD) for thiocyanate binding to HRP was deduced to be 158 mM from the relaxation rate measurements. Chemical shift changes of 1- and 8-ring methyl proton resonances in the presence of various amounts of thiocyanate at pH 4.0 yielded KD values of 166 and 136 mM, respectively. From the pH dependence of KD and the 15N resonance line width, it was observed that thiocyanate binds to HRP only under acidic conditions (pH less than 6). The binding was found to be facilitated by protonation of an acid group on the enzyme with pKa 4.0. The pH dependence of the 15N line width as well as the apparent dissociation constant were quantitatively analyzed on the basis of a reaction scheme in which thiocyanate in deprotonated ionic form binds to the enzyme in protonated acidic form. The KD for thiocyanate binding to HRP was also evaluated in the presence of an excess of exogenous substrates such as resorcinol, cyanide, and iodide ions. It was found that the presence of cyanide (which binds to heme iron at the sixth coordination position) and resorcinol did not have any effect on the binding of thiocyanate, indicating that the binding site of the thiocyanate ion is located away from the ferric center as well as from the aromatic donor binding site. The KD in the presence of iodide, however, showed that iodide competes with thiocyanate for binding at the same site. The distance of the bound thiocyanate ion from the ferric center was deduced from the 15N relaxation time measurements and was found to be a 6.8 A. From the distance as well as the change in the chemical shifts and line width of 1- and 8-methyl proton resonances, it is suggested that the

  8. Nature of the inhibition of horseradish peroxidase and mitochondrial cytochrome c oxidase by cyanyl radical.

    PubMed

    Chen, Y R; Deterding, L J; Tomer, K B; Mason, R P

    2000-04-18

    Previous studies established that the cyanyl radical ((*)CN), detected as 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/(*)CN by the electron spin resonance (ESR) spin-trapping technique, can be generated by horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H(2)O(2)) and by mitochondrial cytochrome c oxidase (CcO) in the absence of H(2)O(2). To investigate the mechanism of inhibition by cyanyl radical, we isolated and characterized the iron protoporphyrin IX and heme a from the reactions of CN(-) with HRP and CcO, respectively. The purified heme from the reaction mixture of HRP/H(2)O(2)/KCN was unambiguously identified as cyanoheme by the observation of the protonated molecule, (M + H)(+), of m/z = 642.9 in the matrix-assisted laser desorption/ionization (MALDI) mass spectrum. The proton NMR spectrum of the bipyridyl ferrous cyanoheme complex revealed that one of the four meso protons was missing and had been replaced with a cyanyl group, indicating that the single, heme-derived product was meso-cyanoheme. The holoenzyme of HRP from the reconstitution of meso-cyanoheme with the apoenzyme of HRP (apoHRP) showed no detectable catalytic activity. The Soret peak of cyanoheme-reconstituted apoHRP was shifted to 411 nm from the 403 nm peak of native HRP. In contrast, the heme a isolated from partially or fully inhibited CcO did not show any change in the structure of the protoporphyrin IX as indicated by its MALDI mass spectrum, which showed an (M + H)(+) of m/z = 853.6, and by its pyridine hemochromogen spectrum. However, a protein-centered radical on the CcO can be detected in the reaction of CcO with cyanide and was identified as the thiyl radical(s) based on inhibition of its formation by N-ethylmaleimide pretreatment, suggesting that the protein matrix rather than protoporphyrin IX was attacked by the cyanyl radical. In addition to the difference in heme structures between HRP and CcO, the available crystallographic data also suggested that the distinct

  9. Which one of the two common reporter systems is more suitable for chemiluminescent enzyme immunoassay: alkaline phosphatase or horseradish peroxidase?

    PubMed

    Yu, Songcheng; Yu, Fei; Liu, Lie; Zhang, Hongquan; Zhang, Zhenzhong; Qu, Lingbo; Wu, Yongjun

    2016-05-01

    Alkaline phosphatase and horseradish peroxidase are the most commonly used reporter systems in chemiluminescent enzyme immunoassay (CLEIA). Which one, therefore, would be better when establishing a CLEIA method for a new target substance? There was no standard answer. In this study, both reporters were compared systematically including luminescence kinetics, conjugation methods, optimal condition and detection performance, using two common drugs, SD-methoxy-pyrimidine and enrofloxacin, as determination objects. The results revealed that there was much difference between the luminescence kinetics of the two systems. However, there was little difference between these systems when detecting the same substance, including in optimal conditions and determination of performance. Both reporters were suitable for establishing chemiluminescent enzyme immunoassays. Therefore, the choice of alkaline phosphatase or horseradish peroxidase as the reporter system in chemiluminescent enzyme immunoassays depends on availability. Conversely, these two report systems could be applied in simultaneous analysis of multicomponents due to their different optical behaviors and similar performances. But attention should be paid to conjugation method and coating buffer, which affected the luminescent intensity of different determination targets. PMID:26552992

  10. An Amperometric Biosensor Utilizing a Ferrocene-Mediated Horseradish Peroxidase Reaction for the Determination of Capsaicin (Chili Hotness)

    PubMed Central

    Mohammad, Rosmawani; Ahmad, Musa; Heng, Lee Yook

    2013-01-01

    Chili hotness is very much dependent on the concentration of capsaicin present in the chili fruit. A new biosensor based on a horseradish peroxidase enzyme-capsaicin reaction mediated by ferrocene has been successfully developed for the amperometric determination of chili hotness. The amperometric biosensor is fabricated based on a single-step immobilization of both ferrocene and horseradish peroxidase in a photocurable hydrogel membrane, poly(2-hydroxyethyl methacrylate). With mediation by ferrocene, the biosensor could measure capsaicin concentrations at a potential 0.22 V (vs. Ag/AgCl), which prevented potential interference from other electroactive species in the sample. Thus a good selectivity towards capsaicin was demonstrated. The linear response range of the biosensor towards capsaicin was from 2.5–99.0 μM with detection limit of 1.94 μM. A good relative standard deviation (RSD) for reproducibility of 6.4%–9.9% was obtained. The capsaicin biosensor demonstrated long-term stability for up to seven months. The performance of the biosensor has been validated using a standard method for the analysis of capsaicin based on HPLC. PMID:23921830

  11. Horseradish peroxidase-immobilized magnetic mesoporous silica nanoparticles as a potential candidate to eliminate intracellular reactive oxygen species.

    PubMed

    Shen, Yajing; Zhang, Ye; Zhang, Xiang; Zhou, Xiuhong; Teng, Xiyao; Yan, Manqing; Bi, Hong

    2015-02-21

    Horseradish peroxidase-immobilized magnetic mesoporous silica nanoparticles (MMSNs-HRP) have been synthesized by a NHS/EDC coupling between the amino groups of horseradish peroxidase (HRP) and the carboxyl groups on the MMSNs surface. It is found that the immobilized HRP on MMSNs still retain high activity and the MMSNs-HRP can eliminate the reactive oxygen species (ROS) in Chinese hamster ovary (CHO) cells induced by the addition of H2O2 aqueous solution. Further, the fluorescent MMSN-HRP-CD nanoparticles have been prepared by attaching biocompatible, fluorescent carbon dots (CDs) to MMSNs-HRP. We have also investigated the effect of an applied magnetic field on cellular uptake of MMSNs-HRP-CDs and found that the internalization of MMSNs-HRP-CDs by CHO cells could be enhanced within 2 hours under the magnetic field. This work provides us with a novel and efficient method to eliminate ROS in living cells by using HRP-immobilized nanoparticles. PMID:25587910

  12. Application of horse-radish peroxidase linked chemiluminescence to determine the production mechanism of Shiga-like toxins by E. coli O157:H7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A sandwiched immunoassay consisting of toxin capture by immunomagnetic beads (IMB) and toxin detection by horseradish peroxidase (HRP) linked chemiluminescence was used to follow the production of Shiga-like toxins (SLT) by E. coli O157:H7. The intensity of luminescence generated by the oxidation o...

  13. A split horseradish peroxidase for the detection of intercellular protein-protein interactions and sensitive visualization of synapses.

    PubMed

    Martell, Jeffrey D; Yamagata, Masahito; Deerinck, Thomas J; Phan, Sébastien; Kwa, Carolyn G; Ellisman, Mark H; Sanes, Joshua R; Ting, Alice Y

    2016-07-01

    Intercellular protein-protein interactions (PPIs) enable communication between cells in diverse biological processes, including cell proliferation, immune responses, infection, and synaptic transmission, but they are challenging to visualize because existing techniques have insufficient sensitivity and/or specificity. Here we report a split horseradish peroxidase (sHRP) as a sensitive and specific tool for the detection of intercellular PPIs. The two sHRP fragments, engineered through screening of 17 cut sites in HRP followed by directed evolution, reconstitute into an active form when driven together by an intercellular PPI, producing bright fluorescence or contrast for electron microscopy. Fusing the sHRP fragments to the proteins neurexin (NRX) and neuroligin (NLG), which bind each other across the synaptic cleft, enabled sensitive visualization of synapses between specific sets of neurons, including two classes of synapses in the mouse visual system. sHRP should be widely applicable to studying mechanisms of communication between a variety of cell types. PMID:27240195

  14. Measuring hydrogen peroxide due to water radiolysis using a modified horseradish peroxidase based biosensor as an alternative dosimetry method.

    PubMed

    Tavakoli, Hassan; Baghbanan, Amin Azam

    2015-08-01

    H2O2 generated during water radiolysis was measured electrochemically as an alternative dosimetry method. A biosensor was fabricated by immobilising modified horseradish peroxidase (HRP) on a glassy carbon electrode (GCE) followed by evaluation of its analytical parameters. Anthraquinone 2-carboxylic acid was used to modify HRP. To assess sensor performance, phosphate buffer solutions were irradiated with 0.510 Gy of gamma ray emitted from (60)Co. The results showed that this sensor can detect low quantities of hydrogen peroxide in water radiolysis. Sensitivity, detection limit and linear range of the biosensor were 260 nA/Gy, 0.392 Gy and 0.5-5 Gy, respectively. Long term stability studies showed that sensor responses were stable for at least a month. The cathodic peak current, as biosensor response, subsequently decreased to 20% of its initial value. PMID:25880374

  15. Immobilization of horseradish peroxidase in phospholipid-templated titania and its applications in phenolic compounds and dye removal.

    PubMed

    Jiang, Yanjun; Tang, Wei; Gao, Jing; Zhou, Liya; He, Ying

    2014-02-01

    In this study, horseradish peroxidase (HRP) was encapsulated in phospholipid-templated titania particles through the biomimetic titanification process and used for the treatment of wastewater polluted with phenolic compounds and dye. The encapsulated HRP exhibited improved thermal stability, a wide range of pH stability and high tolerance against inactivating agents. It was observed an increase in Km value for the encapsulated HRP (8.21 mM) when compared with its free counterpart. For practical applications in the removal of phenolic compounds and dye by the encapsulated HRP, the removal efficiency for phenol, 2-chlorophenol, Direct Black-38 were 92.99%, 87.97%, and 79.72%, respectively, in the first treatment cycle. Additionally, the encapsulated HRP showed better removal efficiency than free HRP and a moderately good capability of reutilization. PMID:24411438

  16. Evaluation of density functional theory methods for the electronic interactions between indole and substituted benzene: Applications to horseradish peroxidase

    NASA Astrophysics Data System (ADS)

    van Sickle, Karina; Culberson, Lori Marie; Holzmacher, Jeremy Levin; Cafiero, Mauricio

    In an effort to evaluate and design fast, accurate density functional theory (DFT) methods for calculating electrostatic and dispersion interactions between proteins and ligands, we have set up a model system examining interactions between mono-substituted benzene and indole in seven different stable conformations. We first optimized the geometries of the monomers at the B3LYP/6-31G level, and then scanned the potential curves with MP2, HF, B3LYP, SVWN, and HCTH407 [all at the 6-311++G(d,p) level] to find the optimum separation. We used the approximate counterpoise method to calculate the basis set superposition error-corrected interaction energies at the optimum geometries. We then applied these methods to the interactions between aromatic active site residues of horseradish peroxidase C with indole-3-propionic acid at two different binding sites.

  17. Comparison of cattle and sheep colonic permeabilities to horseradish peroxidase and hamster scrapie prion protein in vitro

    PubMed Central

    McKie, A; Zammit, P; Naftalin, R

    1999-01-01

    BACKGROUND—Paracellular permeability to solutes across the descending colon is much higher in cattle than sheep. This is a possible route for transmission of infective materials, such as scrapie prion.
AIMS—To compare the permeabilities of labelled scrapie prion protein and other macromolecules in bovine and ovine descending colons in vitro.
METHODS—Using fresh slaughterhouse material, transepithelial fluxes of macromolecules across colonic mucosae mounted in Ussing chambers were measured by monitoring transport of either enzyme activity or radioactivity.
RESULTS—The comparative bovine to ovine permeability ratio of the probes increased with molecular weight: from 3.1 (0.13) for PEG400 to 10.67 (0.20) (p<0.001) for PEG4000; and from 1.64 (0.17) for microperoxidase to 7.03 (0.20) (p<0.001) for horseradish peroxidase (HRP). The permeability of 125I-labelled inactivated Syrian hamster scrapie prion protein (ShaPrPsc) was 7.02 (0.33)-fold higher in bovine than ovine colon (p<0.0025). In each species, the probe permeabilities decreased according to the formula: P = Po.exp(−K.ra). The "ideal" permeabilities, Po are similar, however, K(ovine) = 2.46 (0.20) cm/h/nm exceeds K(bovine) = 0.85 (0.15) cm/h/nm (p<0.001) indicating that bovine colon has a higher proportion of wide pores than ovine. Image analysis confirmed that HRP permeated through the bovine mucosal layer via a pericryptal paracellular route much more rapidly than in sheep.
CONCLUSIONS—These data may imply that scrapie prion is transmitted in vivo more easily across the low resistance bovine colonic barrier than in other species.


Keywords: cattle; sheep; colon; paracellular permeability; horseradish peroxidase; hamster scrapie prion protein PMID:10562587

  18. The Endogenous Calcium Ions of Horseradish Peroxidase C Are Required to Maintain the Functional Nonplanarity of the Heme

    PubMed Central

    Laberge, Monique; Huang, Qing; Schweitzer-Stenner, Reinhard; Fidy, Judit

    2003-01-01

    Horseradish peroxidase C (HRPC) binds 2 mol calcium per mol of enzyme with binding sites located distal and proximal to the heme group. The effect of calcium depletion on the conformation of the heme was investigated by combining polarized resonance Raman dispersion spectroscopy with normal coordinate structural decomposition analysis of the hemes extracted from models of Ca2+-bound and Ca2+-depleted HRPC generated and equilibrated using molecular dynamics simulations. Results show that calcium removal causes reorientation of heme pocket residues. We propose that these rearrangements significantly affect both the in-plane and out-of-plane deformations of the heme. Analysis of the experimental depolarization ratios are clearly consistent with increased B1g- and B2g-type distortions in the Ca2+-depleted species while the normal coordinate structural decomposition results are indicative of increased planarity for the heme of Ca2+-depleted HRPC and of significant changes in the relative contributions of three of the six lowest frequency deformations. Most noteworthy is the decrease of the strong saddling deformation that is typical of all peroxidases, and an increase in ruffling. Our results confirm previous work proposing that calcium is required to maintain the structural integrity of the heme in that we show that the preferred geometry for catalysis is lost upon calcium depletion. PMID:12668462

  19. An efficient proton-coupled electron-transfer process during oxidation of ferulic acid by horseradish peroxidase: coming full cycle.

    PubMed

    Derat, Etienne; Shaik, Sason

    2006-10-25

    Quantum mechanics/molecular mechanics calculations were utilized to study the process of oxidation of a native substrate (ferulic acid) by the active species of horseradish peroxidase (Dunford, H. B. Heme Peroxidases; Wiley-VCH: New York, 1999), Compound I and Compound II, and the manner by which the enzyme returns to its resting state. The results match experimental findings and reveal additional novel features. The calculations demonstrate that both oxidation processes are initiated by a proton-coupled electron-transfer (PCET) step, in which the active species of the enzyme participate only as electron-transfer partners, while the entire proton-transfer event is being relayed from the substrate to and from the His42 residue by a water molecule (W402). The reason for the observed (Henriksen, A; Smith, A. T.; Gajhede, M. J. Biol. Chem. 1999, 274, 35005-35011) similar reactivities of Compound I and Compound II toward ferulic acid is that the reactive isomer of Compound II is the, hitherto unobserved, Por(*)(+)Fe(III)OH isomer that resembles Compound I. The PCET mechanism reveals that His42 and W402 are crucial moieties and they determine the function of the HRP enzyme and account for its ability to perform substrate oxidation (Poulos, T. L. Peroxidases and Cytochrome P450. In The Porphyrin Handbook; Kadish, K. M., Smith, K. M., Guilard, R., Eds.; Academic Press: New York, 2000; Vol. 4, pp 189). In view of the results, the possibility of manipulating substrate oxidation by magnetic fields is an intriguing possibility. PMID:17044722

  20. Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter

    PubMed Central

    Zhu, Bo; Mizoguchi, Takuro; Kojima, Takaaki; Nakano, Hideo

    2015-01-01

    The C1a isoenzyme of horseradish peroxidase (HRP) is an industrially important heme-containing enzyme that utilizes hydrogen peroxide to oxidize a wide variety of inorganic and organic compounds for practical applications, including synthesis of fine chemicals, medical diagnostics, and bioremediation. To develop a ultra-high-throughput screening system for HRP, we successfully produced active HRP in an Escherichia coli cell-free protein synthesis system, by adding disulfide bond isomerase DsbC and optimizing the concentrations of hemin and calcium ions and the temperature. The biosynthesized HRP was fused with a single-chain Cro (scCro) DNA-binding tag at its N-terminal and C-terminal sites. The addition of the scCro-tag at both ends increased the solubility of the protein. Next, HRP and its fusion proteins were successfully synthesized in a water droplet emulsion by using hexadecane as the oil phase and SunSoft No. 818SK as the surfactant. HRP fusion proteins were displayed on microbeads attached with double-stranded DNA (containing the scCro binding sequence) via scCro-DNA interactions. The activities of the immobilized HRP fusion proteins were detected with a tyramide-based fluorogenic assay using flow cytometry. Moreover, a model microbead library containing wild type hrp (WT) and inactive mutant (MUT) genes was screened using fluorescence-activated cell-sorting, thus efficiently enriching the WT gene from the 1:100 (WT:MUT) library. The technique described here could serve as a novel platform for the ultra-high-throughput discovery of more useful HRP mutants and other heme-containing peroxidases. PMID:25993095

  1. Enhanced chemiluminescence of CdTe quantum dots-H2O2 by horseradish peroxidase-mimicking DNAzyme

    NASA Astrophysics Data System (ADS)

    Zhang, Junli; Li, Baoxin

    In this study, it was found that horseradish peroxidase (HRP)-mimicking DNAzyme could effectively enhance the CL emission of CdTe quantum dots (QDs)-H2O2 system, whereas HRP could not enhance the CL intensity. The CL enhancement mechanism was investigated, and the CL enhancement was supposed to originate from the catalysis of HRP-mimicking DNAzyme on the CL reaction between CdTe QDs and H2O2. Meantime, compared with CdTe QDs-H2O2 CL system, H2O2 concentration was markedly decreased in QDs-H2O2-HRP-mimicking DNAzyme CL system, improving the stability of QDs-H2O2 CL system. The QDs-based CL system was used to detect sensitively CdTe QDs and HRP-mimicking DNAzyme (as biologic labels). This work gives a path for enhancing CL efficiency of QDs system, and will be helpful to promote the step of QDs application in various fields such as bioassay and trace detection of analyte.

  2. Ultrasensitive electrochemical immunosensor based on horseradish peroxidase (HRP)-loaded silica-poly(acrylic acid) brushes for protein biomarker detection.

    PubMed

    Zhao, Yan; Zheng, Yiqun; Kong, Rongmei; Xia, Lian; Qu, Fengli

    2016-01-15

    We report an ultrasensitive electrochemical immunosensor designed for the detection of protein biomarkers using horseradish peroxidase (HRP)-loaded silica-poly(acrylic acid) brushes (SiO2-SPAABs) as labels. HRP could be efficiently and stably accommodated in the three-dimensional architecture of the SiO2-SPAABs and the SiO2-SPAABs-HRP exhibited high catalytic performance towards o-phenylenediamine (OPD) oxidation in the presence of H2O2, which resulted in significant differential pulse voltammetric (DPV) response change and color change. Using human IgG (HIgG) as a model analyte, a sandwich-type immunosensor was constructed. In particular, graphene oxide (GO) and SiO2-SPAABs-HRP were used to immobilize capture antibody (Ab1) and bind a layer of detection antibody (Ab2), respectively. The current biosensor exhibited a good linear response of HIgG from 100pg/mL to 100μg/mL with a detection limit of 50pg/mL (S/N=5). The sensitivity was 6.70-fold higher than the conventional enzyme-linked immunosorbent assays. The immunosensor results were validated through the detection of HIgG in serum samples. PMID:26342574

  3. An amperometric biosensor based on multiwalled carbon nanotube-poly(pyrrole)-horseradish peroxidase nanobiocomposite film for determination of phenol derivatives.

    PubMed

    Korkut, Seyda; Keskinler, Bulent; Erhan, Elif

    2008-09-15

    An amperometric biosensor based on horseradish peroxidase (HRP) and carbon nanotube (CNT)/polypyrrole (PPy) nanobiocomposite film on a gold surface has been developed. The HRP was incorporated into the CNT/PPy nanocomposite matrix in one-step electropolymerization process without the aid of cross-linking agent. Amperometric response was measured as a function of concentration of phenol derivatives, at a fixed bias voltage of -50 mV. Optimization of the experimental parameters was performed with regard to pH and concentration of hydrogen peroxide. The linear range, sensitivity and detection limit of the biosensor were investigated for eighteen phenol derivatives. The sensitivity in the linear range increased in this order: 4-methoxyphenol>2-aminophenol>guaiacol=m-cresol>2-chlorophenol=4-chlorophenol=hydroquinone=pyrocatechol>2,6-dimethoxyphenol>3-chlorophenol>p-cresol>p-benzoquinone=4-acetamidophenol>catechol>phenol=pyrogallol=2,4-dimethylphenol. CNTs was shown to enhance the electron transfer as a mediator and capable to carry higher bioactivity owing to its intensified surface area. The biosensor exhibited low detection limits with a short response time (2s) for the tested phenolics compared to the reported working electrodes. It retained 70% of its initial activity after using for 700 measurements in 1 month. PMID:18761169

  4. Catalytic activity and stability of glucose oxidase/horseradish peroxidase co-confined in macroporous silica foam.

    PubMed

    Cao, Xiaodong; Li, Ying; Zhang, Zhiqiang; Yu, Jiachao; Qian, Jing; Liu, Songqin

    2012-12-21

    Investigation of the catalytic activity and stability of enzymes in confined nano/microspace provides valuable contributions to the fundamental understanding of biological reactions taking place on a mesoscopic scale within confined spaces. In this paper, macroporous silica foam (MSF) is used as a nanoreactor to co-confine glucose oxidase (GOD) and horseradish peroxidase (HRP). Then, the enzymatic cascade reactions, which act in tandem inside nanoreactors, for oxidation of glucose and 3,3',5,5'-tetramethylbenzidine (TMB) were studied. The catalytic kinetic parameters of apparent Michaelis constant (K(m)(app)) and maximum rate (V(max)) were obtained from Lineweaver-Burk plot by UV-vis spectrometry. Results showed that the catalytic activity of the co-confined enzymes is reduced compared to that of free enzymes in solution at room temperature. The stabilities of co-confined enzymes in denaturing agents, such as guanidinium chloride (GdmCl) and urea, were higher than those of free enzymes in solution. When employing a co-confined bienzyme system as a biosensor for the detection of glucose, a wider linear range of glucose was obtained for the co-confined bienzyme system than for free enzymes in solution. PMID:23096254

  5. A novel glutathione-S transferase immunosensor based on horseradish peroxidase and double-layer gold nanoparticles.

    PubMed

    Lu, Dingqiang; Lu, Fuping; Pang, Guangchang

    2016-06-01

    GSTs, a biotransformation enzyme group, can perform metabolism, drug transfer and detoxification functions. Rapid detection of the GSTs with more sensitive approaches is of great importance. In the current study, a novel double-layer gold nanoparticles-electrochemical immunosensor electrode (DGN-EIE) immobilized with Glutathione S-Transferase (GST) antibody derived from Balb/c mice was developed. To increase the fixed quantity of antibodies and electrochemical signal, an electrochemical biosensing signal amplification system was utilized with gold nanoparticles-thionine-chitosan absorbing horseradish peroxidase (HRP). In addition, transmission electron microscope (TEM) was used to characterize the nanogold solution. To evaluate the quality of DGN-EIE, the amperometric I-t curve method was applied to determine the GST in PBS. The results showed that the response current had a good linear correlation with the GST concentration ranged from 0.1-10(4) pg/mL. The lowest detection limit was found at 0.03 pg/mL(S/N = 3). The linear equation was deduced as △I/% = 7.386lgC + 22.36 (R(2) = 0.998). Moreover, it was validated with high sensitivity and reproducibility. Apparently, DGN-EIE may be a very useful tool for monitoring the GST. PMID:27220630

  6. Influence of iodothyronine conjugates of bovine serum albumin and horseradish peroxidase on enzyme immunosorbent assay of thyroid hormones.

    PubMed

    Kumari, G Lakshmi; Kumar, Sachin; Gupta, Satish; Saini, Anuradha; Sharma, Sudesh K; Kaur, Navneet

    2014-01-01

    Enzyme-linked immunosorbent assays (ELISA's) reported for thyroxine (T₄) and 3,5,3'-triiodothyronine (T₃), involved coupling of the haptens through (i) carboxylic group to carrier protein for producing antibodies and (ii) amino group to detection labels. To improve the titer and specificity of antibodies, immunogens were prepared by coupling of carboxyl group to bovine serum albumin (BSA) either directly or through adipic acid dihydrazide (ADH), after protecting amino group through acetylation of T₄ and T₃. Direct coupling resulted in the incorporation of 40-50 moles of T₄ and T₃ per BSA molecule and helped in improving immunogenic response and specificity, especially of T₄. High epitope density of immunogens evoked better antibody response, since attachement of ADH as spacer, introduced 18-27 moles of haptens into carrier protein and had less effect on antibody development, with T₃ being exception. Detection labels were prepared by coupling horseradish peroxidase (HRP) to amino group of thyroid hormones directly and after preparing their methyl esters, which provided sensitive displacement curves in combination with the antibodies developed against N-acetylated-T₄ and T₃. Unlike methyl esters, T₄-HRP and T₃-HRP showed higher sensitivity and seemed to be related to the affinity of the labels for binding the antibody. PMID:24295178

  7. Selective determination of phenols and aromatic amines based on horseradish peroxidase-nanoporous gold co-catalytic strategy.

    PubMed

    Wu, Chao; Liu, Zhuang; Sun, Huihui; Wang, Xia; Xu, Ping

    2016-05-15

    Aromatic compounds, such as phenols and aromatic amines, are environmental contaminants suspected of posing human health risks. For phenols and aromatic amines reliable detection, promoting selectivity and sensitivity for phenols and aromatic amines is crucial in biosensor design. Here, a biosensor combined the advantages of both enzymatic and nonenzymatic electrochemical sensors is constructed. Nanoporous gold (NPG) is selected as an enzyme carrier for horseradish peroxidase (HRP) biosensor fabrication due to its three-dimension structure with unique properties. It is firstly discovered that NPG can achieve selective oxidation for phenols and aromatic amines. Thus, the electrochemical reaction on the resulting HRP/NPG/GCE bioelectrode is attributed to the co-catalysis of HRP and NPG. For the detection of catechol (Cat), 4-aminophenol (p-AP), o-phenylenediamine (o-PD), and p-phenylenediamine (p-PD), linear responses are observed in large concentration ranges with high sensitivities and low detection limits. Further, the HRP/NPG/GCE bioelectrode presents strong reproducibility, specificity, selectivity and anti-interference capability in detecting the mixture of phenols and aromatic amines along with a long shelf-life, and the real sea water sample analysis was achieved. These unique properties make the HRP/NPG/GCE bioelectrode an excellent choice for phenols and aromatic amines reliable detection. PMID:26780372

  8. Localisation of motoneurons supplying the extra-ocular muscles of the rat using horseradish peroxidase and fluorescent double labelling.

    PubMed Central

    Labandeira Garcia, J L; Gomez Segade, L A; Suarez Nuñez, J M

    1983-01-01

    This paper describes a qualitative and quantitative investigation into the location of the motoneurons innervating the extra-ocular muscles of the rat. Injections of horseradish peroxidase, bisbenzimide, propidium iodide and DAPI-primuline were made either in one or simultaneously in two muscles. Unlike those of the cat, rabbit and monkey, the motoneurons which make up the oculomotor nucleus of the rat are not arranged in spatially separate subgroups belonging each to its corresponding extra-ocular muscle, but instead allow a high degree of superposition among the motor pools which they compose. The motoneurons innervating the lateral rectus and inferior oblique muscles are all homolateral; those of the medial and inferior rectus muscles are mainly homolateral with a few contralateral exceptions; and those of the superior rectus, levator palpebrae and superior oblique muscles are mainly contralateral with a small minority of homolateral exceptions. As well as from the main motor pools with which they are associated, the medial rectus, inferior rectus, superior rectus, levator palpebrae, superior oblique and lateral rectus muscles all receive innervation from motoneurons lying among the fibres of the fasciculus longitudinalis medialis. All these observations are supported by quantitative data. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:6195140

  9. Antibody-targeted horseradish peroxidase associated with indole-3-acetic acid induces apoptosis in vitro in hematological malignancies.

    PubMed

    Dalmazzo, Leandro F F; Santana-Lemos, Bárbara A; Jácomo, Rafael H; Garcia, Aglair B; Rego, Eduardo M; da Fonseca, Luiz M; Falcão, Roberto P

    2011-05-01

    Indole-3-acetic acid (IAA), when oxidized by horseradish peroxidase (HRP), is transformed into cytotoxic molecules capable of inducing cell injury. The aim of this study was to test if, by targeting hematopoietic tumors with HRP-conjugated antibodies in association with IAA treatment, there is induction of apoptosis. We used two lineages of hematologic tumors: NB4, derived from acute promyelocytic leukemia (APL) and Granta-519 from mantle cell lymphoma (MCL). We also tested cells from 12 patients with acute myeloid leukemia (AML) and from 10 patients with chronic lymphocytic leukemia (CLL). HRP targeting was performed with anti-CD33 or anti-CD19 antibodies (depending on the origin of the cell), followed by incubation with goat anti-mouse antibody conjugated with HRP. Eight experimental groups were analyzed: control, HRP targeted, HRP targeted and incubated with 1, 5 and 10mM IAA, and cells not HRP targeted but incubated with 1, 5 and 10mM IAA. Apoptosis was analyzed by flow cytometry using annexin V-FITC and propidium iodide labeling. Results showed that apoptosis was dependent on the dose of IAA utilized, the duration of exposure to the prodrug and the origin of the neoplasia. Targeting HRP with antibodies was efficient in activating IAA and inducing apoptosis. PMID:21168913

  10. Electrochemical immunosensor for α-fetoprotein detection using ferroferric oxide and horseradish peroxidase as signal amplification labels.

    PubMed

    Wang, Huan; Li, Xiaojian; Mao, Kexia; Li, Yan; Du, Bin; Zhang, Yihe; Wei, Qin

    2014-11-15

    An electrochemical immunosensor for quantitative detection of α-fetoprotein (AFP) in human serum was developed using graphene sheets (GS) and thionine (TH) as electrode materials and mesoporous silica nanoparticles (MSNs) loaded with ferroferric oxide (Fe3O4) nanoparticles and horseradish peroxidase (HRP) as labels for signal amplification. In this study, the compound of GS and TH (GS-TH) was used as a substrate for promoting electron transfer and immobilization of primary antibody of AFP (Ab1). MSNs were used as a carrier for immobilization of secondary antibody of AFP (Ab2), Fe3O4, and HRP. The synergistic effect occurred between Fe3O4 and HRP and greatly improved the sensitivity of the immunosensor. This method could detect AFP over a wide concentration range from 0.01 to 25 ng ml(-1) with a detection limit of 4 pg ml(-1). This strategy may find wide potential application in clinical analysis or detection of other tumor markers. PMID:25168193

  11. Horseradish peroxidase-catalyzed formation of hydrogels from chitosan and poly(vinyl alcohol) derivatives both possessing phenolic hydroxyl groups.

    PubMed

    Sakai, Shinji; Khanmohammadi, Mehdi; Khoshfetrat, Ali Baradar; Taya, Masahito

    2014-10-13

    Horseradish peroxidase-catalyzed cross-linking was applied to prepare hydrogels from aqueous solutions containing chitosan and poly(vinyl alcohol) derivatives both possessing phenolic hydroxyl groups (denoted as Ph-chitosan and Ph-PVA, respectively). Comparing the hydrogels prepared from the solution of 1.0% (w/v) Ph-chitosan and 3.0% (w/v) Ph-PVA and that of 3.0% (w/v) Ph-chitosan and 1.0% (w/v) Ph-PVA, the gelation time of the former hydrogel was 47 s, while was 10s longer than that of the latter one. The breaking point for the former hydrogel under stretching (114% strain) was approximately twice larger than that for the latter one. The swelling ratio of the former hydrogel in saline was about half of the latter one. Fibroblastic cells did not adhere on the former hydrogel but adhered and spread on the latter one. The growth of Escherichia coli cells was fully suppressed on the latter hydrogel during 48 h cultivation. PMID:25037368

  12. Evaluation of textile dye degradation due to the combined action of enzyme horseradish peroxidase and hydrogen peroxide.

    PubMed

    Pereira, A R; da Costa, R S; Yokoyama, L; Alhadeff, E M; Teixeira, L A C

    2014-12-01

    The kinetic parameters of the oxidant action of the combination of enzyme horseradish peroxidase (HRP) with hydrogen peroxide in the degradation of methylene blue dye were investigated. Twenty-one percent of color removal was obtained at pH 5.0 and temperature of 30 °C. Under these conditions, the kinetic parameters K m and V max of enzymatic reactions were determined for hydrogen peroxide in the absence of methylene blue dye (K m = 17.3 mM; V max = 1.97 mM/min) and in the presence of methylene blue dye (K m = 0.27 mM, V max = 0.29 μM/min). By means of analysis of phosphorescence, the presence of reactive oxygen species was detected in the form of singlet oxygen through the redox reaction between HRP and hydrogen peroxide. The existence of this reactive species is directly dependent on the concentration of hydrogen peroxide in the aqueous solution. PMID:25248990

  13. Horseradish peroxidase and aptamer dual-functionalized nanoprobe for the amplification detection of alpha-methylacyl-CoA racemase.

    PubMed

    Zeng, Yongyi; Zheng, Aixian; Wu, Jing; Cai, Zhixiong; Huang, Aimin; Liu, Xiaolong

    2015-10-29

    Alpha-methylacyl-CoA racemase (AMACR) is over-expressed in many cancer types and can serve as a novel diagnostic biomarker. Development of convenient and sensitive detection methods of AMACR is of particular importance for cancer diagnosis. Aptamers are a type of recognition elements, which possess many advantages over antibody, making them suitable for applications in biosensing and biotechnology. In this work, we use the efficient surface modification of gold nanoparticles (AuNPs) to prepare the horseradish peroxidase (HRP) and aptamer dual-functionalized nanoprobe. The immobilization of HRP and thiol-terminated aptamer on the surface of AuNPs can be achieved through electrostatic interaction and the formation of Au-S bond, respectively. This nanoprobe, which is used as discriminating and catalytic probe, can be combined with enzyme immunoassay method to increase the detection sensitivity of AMACR. The detection limit can reach as low as 4.6 pg mL(-1) due to the dual signal amplification from enzymatic cycling and the high loading of enzymes on AuNPs. This sensitivity is about three orders of magnitude higher than that of AMACR aptamer based fluorescence method, which is also comparable to or one order of magnitude higher than that of ELISA. Furthermore, this method is more simple and effective, which not only avoids the conjugation between recognition element and the catalytic enzyme, but also achieves greater signal amplification. This assay could be used as a sensitive and selective platform for the detection of target protein. PMID:26547498

  14. Enhanced chemiluminescence of CdTe quantum dots-H₂O₂ by horseradish peroxidase-mimicking DNAzyme.

    PubMed

    Zhang, Junli; Li, Baoxin

    2014-05-01

    In this study, it was found that horseradish peroxidase (HRP)-mimicking DNAzyme could effectively enhance the CL emission of CdTe quantum dots (QDs)-H2O2 system, whereas HRP could not enhance the CL intensity. The CL enhancement mechanism was investigated, and the CL enhancement was supposed to originate from the catalysis of HRP-mimicking DNAzyme on the CL reaction between CdTe QDs and H2O2. Meantime, compared with CdTe QDs-H2O2 CL system, H2O2 concentration was markedly decreased in QDs-H2O2-HRP-mimicking DNAzyme CL system, improving the stability of QDs-H2O2 CL system. The QDs-based CL system was used to detect sensitively CdTe QDs and HRP-mimicking DNAzyme (as biologic labels). This work gives a path for enhancing CL efficiency of QDs system, and will be helpful to promote the step of QDs application in various fields such as bioassay and trace detection of analyte. PMID:24556131

  15. Mixed Monolayers of Ferrocenylalkanethiol and Encapsulated Horseradish Peroxidase for Sensitive and Durable Electrochemical Detection of Hydrogen Peroxide

    PubMed Central

    Peng, Yong; Jiang, Dianlu; Su, Lei; Zhang, Lin; Yan, Ming; Du, Juanjuan; Lu, Yufeng; Liu, You-Nian; Zhou, Feimeng

    2009-01-01

    This paper describes the construction of a mixed monolayer of ferrocenylalkanethiol and encapsulated horseradish peroxidase (HRP) at a gold electrode for amperometric detection of H2O2 at trace levels. By tuning the alkanethiol chain lengths that tether the HRP enzyme and the ferrocenylalkanethiol (FcC11SH) mediator, facile electron transfer between FcC11SH and HRP can be achieved. Unlike most HRP-based electrochemical sensors, which rely on HRP-facilitated H2O2 reduction (to H2O), the electrocatalytic current is resulted from an HRP-catalyzed oxidation reaction of H2O2 (to O2). Upon optimizing other experimental conditions (surface coverage ratio, pH, and flow rate), the electrocatalytic reaction proceeding at the electrode was used to attain a low amperometric detection level (0.64 nM) and a dynamic range spanning over three orders of magnitude. Not only does the thin hydrophilic porous HRP capsule allow facile electron transfer, it also enables H2O2 to permeate. More significantly, the enzymatic activity of the encapsulated HRP is retained for a considerably longer period (> three weeks) than naked HRP molecules attached to an electrode or those wired to a redox polymer thin film. By comparing to electrodes modified with denatured HRP that are subsequently encapsulated or embedded in a poly-L-lysine matrix, it is concluded that the encapsulation has significantly preserved the native structure of HRP and therefore its enzymatic activity. The electrode covered with FcC11SH and encapsulated HRP is shown to be capable of rapidly and reproducibly detecting H2O2 present in complex sample media. PMID:19928778

  16. Proton nuclear magnetic resonance spectroscopy of horseradish peroxidase isoenzymes: correlation of distinctive spectra with isoenzyme specific activities.

    PubMed

    Gonzalez-Vergara, E; Meyer, M; Goff, H M

    1985-11-01

    High-resolution proton NMR spectra are reported for the paramagnetic ferric native and cyano complexes of the five major horseradish root peroxidase (HRP) isoenzymes (A1, A2, A3, B, and C). Axial imidazole resonances are observed in the native and cyano-complex spectra of all the isoenzymes, thus indicating the presence of a common axial histidine ligand. Proton NMR spectra outside the usual diamagnetic region are identical for sets of A1 and A2 isoenzymes and for the B and C isoenzyme set. Variation in heme residue chemical shift positions may be controlled in part by porphyrin vinyl side chain-protein interactions. Diverse upfield spectra among the isoenzymes reflect amino acid substitutions and/or conformational differences near the prosthetic group, as signals in this region must result from amino acid residues in proximity to the heme center. Acid-base dependence studies reveal an "alkaline" transition that converts the native high-spin iron (III) porphyrin to the low-spin state. The transition occurs at pH 9.3, 9.4, 9.8, and 10.9 for respective HRP A1, A2, A3, and C isoenzymes, respectively. Significantly, this ordering also reflects specific activities for the isoenzymes in the order A1 = A2 greater than A3 greater than B = C. Identical proton NMR spectra for A1/A2 and B/C isoenzyme sets parallel equivalent specific activities for members of a particular set. Proton NMR spectra thus appear to be highly sensitive to protein modifications that affect catalytic activity. PMID:4084538

  17. Conjugation of poly-L-lysine to albumin and horseradish peroxidase: a novel method of enhancing the cellular uptake of proteins.

    PubMed Central

    Shen, W C; Ryser, H J

    1978-01-01

    The carbodiimide-catalyzed conjugation of a 6700 molecular weight fragment of poly-L-lysine to radiolabeled human serum albumin or to horseradish peroxidase enhances the membrane transport of each protein into cultured mouse fibroblasts approximately 11- and 200-fold, respectively. At least 50% of the peroxidase activity remained after conjugation. Trypsinization and carbamylation of the two conjugates demonstrates that the enhancement of their cellular uptake is related to their poly-L-lysine content. Simple addition to the medium of comparable amounts of free poly-L-lysine has no effect on the transport of either native protein. Addition of poly-L-ornithine (molecular weight 200,000) at 3-30 microgram/ml, a condition known to cause enhancement of 125I-labeled human serum albumin uptake by mouse sarcoma cells, has no visible effect on the cellular uptake of native horseradish peroxidase. The intracellular localization of the enzyme-poly-L-lysine conjugate can be demonstrated cytochemically by either light or transmission electron microscopy. A concentration of conjugate that increases the uptake more than 200-fold does not cause any detectable morphological change suggestive of cell toxicity. Furthermore, because poly-L-lysine is an excellent substrate for intracellular proteolytic enzymes, it can be expected to be broken down and reutilized in the cell. Images PMID:273916

  18. Propagation of human iPS cells in alginate-based microcapsules prepared using reactions catalyzed by horseradish peroxidase and catalase.

    PubMed

    Ashida, Tomoaki; Sakai, Shinji; Taya, Masahito

    2016-09-01

    Cell encapsulation has been investigated as a bioproduction system in the biomedical and pharmaceutical fields. We encaps-ulated human induced pluripotent stem (hiPS) cells in duplex microcapsules prepared from an alginate derivative possessing phenolic hydroxyl moieties, in a single-step procedure based on two competing enzymatic reactions catalyzed by horseradish peroxidase (HRP) and catalase. The encapsulated cells maintained 91.4% viability and proliferated to fill the microcapsules following 19 days of culture. Encapsulated hiPS cells showed pluripotency comparable to that of unencapsulated cells during the cultures, as demonstrated by the expression of the SSEA-4 marker. PMID:26148179

  19. Studies on the oxidation reaction of tyrosine (Tyr) with H 2O 2 catalyzed by horseradish peroxidase (HRP) in alcohol-water medium by spectrofluorimetry and differential spectrophotometry

    NASA Astrophysics Data System (ADS)

    Tang, Bo; Wang, Yan; Liang, Huiling; Chen, Zhenzhen; He, Xiwen; Shen, Hanxi

    2006-03-01

    An oxidation reaction of tyrosine (Tyr) with H 2O 2 catalyzed by horseradish peroxidase (HRP) was studied by spectrofluorimetry and differential spectrophotometry in the alcohol(methanol, ethanol, 1-propanol and isopropanol)-water mutual solubility system. Compared with the enzymatic-catalyzed reaction in the water medium, the fluorescence intensities of the product weakened, even extinguished. Because the addition of alcohols made the conformation of HRP change, the catalytic reaction shifted to the side of polymerization and the polymer (A nH 2, n ≥ 3) exhibited no fluorescence. The four alcohols cannot deactivate HRP. Moreover isopropanol activated HRP remarkably.

  20. A general method for immobilization of glycoproteins on regenerable immobilized metal-ion carriers: application to glucose oxidase from Penicillium chrysogenum and horseradish peroxidase.

    PubMed

    Chaga, G

    1994-08-01

    A general method for immobilization of glycoproteins on immobilized metal-ion carriers is described. The method includes oxidation of the carbohydrate moiety of the glycoprotein with potassium periodate, covalent modification with histidine and immobilization on bivalent metal ion-iminodiacetic acid-agarose. The method is exemplified with horseradish peroxidase and glucose oxidase from Penicillium chrysogenum. The modified and immobilized enzymes are stable for at least 2 months at 4 degrees C. The immobilized enzymes were used as enzyme electrodes for the determination of the glucose concentration. PMID:7917065

  1. Direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized in hybrid organic-inorganic film of chitosan/sol-gel/carbon nanotubes

    SciTech Connect

    Kang, Xinhuang; Wang, Jun; Tang, Zhiwen; Wu, Hong; Lin, Yuehe

    2009-04-15

    A hybrid organic-inorganic nanocomposite film of chitosan/sol-gel/multi-walled carbon nanotubes was constructed for the immobilization of horseradish peroxidase (HRP). This film was characterized by scanning electron microscopy. Direct electron transfer (DET) and bioelectrocatalysis of HRP incorporated into the composite film were investigated. The results indicate that the film can provide a favorable microenvironment for HRP to perform DET on the surface of glassy carbon electrodes with a pair of quasi-reversible redox waves and to retain its bioelectrocatalytic activity toward hydrogen peroxide.

  2. Amperometric determination of cadmium, lead, and mercury metal ions using a novel polymer immobilised horseradish peroxidase biosensor system.

    PubMed

    Silwana, Bongiwe; Van Der Horst, Charlton; Iwuoha, Emmanuel; Somerset, Vernon

    2014-01-01

    This work was undertaken to develop a novel Pt/PANI-co-PDTDA/HRP biosensor system for environmental applications to investigate the inhibition studies by specific heavy metals, to provide data suitable for kinetic studies and further application of the biosensor to environmental samples. The newly constructed biosensor was compared to the data of the well-researched Pt/PANI/HRP biosensor. Optimised experimental conditions, such as the working pH for the biosensor was evaluated. The functionality of the amperometric enzyme sensor system was demonstrated by measuring the oxidation current of hydrogen peroxide followed by the development of an assay for determination of metal concentration in the presence of selected metal ions of Cd(2+), Pb(2+) and Hg(2+). The detection limits were found to be 8 × 10(-4) μg L(-1) for cadmium, 9.38 × 10(-4) μg L(-1) for lead and 7.89 × 10(-4) μg L(-1) for mercury. The World Health Organisation recommended that the maximum safety level of these metals should not exceed 0.005 mg L(-1) of Cd(2+), 0.01 mg L(-1) of Pb(2+) and 0.001 mg L(-1) of Hg(2+.), respectively. The analytical and detection data for the metals investigated were observed to be lower than concentrations recommended by several bodies including World Health Organisation and Environmental Protection Agencies. Therefore the biosensors developed in this study can be used to screen the presence of these metals in water samples because of its low detection limit. The modes of inhibition of horseradish peroxidase by Pb(2+), Cd(2+) and Hg(2+) as analysed using the double reciprocal plots of the Michaelis-Menten equation was found to be reversible and uncompetitive inhibition. Based on the Km(app) and Imax values for both biosensors the results have shown smaller values. These results also proved that the enzyme modified electrode is valuable and can be deployed for the determination or screening of heavy metals. PMID:25137538

  3. Production and characterization of recombinant lignin peroxidase isozyme H2 from Phanerochaete chrysosporium using recombinant baculovirus.

    PubMed

    Johnson, T M; Pease, E A; Li, J K; Tien, M

    1992-08-01

    Recombinant Phanerochaete chrysosporium lignin peroxidase isozyme H2 (pI 4.4) was produced in insect cells infected with a genetically engineered baculovirus containing a copy of the cDNA clone lambda ML-6. The recombinant enzyme was purified to near homogeneity and is capable of oxidizing veratryl alcohol, iodide, and, to a lesser extent, guaiacol. The Km of the recombinant enzyme for veratryl alcohol and H2O2 is similar to that of the fungal enzyme. The guaiacol oxidation activity or any other activity is not dependent upon Mn2+. The purified recombinant peroxidase is glycosylated with N-linked carbohydrate(s). The recombinant lignin peroxidase eluted from an anion exchange resin similar to that of native isozyme H1 rather than H2. However, the pI of the recombinant enzymes is different from both H1 and H2 isozymes. Further characterization of native isozymes H1 and H2 from the fungal cultures revealed identical N-terminus residues. This indicates that isozymes H1 and H2 differ in post-translational modification. PMID:1632652

  4. [Catalytic activity and the stability of horseradish peroxidase increase as a result of its incorporation into a polyelectrolyte complex with chitosan].

    PubMed

    Veselova, I A; Koreĭko, A V; Shekhovtsova, T N

    2009-01-01

    The incorporation of horseradish peroxidase into polyelectrolyte complexes with chitosans of different molecular weights (MW 5-150 kDa) yielded highly active and stable enzyme preparations. As a result of the selection of optimal conditions for the formation of peroxidase-chitosan complexes, it was found that 0.1% chitosan with a MW of 10 kDa had the strongest activatory effect on peroxidase (activation degree, > 70%) in the reaction of o-dianisidine oxidation by hydrogen peroxide. The complex formed by 0.001% chitosan with a molecular weight of 150 kDa was most stable: when immobilized on foamed polyurethane, it retained at least 50% of the initial activity for 550 days. The highest catalytic activity was exhibited in a 0.05 M phthalate buffer (pH 5.9-6.2) by the complex containing 0.006-0.009% chitosan in the indicator reaction. The activatory effect of the polysaccharide on the enzyme was determined by its influence on the binding and conversion of the reducting substrate peroxidase. PMID:19382699

  5. Combined experimental and theoretical study on the reactivity of compounds I and II in horseradish peroxidase biomimetics.

    PubMed

    Ji, Li; Franke, Alicja; Brindell, Małgorzata; Oszajca, Maria; Zahl, Achim; van Eldik, Rudi

    2014-10-27

    For the exploration of the intrinsic reactivity of two key active species in the catalytic cycle of horseradish peroxidase (HRP), Compound I (HRP-I) and Compound II (HRP-II), we generated in situ [Fe(IV) O(TMP(+.) )(2-MeIm)](+) and [Fe(IV) O(TMP)(2-MeIm)](0) (TMP=5,10,15,20-tetramesitylporphyrin; 2-MeIm=2-methylimidazole) as biomimetics for HRP-I and HRP-II, respectively. Their catalytic activities in epoxidation, hydrogen abstraction, and heteroatom oxidation reactions were studied in acetonitrile at -15 °C by utilizing rapid-scan UV/Vis spectroscopy. Comparison of the second-order rate constants measured for the direct reactions of the HRP-I and HRP-II mimics with the selected substrates clearly confirmed the outstanding oxidizing capability of the HRP-I mimic, which is significantly higher than that of HRP-II. The experimental study was supported by computational modeling (DFT calculations) of the oxidation mechanism of the selected substrates with the involvement of quartet and doublet HRP-I mimics ((2,4) Cpd I) and the closed-shell triplet spin HRP-II model ((3) Cpd II) as oxidizing species. The significantly lower activation barriers calculated for the oxidation systems involving (2,4) Cpd I than those found for (3) Cpd II are in line with the much higher oxidizing efficiency of the HRP-I mimic proven in the experimental part of the study. In addition, the DFT calculations show that all three reaction types catalyzed by HRP-I occur on the doublet spin surface in an effectively concerted manner, whereas these reactions may proceed in a stepwise mechanism with the HRP-II mimic as oxidant. However, the high desaturation or oxygen rebound barriers during CH bond activation processes by the HRP-II mimic predict a sufficient lifetime for the substrate radical formed through hydrogen abstraction. Thus, the theoretical calculations suggest that the dissociation of the substrate radical may be a more favorable pathway than desaturation or

  6. Homologous expression of recombinant lignin peroxidase in Phanerochaete chrysosporium

    SciTech Connect

    Sollewijn Gelpke, M.D.; Mayfield-Gambill, M.; Lin Cereghino, G.P.; Gold, M.H.

    1999-04-01

    The glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter was used to drive expression of lip2, the gene encoding lignin peroxidase (LiP) isozyme H8, in primary metabolic cultures of Phanerochaete chrysosporium. The expression vector, pUGL, also contained the Schizophyllum commune ura1 gene as a selectable marker. pUGL was used to transform a P. chrysosporium Ura11 auxotroph to prototrophy. Ura{sup +} transformants were screened for peroxidase activity in liquid cultures containing high-carbon and high-nitrogen medium. Recombinant LiP (rLiP) was secreted in active form by the transformants after 4 days of growth, whereas endogenous lip genes were not expressed under these conditions. Approximately 2 mg of homogeneous rLiP/liter was obtained after purification. The molecular mass, pI, and optical absorption spectrum of rLiPH8 were essentially identical to those of the wild-type LiPH8 (wt LiPH8), indicating that heme insertion, folding, and secretion functioned normally in the transformant. Steady-state and transient-state kinetic properties for the oxidation of veratryl alcohol between wtLiPH8 and rLiPH8 were also identical.

  7. Catalytic spectrofluorimetric determination of superoxide anion radical and superoxide dismutase activity using N, N-dimethylaniline as the substrate for horseradish peroxidase (HRP)

    NASA Astrophysics Data System (ADS)

    Tang, Bo; Wang, Yan; Chen, Zhen-zhen

    2002-10-01

    The coupled reaction of N, N-dimethylaniline (DMA) with 4-aminoantipyrine (4-AAP) using superoxide anion radical (O 2-) as oxidizing agent under the catalysis of horseradish peroxidase (HRP) was studied. Based on the reaction, O 2- produced by irradiating Vitamin B 2, (V B2) was spectrophotometricly determined at 554 nm. The linear range of this method was 1.8×10 -6-1.2×10 -4 mol l -1 with a detection limit of 5.3×10 -7 mol l -1. The effect of interferences on the determination of O 2- was investigated. The proposed method was successfully applied to the determination of superoxide dismutase (SOD) activity in human blood and mouse blood.

  8. Retention and extractability of phenol, cresol, and dichlorophenol exposed to two surface soils in the presence of horseradish peroxidase enzyme.

    PubMed

    Xu, Fangxiang; Bhandari, Alok

    2003-01-01

    The retention of phenol, o-cresol, 2,4-dichlorophenol (DCP), and their peroxidase-catalyzed polymerization products was evaluated on two surface soils. The extractability of the parent solutes and their polymerization products was also investigated. (14)C-Labeled radioisotopes were used to quantify the contaminant retained on soil as water-extractable, methanol-extractable, humic/fulvic (HA/FA) acid-bound, and soil/humin bound. Between 2 and 20% of the solute retained on soil after a 7-day contact period remained bound to the HA/FA and soil/humin components in unamended soils; in the presence of peroxidase this amount was as high as 40-75%. The alkali-extractable HA/FA component contained the largest fraction of radioactivity in peroxidase-amended soils. Whereas the soil organic matter content was the predominant factor controlling the extent of sorption of the parent phenols, the clay content and particle surface area appeared to contribute to the retention of the polymerization products. High molecular weight oligomers produced during peroxidase-mediated polymerization of phenols associate strongly with soil components and are likely incorporated into the soil organic matter via oxidative coupling reactions. PMID:12502405

  9. Glycopeptide analysis by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry reveals novel features of horseradish peroxidase glycosylation.

    PubMed

    Wuhrer, Manfred; Hokke, Cornelis H; Deelder, André M

    2004-01-01

    We explored matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight (TOF/TOF) mass spectrometry for the analysis of N-glycosylated peptides, using horseradish peroxidase (HRP) as a test case. Two different types of cleavage were observed in the TOF/TOF fragmentation spectra: Firstly, cleavages of peptide bonds yielded fragments with the attached N-glycans staying intact, thus revealing information on peptide sequence and glycan attachment site. Secondly, fragmentation of the glycan moiety was characterized by cleavage of glycosidic bonds as well as a (0,2)X-ring fragmentation of the innermost N-acetylglucosamine of the chitobiose core. Loss of the complete N-glycan moiety occurred by cleavage of both the N-glycosidic bond and the side-chain amide group of the N-glycosylated asparagine, yielding a characteristic peak doublet with a mass difference of 17 Da, which revealed the individual masses of the N-glycan and the peptide moiety. Analysis of a HRP tryptic digest at the sub-picomole level allowed the characterization of various N-glycosylated peptides including those with internal disulfide linkages, a glycopeptide linked via a disulfide bond to another peptide, and a 5 kDa glycopeptide carrying two N-glycans. The potential of our approach was illustrated by the detection of the following novel features of HRP glycosylation: (i) The conjugation of a xylosylated trimannosyl N-glycan without core-fucosylation to site Asn316, showing for the first time unambiguously the occupation of this site; and (ii) A disaccharide N-acetylhexosamine1deoxyhexose1 attached to N-glycosylation sites Asn285 and Asn298, which might represent a Fuc(alpha1-3)GlcNAc- moiety arising from the processing of N-glycans by a horse-radish endoglycosidase during biosynthesis of HRP. PMID:15282773

  10. Application of horse-radish peroxidase linked chemiluminescence to determine the production mechanism of Shiga-like toxins by E. coli O157:H7

    NASA Astrophysics Data System (ADS)

    Tu, Shu-I.; Uknalis, Joseph; Gehring, Andrew; He, Yiping

    2007-09-01

    A sandwiched immunoassay consisting of toxin capture by immunomagnetic beads (IMB) and toxin detection by horseradish peroxidase (HRP) linked chemiluminescence was used to follow the production of Shiga-like toxins (SLT) by E. coli O157:H7. The intensity of luminescence generated by the oxidation of luminol-liked compounds was used to represent the concentration of toxins produced. The time-course of SLT production by E. coli O157:H7 under different conditions was investigated. In pure culture, optimal generation of SLT showed a significant delay than the steady state of cell growth. In mixed cultures of SLT producing E. coli O157:H7 and non-SLT producing E. coli K-12 strain, the production of toxins was substantially decreased. However, the growth of E. coli O157:H7 was not affected by the presence of K-12 strain. This decrease in SLT production was also observed in radiation-sterile ground beef. In regular ground beef that might contain numerous other bacteria, the growth of E. coli O157:H7 in EC media was not significantly affected but the lowered production of SLT was observed. The results showed that mechanism of inducing SLT production was complex with both the growth time and growth environment could influence SLT production. The addition of homo-serine lactone to the growth media enhanced the production of SLT. Thus, possibly cell-cell communication may have a role in SLT production by E. coli O157:H7.

  11. Alterations in blood-brain barrier function following acute hypertension: comparison of the blood-to-brain transfer of horseradish peroxidase with that of alpha-aminisobutyric acid

    SciTech Connect

    Ellison, M.D.B.

    1985-01-01

    The blood-brain barrier (BBB) selectively restricts the blood-to-brain passage of many solutes owing to unique properties of cerebrovascular endothelial cell membranes. To date, experimental study of the BBB has been accomplished primarily through the use of two different methodological approaches. Morphological studies have mostly employed large molecular weight (MW) tracers to detect morphological alterations underlying increased permeability. Physiological studies, employing smaller, more physiologic tracers have successfully described, quantitatively, certain functional aspects of blood-to-brain transfer. The current work attempts to merge these two approaches and to consider barrier function/dysfunction from both a morphological and a functional perspective. Specifically, the study compares in rats, following acute hypertension, the cerebrovascular passage of /sup 14/C-alpha-aminoisobutyric acid (AIB) and that of horseradish peroxidase (HRP). The blood-to-brain passage of AIB and HRP were compared following acute hypertension, with regard to both the distributions of the tracer extravasation patterns and the magnitude of tracer extravasation. The results of this study suggest that traditional morphological barrier studies alone do not reveal all aspects of altered barrier status and that multiple mechanisms underlying increased BBB permeability may operate simultaneously during BBB dysfunction.

  12. Distribution of cardiac sympathetic afferent fibers in the guinea pig heart labeled by anterograde transport of wheat germ agglutinin-horseradish peroxidase.

    PubMed

    Quigg, M; Elfvin, L G; Aldskogius, H

    1988-12-01

    Anterogradely transported wheat germ agglutinin-horseradish peroxidase (WGA-HRP) was used to selectively label the distribution within the guinea pig heart of cardiac sympathetic afferent fibers whose cell bodies lie in the dorsal root ganglia (DRGs) of C6, C8, T1-3. The majority of fibers were seen in the posterior atrial wall, the pulmonary arterial walls, and along the major branches of the coronary arteries. Labeled fibers were also found in the parietal pericardium and associated with the atrioventricular and aortic valves. The labeling pattern was dependent upon segmental level: the most general labeling followed upper thoracic DRG injection, while labeled fibers associated with the coronary arteries were nearly absent after lower cervical DRG injection. Comparison of heart labeling among chemically sympathectomized and untreated animals demonstrated no difference in the distribution of frequency of WGA-HRP labeled fibers, indicating the specificity of this technique. The present findings indicate that the spinal sensory innervation of the heart has its major origins in the uppermost thoracic dorsal root ganglia and has a highly selective regional distribution. The implications of these findings in relation to cardiac autonomic dysfunction and pain are discussed. PMID:3148648

  13. Chemiluminescence immunoassay for the rapid and sensitive detection of antibody against porcine parvovirus by using horseradish peroxidase/detection antibody-coated gold nanoparticles as nanoprobes.

    PubMed

    Zhou, Yuan; Zhou, Tao; Zhou, Rui; Hu, Yonggang

    2014-06-01

    A rapid, simple, facile, sensitive and enzyme-amplified chemiluminescence immunoassay (CLIA) method to detect antibodies against porcine parvovirus has been developed. Horseradish peroxidase (HRP) and the detection antibody were simultaneously co-immobilized on the surface of gold nanoparticles using the electrostatic method to form gold nanoparticle-based nanoprobes. This nanoprobe was employed in a sandwich-type CLIA, which enables CL signal readout from enzymatic catalysis and results in signal amplification. The presence of porcine parvovirus infection was determined in porcine parvovirus antibodies by measuring the CL intensity caused by the reaction of HRP-luminol with H2 O2 . Under optimal conditions, the obtained calibration plot for the standard positive serum was approximately linear within the dilution range of 1:80 to 1:5120. The limit of detection for the assay was 1:10,240 (S/N = 3), which is much lower than that typically achieved with an enzyme-linked immunosorbent assay (1:160; S/N = 3). A series of repeatability measurements using 1:320-fold diluted standard positive serum gave reproducible results with a relative standard deviation of 4.9% (n = 11). The ability of the immunosensor to analyze clinical samples was tested on porcine sera. The immunosensor had an efficiency of 90%, a sensitivity of 93.3%, and a specificity of 87.5% relative to the enzyme-linked immunosorbent assay results. PMID:23832716

  14. Horseradish peroxidase and antibody labeled gold nanoparticle probe for amplified immunoassay of ciguatoxin in fish samples based on capillary electrophoresis with electrochemical detection.

    PubMed

    Zhang, Zhaoxiang; Liu, Ying; Zhang, Chaoying; Luan, Wenxiu

    2015-03-01

    This paper describes a new amplified immunoassay with horseradish peroxidase (HRP) and antibody (Ab) labeled gold nanoparticles (AuNPs) probe hyphenated to capillary electrophoresis (CE) with electrochemical (EC) detection for ultrasensitive determination of ciguatoxin CTX1B. AuNPs were conjugated with HRP and Ab, and then incubated with limited amount of CTX1B to produce immunocomplex. The immunoreactive sample was injected into capillary for CE separation and EC detection. Enhanced sensitivity was obtained by adopting the AuNPs as carriers of HRP and Ab at high HRP/Ab molar ratio. The calibration curve of CTX1B was in the range of 0.06-90 ng/mL. The detection limit was 0.045 ng/mL, which is 38-fold lower than that of HPLC-MS method for CTX1B analysis. The proposed method was successfully applied for the quantification of CTX1B in contamined fish samples by simultaneously labeling Ab and HRP on AuNPs. The amplified IA with HRP and Ab labeled AuNPs probe hyphenated to CE and EC detection provides a sensitive analytical approach for the determination of trace ciguatoxin in complex samples. PMID:25637767

  15. Immobilization of horseradish peroxidase on NH2-modified magnetic Fe3O4/SiO2 particles and its application in removal of 2,4-dichlorophenol.

    PubMed

    Chang, Qing; Tang, Heqing

    2014-01-01

    Fe3O4 nanoparticles were prepared by a co-precipitation method with the assistance of ultrasound irradiation, and then coated with silica generated by hydrolysis and condensation of tetraethoxysilane. The silica-coated Fe3O4 nanoparticles were further modified with 3-aminopropyltriethoxysilane, resulting in anchoring of primary amine groups on the surface of the particles. Horseradish peroxidase (HRP) was then immobilized on the magnetic core-shell particles by using glutaraldehyde as a crosslinking agent. Immobilization conditions were optimized to obtain the highest relative activity of the immobilized enzyme. It was found the durability of the immobilized enzyme to heating and pH variation were improved in comparison with free HRP. The apparent Michaelis constants of the immobilized HRP and free HRP with substrate were compared, showing that the enzyme activity of the immobilized HRP was close to that of free HRP. The HRP immobilized particles, as an enzyme catalyst, were used to activate H2O2 for degrading 2,4-dichlorophenol. The rapid degradation of 2,4-dichlorophenol indicated that the immobilized enzyme has potential applications for removing organic pollutants. PMID:25268726

  16. Thermo- and sulfate-controllable bioelectrocatalysis of glucose based on horseradish peroxidase and glucose oxidase embedded in poly(N,N-diethylacrylamide) hydrogel films.

    PubMed

    Yao, Huiqin; Lin, Ling; Wang, Peng; Liu, Hongyun

    2014-08-01

    Dual-responsive poly(N,N-diethylacrylamide) (PDEA) hydrogel films with entrapped horseradish peroxidase (HRP) and glucose oxidase (GOD) were successfully prepared on electrode surface with a simple one-step polymerization procedure under mild conditions, designated as PDEA-HRP-GOD. Cyclic voltammetric (CV) response of electroactive probe K3Fe(CN)6 at the film electrodes displayed reversible thermo- and sulfate-responsive switching behavior. For example, at 25 °C, the K3Fe(CN)6 demonstrated a well-defined CV peak pair with large peak currents for the films, showing the on state, while at 40 °C, the CV response was greatly suppressed and the system was at the off state. The influence of temperature and Na2SO4 concentration on the switching behavior of the film system was not independent or separated, but was synergetic. The responsive mechanism of the system was ascribed to the structure change of PDEA component in the films with temperature and sulfate concentration. This switching property of the PDEA-HRP-GOD films could be further used to realize dual-responsive catalytic oxidation of glucose sequentially by HRP and GOD entrapped in the films with Fe(CN)6 (3-) as the mediator through changing the surrounding temperature and Na2SO4 concentration. This system may establish a foundation for fabricating a new type of multi-switchable electrochemical biosensors based on bienzyme electrocatalysis. PMID:24888410

  17. A third-generation hydrogen peroxide biosensor based on horseradish peroxidase immobilized in a tetrathiafulvalene-tetracyanoquinodimethane/multiwalled carbon nanotubes film.

    PubMed

    Cao, Zhijun; Jiang, Xueqin; Xie, Qingji; Yao, Shouzhuo

    2008-10-15

    A new third-generation biosensor for H(2)O(2) assay was developed on the basis of the immobilization of horseradish peroxidase (HRP) in a nanocomposite film of tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ)/multiwalled carbon nanotubes (MWCNTs) modified gold electrode. The prepared HRP/TTF-TCNQ/MWCNTs/Au electrode was used for the bioelectrocatalytic reduction of H(2)O(2), with a linear range from 0.005 to 1.05 mM and a detection limit of 0.5 microM for amperometric sensing of H(2)O(2). In addition, a novel method on the basis of electrochemical quartz crystal microbalance (EQCM) measurements was proposed to determine the effective enzymatic specific activity (ESA) of the immobilized HRP for the first time, and the ESA was found to be greater at the TTF-TCNQ/MWCNTs/Au electrode than that at the MWCNTs/Au or TTF-TCNQ/Au electrode, indicating that the TTF-TCNQ/MWCNTs film is a good HRP-immobilization matrix to achieve the direct electron transfer between the enzyme and the electrode. PMID:18479905

  18. A novel biosensor based on the direct electrochemistry of horseradish peroxidase immobilized in the three-dimensional flower-like Bi2WO6 microspheres.

    PubMed

    Liu, Hui; Guo, Kai; Duan, Congyue; Chen, Xianjin; Zhu, Zhenfeng

    2016-07-01

    Three-dimensional flower-like Bi2WO6 microspheres (3D-Bi2WO6 MSs) have been synthesized through a simple hydrothermal method. The morphology and structure of 3D-Bi2WO6 MSs were characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The 3D-Bi2WO6 MSs subsequently were used to immobilize horseradish peroxidase (HRP) and fabricate a mediator-free biosensor for the detection of H2O2. Spectroscopic and electrochemical results reveal that 3D-Bi2WO6 MSs constitute an excellent immobilization matrix with biocompatibility for enzymes. Meanwhile, due to unique morphology of the flower-like microspheres, the direct electron transfer of HRP is facilitated and the prepared biosensors display good performances for the detection of H2O2 with a wide linear range, including two linear sections: 0.5-100μM (R(2)=0.9983) and 100-250μM (R(2)=0.9981), as well as an extremely low method detection limit of 0.18μM. PMID:27127050

  19. Investigations of an electrochemical platform based on the layered MoS2-graphene and horseradish peroxidase nanocomposite for direct electrochemistry and electrocatalysis.

    PubMed

    Song, Haiyan; Ni, Yongnian; Kokot, Serge

    2014-06-15

    The self-assembly of layered molybdenum disulfide-graphene (MoS2-Gr) and horseradish peroxidase (HRP) by electrostatic attraction into a novel hybrid nanomaterial (HRP-MoS2-Gr) is reported. The properties of the MoS2-Gr were characterized by X-ray diffraction (XRD), high-resolution transmission electron microscopy (TEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). UV-vis and Fourier transform infrared spectroscopy (FT-IR) indicate that the native structure of the HRP is maintained after the assembly, implying good biocompatibility of MoS2-Gr nanocomposite. Furthermore, the HRP-MoS2-Gr composite is utilized as a biosensor, which displays electrocatalytic activity to hydrogen peroxide (H2O2) with high sensitivity (679.7 μA mM(-1)cm(-2)), wide linear range (0.2 μM-1.103 mM), low detection limit (0.049 μM), and fast amperometric response. In addition, the biosensor also exhibits strong anti-interference ability, satisfactory stability and reproducibility. These desirable electrochemical properties are attributed to the good biocompatibility and electron transport efficiency of the MoS2-Gr composite, as well as the high loading of HRP. Therefore, this biosensor is potentially suitable for H2O2 analysis in environmental, pharmaceutical, food or industrial applications. PMID:24480133

  20. Sensory nerve endings in the rat oro-facial region labeled by the anterograde and transganglionic transport of horseradish peroxidase: a new method for tracing peripheral nerve fibers.

    PubMed

    Marfurt, C F; Turner, D F

    1983-02-14

    The purpose of the present investigation is to introduce the enzyme horseradish peroxidase (HRP) for the study of the morphology and peripheral distribution of sensory nerve endings. HRP was injected into the trigeminal ganglion or trigeminal brainstem nuclear complex (TBNC) in separate adult rats. HRP injected into the trigeminal ganglion was taken up by the neuronal perikarya and transported anterogradely in massive amounts to sensory nerve endings in the cornea, vibrissal hair follicles, tooth pulps, and periodontal ligaments. HRP injected into the TBNC was taken up by trigeminal primary afferent fibers that terminated there and transported transganglionically, i.e., past or through the trigeminal ganglion, to peripheral sensory endings. The results of the present study demonstrate for the first time that: (1) anterograde HRP transport is a highly successful method of labeling with an intracellular marker trigeminal sensory endings in a variety of oro-facial tissues, and (2) trigeminal primary sensory neurons possess intra-axonal transport mechanisms by which HRP, and possibly other substances, taken up in the central nervous system may be transported to the periphery. PMID:6601506

  1. Enzymatic biosensor of horseradish peroxidase immobilized on Au-Pt nanotube/Au-graphene for the simultaneous determination of antioxidants.

    PubMed

    Wu, Long; Yin, Wenmin; Tang, Kun; Li, Dian; Shao, Kang; Zuo, Yunpeng; Ma, Jing; Liu, Jiawei; Han, Heyou

    2016-08-24

    A new electrochemical method has been proposed for the simultaneous determination of butylated hydroxyanisole (BHA) and propyl gallate (PG) in food matrices based on enzymatic biosensors. Spiny Au-Pt nanotubes (SAP NTs) was first synthesized and demonstrated to exhibit intrinsic peroxidase and catalase-like activity. The structure of SAP NTs provides large surface area and favorable medium for electron transfer, on which HRP were immobilized and acted as enzymatic biosensor for the simultaneous detection of BHA and PG. The results revealed that BHA and PG both have well-defined oxidation waves with peak potentials of 624 and 655 mV, respectively. Under the optimal conditions, the method behaved satisfactory analytical performance towards BHA and PG with a wide linear range of 0.3-50 mg L(-1) and 0.1-100 mg L(-1), as well as a detection limit of 0.046 mg L(-1) and 0.024 mg L(-1) (3σ/slope), respectively. Besides, the proposed method exhibits good sensitivity, stability and reproducibility, providing an alternative to fabricate electrode and construct sensitive biosensors. PMID:27497001

  2. Coupled enzyme reactions performed in heterogeneous reaction media: experiments and modeling for glucose oxidase and horseradish peroxidase in a PEG/citrate aqueous two-phase system.

    PubMed

    Aumiller, William M; Davis, Bradley W; Hashemian, Negar; Maranas, Costas; Armaou, Antonios; Keating, Christine D

    2014-03-01

    The intracellular environment in which biological reactions occur is crowded with macromolecules and subdivided into microenvironments that differ in both physical properties and chemical composition. The work described here combines experimental and computational model systems to help understand the consequences of this heterogeneous reaction media on the outcome of coupled enzyme reactions. Our experimental model system for solution heterogeneity is a biphasic polyethylene glycol (PEG)/sodium citrate aqueous mixture that provides coexisting PEG-rich and citrate-rich phases. Reaction kinetics for the coupled enzyme reaction between glucose oxidase (GOX) and horseradish peroxidase (HRP) were measured in the PEG/citrate aqueous two-phase system (ATPS). Enzyme kinetics differed between the two phases, particularly for the HRP. Both enzymes, as well as the substrates glucose and H2O2, partitioned to the citrate-rich phase; however, the Amplex Red substrate necessary to complete the sequential reaction partitioned strongly to the PEG-rich phase. Reactions in ATPS were quantitatively described by a mathematical model that incorporated measured partitioning and kinetic parameters. The model was then extended to new reaction conditions, i.e., higher enzyme concentration. Both experimental and computational results suggest mass transfer across the interface is vital to maintain the observed rate of product formation, which may be a means of metabolic regulation in vivo. Although outcomes for a specific system will depend on the particulars of the enzyme reactions and the microenvironments, this work demonstrates how coupled enzymatic reactions in complex, heterogeneous media can be understood in terms of a mathematical model. PMID:24517887

  3. Multi-input and -output logic circuits based on bioelectrocatalysis with horseradish peroxidase and glucose oxidase immobilized in multi-responsive copolymer films on electrodes.

    PubMed

    Yu, Xue; Lian, Wenjing; Zhang, Jiannan; Liu, Hongyun

    2016-06-15

    Herein, poly(N-isopropylacrylamide-co-N,N'-dimethylaminoethylmethacrylate) copolymer films were polymerized on electrode surface with a simple one-step method, and the enzyme horseradish peroxidase (HRP) was embedded in the films simultaneously, which were designated as P(NiPAAm-co-DMEM)-HRP. The films exhibited a reversible structure change with the external stimuli, such as pH, CO2, temperature and SO4(2-), causing the cyclic voltammetric (CV) response of electroactive K3Fe(CN)6 at the film electrodes to display the corresponding multi-stimuli sensitive ON-OFF behavior. Based on the switchable CV property of the system and the electrochemical reduction of H2O2 catalyzed by HRP in the films and mediated by Fe(CN)6(3-) in solution, a 5-input/3-output logic gate was established. To further increase the complexity of the logic system, another enzyme glucose oxidase (GOD) was added into the films, designated as P(NiPAAm-co-DMEM)-HRP-GOD. In the presence of oxygen, the oxidation of glucose in the solution was catalyzed by GOD in the films, and the produced H2O2 in situ was recognized and electrocatalytically reduced by HRP and mediated by Fe(CN)6(3-). Based on the bienzyme films, a cascaded or concatenated 4-input/3-output logic gate system was proposed. The present work combined the multi-responsive interface with bioelectrocatalysis to construct cascaded logic circuits, which might open a new avenue to develop biocomputing elements with more sophisticated functions and design novel glucose biosensors. PMID:26901460

  4. Radical Energies and the Regiochemistry of Addition to Heme Groups. Methylperoxy and Nitrite Radical Additions to the Heme of Horseradish Peroxidase

    PubMed Central

    Wojciechowski, Grzegorz; Ortiz de Montellano, Paul R.

    2008-01-01

    The heme of hemoproteins, as exemplified by horseradish peroxidase (HRP), can undergo additions at the meso carbons and/or vinyl groups of the electrophilic or radical species generated in the catalytic oxidation of halides, pseudohalides, carboxylic acids, aryl and alkyl hydrazines, and other substrates. The determinants of the regiospecificity of these reactions, however, are unclear. We report here modification of the heme of HRP by autocatalytically generated, low energy NO2• and CH3OO• radicals. The NO2• radical adds regioselectively to the 4- over the 2-vinyl group but does not add to the meso positions. Reaction of HRP with tert-BuOOH does not lead to heme modification, but reaction with the F152M mutant, in which the heme vinyls are more sterically accessible, results in conversion of the heme 2-vinyl into a 1-hydroxy-2-(methylperoxy)-ethyl group [-CH(OH)CH2OOCH3]. [18O]-labeling studies indicate that the hydroxyl group in this adduct derives from water and the methylperoxide oxygens from O2. Under anaerobic conditions, methyl radicals formed by fragmentation of the autocatalytically generated tert-BuO• radical add to both the δ-meso-carbon and the 2-vinyl group. The regiochemistry of these and the other known additions to the heme indicate that only high-energy radicals (e.g., CH3•) add to the meso-carbon. Less energetic radicals, including NO2• and CH3OO•, add to heme vinyl groups if they are small enough but do not add to the meso-carbons. Electrophilic species such as HOBr, HOCl, and HOSCN add to vinyl groups but do not react with the meso-carbons. This meso- versus vinyl-reactivity paradigm, which appears to be general for autocatalytic additions to heme prosthetic groups, suggests that meso-hydroxylation of the heme by heme oxygenase occurs by a controlled radical reaction rather than by electrophilic addition. PMID:17249668

  5. Amperometric carbohydrate antigen 19-9 immunosensor based on three dimensional ordered macroporous magnetic Au film coupling direct electrochemistry of horseradish peroxidase.

    PubMed

    Zhang, Qi; Chen, Xiaojun; Tang, Yin; Ge, Lingna; Guo, Buhua; Yao, Cheng

    2014-03-01

    A sandwich-type electrochemical immunosensor for the detection of carbohydrate antigen 19-9 (CA 19-9) antigen based on the immobilization of primary antibody (Ab1) on three dimensional ordered macroporous magnetic (3DOMM) electrode, and the direct electrochemistry of horseradish peroxidase (HRP) that was used as both the label of secondary antibody (Ab2) and the blocking reagent. The 3DOMM electrode was fabricated by introducing core-shell Au-SiO2@Fe3O4 nanospheres onto the surface of three dimensional ordered macroporous (3DOM) Au electrode via the application of an external magnet. Au nanoparticles functionalized SBA-15 (Au@SBA-15) was conjugated to the HRP labeled secondary antibody (HRP-Ab2) through the Au-SH or Au-NH3(+) interaction, and HRP was also used as the block reagent. The formation of antigen-antibody complex made the combination of Au@SBA-15 and 3DOMM exhibit remarkable synergistic effects for accelerating direct electron transfer (DET) between HRP and the electrode. Under the optimal conditions, the DET current signal increased proportionally to CA 19-9 concentration in the range of 0.05 to 15.65 U mL(-1) with a detection limit of 0.01 U mL(-1). Moreover, the immunosensor showed high selectivity, good stability, satisfactory reproducibility and regeneration. Importantly, the developed method was used to assay clinical serum specimens, achieving a good relation with those obtained from the commercialized electrochemiluminescent method. PMID:24560371

  6. Steady-state oxidation model by horseradish peroxidase for the estimation of the non-inactivation zone in the enzymatic removal of pentachlorophenol.

    PubMed

    Choi, Y J; Chae, H J; Kim, E Y

    1999-01-01

    A theoretical model for the rate of oxidation of pentachlorophenol (PCP) catalyzed by horseradish peroxidase (HRP), was investigated to account for the influence of hydrogen peroxide (H2O2) concentration on the catalytic activity. To evaluate the maximum allowable H2O2 concentration, a relatively simple steady-state model was developed based on the Ping-Pong Bi-Bi mechanism considering the effect of excess H2O2. Several sets of experimental data obtained from batch reactions using an equimolar concentration of H2O2 and PCP were used to estimate the kinetic parameters by a nonlinear regression method. The model profiles acquired using the estimated parameters were in good agreement with experimental data at different initial enzyme and substrate concentrations. The best-fitted parameters were used to predict the initial rate of the enzyme reaction. The model prediction was coincident with the experimental results of other studies, indicating that the proposed model could be used for the optimization of reaction conditions. The maximum allowable H2O2 concentration to prevent H2O2 inhibition was calculated from the proposed model equation: [H2O2](0,max) = (square root)KmH2O2Ki[PCP]0/KmPCP+[PCP]0. Using this equation, a curve depicting the non-inactivation zone for the two substrates (hydrogen peroxide and PCP) was plotted and it could be used for experimental design and optimal process operation. To minimize enzyme inactivation by H2O2, it was determined that the concentration of H2O2 should be lower than 2.78 mM, regardless of the stoichiometric ratio. PMID:16232630

  7. Horseradish peroxidase-catalyzed polymerization of L-DOPA for mono-/bi-enzyme immobilization and amperometric biosensing of H2O2 and uric acid.

    PubMed

    Dai, Mengzhen; Huang, Ting; Chao, Long; Xie, Qingji; Tan, Yueming; Chen, Chao; Meng, Wenhua

    2016-03-01

    Horseradish peroxidase (HRP)-catalyzed polymerization of L-DOPA (vs. dopamine) in the presence of H2O2 (and uricase (UOx)) was exploited to immobilize mono-/bi-enzymes for hydroquinone-mediated amperometric biosensing of H2O2 and uric acid (UA). The relevant polymeric biocomposites (PBCs) were prepared in phosphate buffer solution containing HRP and L-DOPA (or plus UOx) after adding H2O2. The mono-/bi-enzyme amperometric biosensors were prepared simply by casting some of the PBCs on Au-plated Au (Au(plate)/Au) electrodes, followed by coating with an outer-layer chitosan (CS) film for each. UV-vis spectrophotometry, scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy were used for film characterization and/or process monitoring. The HRP immobilized by enzyme catalysis well preserved its bioactivity, as confirmed by UV-vis spectrophotometry. Under optimized conditions, the monoenzyme CS/HRP-poly(L-DOPA) (PD)/Au(plate)/Au electrode potentiostated at -0.1V responded linearly to H2O2 concentration from 0.001 to 1.25mM with a sensitivity of 700μA mM(-1)cm(-2) and a limit of detection (LOD) of 0.1μM, and the bienzyme CS/UOx-HRP-PD/Au(plate)/Au electrode at -0.1V responded linearly to UA concentration from 0.001 to 0.4mM with a sensitivity of 349μA mM(-1)cm(-2) and a LOD of 0.1μM. The mono-/bi-enzyme biosensors based on biosynthesized PD performed better than many reported analogues and those based on similarly biosynthesized polydopamine. PMID:26717822

  8. A dopaminergic projection to the rat mammillary nuclei demonstrated by retrograde transport of wheat germ agglutinin-horseradish peroxidase and tyrosine hydroxylase immunohistochemistry

    NASA Technical Reports Server (NTRS)

    Gonzalo-Ruiz, A.; Alonso, A.; Sanz, J. M.; Llinas, R. R.

    1992-01-01

    The presence and distribution of dopaminergic neurons and terminals in the hypothalamus of the rat were studied by tyrosine hydroxylase (TH) immunohistochemistry. Strongly labelled TH-immunoreactive neurons were seen in the dorsomedial hypothalamic nucleus, periventricular region, zona incerta, arcuate nucleus, and supramammillary nucleus. A few TH-positive neurons were also identified in the dorsal and ventral premammillary nucleus, as well as the lateral hypothalamic area. TH-immunoreactive fibres and terminals were unevenly distributed in the mammillary nuclei; small, weakly labelled terminals were scattered in the medial mammillary nucleus, while large, strongly labelled, varicose terminals were densely concentrated in the internal part of the lateral mammillary nucleus. A few dorsoventrally oriented TH-positive axon bundles were also identified in the lateral mammillary nucleus. A dopaminergic projection to the mammillary nuclei from the supramammillary nucleus and lateral hypothalamic area was identified by double labelling with retrograde transport of wheat germ agglutinin-horseradish peroxidase and TH-immunohistochemistry. The lateral mammillary nucleus receives a weak dopaminergic projection from the medial, and stronger projections from the lateral, caudal supramammillary nucleus. The double-labelled neurons in the lateral supramammillary nucleus appear to encapsulate the caudal end of the mammillary nuclei. The medial mammillary nucleus receives a very light dopaminergic projection from the caudal lateral hypothalamic area. These results suggest that the supramammillary nucleus is the principal source of the dopaminergic input to the mammillary nuclei, establishing a local TH-pathway in the mammillary complex. The supramammillary cell groups are able to modulate the limbic system through its dopaminergic input to the mammillary nuclei as well as through its extensive dopaminergic projection to the lateral septal nucleus.

  9. Spectroscopic characterization of mutations at the Phe41 position in the distal haem pocket of horseradish peroxidase C: structural and functional consequences.

    PubMed Central

    Heering, Hendrik A; Smith, Andrew T; Smulevich, Giulietta

    2002-01-01

    Three mutants of horseradish peroxidase isoenzyme C (HRPC) have been constructed in which the conserved distal aromatic residue Phe(41) has been substituted by Trp, Val or Ala and the properties of the mutant proteins have been compared with that of the wild-type. The ferric and ferrous states have been studied by resonance Raman, electronic absorption and Fourier-transform infrared spectroscopies, together with their respective fluoride and CO complexes as probes for the integrity of the distal haem-pocket hydrogen-bonding network. The catalytic properties of the mutants, most notably the HRPC-mutant Phe(41)-->Trp (F41W) variant, were also affected. Structural modelling suggests that the bulky indole group of the F41W mutant blocks the distal cavity, inhibiting the binding of fluoride and CO to the haem iron, severely impairing the reaction of the enzyme with H(2)O(2) to form Compound I. Substitution with the smaller side-chain residues Val or Ala resulted in a 2-fold increase in the affinity of the mutants for the aromatic donor benzhydroxamic acid (BHA) compared with the wild-type, whereas the sterically hindered F41W mutant was not able to bind BHA at all. All the mutations studied increased the amount of a ferric six-coordinate aquo-high-spin species. On the other hand, the similarity in the Fe-Im stretching frequencies of the mutants and wild-type protein suggests that the distal haem-pocket mutations do not cause any substantive changes on the proximal side of the haem. Spectra of the HRPC mutant Phe(41)-->Ala-CO and the HRPC mutant Phe(41)-->Val-CO complexes strongly suggested a weakening of the interaction between CO and Arg(38) due to a secondary rearrangement of the haem relative to helix B. The effects observed for these HRP mutants were somewhat different from those noted recently for the analogous Coprinus cinereus peroxidase (CIP) mutants, particularly the Trp mutant. These differences can be reconciled in part as being due to the smaller size of the

  10. NMR Studies of Peroxidases.

    NASA Astrophysics Data System (ADS)

    Veitch, Nigel Charles

    Available from UMI in association with The British Library. Requires signed TDF. Peroxidases are a haem-containing group of enzymes with a wide diversity of function within biological systems. While a common characteristic is the ability to catalyse the conversion of hydrogen peroxide to water, it is the accompanying processes of hormone synthesis and degradation which have generated such a high level of interest. However, information at the molecular level is limited to a single well-resolved crystal structure, that of yeast cytochrome c peroxidase. This thesis presents a strategy for the investigation of peroxidase structure and function based on proton nuclear magnetic resonance spectroscopy, a technique which has the ability to address aspects of both protein structure and protein dynamics in solution. The application of one- and two-dimensional NMR techniques has been developed in the context of plant peroxidases, notably the isoenzyme HRP-C derived from the horseradish root. Characterisation of the proton NMR spectra of HRP -C in resting and ligated states provided new information enabling the structure of the binding site for aromatic donor molecules, such as indole-3-propionic, ferulic and benzhydroxamic acids, to be resolved. In order to overcome difficulties encountered with a protein of the complexity of peroxidase, additional information was obtained from chemical shift parameters and the use of peroxidase variants produced by site-directed mutagenesis. A comparative study using NMR spectroscopy was undertaken for wild-type recombinant HRP-C expressed in Escherichia coli, and two protein variants with substitutions made to residues located on the distal side of the haem pocket, Phe41 to Val and Arg38 to Lys. NMR analyses of a plant peroxidase from barley grains and the fungal peroxidase from Coprinus cinereus were also successful using methods conceived with HRP-C. Examination of three specifically constructed recombinant protein variants of C. cinereus

  11. Development of a fed-batch process for a recombinant Pichia pastoris Δoch1 strain expressing a plant peroxidase.

    PubMed

    Gmeiner, Christoph; Saadati, Amirhossein; Maresch, Daniel; Krasteva, Stanimira; Frank, Manuela; Altmann, Friedrich; Herwig, Christoph; Spadiut, Oliver

    2015-01-01

    Pichia pastoris is a prominent host for recombinant protein production, amongst other things due to its capability of glycosylation. However, N-linked glycans on recombinant proteins get hypermannosylated, causing problems in subsequent unit operations and medical applications. Hypermannosylation is triggered by an α-1,6-mannosyltransferase called OCH1. In a recent study, we knocked out OCH1 in a recombinant P. pastoris CBS7435 Mut(S) strain (Δoch1) expressing the biopharmaceutically relevant enzyme horseradish peroxidase. We characterized the strain in the controlled environment of a bioreactor in dynamic batch cultivations and identified the strain to be physiologically impaired. We faced cell cluster formation, cell lysis and uncontrollable foam formation.In the present study, we investigated the effects of the 3 process parameters temperature, pH and dissolved oxygen concentration on 1) cell physiology, 2) cell morphology, 3) cell lysis, 4) productivity and 5) product purity of the recombinant Δoch1 strain in a multivariate manner. Cultivation at 30°C resulted in low specific methanol uptake during adaptation and the risk of methanol accumulation during cultivation. Cell cluster formation was a function of the C-source rather than process parameters and went along with cell lysis. In terms of productivity and product purity a temperature of 20°C was highly beneficial. In summary, we determined cultivation conditions for a recombinant P. pastoris Δoch1 strain allowing high productivity and product purity. PMID:25567661

  12. Enzymatic hydrolysate-induced displacement reaction with multifunctional silica beads doped with horseradish peroxidase-thionine conjugate for ultrasensitive electrochemical immunoassay.

    PubMed

    Lin, Youxiu; Zhou, Qian; Lin, Yuping; Tang, Dianping; Niessner, Reinhard; Knopp, Dietmar

    2015-08-18

    A novel (invertase) enzymatic hydrolysate-triggered displacement reaction strategy with multifunctional silica beads, doped with horseradish peroxidase-thionine (HRP-Thi) conjugate, was developed for competitive-type electrochemical immunoassay of small molecular aflatoxin B1 (AFB1). The competitive-type displacement reaction was carried out on the basis of the affinity difference between enzymatic hydrolysate (glucose) and its analogue (dextran) for concanavalin A (Con A) binding sites. Initially, thionine-HRP conjugates were doped into nanometer-sized silica beads using the reverse micelle method. Then monoclonal anti-AFB1 antibody and Con A were covalently conjugated to the silica beads. The immunosensor was prepared by means of immobilizing the multifunctional silica beads on a dextran-modified sensing interface via the dextran-Con A binding reaction. Gold nanoparticles functionalized with AFB1-bovine serum albumin conjugate (AFB1-BSA) and invertase were utilized as the trace tag. Upon target AFB1 introduction, a competitive-type immunoreaction was implemented between the analyte and the labeled AFB1-BSA on the nanogold particles for the immobilized anti-AFB1 antibody on the electrode. The invertase followed by gold nanoparticles hydrolyzed sucrose into glucose and fructose. The produced glucose displaced the multifunctional silica beads from the electrode based on the classical dextran-Con A-glucose system, thus decreasing the catalytic efficiency of the immobilized HRP on the electrode relative to that of the H2O2-thionine system. Under optimal conditions, the detectable electrochemical signal increased with the increasing target AFB1 in a dynamic working range from 3.0 pg mL(-1) to 20 ng mL(-1) with a detection limit of 2.7 pg mL(-1). The strong bioconjugation with two nanostructures also resulted in a good repeatability and interassay precision down to 9.3%. Finally, the methodology was further validated for analysis of naturally contaminated or spiked AFB1

  13. Horseradish peroxidase-catalyzed synthesis of poly(thiophene-3-boronic acid) biocomposites for mono-/bi-enzyme immobilization and amperometric biosensing.

    PubMed

    Huang, Yi; Wang, Wen; Li, Zou; Qin, Xiaoli; Bu, Lijuan; Tang, Zhiyong; Fu, Yingchun; Ma, Ming; Xie, Qingji; Yao, Shouzhuo; Hu, Jiming

    2013-06-15

    We report here on a facile enzymatic polymerization protocol to prepare enzyme-poly(thiophene-3-boronic acid) (PTBA) polymeric biocomposites (PBCs) for high-performance mono-/bi-enzyme amperometric biosensing. Horseradish peroxidase (HRP)-catalyzed polymerization of thiophene-3-boronic acid (TBA) monomer was conducted in aqueous solution containing HRP (or plus glucose oxidase (GOx)) by either directly added or GOx-glucose generated oxidant H2O2. The mono-/bi-enzyme amperometric biosensors were prepared simply by casting the dialysis-isolated PBCs on Au-plated Au electrode (Auplate/Au), followed by coating with an outer-layer chitosan (CS) film. The boronic acid residues are capable of covalent bonding with enzyme at the glycosyl sites (boronic acid-diols interaction), which should less affect the enzymatic activity as compared with the common cases of covalent bonding at the peptide chains, and UV-vis spectrophotometric tests confirmed that the encapsulated HRP almost possesses its pristine enzymatic specific activity. The enzyme electrodes were studied by cyclic voltammetry, electrochemical impedance spectroscopy and chronoamperometry in the presence of Fe(CN)6(4-) mediator. The CS/HRP-PTBA/Auplate/Au electrode responded linearly to H2O2 concentration from 1 to 300 μM with a sensitivity of 390 μA mM(-1)cm(-2) and a limit of detection (LOD) of 0.1 μM. The bienzyme CS/GOx-HRP-PTBA(H2O2)/Auplate/Au electrode responded linearly to glucose concentration from 5 μM to 0.83 mM with a sensitivity of 75.1 μA mM(-1)cm(-2) and a LOD of 1 μM, and it is found here that the use of Fe(CN)6(4-) that can only efficiently mediate HRP favorably avoids the "unusual amperometric responses" observed when other mediators that can efficiently turn over both HRP and GOx are used. PMID:23391705

  14. Use of immobilized metal ions as a negative adsorbent for purification of enzymes: application to phosphoglycerate mutase from chicken muscle extract and horseradish peroxidase.

    PubMed

    Chaga, G; Andersson, L; Ersson, B; Berg, M

    1992-01-01

    Two enzymes, phosphoglycerate mutase and peroxidase, were purified by using an immobilized metal ion adsorbent for the removal of unwanted proteins. The mutase was obtained pure from a single column, whereas the purification of peroxidase required the use of a thiophilic adsorbent in a tandem. The capacity was 2.5 mg pure peroxidase per mL gel. PMID:1386542

  15. Purification and characterization of recombinant catalase-peroxidase, which confers isoniazid sensitivity in Mycobacterium tuberculosis.

    PubMed

    Nagy, J M; Cass, A E; Brown, K A

    1997-12-12

    The Mycobacterium tuberculosis katG gene encodes a dual-function enzyme called catalase-peroxidase, which confers sensitivity in M. tuberculosis to isonicotinic acid hydrazide. We have constructed a system for the high level expression of a recombinant form of this enzyme by amplifying the katG gene from the pYZ56 construct (1) and subcloning into a vector suitable for expression in Escherichia coli. The resulting plasmid, pTBCP, produced the catalase-peroxidase in large quantities, corresponding to 30% of total cell protein. The enzyme has been purified to homogeneity and appears to be a dimer in the native form. Using either hydrogen peroxide or t-butyl hydroperoxide and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) as substrates, kcat and Km values have been obtained for both catalatic and peroxidatic activities, respectively. The availability of significant quantities of an active, folded, recombinant form of M. tuberculosis catalase-peroxidase should thus facilitate future studies of its role in drug activation and antibiotic resistance. PMID:9395452

  16. Effects of pH and Temperature on Recombinant Manganese Peroxidase Production and Stability

    NASA Astrophysics Data System (ADS)

    Jiang, Fei; Kongsaeree, Puapong; Schilke, Karl; Lajoie, Curtis; Kelly, Christine

    The enzyme manganese peroxidase (MnP) is produced by numerous white-rot fungi to overcome biomass recalcitrance caused by lignin. MnP acts directly on lignin and increases access of the woody structure to synergistic wood-degrading enzymes such as cellulases and xylanases. Recombinant MnP (rMnP) can be produced in the yeast Pichia pastoris αMnP1-1 in fed-batch fermentations. The effects of pH and temperature on recombinant manganese peroxidase (rMnP) production by P. pastoris αMnP1-1 were investigated in shake flask and fed-batch fermentations. The optimum pH and temperature for a standardized fed-batch fermentation process for rMnP production in P. pastoris ctMnP1-1 were determined to be pH 6 and 30 °C, respectively. P. pastoris αMnP1-1 constitutively expresses the manganese peroxidase (mnp1) complementary DNA from Phanerochaete chrysosporium, and the rMnP has similar kinetic characteristics and pH activity and stability ranges as the wild-type MnP (wtMnP). Cultivation of P. chrysosporium mycelia in stationary flasks for production of heme peroxidases is commonly conducted at low pH (pH 4.2). However, shake flask and fed-batch fermentation experiments with P. pastoris αMnP1-1 demonstrated that rMnP production is highest at pH 6, with rMnP concentrations in the medium declining rapidly at pH less than 5.5, although cell growth rates were similar from pH 4-7. Investigations of the cause of low rMnP production at low pH were consistent with the hypothesis that intracellular proteases are released from dead and lysed yeast cells during the fermentation that are active against rMnP at pH less than 5.5.

  17. rhEPO (recombinant human eosinophil peroxidase): expression in Pichia pastoris and biochemical characterization

    PubMed Central

    Ciaccio, Chiara; Gambacurta, Alessandra; Sanctis, Giampiero DE; Spagnolo, Domenico; Sakarikou, Christina; Petrella, Giovanni; Coletta, Massimo

    2006-01-01

    A Pichia pastoris expression system has for the first time been successfully developed to produce rhEPO (recombinant human eosinophil peroxidase). The full-length rhEPO coding sequence was cloned into the pPIC9 vector in frame with the yeast α-Factor secretion signal under the transcriptional control of the AOX (acyl-CoA oxidase) promoter, and transformed into P. pastoris strain GS115. Evidence for the production of rhEPO by P. pastoris as a glycosylated dimer precursor of approx. 80 kDa was determined by SDS/PAGE and gel filtration chromatography. Recombinant hEPO undergoes proteolytic processing, similar to that in the native host, to generate two chains of approx. 50 and 20 kDa. A preliminary biochemical characterization of purified rhEPO demonstrated that the spectral and kinetic properties of the recombinant wild-type EPO are comparable with those of the native enzyme and are accompanied by oxidizing activity towards several physiological anionic substrates such as SCN−, Br− and Cl−. On the basis of the estimated Km and kcat values it is evident that the pseudohalide SCN− is the most specific substrate for rhEPO, consistent with the catalytic properties of other mammalian EPOs purified from blood. PMID:16396635

  18. Exploring the process-structure-function relationship of horseradish peroxidase through investigation of pH- and heat induced conformational changes

    NASA Astrophysics Data System (ADS)

    Stănciuc, Nicoleta; Aprodu, Iuliana; Ioniță, Elena; Bahrim, Gabriela; Râpeanu, Gabriela

    2015-08-01

    Given the importance of peroxidase as an indicator for the preservation of vegetables by heat treatment, the present study is focused on enzyme behavior under different pH and temperature conditions, in terms of process-structure-function relationships. Thus, the process-structure-function relationship of peroxidase was investigated by combining fluorescence spectroscopy, in silico prediction methods and inactivation kinetic studies. The fluorescence spectra indicated that at optimum pH value, the Trp117 residue is not located in the hydrophobic core of the protein. Significant blue- and red-shifts were obtained at different pH values, whereas the heat-treatment did not cause significant changes in Trp and Tyr environment. The ANS and quenching experiments demonstrated a more flexible conformation at lower pH and respectively at higher temperature. On the other hand molecular dynamics simulations at different temperatures highlighted that the secondary structure appeared better preserved against temperature, whereas the tertiary structure around the heme was more affected. Temperature dependent changes in the hydrogen bonding and ion paring involving amino acids from the heme-binding region (His170 and Asp247) might trigger miss-coordination of the heme iron atom by His170 residue and further enzyme activity loss.

  19. Employment of 4-(1,2,4-triazol-1-yl)phenol as a signal enhancer of the chemiluminescent luminol-H2O2-horseradish peroxidase reaction for detection of hepatitis C virus in real samples.

    PubMed

    Liu, Jian; Zhang, Lili; Fu, Chuanyun; Wang, Yunshan; Sun, Shanhui

    2015-12-01

    Highly sensitive detection of hepatitis C virus (HCV) in serum is a key method for diagnosing and classifying the extent of HCV infection. In this study, a p-phenol derivative, 4-(1,2,4-triazol-1-yl)phenol (4-TRP), was employed as an efficient enhancer of the luminol-hydrogen peroxide (H2O2)-horseradish peroxidase (HRP) chemiluminescence (CL) system for detection of HCV. Compared with a traditional enhancer, 4-TRP strongly enhanced CL intensity with the effect of prolonging and stabilizing light emission. The developed CL system was applied to detecting HCV core antigen (HCV-cAg) using a sandwich structure inside microwells. Our experimental results showed that there was good linear relationship between CL intensity and HCV-cAg concentration in the 0.6-3.6 pg/mL range (R = 0.99). The intra- and inter-assay coefficients of variation were 4.5-5.8% and 5.0-7.3%, respectively. In addition, sensitive determination of HCV-cAg in serum samples using the luminol-H2O2-HRP-4-TRP CL system was also feasible in clinical settings. PMID:25820800

  20. Development of a novel method for determination of mercury based on its inhibitory effect on horseradish peroxidase activity followed by monitoring the surface plasmon resonance peak of gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Khodaveisi, Javad; Shabani, Ali Mohammad Haji; Dadfarnia, Shayessteh; Moghadam, Masoud Rohani; Hormozi-Nezhad, Mohammad Reza

    2016-01-01

    A highly sensitive and simple indirect spectrophotometric method has been developed for the determination of trace amounts of inorganic mercury (Hg2 +) in aqueous media. The method is based on the inhibitory effect of Hg2 + on the activity of horseradish peroxidase (HRP) in the oxidation of ascorbic acid by hydrogen peroxide followed by the reduction of Au3 + to Au-NPs by unreacted ascorbic acid and the measurement of the absorbance of localized surface plasmon resonance (LSPR) peak of gold nanoparticles (at 530 nm) which is directly proportional to the concentration of Hg2 +. Under the optimum conditions, the calibration curve was linear in the concentration range of 1-220 ng mL- 1. Limits of detection (LOD) and quantification (LOQ) were 0.2 and 0.7 ng mL- 1, respectively and the relative standard deviation at 100 ng mL- 1 level of Hg2 + was 2.6%. The method was successfully applied to the determination of mercury in different water samples.

  1. Isoenzyme Multiplicity and Characterization of Recombinant Manganese Peroxidases from Ceriporiopsis subvermispora and Phanerochaete chrysosporium

    PubMed Central

    Larrondo, Luis F.; Lobos, Sergio; Stewart, Phillip; Cullen, Dan; Vicuña, Rafael

    2001-01-01

    We expressed cDNAs coding for manganese peroxidases (MnPs) from the basidiomycetes Ceriporiopsis subvermispora (MnP1) and Phanerochaete chrysosporium (H4) under control of the α-amylase promoter from Aspergillus oryzae in Aspergillus nidulans. The recombinant proteins (rMnP1 and rH4) were expressed at similar levels and had molecular masses, both before and after deglycosylation, that were the same as those described for the MnPs isolated from the corresponding parental strains. Isoelectric focusing (IEF) analysis of rH4 revealed several isoforms with pIs between 4.83 and 4.06, and one of these pIs coincided with the pI described for H4 isolated from P. chrysosporium (pI 4.6). IEF of rMnP1 resolved four isoenzymes with pIs between 3.45 and 3.15, and the pattern closely resembled the pattern observed with MnPs isolated from C. subvermispora grown in solid-state cultures. We compared the abilities of recombinant MnPs to use various substrates and found that rH4 could oxidize o-dianisidine and p-anisidine without externally added manganese, a property not previously reported for this MnP isoenzyme from P. chrysosporium. PMID:11319083

  2. EFFECTS OF BACTERIAL LIGNIN PEROXIDASE ON ORGANIC CARBON MINERALIZATION IN SOIL, USING RECOMBINANT STREPTOMYCES STRAINS

    EPA Science Inventory

    Purified lignin peroxidase was added to sterile and nonsterile silt loam soil to study the effects of bacterial lignin peroxidase ALip-P3 of Streptomyces viridosporus T7A on the rate of organic carbon turnover in soil. ignin peroxidase ALip-P3 appears to affect the short-term tur...

  3. Covalent immobilization of lipase, glycerol kinase, glycerol-3-phosphate oxidase & horseradish peroxidase onto plasticized polyvinyl chloride (PVC) strip & its application in serum triglyceride determination

    PubMed Central

    Chauhan, Nidhi; Narang, Jagriti; Pundir, Chandra Shekhar

    2014-01-01

    Background & objectives: Reusable biostrip consisting enzymes immobilized onto alkylamine glass beads affixed on plasticized PVC strip for determination of triglyceride (TG) suffers from high cost of beads and their detachments during washings for reuse, leading to loss of activity. The purpose of this study was to develop a cheaper and stable biostrip for investigation of TG levels in serum. Methods: A reusable enzyme-strip was prepared for TG determination by co-immobilizing lipase, glycerol kinase (GK), glycerol-3-phosphate oxidase (GPO) and peroxidase (HRP) directly onto plasticized polyvinyl chloride (PVC) strip through glutaraldehyde coupling. The method was evaluated by studying its recovery, precision and reusability. Results: The enzyme-strip showed optimum activity at pH 7.0, 35°C and a linear relationship between its activity and triolein concentration in the range 0.1 to 15 mM. The strip was used for determination of serum TG. The detection limit of the method was 0.1 mM. Analytical recovery of added triolein was 96 per cent. Within and between batch coefficients of variation (CV) were 2.2 and 3.7 per cent, respectively. A good correlation (r=0.99) was found between TG values by standard enzymic colrimetric method employing free enzymes and the present method. The strip lost 50 per cent of its initial activity after its 200 uses during the span of 100 days, when stored at 4°C. Interpretation & conclusions: The nitrating acidic treatment of plasticized PVC strip led to glutaraldehyde coupling of four enzymes used for enzymic colourimetric determination of serum TG. The strip provided 200 reuses of enzymes with only 50 per cent loss of its initial activity. The method could be used for preparation of other enzyme strips also. PMID:24927348

  4. Unglycosylated recombinant human glutathione peroxidase 3 mutant from Escherichia coli is active as a monomer.

    PubMed

    Song, Jian; Yu, Yang; Xing, Ruiqing; Guo, Xiao; Liu, Dali; Wei, Jingyan; Song, Hongwei

    2014-01-01

    Glutathione peroxidase 3 (GPx3) is a glycosylated member of GPx family and can catalyze the reaction of different types of peroxides with GSH to form their corresponding alcohols in vitro. The active center of GPx3 is selenocysteine (Sec), which is incorporated into proteins by a specific mechanism. In this study, we prepared a recombinant human GPx3 (rhGPx3) mutant with all Cys changed to Ser from a Cys auxotrophic strain of E. coli, BL21(DE3)cys. Although lacking post-translational modification, rhGPx3 mutant still retained the ability to reduce H2O2 and PLPC-OOH. Study on the quaternary structure suggested that rhGPx3 mutant existed as a monomer in solution, which is different from native tetrameric GPx3. Loss of the catalytic activity was considered to be attributed to both the absence of glycosylation and the failure of the tetramer. Further analysis was performed to compare the structures of rhGPx3 and GPx4 mutant, which were quite similar except for oligomerization loop. The differences of amino acid composition and electrostatic potentials on the oligomerization loop may affect the binding of large substrates to rhGPx3 mutant. This research provides an important foundation for biosynthesis of functionally selenium-containing GPx3 mutant in E.coli. PMID:25331785

  5. Structure of poly(ethylene glycol)-modified horseradish peroxidase in organic solvents: infrared amide I spectral changes upon protein dehydration are largely caused by protein structural changes and not by water removal per se.

    PubMed Central

    Al-Azzam, Wasfi; Pastrana, Emil A; Ferrer, Yancy; Huang, Qing; Schweitzer-Stenner, Reinhard; Griebenow, Kai

    2002-01-01

    Fourier transform infrared (FTIR) spectroscopy has emerged as a powerful tool to guide the development of stable lyophilized protein formulations by providing information on the structure of proteins in amorphous solids. The underlying assumption is that IR spectral changes in the amide I and III region upon protein dehydration are caused by protein structural changes. However, it has been claimed that amide I IR spectral changes could be the result of water removal per se. Here, we investigated whether such claims hold true. The structure of horseradish peroxidase (HRP) and poly(ethylene glycol)-modified HRP (HRP-PEG) has been investigated under various conditions (in aqueous solution, the amorphous dehydrated state, and dissolved/suspended in toluene and benzene) by UV-visible (UV-Vis), FTIR, and resonance Raman spectroscopy. The resonance Raman and UV-Vis spectra of dehydrated HRP-PEG dissolved in neat toluene or benzene were very similar to that of HRP in aqueous buffer, and thus the heme environment (heme iron spin, coordination, and redox state) was essentially the same under both conditions. Therefore, the three-dimensional structure of HRP-PEG dissolved in benzene and toluene was similar to that in aqueous solution. The amide I IR spectra of HRP-PEG in aqueous buffer and of dehydrated HRP-PEG dissolved in neat benzene and toluene were also very similar, and the secondary structure compositions (percentages of alpha-helices and beta-sheets) were within the standard error the same. These results are irreconcilable with recent claims that water removal per se could cause substantial amide I IR spectral changes (M. van de Weert, P.I. Haris, W.E. Hennink, and D.J. Crommelin. 2001. Anal. Biochem. 297:160-169). On the contrary, amide I IR spectral changes upon protein dehydration are caused by perturbations in the secondary structure. PMID:12496131

  6. Ultrastructural characterization of gerbil olivocochlear neurons based on differential uptake of /sup 3/H-D-aspartic acid and a wheatgerm agglutinin-horseradish peroxidase conjugate from the cochlea

    SciTech Connect

    Helfert, R.H.; Schwartz, I.R.; Ryan, A.F.

    1988-09-01

    Two populations of olivocochlear (OC) neurons have been identified in the gerbil brain stem on the basis of differential labeling patterns of 3H-D-aspartic acid (D-ASP) and wheatgerm agglutinin-horseradish peroxidase conjugate (WGA/HRP) from the cochlear perilymph. While both populations are capable of uptake and retrograde uptake of WGA/HRP, one population accumulates and retrogradely transports D-ASP (D-ASP OC neurons) and the other does not (non-D-ASP OC neurons). D-ASP OC neurons are found in or near the lateral superior olive, are small in size, and receive very few synaptic contacts. The vast majority of these synapses contain small, mildly pleomorphic vesicles with scattered dense core vesicles. Synapses with distinctly larger pleomorphic vesicles have also been observed. These neurons possess all of the features common to neurons of the lateral olivocochlear system. Non-D-ASP OC neurons are found primarily in the ventral nucleus of the trapezoid body, as well as in the area between the medial superior olive and the medial nucleus of the trapezoid body. These neurons are larger and receive greater numbers and types of synaptic contacts than those found on D-ASP OC neurons. The 2 most common synapses found on non-D-ASP OC neurons are axosomatic ones containing small, mildly pleomorphic vesicles and scattered dense core vesicles similar to those seen on the D-ASP OC neurons, and axodendritic synapses containing large, round vesicles. Much less frequently observed are synapses containing small, round vesicles or ones containing predominantly flat vesicles. The ultrastructural features of the non-D-ASP OC neurons correspond to those described for neurons of the medial olivocochlear system.

  7. Development of multianalyte flow-through and lateral-flow assays using gold particles and horseradish peroxidase as tracers for the rapid determination of carbaryl and endosulfan in agricultural products.

    PubMed

    Zhang, Can; Zhang, Yan; Wang, Shuo

    2006-04-01

    Membrane-based competitive immunoassays using gold particles and horseradish peroxidase (HRP) as tracers in flow-through and lateral-flow formats for multianalysis of carbaryl and endosulfan were developed. For gold-based immunoassay, membrane strips were coated with goat anti-rabbit IgG (control line) and carbaryl hapten-ovalbumin (OVA) and endosulfan hapten-OVA (test lines). The visual detection limits for carbaryl and endosulfan were 100 and 10 microg/L in gold-based assays, respectively. For immunoassay using HRP as tracer, anti-carbaryl and anti-endosulfan antibodies were separately coated on the membrane as test lines, and the visual detection limits were 10 microg/L for carbaryl and 1 microg/L for endosulfan. The developed assays used gold particles and HRP as labels, respectively; 10 times enhancement in the visual detection limit using HRP label was obtained in the study. Matrix interference was eliminated by appropriate dilution of sample extracts with buffer. For the validation of the multianalyte assay, the samples were screened by multianalyte gold-based assay and confirmed by HPLC for carbaryl determination and by GC for endosulfan determination. The results of multianalyte gold-based flow-through assay for the determination of carbaryl and endosulfan were in good agreement with the results of instrumental analysis (HPLC with ultraviolet detection and GC with electron capture detection). The developed multianalyte immunoassays for which the results were interpreted visually can be used as convenient qualitative tools for on-site rapid screening of carbaryl and endosulfan simultaneously in agricultural products. PMID:16569035

  8. Analysis of the Peroxidase Activity of Rice (Oryza Sativa) Recombinant Hemoglobin 1: Implications for the In Vivo Function of Hexacoordinate Non-Symbiotic Hemoglobins in Plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In plants, it has been proposed that hexacoordinate (class 1) non-symbiotic Hbs (nsHb-1) function in vivo as peroxidases. However, little is known about the peroxidase activity of nsHb-1. We evaluated the peroxidase activity of rice recombinant Hb1 (a nsHb-1) by using the guaiacol/H2O2 system at pH ...

  9. pH-dependent one- and two-electron oxidation of 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine catalyzed by horseradish peroxidase.

    PubMed

    Sugiyama, K; Correia, M A; Thummel, K E; Nagata, K; Darbyshire, J F; Osawa, Y; Gillette, J R

    1994-01-01

    The porphyrinogenic agent 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) is known to inactivate hepatic cytochrome P450 (P450) enzymes 2C11, 2C6, and 3A1 [Correia et al. (1987) Arch. Biochem. Biophys. 258, 436-451] by different mechanisms. The inactivation of P450 2C11 and 2C6 appears to be due to the ethylation of the heme in the active sites of the enzymes [Augusto et al. (1982) J. Biol. Chem. 257, 11288-11295], whereas the inactivation of P450 3A1 appears to involve the covalent binding of the heme to the apoprotein [Correia et al. (1987)]. Moreover, we have found that DDEP inactivates horseradish peroxidase (HRP) pretreated with hydrogen peroxide. In this system, DDEP was oxidized predominately to 3,5-dicarbethoxy-2,6-dimethyl-4-ethylpyridine (EDP) under weakly acidic conditions and predominately to 3,5-dicarbethoxy-2,6-dimethylpyridine (DP) under basic conditions. The loss of heme and the formation of altered heme products were also pH-dependent and were correlated with the formation of DP and the inactivation of HRP. Thus the inactivation of HRP appears to depend on the formation of an ethyl radical, which presumably reacts with the heme in the active site of the enzyme. Similar product ratios were obtained for the oxidation of DDEP by K3Fe(CN)6, indicating that product ratios of DP over EDP are mainly determined by the pH of buffer. These results, in addition to semiemperical calculations (AM1) for the oxidation of DDEP in the gas phase, are consistent with the idea that the inhibitor undergoes a single-electron oxidation to form the DDEP radical cation, the fate of which depends on the environment of the active site of the enzyme. The proposed formation of a radical cation by the abstraction of an electron from nitrogen is consistent with the finding of low intramolecular isotope effects of the metabolism of 3,5-dicarbethoxy-2,6-dimethyl-[4-2H,4-1H]-1,4-dihydropyridine by P450 2C11 and 3A4. Under basic or aprotic conditions, the radical

  10. Biotechnological advances towards an enhanced peroxidase production in Pichia pastoris.

    PubMed

    Krainer, Florian W; Gerstmann, Michaela A; Darnhofer, Barbara; Birner-Gruenberger, Ruth; Glieder, Anton

    2016-09-10

    Horseradish peroxidase (HRP) is a high-demand enzyme for applications in diagnostics, bioremediation, biocatalysis and medicine. Current HRP preparations are isolated from horseradish roots as mixtures of biochemically diverse isoenzymes. Thus, there is a strong need for a recombinant production process enabling a steady supply with enzyme preparations of consistent high quality. However, most current recombinant production systems are limited at titers in the low mg/L range. In this study, we used the well-known yeast Pichia pastoris as host for recombinant HRP production. To enhance recombinant enzyme titers we systematically evaluated engineering approaches on the secretion process, coproduction of helper proteins, and compared expression from the strong methanol-inducible PAOX1 promoter, the strong constitutive PGAP promoter, and a novel bidirectional promoter PHTX1. Ultimately, coproduction of HRP and active Hac1 under PHTX1 control yielded a recombinant HRP titer of 132mg/L after 56h of cultivation in a methanol-independent and easy-to-do bioreactor cultivation process. With regard to the many versatile applications for HRP, the establishment of a microbial host system suitable for efficient recombinant HRP production was highly overdue. The novel HRP production platform in P. pastoris presented in this study sets a new benchmark for this medically relevant enzyme. PMID:27432633

  11. EFFECTS OF A LIGNIN PEROXIDASE-EXPRESSING RECOMBINANT STREPTOMYCES LIVIDANS TK23.1 ON BIOGEOCHEMICAL CYCLING AND THE NUMBERS AND ACTIVITIES OF MICROORGANISMS IN SOIL

    EPA Science Inventory

    A recombinant actinomycete, Streptomyces lividans TK23.1, expressing a pIJ702-encoded extracellular lignin peroxidase gene cloned from the chromosome of Streptomyces virodosporus T7A, was released into soil in flask- and microcosm-scale studies to determine its effects on humific...

  12. Detection of Q Fever Specific Antibodies Using Recombinant Antigen in ELISA with Peroxidase Based Signal Amplification

    PubMed Central

    Chen, Hua-Wei; Zhang, Zhiwen; Glennon, Erin; Ching, Wei-Mei

    2014-01-01

    Currently, the accepted method for Q fever serodiagnosis is indirect immunofluorescent antibody assay (IFA) using the whole cell antigen. In this study, we prepared the recombinant antigen of the 27-kDa outer membrane protein (Com1) which has been shown to be recognized by Q fever patient sera. The performance of recombinant Com1 was evaluated in ELISA by IFA confirmed serum samples. Due to the low titers of IgG and IgM in Q fever patients, the standard ELISA signals were further amplified by using biotinylated anti-human IgG or IgM plus streptavidin-HRP polymer. The modified ELISA can detect 88% (29 out of 33) of Q fever patient sera collected from Marines deployed to Iraq. Less than 5% (5 out of 156) of the sera from patients with other febrile diseases reacted with the Com1. These results suggest that the modified ELISA using Com1 may have the potential to improve the detection of Q fever specific antibodies. PMID:26904739

  13. New insights into the heme cavity structure of catalase-peroxidase: a spectroscopic approach to the recombinant synechocystis enzyme and selected distal cavity mutants.

    PubMed

    Heering, Hendrik A; Indiani, Chiara; Regelsberger, Günther; Jakopitsch, Christa; Obinger, Christian; Smulevich, Giulietta

    2002-07-23

    Catalase-peroxidases (KatGs) are heme peroxidases with homology to yeast cytochrome cperoxidase (CCP) and plant ascorbate peroxidases (APXs). KatGs exhibit a peroxidase activity of broad specificity and a high catalase activity, which strongly depends on the presence of a distal Trp as part of the conserved amino acid triad Arg-Trp-His. By contrast, both CCP and APX do not have a substantial catalase activity despite the presence of the same triad. Thus, to elucidate structure-function relationships of catalase-peroxidases (for which no crystal structure is available at the moment), we performed UV-Vis and resonance Raman studies of recombinant wild-type KatG from the cyanobacterium SynechocystisPCC 6803 and the distal side variants (His123-->Gln, Glu; Arg119-->Ala, Asn; Trp122-->Phe, Ala). The distal cavity of KatG is very similar to that of the other class I peroxidases. A H-bond network involving water molecules and the distal Trp, Arg, and His is present, which connects the distal and proximal sides of the heme pocket. However, distal mutation not only affects the heme Fe coordination state and perturbs the proximal Fe-Im bond, as previously observed for other peroxidases, but also alters the stability of the heme architecture. The charge of the distal residues appears particularly important for maintaining the heme architecture. Moreover, the Trp plays a significant role in the distal H-bonding, much more pronounced than in CCP. The relevance of these findings for the catalase activity of KatG is discussed in light of the complete loss of catalase activity in the distal Trp mutants. PMID:12119039

  14. Advanced Recombinant Manganese Peroxidase for Biosynthesis of Lignin Bioproducts, Phase I Final Report, STTR Grant #: DE-SC0007503.

    SciTech Connect

    Beatty, Christopher; Kitner, Joshua; Lajoie, Curtis; McClain, Sean; Potochnik, Steve

    2012-12-13

    The core purpose of this Phase I STTR was to evaluate the feasibility of a new method of producing a recombinant version of manganese peroxidase (MnP) enzyme. MnP is a potentially valuable enzyme for producing high value lignin products and also for industrial de-coloring operations such as biobleaching of pulp and color removal from textile dye effluents. This lignin-modifying enzyme is produced in small amounts by the native host, a white rot fungus. Previous work by Oregon State University developed a secreted recombinant version of the enzyme in the yeast Pichia pastoris. Unfortunately, the expression is barely moderate and the enzyme is heavily glycosylated, which inhibits purification. In this work, the gene for the enzyme is given a tag which targets production of the enzyme to the peroxisome. This is a promising approach since this location is also where heme and hydrogen peroxide are sequestered, which are both necessary cofactors for MnP. More than ten recombinant strains were constructed, verified, and expressed in the Pichia system. Constitutive (GAP) and methanol-induced promoters (AOX) were tried for peroxisomal targeted, cytosolic, and secreted versions of MnP. Only the secreted strains showed activity. The amount of expression was not significantly changed. The degree of glycosylation was lessened using the AOX (methanol) promotoer, but the resulting enzyme was still not able to be purified using immobilized metal affinity chromatography. Additional work beyond the scope of the defined Phase I project was undertaken to construct, verify, and express Pichia strains that mutated the MnP glycosylation sites to inhibit this process. These strains did not show significant activity. The cause is not known, but it is possible that these sites are important to the structure of the enzyme. Also beyond the scope proposed for our Phase I STTR, the team collaborated with AbSci, a startup with a new E. coli based expression system focused on the production of

  15. Screening Actinomycetes for Extracellular Peroxidase Activity

    PubMed Central

    Mercer, D. K.; Iqbal, M.; Miller, P.; McCarthy, A. J.

    1996-01-01

    A diverse collection of actinomycete strains were screened for production of extracellular peroxidase activity by adapting a chemiluminescence analysis system developed for horseradish peroxidase-based enzyme-linked immunosorbent assay. Extracellular peroxidase activity was found to be common but quantitatively variable, and this rapid and sensitive screening system permitted identification of a small group of high-producing strains. A range of spectrophotometric assays were compared for the measurement of peroxidase activity in concentrated culture supernatants of two selected thermophilic streptomycetes. Of these, the peroxide-dependent oxidation of 2,4-dichlorophenol was identified as the most robust and reproducible assay for quantitative studies. PMID:16535344

  16. Lignin-degrading peroxidases of Phanerochaete chrysosporium.

    PubMed

    Cai, D; Tien, M

    1993-07-01

    Lignin and manganese peroxidases are secreted by the basidiomycete Phanerochaete chrysosporium during secondary metabolism. These enzymes play major roles in lignin degradation. The active site amino acid sequence of these lignin-degrading peroxidases is similar to that of horseradish peroxidase (HRP) and cytochrome c peroxidase (CcP). The mechanism by which they oxidize substrates also appears to be the similar. pH has a similar effect on lignin peroxidase compound I formation as on HRP or CcP; however, the pKa controlling compound I formation for lignin peroxidase appears to be much lower. Lignin-degrading peroxidases are able to catalyze the oxidation of substrates with high redox potential. This unique ability is consistent with a heme active site of low electron density, which is indicated by high redox potential. PMID:7763834

  17. Graft copolymers with immobilized peroxidase for organic synthesis

    NASA Astrophysics Data System (ADS)

    De Queiroz, Alvaro Antonio Alencar; Vargas, Reinaldo Romero; Higa, Olga Zazuco; Barrak, Élcio Rogério; Bechara, Etelvino J. H.; Wlasdislaw, Blanka; Marzorati, Liliana

    1999-07-01

    The graft copolymer poly(propylene)-graft-poly(acrylic acid) (PP-G-AA) was prepared by radiation-induced graft copolymerization of acrylic acid onto polypropylene spheres and characterized by thermal analysis and scanning electron microscopy (SEM). Maximum percentage of grafting (70%) was obtained at a total dose of 12 kGy using 30% (w/w) of acrylic acid. The Michaelis constant, KM, and the maximum reaction velocity, VMax, were determined for the free horseradish peroxidase and for the immobilized horseradish peroxidase. The enzyme affinity for the substrate ( KM/ Vmax) remains quite good after immobilization. The sulfoxidation reaction of a ketosulfide was investigated with the immobilized peroxidase.

  18. Distance-Independent Charge Recombination Kinetics in Cytochrome c - Cytochrome c Peroxidase Complexes: Compensating Changes in the Electronic Coupling and Reorganization Energies

    PubMed Central

    Jiang, Nan; Kuznetsov, Aleksey; Nocek, Judith M.; Hoffman, Brian M.; Crane, Brian R.; Hu, Xiangqian; Beratan, David N.

    2013-01-01

    Charge recombination rate constants vary no more than three-fold for inter-protein ET in the Zn-substituted wild type (WT) cytochrome c peroxidase (CcP):cytochrome c (Cc) complex and in complexes with four mutants of the Cc protein (i.e., F82S, F82W, F82Y and F82I), despite large differences in the ET distance. Theoretical analysis indicates that charge recombination for all complexes involves a combination of tunneling and hopping via Trp191. For three of the five structures (WT and F82S(W)), the protein favors hopping more than that in the other two structures that have longer heme→ZnP distances (F82Y(I)). Experimentally observed biexponential ET kinetics is explained by the complex locking in alternative coupling pathways, where the acceptor hole state is either primarily localized on ZnP (slow phase) or on Trp191 (fast phase). The large conformational differences between the CcP:Cc interface for the F82Y(I) mutants compared to the WT and F82S(W) complexes are predicted to change the reorganization energies for the CcP:Cc ET reactions because of changes in solvent exposure and inter-protein ET distances. Since the recombination reaction is likely to occur in the inverted Marcus regime, an increased reorganization energy compensates the decreased role for hopping recombination (and the longer transfer distance) in the F82Y(I) mutants. Taken together, coupling pathway and reorganization energy effects for the five protein complexes explains the observed insensitivity of recombination kinetics to donor-acceptor distance and docking pose and also reveals how hopping through aromatic residues can accelerate long-range ET. PMID:23895339

  19. Cooxidation of styrene by horseradish peroxidase (HRP) and 4-methylphenol

    SciTech Connect

    Grab, L.A.; Ortiz, P.R.

    1987-05-01

    Styrene is cooxidized to styrene oxide in a system containing HRP/H/sub 2/O/sub 2/ and 4-methylphenol. Styrene oxide is not formed in the absence of any of these components, or if the reaction is run under anaerobic conditions. Styrene oxide formation is inhibited by ascorbic acid and catalase but not mannitol or superoxide dismutase. Incubation with /sup 18/O/sub 2/ resulted in more than 90% incorporation of label into styrene oxide. The epoxidation of trans-(1-/sup 2/H)styrene occurred with partial loss of stereochemistry. The products expected from addition of the phenoxy radical to styrene were synthesized and shown not to be formed. Finally, EPR evidence was obtained for formation of 4-methyl catechol in the presence, but not absence, of styrene. The results imply that a peroxy radical is formed by addition of oxygen to the HRP-generated 4-methylphenoxy radical, and that this peroxy radical then cooxidizes styrene.

  20. Soybean peroxidase as an industrial catalyst

    SciTech Connect

    Pokora, A.R.

    1995-12-01

    Peroxidases are a large class of enzymes which are very efficient at catalysing oxidation reactions. Horseradish peroxidase, the most abundant and commercially available peroxidase, has been utilized for many years in medical diagnostic test kits but has never been used successfully in an industrial application. One of the major drawbacks associated with the peroxidases cost and has been their lack of the thermal stability required in an industrial process. Recently, we isolated has been their lack of the peroxidase from soybean seed coats. Soybean seed coats are a commodity product available year round in very large volumes. The useful operational temperature for the soy peroxidase is 40{degrees}C higher than for horseradish peroxidase resulting in shorter reaction times and greater reactor efficiency. This process can be used to produce formaldehyde-free polyphenols as well as numerous phenolic dimers used in the manufacture of anti-oxidants, U-V absorbers, epoxies as well as other materials. The process to manufacture resins and dimers will be discussed.

  1. Novel Applications of Peroxidase

    NASA Astrophysics Data System (ADS)

    Rob, Abdul; Ball, Andrew S.; Tuncer, Munir; Wilson, Michael T.

    1997-02-01

    The article entitled "Novel Biocatalysts Will Work Even Better for Industry" published recently in this Journal (1) was informative and interesting. However it touched only briefly on the application of peroxidase as catalyst. Here, we would like to mention in more detail the novel applications of peroxidase in agricultural, paper pulp, water treatment, pharmaceutical, and medical situations. Firstly, the peroxidase isolated from Phanerochaete chyrosporium has been shown to detoxify herbicides such as atrazine to less toxic compounds and would certainly find potential application in agriculture (2). Secondly, the peroxidase produced by Streptomyces thermoviolaceus may find application in the paper pulp industry as a delignifying agent (3). Thirdly, it has been shown that extracellular peroxidase produced by Streptomyces avermitilis can remove the intense color from paper-mill effluent obtained after semichemical alkaline pulping of wheat straw (4), and thus this enzyme might find application as a catalyst in water treatment plants. Fourthly, the heme-containing horseradish peroxidase enzyme has been exploited in several diagnostic applications in pharmaceutics and medicine, such as the detection of human immunodeficiency virus and cystic fibrosis (5-10). Finally, recent work from our laboratory has suggested that thermophilic nonheme peroxidase produced by Thermomonospora fusca BD25 may find medical use in the diagnosis of myocardial infarction (11, 12). Literature Cited 1. Wiseman, A. J. Chem. Educ. 1996, 73, 55-58. 2. Mougin, C. Appl. Environ. Microbiol. 1994, 60, 705-708. 3. McCarthy A. J.; Peace, W.; Broda, P. Appl. Microbiol. Technol. 1985, 23, 238-244. 4. Hernandez, M; Rodriguez J; Soliveri, J; Copa, J. L; Perez, M. I; Arias, M. E. Appl. Environ. Microbiol. 1994, 60, 3909-3913. 5. Hopfer, S. M.; Aslanzadeh, J. Ann. Clin. Lab. Sci. 1995, 25, 475-480. 6. Suzuki, K; Iman, M. J. Virol. Methods 1995, 55, 347-356. 7. Nielsen, K. J. Immunoassay 1995, 16, 183-197. 8

  2. Hydroxyl-radical production in physiological reactions. A novel function of peroxidase.

    PubMed

    Chen, S X; Schopfer, P

    1999-03-01

    Peroxidases catalyze the dehydrogenation by hydrogen peroxide (H2O2) of various phenolic and endiolic substrates in a peroxidatic reaction cycle. In addition, these enzymes exhibit an oxidase activity mediating the reduction of O2 to superoxide (O2.-) and H2O2 by substrates such as NADH or dihydroxyfumarate. Here we show that horseradish peroxidase can also catalyze a third type of reaction that results in the production of hydroxyl radicals (.OH) from H2O2 in the presence of O2.-. We provide evidence that to mediate this reaction, the ferric form of horseradish peroxidase must be converted by O2.- into the perferryl form (Compound III), in which the haem iron can assume the ferrous state. It is concluded that the ferric/perferryl peroxidase couple constitutes an effective biochemical catalyst for the production of .OH from O2.- and H2O2 (iron-catalyzed Haber-Weiss reaction). This reaction can be measured either by the hydroxylation of benzoate or the degradation of deoxyribose. O2.- and H2O2 can be produced by the oxidase reaction of horseradish peroxidase in the presence of NADH. The .OH-producing activity of horseradish peroxidase can be inhibited by inactivators of haem iron or by various O2.- and .OH scavengers. On an equimolar Fe basis, horseradish peroxidase is 1-2 orders of magnitude more active than Fe-EDTA, an inorganic catalyst of the Haber-Weiss reaction. Particularly high .OH-producing activity was found in the alkaline horseradish peroxidase isoforms and in a ligninase-type fungal peroxidase, whereas lactoperoxidase and soybean peroxidase were less active, and myeloperoxidase was inactive. Operating in the .OH-producing mode, peroxidases may be responsible for numerous destructive and toxic effects of activated oxygen reported previously. PMID:10103001

  3. Proton NMR investigation into the basis for the relatively high redox potential of lignin peroxidase

    SciTech Connect

    Banci, L.; Bertini, I.; Turano, P. ); Ming Tien ); Kirk, T.K. )

    1991-08-15

    Lignin peroxidase shares several structural features with the well-studied horseradish peroxidase and cytochrome c peroxidase but carries a higher redox potential. Here the heme domain of lignin peroxidase and the lignin peroxidase cyanide adduct was examined by {sup 1}H NMR spectroscopy, including nuclear Overhauser effect and two-dimensional measurements, and the findings were compared with those for horseradish peroxidase and cytochrome c peroxidase. Structural information was obtained on the orientation of the heme vinyl and propionate groups and the proximal and distal histidines. The shifts of the {var epsilon}1 proton of the proximal histidine were found to be empirically related to the Fe{sup 3+}/Fe{sup 2+} redox potentials.

  4. Generation of recombinant, enzymatically active human thyroid peroxidase and its recognition by antibodies in the sera of patients with Hashimoto's thyroiditis.

    PubMed Central

    Kaufman, K D; Rapoport, B; Seto, P; Chazenbalk, G D; Magnusson, R P

    1989-01-01

    A full-length cDNA clone for human thyroid peroxidase (TPO) inserted into the mammalian cell expression vector pECE was stably transfected into Chinese hamster ovary (CHO) cells. Clones were assayed for human TPO mRNA, TPO protein, and TPO enzymatic activity. One subclone, expressing the highest TPO enzymatic activity, was used in further studies. FACS analysis of these cells preincubated in Hashimoto's serum revealed approximately 100-fold greater fluorescence compared with controls, indicating that recombinant TPO is expressed on the cell surface. Particulate antigen was extracted from these cells and studied by Western blot analysis using a panel of Hashimoto's sera of known antimicrosomal antibody (anti-MSA) titer. Under nonreducing conditions a broad, immunoreactive band of approximately 200 kD was observed, as well as a doublet of approximately 110 kD. All of the 36 Hashimoto's sera tested reacted with these bands, most in proportion to their anti-MSA titer. Six normal sera tested against this antigen(s) were nonreactive, as were the Hashimoto's sera tested against nontransfected CHO cells. Western blots under reducing conditions revealed a considerably diminished signal, with some of the sera of lower anti-MSA titer becoming negative, the loss of the 200-kD broad band, and the apparent conversion of the 110-kD doublet into a single band. Preincubation of cells in tunicamycin revealed no decrease in TPO immunoreactivity. In conclusion, we expressed enzymatically active human TPO in nonthyroidal eukaryotic cells. Our data prove that functionally active TPO is a major component of the thyroid microsomal antigen. Images PMID:2474568

  5. The ultraviolet filter benzophenone 2 interferes with the thyroid hormone axis in rats and is a potent in vitro inhibitor of human recombinant thyroid peroxidase.

    PubMed

    Schmutzler, Cornelia; Bacinski, Anja; Gotthardt, Inka; Huhne, Katrin; Ambrugger, Petra; Klammer, Holger; Schlecht, Christiane; Hoang-Vu, Cuong; Grüters, Annette; Wuttke, Wolfgang; Jarry, Hubertus; Köhrle, Josef

    2007-06-01

    Endocrine disrupting chemicals (EDCs), either plant constituents or contaminants deriving from industrial products, may interfere with the thyroid hormone (TH) axis. Here, we examined whether selected EDCs inhibit the key reactions of TH biosynthesis catalyzed by thyroid peroxidase (TPO). We used a novel in vitro assay based on human recombinant TPO (hrTPO) stably transfected into the human follicular thyroid carcinoma cell line FTC-238. F21388 (synthetic flavonoid), bisphenol A (building block for polycarbonates), and the UV filter benzophenone 2 (BP2) inhibited hrTPO. BP2 is contained in numerous cosmetics of daily use and may be in regular contact with human skin. Half-maximal inhibition in the guaiacol assay occurred at 450 nmol/liter BP2, a concentration 20- and 200-fold lower than those required in case of the TPO-inhibiting antithyroid drugs methimazole and propylthiouracil, respectively. BP2 at 300 nmol/liter combined with the TPO substrate H(2)O(2) (10 mumol/liter) inactivated hrTPO; this was, however, prevented by micromolar amounts of iodide. BP2 did not inhibit iodide uptake into FRTL-5 cells. In BP2-treated rats (333 and 1000 mg/kg body weight), serum total T(4) was significantly decreased and serum thyrotropin was significantly increased. TPO activities in the thyroids of treated animals were unchanged, a finding also described for methimazole and propylthiouracil. Thus, EDCs, most potently BP2, may disturb TH homeostasis by inhibiting or inactivating TPO, effects that are even more pronounced in the absence of iodide. This new challenge for endocrine regulation must be considered in the context of a still prevailing iodide deficiency in many parts of the world. PMID:17379648

  6. Two cationic peroxidases from cell walls of Araucaria araucana seeds.

    PubMed

    Riquelme, A; Cardemil, L

    1995-05-01

    We have previously reported the purification and partial characterization of two cationic peroxidases from the cell walls of seeds and seedlings of the South American conifer, Araucaria araucana. In this work, we have studied the amino acid composition and NH2-terminal sequences of both enzymes. We also compare the data obtained from these analyses with those reported for other plant peroxidases. The two peroxidases are similar in their amino acid compositions. Both are particularly rich in glycine, which comprises more than 30% of the amino acid residues. The content of serine is also high, ca 17%. The two enzymes are different in their content of arginine, alanine, valine, phenylalanine and threonine. Both peroxidases have identical NH2-terminal sequences, indicating that the two proteins are genetically related and probably are isoforms of the same kind of peroxidase. The amino acid composition and NH2-terminal sequence analyses showed marked differences from the cationic peroxidases from turnip and horseradish. PMID:7786490

  7. Peroxidase catalyzed polymerization of phenol

    SciTech Connect

    Vasudevan, P.T.; Li, L.O.

    1996-07-01

    The effect of horseradish peroxidase (HRP) and H{sub 2}O{sub 2} concentrations on the removal efficiency of phenol, defined as the percentage of phenol removed from solution as a function of time, has been investigated. When phenol and H{sub 2}O{sub 2} react with an approximately one-to-one stoichiometry, the phenol is almost completely precipitated within 10 min. The reaction is inhibited at higher concentrations of H{sub 2}O{sub 2}. The removal efficiency increases with an increase in the concentration of HRP, but an increase in the time of treatment cannot be used to offset the reduction in removal efficiency at low concentrations of the enzyme, because of inactivation of the enzyme. One molecule of HRP is needed to remove approximately 1100 molecules of phenol when the reaction is conducted at pH 8.0 and at ambient temperature. 9 refs., 5 figs.

  8. Identification of core alpha 1,3-fucosylated glycans and cloning of the requisite fucosyltransferase cDNA from Drosophila melanogaster. Potential basis of the neural anti-horseadish peroxidase epitope.

    PubMed

    Fabini, G; Freilinger, A; Altmann, F; Wilson, I B

    2001-07-27

    For many years, polyclonal antibodies raised against the plant glycoprotein horseradish peroxidase have been used to specifically stain the neural and male reproductive tissue of Drosophila melanogaster. This epitope is considered to be of carbohydrate origin, but no glycan structure from Drosophila has yet been isolated that could account for this cross-reactivity. Here we report that N-glycan core alpha1,3-linked fucose is, as judged by preabsorption experiments, indispensable for recognition of Drosophila embryonic nervous system by anti-horseradish peroxidase antibody. Further, we describe the identification by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry and high performance liquid chromatography of two Drosophila N-glycans that, as already detected in other insects, carry both alpha1,3- and alpha1,6-linked fucose residues on the proximal core GlcNAc. Moreover, we have isolated three cDNAs encoding alpha1,3-fucosyltransferase homologues from Drosophila. One of the cDNAs, when transformed into Pichia pastoris, was found to direct expression of core alpha1,3-fucosyltransferase activity. This recombinant enzyme preferred as substrate a biantennary core alpha1,6-fucosylated N-glycan carrying two non-reducing N-acetylglucosamine residues (GnGnF6; Km 11 microm) over the same structure lacking a core fucose residue (GnGn; Km 46 microm). The Drosophila core alpha1,3-fucosyltransferase enzyme was also shown to be able to fucosylate N-glycan structures of human transferrin in vitro, this modification correlating with the acquisition of binding to anti-horseradish peroxidase antibody. PMID:11382750

  9. Sequence analysis and homology modeling of peroxidase from Medicago sativa

    PubMed Central

    Hooda, Vinita; Gundala, Prasada babu; Chinthala, Paramageetham

    2012-01-01

    Plant peroxidases are one of the most extensively studied group of enzymes which find applications in the environment, health, pharmaceutical, chemical and biotechnological processes. Class III secretary peroxidase from alfalfa (Medicago sativa) has been characterized using bioinformatics approach Physiochemical properties and topology of alfalfa peroxidase were compared with that of soybean and horseradish peroxidase, two most popular commercially available peroxidase preparations. Lower value of instability index as predicted by ProtParam and presence of extra disulphide linkages as predicted by Cys_REC suggested alfalfa peroxidase to be more stable than either of the commercial preparations. Multiple Sequence Alignment (MSA) with other functionally similar proteins revealed the presence of highly conserved catalytic residues. Three dimensional model of alfalfa peroxidase was constructed based on the crystal structure of soybean peroxidase (PDB Id: 1FHF A) by homology modelling approach. The model was checked for stereo chemical quality by PROCHECH, VERIFY 3D, WHAT IF, ERRAT, 3D MATCH AND ProSA servers. The best model was selected, energy minimized and used to analyze structure function relationship with substrate hydrogen peroxide by Autodock 4.0. The enzyme substrate complex was viewed with Swiss PDB viewer and one residue ASP43 was found to stabilize the interaction by hydrogen bonds. The results of the study may be a guiding point for further investigations on alfalfa peroxidase. PMID:23275690

  10. High-yield production of manganese peroxidase, lignin peroxidase, and versatile peroxidase in Phanerochaete chrysosporium.

    PubMed

    Coconi-Linares, Nancy; Magaña-Ortíz, Denis; Guzmán-Ortiz, Doralinda A; Fernández, Francisco; Loske, Achim M; Gómez-Lim, Miguel A

    2014-11-01

    The white-rot fungus Phanerochaete chrysosporium secretes extracellular oxidative enzymes during secondary metabolism, but lacks versatile peroxidase, an enzyme important in ligninolysis and diverse biotechnology processes. In this study, we report the genetic modification of a P. chrysosporium strain capable of co-expressing two endogenous genes constitutively, manganese peroxidase (mnp1) and lignin peroxidase (lipH8), and the codon-optimized vpl2 gene from Pleurotus eryngii. For this purpose, we employed a highly efficient transformation method based on the use of shock waves developed by our group. The expression of recombinant genes was verified by PCR, Southern blot, quantitative real-time PCR (qRT-PCR), and assays of enzymatic activity. The production yield of ligninolytic enzymes was up to four times higher in comparison to previously published reports. These results may represent significant progress toward the stable production of ligninolytic enzymes and the development of an effective fungal strain with promising biotechnological applications. PMID:25269601

  11. ATP-enhanced peroxidase-like activity of gold nanoparticles.

    PubMed

    Shah, Juhi; Purohit, Rahul; Singh, Ragini; Karakoti, Ajay Singh; Singh, Sanjay

    2015-10-15

    Gold nanoparticles (AuNPs) are known to possess intrinsic biological peroxidase-like activity that has applications in development of numerous biosensors. The reactivity of the Au atoms at the surface of AuNPs is critical to the performance of such biosensors, yet little is known about the effect of biomolecules and ions on the peroxidase-like activity. In this work, the effect of ATP and other biologically relevant molecules and ions over peroxidase-like activity of AuNPs are described. Contrary to the expectation that nanoparticles exposed to biomolecules may lose the catalytic property, ATP and ADP addition enhanced the peroxidase-like activity of AuNPs. The catalytic activity was unaltered by the addition of free phosphate, sulphate and carbonate anions however, addition of ascorbic acid to the reaction mixture diminished the intrinsic peroxidase-like activity of AuNPs, even in the presence of ATP and ADP. In contrast to AuNPs, ATP did not synergize and improve the peroxidase activity of the natural peroxidase enzyme, horseradish peroxidase. PMID:26111515

  12. Peroxidase mediated conjugation of corn fibeer gum and bovine serum albumin to improve emulsifying properties

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The emulsifying properties of corn fiber gum (CFG), a naturally-occurring polysaccharide protein complex, were improved by kinetically controlled formation of hetero-covalent linkages with bovine serum albumin (BSA), using horseradish peroxidase. The formation of hetero-crosslinked CFG-BSA conjugate...

  13. Magnetic resonance spectral characterization of the heme active site of Coprinus cinereus peroxidase

    SciTech Connect

    Lukat, G.S.; Rodgers, K.R.; Jabro, M.N.; Goff, H.M. )

    1989-04-18

    Examination of the peroxidase isolated from the inkcap Basidiomycete Coprinus cinereus shows that the 42,000-dalton enzyme contains a protoheme IX prosthetic group. Reactivity assays and the electronic absorption spectra of native Coprinus peroxidase and several of its ligand complexes indicate that this enzyme has characteristics similar to those reported for horseradish peroxidase. In this paper, the authors characterize the H{sub 2}O{sub 2}-oxidized forms of Coprinus peroxidase compounds I, II, and III by electronic absorption and magnetic resonance spectroscopies. Electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) studies of this Coprinus peroxidase indicate the presence of high-spin Fe(III) in the native protein and a number of differences between the heme site of Coprinus peroxidase and horseradish peroxidase. Carbon-13 (of the ferrous CO adduct) and nitrogen-15 (of the cyanide complex) NMR studies together with proton NMR studies of the native and cyanide-complexed Caprinus peroxidase are consistent with coordination of a proximal histidine ligand. The EPR spectrum of the ferrous NO complex is also reported. Protein reconstitution with deuterated hemin has facilitated the assignment of the heme methyl resonances in the proton NMR spectrum.

  14. X-ray structures of recombinant yeast cytochrome c peroxidase and three heme-cleft mutants prepared by site-directed mutagenesis

    SciTech Connect

    Wang, J.; Mauro, J.M.; Edwards, S.L.; Oatley, S.J.; Fishel, L.A.; Ashford, V.A.; Xuong, Nguyenhuu; Kraut, J. )

    1990-08-07

    The 2.2-{angstrom} x-ray structure for CCP(MI), a plasmid-encoded form of Saccharomyces cerevisiae cytochrome c peroxidase (CCP) expressed in Escherichia coli has been solved, together with the structures of three specifically designed single-site heme-cleft mutants. The structure of CCP(MI) was solved by using molecular replacement methods, since its crystals grow differently from the crystals of CCP isolated from bakers' yeast used previously for structural solution. Small distal-side differences between CCP(MI) and bakers' yeast CCP are observed, presumably due to a strain-specific Thr-53 {yields} Ile substitution in CCP(MI). The observation of a vacant sixth coordination site in this structure differs from the results of solution resonance Raman studies, which predict hexacoordinated high-spin iron. The coordination behavior of this W51F mutant is apparently altered in the presence of a precipitating agent, 30% 2-methyl-2,4-pentanediol. A proximal Trp-191 {yields} Phe mutant that has substantially diminished enzyme activity and altered magnetic properties accommodates the substitution by allowing the side chain of Phe-191, together with the segment of backbone to which it is attached, to move toward the heme. This relatively large local perturbation is accompanied by numerous small adjustments resulting in a slight overall compression of the enzyme's proximal domain; however, the iron coordination sphere is essentially unchanged. This structures rules out a major alteration in protein conformation as a reason for the dramatically decreased activity of the W191F mutant. From the alteration of local structure that occurs in this mutant, coupled with the results of preliminary functional studies, the authors infer that Asp-235 exerts influence on the heme iron so as to keep its sixth coordination site vacant, and hence reactive with peroxide substrate, over a wide pH range.

  15. Applications and Prospective of Peroxidase Biocatalysis in the Environmental Field

    NASA Astrophysics Data System (ADS)

    Torres-Duarte, Cristina; Vazquez-Duhalt, Rafael

    Environmental protection is, doubtless, one of the most important challenges for the human kind. The huge amount of pollutants derived from industrial activities represents a threat for the environment and ecologic equilibrium. Phenols and halogenated phenols, polycyclic aromatic hydrocarbons, endocrine disruptive chemicals, pesticides, dioxins, polychlorinated biphenyls, industrial dyes, and other xenobiotics are among the most important pollutants. A large variety of these xenobiotics are substrates for peroxidases and thus susceptible to enzymatic transformation. The literature reports mainly the use of horseradish peroxidase, manganese peroxidase, lignin peroxidase, and chloroperoxidase on the transformation of these pollutants. Peroxidases are enzymes able to transform a variety of compounds following a free radical mechanism, giving oxidized or polymerized products. The peroxidase transformation of these pollutants is accompanied by a reduction in their toxicity, due to a biological activity loss, a reduction in the bioavailability or due to the removal from aqueous phase, especially when the pollutant is found in water. In addition, when the pollutants are present in soil, peroxidases catalyze a covalent binding to soil organic matter. In most of cases, oxidized products are less toxic and easily biodegradable than the parent compounds. In spite of their versatility and potential use in environmental processes, peroxidases are not applied at large scale yet. Diverse challenges, such as stability, redox potential, and the production of large amounts, should be solved in order to apply peroxidases in the pollutant transformation. In this chapter, we critically review the transformation of different xenobiotics by peroxidases, with special attention on the identified transformation products, the probable reaction mechanisms, and the toxicity reports. Finally, the design and development of an environmental biocatalyst is discussed. The design challenges are

  16. Structural diversity and transcription of class III peroxidases from Arabidopsis thaliana.

    PubMed

    Welinder, Karen G; Justesen, Annemarie F; Kjaersgård, Inger V H; Jensen, Rikke B; Rasmussen, Søren K; Jespersen, Hans M; Duroux, Laurent

    2002-12-01

    Understanding peroxidase function in plants is complicated by the lack of substrate specificity, the high number of genes, their diversity in structure and our limited knowledge of peroxidase gene transcription and translation. In the present study we sequenced expressed sequence tags (ESTs) encoding novel heme-containing class III peroxidases from Arabidopsis thaliana and annotated 73 full-length genes identified in the genome. In total, transcripts of 58 of these genes have now been observed. The expression of individual peroxidase genes was assessed in organ-specific EST libraries and compared to the expression of 33 peroxidase genes which we analyzed in whole plants 3, 6, 15, 35 and 59 days after sowing. Expression was assessed in root, rosette leaf, stem, cauline leaf, flower bud and cell culture tissues using the gene-specific and highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR). We predicted that 71 genes could yield stable proteins folded similarly to horseradish peroxidase (HRP). The putative mature peroxidases derived from these genes showed 28-94% amino acid sequence identity and were all targeted to the endoplasmic reticulum by N-terminal signal peptides. In 20 peroxidases these signal peptides were followed by various N-terminal extensions of unknown function which are not present in HRP. Ten peroxidases showed a C-terminal extension indicating vacuolar targeting. We found that the majority of peroxidase genes were expressed in root. In total, class III peroxidases accounted for an impressive 2.2% of root ESTs. Rather few peroxidases showed organ specificity. Most importantly, genes expressed constitutively in all organs and genes with a preference for root represented structurally diverse peroxidases (< 70% sequence identity). Furthermore, genes appearing in tandem showed distinct expression profiles. The alignment of 73 Arabidopsis peroxidase sequences provides an easy access to the identification of orthologous peroxidases

  17. Heterologous Expression of Peroxidases

    NASA Astrophysics Data System (ADS)

    de Weert, Sandra; Lokman, B. Christien

    The industrial importance of peroxidases has led to much research in the past two decades on the development of a cost effective and efficient production process for peroxidases. Unfortunately, even today, no clear answers can be given to questions such as (1) should the peroxidase be expressed in bacteria, yeast, or fungi? (2) which is the optimal production strain (e.g., protease deficient, heme overproducing)? (3) which expression vector should be chosen? and (4) what purification method should be used? Strategies that have proven successful for one peroxidase can fail for another one; for each individual peroxidase, a new strategy has to be developed. This chapter gives an overview of the heterologous production of heme containing peroxidases in various systems. It focuses on the heterologous production of fungal peroxidases as they have been subject of considerable research for their industrial and environmental applications. An earlier study has also been performed by Conesa et al. [1] and is extended with recent proceedings.

  18. Characterization of lignin and Mn peroxidases from Phanerochaete chrysosporium

    SciTech Connect

    Not Available

    1991-01-01

    Long-term objectives are to elucidate the role and mechanism of the various isozymes in lignin biodegradation. Work is described on electrochemical studies on lignin and Mn peroxidases. This study was performed to investigate the structural aspects which confer the lignin and Mn peroxidases with their high reactivity. The experimentally determined redox potential of the Fe{sup 3+}/Fe{sup 2+} couple for the lignin peroxidase isozymes H1, H2, H8 and H10 are very similar, near-130 mV. The redox potential for the Mn peroxidase isozymes H3 and H4 are similar to each other ({minus}88 mV and {minus}95 mV, respectively) and are more positive than the lignin peroxidases. The higher redox potential for the Fe{sup 3+}/Fe{sup 2+} couple is consistent with the heme active site of these fungal peroxidases being more electron deficient. To investigate the accessibility of the heme active site to the substrate which is oxidized (veratryl alcohol and Mn (II)), we investigated whether these substrates had any affect on the redox potential of the heme. The E{sub m7} value for lignin and Mn peroxidases are not affected by their respective substrates, veratryl alcohol and Mn (II). These results suggest that substrates do not directly interact with the ferric heme-iron as axial ligands. This is consistent with the present model for peroxidase catalysis. Suicide inhibitor (1) and nmr studies (2) indicate that the heme-iron of horseradish peroxidase (HRP) is not fully accessible to bulky substrates occur at the periphery of the heme.

  19. Redox Potential of Peroxidases

    NASA Astrophysics Data System (ADS)

    Ayala, Marcela

    Redox potential of peroxidases greatly influences the range of oxidizable substrates: in principle, peroxidases may only catalyze the oxidation of substrates with lower redox potential. There is substantial information on the factors that modulate the redox potential of heme proteins. Both theoretical and experimental evidence highlight the most significant contributions arising from the interaction of heme iron with the axial ligands, as well as the electrostatic interactions surrounding the heme group. However, for different proteins, the factors contribute to different extents. Understanding the electrochemistry of heme peroxidases is fundamental in order to design enhanced biocatalysts. In this chapter, current knowledge of the forces influencing redox potential of heme peroxidases is reviewed.

  20. Nanostructures for peroxidases

    PubMed Central

    Carmona-Ribeiro, Ana M.; Prieto, Tatiana; Nantes, Iseli L.

    2015-01-01

    Peroxidases are enzymes catalyzing redox reactions that cleave peroxides. Their active redox centers have heme, cysteine thiols, selenium, manganese, and other chemical moieties. Peroxidases and their mimetic systems have several technological and biomedical applications such as environment protection, energy production, bioremediation, sensors and immunoassays design, and drug delivery devices. The combination of peroxidases or systems with peroxidase-like activity with nanostructures such as nanoparticles, nanotubes, thin films, liposomes, micelles, nanoflowers, nanorods and others is often an efficient strategy to improve catalytic activity, targeting, and reusability. PMID:26389124

  1. Nanostructures for peroxidases.

    PubMed

    Carmona-Ribeiro, Ana M; Prieto, Tatiana; Nantes, Iseli L

    2015-01-01

    Peroxidases are enzymes catalyzing redox reactions that cleave peroxides. Their active redox centers have heme, cysteine thiols, selenium, manganese, and other chemical moieties. Peroxidases and their mimetic systems have several technological and biomedical applications such as environment protection, energy production, bioremediation, sensors and immunoassays design, and drug delivery devices. The combination of peroxidases or systems with peroxidase-like activity with nanostructures such as nanoparticles, nanotubes, thin films, liposomes, micelles, nanoflowers, nanorods and others is often an efficient strategy to improve catalytic activity, targeting, and reusability. PMID:26389124

  2. Disulfide bonds and glycosylation in fungal peroxidases.

    PubMed

    Limongi, P; Kjalke, M; Vind, J; Tams, J W; Johansson, T; Welinder, K G

    1995-01-15

    Four conserved disulfide bonds and N-linked and O-linked glycans of extracellular fungal peroxidases have been identified from studies of a lignin and a manganese peroxidase from Trametes versicolor, and from Coprinus cinereus peroxidase (CIP) and recombinant C. cinereus peroxidase (rCIP) expressed in Aspergillus oryzae. The eight cysteine residues are linked 1-3, 2-7, 4-5 and 6-8, and are located differently from the four conserved disulfide bridges present in the homologous plant peroxidases. CIP and rCIP were identical in their glycosylation pattern, although the extent of glycan chain heterogeneity depended on the fermentation batch. CIP and rCIP have one N-linked glycan composed only of GlcNAc and Man at residue Asn142, and two O-linked glycans near the C-terminus. The major glycoform consists of single Man residues at Thr331 and at Ser338. T. versicolor lignin isoperoxidase TvLP10 contains a single N-linked glycan composed of (GlcNAc)2Man5 bound to Asn103, whereas (GlcNAc)2Man3 was found in T. versicolor manganese isoperoxidase TvMP2 at the same position. In addition, mass spectrometry of the C-terminal peptide of TvMP2 indicated the presence of five Man residues in O-linked glycans. No phosphate was found in these fungal peroxidases. PMID:7851395

  3. Peroxidase(s) in Environment Protection

    PubMed Central

    Bansal, Neelam; Kanwar, Shamsher S.

    2013-01-01

    Industrial discharges of untreated effluents into water bodies and emissions into air have deteriorated the quality of water and air, respectively. The huge amount of pollutants derived from industrial activities represents a threat for the environment and ecologic equilibrium. Phenols and halogenated phenols, polycyclic aromatic hydrocarbons (PAH), endocrine disruptive chemicals (EDC), pesticides, dioxins, polychlorinated biphenyls (PCB), industrial dyes, and other xenobiotics are among the most important pollutants. Peroxidases are enzymes that are able to transform a variety of compounds following a free radical mechanism, thereby yielding oxidized or polymerized products. The peroxidase transformation of these pollutants is accompanied by a reduction in their toxicity, due to loss of biological activity, reduction in the bioavailability, or the removal from aqueous phase, especially when the pollutant is found in water. The review describes the sources of peroxidases, the reactions catalyzed by them, and their applications in the management of pollutants in the environment. PMID:24453894

  4. Bacterial extracellular lignin peroxidase

    DOEpatents

    Crawford, Donald L.; Ramachandra, Muralidhara

    1993-01-01

    A newly discovered lignin peroxidase enzyme is provided. The enzyme is obtained from a bacterial source and is capable of degrading the lignin portion of lignocellulose in the presence of hydrogen peroxide. The enzyme is extracellular, oxidative, inducible by lignin, larch wood xylan, or related substrates and capable of attacking certain lignin substructure chemical bonds that are not degradable by fungal lignin peroxidases.

  5. The Effect of α-Mating Factor Secretion Signal Mutations on Recombinant Protein Expression in Pichia pastoris

    PubMed Central

    Lin-Cereghino, Geoff P.; Stark, Carolyn M.; Kim, Daniel; Chang, Jennifer; Shaheen, Nadia; Poerwanto, Hansel; Agari, Kimiko; Moua, Pachai; Low, Lauren K.; Tran, Namphuong; Huang, Amy D.; Nattestad, Maria; Oshiro, Kristin T.; Chang, John William; Chavan, Archana; Tsai, Jerry W.; Lin-Cereghino, Joan

    2013-01-01

    The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins are initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57-70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future. PMID:23454485

  6. Immobilized Horseradish Peroxidase on Discs of Polyvinyl Alcohol-Glutaraldehyde Coated with Polyaniline

    PubMed Central

    Caramori, Samantha Salomão; Fernandes, Kátia Flávia; de Carvalho Junior, Luiz Bezerra

    2012-01-01

    Discs of network polyvinyl alcohol-glutaraldehyde (PVAG) were synthesized and coated with polyaniline (PANI) using glutaraldehyde as a chemical arm (PVAG-PANIG-HRP disc). The best conditions for the immobilization were established as about 1.0 mg mL−1 of protein, for 60 min and pH 5.5. The soluble enzyme lost all of its activity after incubation at 70°C for 15 min, whereas the PVAG-PANIG-HRP disc retained about half of the initial activity for pyrogallol. The same PVAG-PANIG-HRP disc was used consecutively three times without any activity lossbut presented 25% of the initial activity after the 7th use. PVAG-PANIG-HRP disc retained approximately 80% and 60% of its initial activity after 60 and 80 days of storage, respectively. Resorcinol, m-cresol, catechol, pyrogallol, α-naphthol, βnaphthol, and 4, 4′-diaminodiphenyl benzidine were efficiently oxidized by the PVAG-PANIG-HRP disc (from about 70% to 90%), and it was less efficient towards aniline, phenol, and 2-nitrosonaphthol. PMID:22619582

  7. Human myeloperoxidase (MPO) and horseradish peroxidase (HRP) catalyzed oxidation of phenol

    SciTech Connect

    Ross, D.; Eastmond, D.A.; Ruzo, L.O.; Smith, M.T.

    1986-03-01

    MPO-catalyzed conversion of phenolic metabolites of benzene may be involved in benzene-induced myelotoxicity. The authors have studied the metabolism and protein binding of phenol - the major metabolite of benzene - during peroxidatic oxidation. The major metabolite observed during MPO- and HRP- catalyzed oxidation was characterized as 4,4 biphenol using HPLC and combined GC-MS. When glutathione (GSH) was added to the incubation mixtures, two additional compounds were observed during HPLC analysis which were characterized as GSH-conjugates of 4,4-diphenoquinone by fast atom bombardment MS and by NMR. ESR spectroscopy showed that both MPO-and HRP-catalyzed oxidation of phenol proceeded via the generation of free radical intermediates. Using /sup 14/C-phenol, both MPO- and HRP-catalyzed oxidations resulted in the production of species which bound covalently to boiled liver microsomal protein. The increase in binding correlated well with removal of substrate. Thus, peroxidatic oxidation of phenolic metabolites of benzene in the bone marrow may be involved in benzene-induced myelotoxicity.

  8. Recombinant Exon-Encoded Resilins for Elastomeric Biomaterials

    PubMed Central

    Qin, Guokui; Rivkin, Amit; Lapidot, Shaul; Hu, Xiao; Arinus, Shira B.; Dgany, Or; Shoseyov, Oded; Kaplan, David L.

    2011-01-01

    Resilin is an elastomeric protein found in specialized regions of the cuticle of most insects, providing outstanding material properties including high resilience and fatigue lifetime for insect flight and jumping needs. Two exons (1 and 3) from the resilin gene in Drosophila melanogaster were cloned and the encoded proteins expressed as soluble products in Escherichia coli. A heat and salt precipitation method was used for efficient purification of the recombinant proteins. The proteins were solution cast from water and formed into rubber-like biomaterials via horseradish peroxidase-mediated cross-linking. Comparative studies of the two proteins expressed from the two different exons were investigated by Fourier Transform Infrared Spectroscopy (FTIR) and Circular Dichrosim (CD) for structural features. Little structural organization was found, suggesting structural order was not induced by the enzyme-mediateed dityrosine cross-links. Atomic Force Microscopy (AFM) was used to study the elastomeric properties of the uncross-linked and cross-linked proteins. The protein from exon 1 exhibited 90% resilience in comparison to 63% for the protein from exon 3, and therefore may be the more critical domain for functional materials to mimic native resilin. Further, the cross-linking of the recombinant exon 1 via the citrate-modified photo-Fenton reaction was explored as an alternative dityrosine mediated polymerization method and resulted in both highly elastic and adhesive materials. The citrate-modified photo-Fenton system may be suitable for in-vivo applications of resilin biomaterials. PMID:21963157

  9. Recombinant exon-encoded resilins for elastomeric biomaterials.

    PubMed

    Qin, Guokui; Rivkin, Amit; Lapidot, Shaul; Hu, Xiao; Preis, Itan; Arinus, Shira B; Dgany, Or; Shoseyov, Oded; Kaplan, David L

    2011-12-01

    Resilin is an elastomeric protein found in specialized regions of the cuticle of most insects, providing outstanding material properties including high resilience and fatigue lifetime for insect flight and jumping needs. Two exons (1 and 3) from the resilin gene in Drosophila melanogaster were cloned and the encoded proteins expressed as soluble products in Escherichia coli. A heat and salt precipitation method was used for efficient purification of the recombinant proteins. The proteins were solution cast from water and formed into rubber-like biomaterials via horseradish peroxidase-mediated cross-linking. Comparative studies of the two proteins expressed from the two different exons were investigated by Fourier Transform Infrared Spectroscopy (FTIR) and Circular Dichrosim (CD) for structural features. Little structural organization was found, suggesting structural order was not induced by the enzyme-mediated di-tyrosine cross-links. Atomic Force Microscopy (AFM) was used to study the elastomeric properties of the uncross-linked and cross-linked proteins. The protein from exon 1 exhibited 90% resilience in comparison to 63% for the protein from exon 3, and therefore may be the more critical domain for functional materials to mimic native resilin. Further, the cross-linking of the recombinant exon 1 via the citrate-modified photo-Fenton reaction was explored as an alternative di-tyrosine mediated polymerization method and resulted in both highly elastic and adhesive materials. The citrate-modified photo-Fenton system may be suitable for in vivo applications of resilin biomaterials. PMID:21963157

  10. Hydrogen peroxide-independent generation of superoxide catalyzed by soybean peroxidase in response to ferrous ion

    PubMed Central

    Kimura, Makoto; Kawano, Tomonori

    2015-01-01

    It is well documented that extracellular alkalization occurs in plants under the challenges by pathogenic microbes. This may eventually induce the pH-dependent extracellular peroxidase-mediated oxidative burst at the site of microbial challenges. By employing the purified proteins of horseradish peroxidase as a model, we have recently proposed a likely role for free Fe2+ in reduction of ferric enzyme of plant peroxidases into ferrous intermediate and oxygen-bound form of enzyme known as Compound III which may eventually releases superoxide anion radical (O2•−), especially under alkaline condition, possibly contributing to the plant defense mechanism. In the present study, we employed the purified protein of soybean peroxidase (SBP) as an additional model, and examined the changes in the redox status of enzyme accompanying the generation of O2•− in response to Fe2+ under alkaline condition. PMID:26417938

  11. Mechanism of reaction of chlorite with mammalian heme peroxidases

    PubMed Central

    Jakopitsch, Christa; Pirker, Katharina F.; Flemmig, Jörg; Hofbauer, Stefan; Schlorke, Denise; Furtmüller, Paul G.; Arnhold, Jürgen; Obinger, Christian

    2014-01-01

    This study demonstrates that heme peroxidases from different superfamilies react differently with chlorite. In contrast to plant peroxidases, like horseradish peroxidase (HRP), the mammalian counterparts myeloperoxidase (MPO) and lactoperoxidase (LPO) are rapidly and irreversibly inactivated by chlorite in the micromolar concentration range. Chlorite acts as efficient one-electron donor for Compound I and Compound II of MPO and LPO and reacts with the corresponding ferric resting states in a biphasic manner. The first (rapid) phase is shown to correspond to the formation of a MPO-chlorite high-spin complex, whereas during the second (slower) phase degradation of the prosthetic group was observed. Cyanide, chloride and hydrogen peroxide can block or delay heme bleaching. In contrast to HRP, the MPO/chlorite system does not mediate chlorination of target molecules. Irreversible inactivation is shown to include heme degradation, iron release and decrease in thermal stability. Differences between mammalian peroxidases and HRP are discussed with respect to differences in active site architecture and heme modification. PMID:24632343

  12. Removal of phenolic compounds from wastewaters using soybean peroxidase

    SciTech Connect

    Wright, H.; Nicell, J.A.

    1996-11-01

    Toxic and odiferous phenolic compounds are present in wastewaters generated by a variety of industries including petroleum refining, plastics, resins, textiles, and iron and steel manufacturing among others. Due to its commercial availability in purified form, its useful presence in raw plant material, and its proven ability to remove a variety of phenolic contaminants from wastewaters over a wide range of pH and temperature, horseradish peroxidase (HRP) appears to be the peroxidase enzyme of choice in enzymatic wastewater treatment studies. Problems with HRP catalyzed phenol removal, however, include the formation of toxic soluble reaction by-products, the cost of the enzyme, and costs associated with disposal of the phenolic precipitate generated. Enzyme costs are incurred because the enzyme is inactivated during the phenol removal process by various side reactions. While recent work has shown that enzyme inactivation can be reduced using chemical additives, the problem of enzyme cost could be circumvented by using a less expensive source of enzyme. In 1991, the seed coat of the soybean was identified as a very rich source of peroxidase enzyme. Since the seed coat of the soybean is a waste product of the soybean food industry, soybean peroxidase (SBP) has the potential of being a cost effective alternative to HRP in wastewater treatment. In this study, SBP is characterized in terms of its catalytic activity, its stability, and its ability to promote removal of phenolic compounds from synthetic wastewaters. Results obtained are discussed and compared to similar investigations using HRP.

  13. 18O studies of the peroxidase-catalyzed oxidation of N-methylcarbazole. Mechanisms of carbinolamine and carboxaldehyde formation.

    PubMed

    Kedderis, G L; Rickert, D E; Pandey, R N; Hollenberg, P F

    1986-12-01

    Chloroperoxidase, horseradish peroxidase, hemoglobin, myoglobin, lactoperoxidase, and microperoxidase catalyzed the ethyl hydroperoxide-dependent oxidation of N-methylcarbazole to N-(hydroxymethyl)carbazole and N-formylcarbazole as major products. Mass spectral analysis of the N-(hydroxymethyl)carbazole formed during the peroxidase-catalyzed N-demethylation of N-methylcarbazole in 18O-enriched medium indicated partial incorporation (7.5-25.9%) of solvent water oxygen into the carbinolamine intermediate in all systems investigated, suggesting that the peroxidase active site is partially accessible to solvent water during N-demethylation. In contrast, solvent water oxygen was not incorporated into the N-formylcarbazole formed during the peroxidase-catalyzed oxidation of N-methylcarbazole. N-(Hydroxymethyl)carbazole was not further metabolized by the peroxidases in the presence of ethyl hydroperoxide, indicating that it is not an intermediate in N-formylcarbazole formation. The horseradish peroxidase-catalyzed formation of N-formylcarbazole was decreased by 77% when the hydroperoxide-supported reactions were carried out in a nitrogen atmosphere, while the formation of N-(hydroxymethyl)carbazole was decreased by 46%. When the horseradish peroxidase-catalyzed reactions were carried out in a 18O2 atmosphere, 18O incorporation into N-(hydroxymethyl)carbazole was 64.4% of the total oxygen, while 81.8% of the oxygen incorporated into N-formylcarbazole came from 18O2. These results suggest that there are two different mechanisms for the formation of N-(hydroxymethyl)carbazole, both involving the initial oxidation of N-methylcarbazole to a neutral carbon-centered radical. The radical can be further oxidized in the enzyme active site to an iminium cation, which reacts with water derived from either the oxidant or the medium to form the carbinolamine. Alternatively, the substrate radical can react with molecular oxygen to form a hydroperoxy radical, which decomposes to form the

  14. Catalase-peroxidases (KatG) exhibit NADH oxidase activity.

    PubMed

    Singh, Rahul; Wiseman, Ben; Deemagarn, Taweewat; Donald, Lynda J; Duckworth, Harry W; Carpena, Xavi; Fita, Ignacio; Loewen, Peter C

    2004-10-01

    Catalase-peroxidases (KatG) produced by Burkholderia pseudomallei, Escherichia coli, and Mycobacterium tuberculosis catalyze the oxidation of NADH to form NAD+ and either H2O2 or superoxide radical depending on pH. The NADH oxidase reaction requires molecular oxygen, does not require hydrogen peroxide, is not inhibited by superoxide dismutase or catalase, and has a pH optimum of 8.75, clearly differentiating it from the peroxidase and catalase reactions with pH optima of 5.5 and 6.5, respectively, and from the NADH peroxidase-oxidase reaction of horseradish peroxidase. B. pseudomallei KatG has a relatively high affinity for NADH (Km=12 microm), but the oxidase reaction is slow (kcat=0.54 min(-1)) compared with the peroxidase and catalase reactions. The catalase-peroxidases also catalyze the hydrazinolysis of isonicotinic acid hydrazide (INH) in an oxygen- and H2O2-independent reaction, and KatG-dependent radical generation from a mixture of NADH and INH is two to three times faster than the combined rates of separate reactions with NADH and INH alone. The major products from the coupled reaction, identified by high pressure liquid chromatography fractionation and mass spectrometry, are NAD+ and isonicotinoyl-NAD, the activated form of isoniazid that inhibits mycolic acid synthesis in M. tuberculosis. Isonicotinoyl-NAD synthesis from a mixture of NAD+ and INH is KatG-dependent and is activated by manganese ion. M. tuberculosis KatG catalyzes isonicotinoyl-NAD formation from NAD+ and INH more efficiently than B. pseudomallei KatG. PMID:15280362

  15. Recombinant human diamine oxidase activity is not inhibited by ethanol, acetaldehyde, disulfiram, diethyldithiocarbamate or cyanamide.

    PubMed

    Bartko, Johann; Gludovacz, Elisabeth; Petroczi, Karin; Borth, Nicole; Jilma, Bernd; Boehm, Thomas

    2016-08-01

    Human diamine oxidase (hDAO, EC 1.4.3.22) is the key enzyme in the degradation of extracellular histamine. Consumption of alcohol is a known trigger of mast cell degranulation in patients with mast cell activation syndrome. Ethanol may also interfere with enzymatic histamine degradation, but reports on the effects on DAO activity are controversial. There are also conflicting reports whether disulfiram, an FDA-approved agent in the treatment of alcohol dependence, inhibits DAO. We therefore investigated the inhibitory potential of ethanol and disulfiram and their metabolites on recombinant human DAO (rhDAO) in three different assay systems. Relevant concentrations of ethanol, acetaldehyde, and acetate did not inhibit rhDAO activity in an in vitro assay system using horseradish peroxidase (HRP) -mediated luminol oxidation. The aldehyde dehydrogenase (ALDH; EC 1.2.1.3) inhibitors cyanamide and its dimer dicyanamide also had no effect on DAO activity. In one assay system, the irreversible ALDH inhibitor disulfiram and its main metabolite diethyldithiocarbamate seemed to inhibit DAO activity. However, the decreased product formation was not due to a direct block of DAO activity but resulted from inhibition of peroxidase employed in the coupled system. Our in vitro data do not support a direct blocking effect of ethanol, disulfiram, and their metabolites on DAO activity in vivo. PMID:27401969

  16. Effect of Low and Very Low Doses of Simple Phenolics on Plant Peroxidase Activity

    PubMed Central

    Malarczyk, Elżbieta; Kochmańska-Rdest, Janina; Paździoch-Czochra, Marzanna

    2004-01-01

    Changes in the activity of horseradish peroxidase resulting from an addition of ethanol water dilutions of 19 phenolic compounds were observed. For each compound, the enzyme activity was plotted against the degree of dilution expressed as n = –log100 (mol/L) in the range 0 ≤ n ≥ 20. All the curves showed sinusoidal activity, more or less regular, with two to four peaks on average. Each analyzed compound had a characteristic sinusoidal shape, which was constant for samples of peroxidase from various commercial firms. This was clearly visible after function fitting to experimental results based on the Marquadt–Levenberg algorithm using the least-squares method. Among the 19 phenolics, the highest amplitudes were observed for phenol and iso- and vanillate acids and aldehydes. The specific character of each of the analyzed curves offers a possibility of choosing proper dilutions of phenolic compound for activating or inhibiting of peroxidase activity. PMID:19330128

  17. [Thermostabilities of plant phenol oxidase and peroxidase, determining the technology of their use in food industry].

    PubMed

    Mchedlishvili, N I; Omiadze, N T; Gulua, L K; Sadunishvili, T A; Zamtaradze, R K; Abutidze, M O; Bendeliani, E G; Kvesitadze, G I

    2005-01-01

    Stabilities of phenol oxidase and peroxidase from tea plant (Camellia sinensis L.) clone Kolkhida leaves, apple (Malus domestica L.) cultivar Kekhura fruits, walnut (Juglans regia L.) green pericarp, and horseradish (Armoracia lapathifolia Gilib) roots were studied using different storage temperature modes and storage duration. It was demonstrated that both enzymes retained residual activities (approximately 10%) upon 20-min incubation at 80 degrees C. Phenol oxidases from tea, walnut, and, especially, apple, as well as tea peroxidase were stable during storage. A technology for treatment of plant oxidases was proposed, based on the use of a natural inhibitor phenol oxidase and peroxidase, isolated from tea leaves, which solving the problem of residual activities of these enzymes, arising during pasteurization and storage of beverages and juices. It was demonstrated that browning of apple juice during pasteurization and beer turbidity during storage could be efficiently prevented using the natural inhibitor of these enzymes. PMID:15859458

  18. Broccoli processing wastes as a source of peroxidase.

    PubMed

    Duarte-Vázquez, Miguel A; García-Padilla, Sandra; García-Almendárez, Blanca E; Whitaker, John R; Regalado, Carlos

    2007-12-12

    A peroxidase isozyme (BP) was purified to homogeneity from broccoli stems ( Brassica oleraceae var. maraton) discarded from industrial processing wastes. BP specific activity was 1216 ABTS [2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)] units/mg, representing 466-fold that of crude extract. BP is a monomeric glycoprotein containing 16% carbohydrates, with a molecular mass of 49 kDa and an isoelectric point close to 4.2. From kinetic data it showed a two-substrate ping-pong mechanism, and the catalytic efficiency measured as the rate-limiting step of free BP regeneration was 3.4 x 10(6) M(-1) s(-1). The ABTS K m value was 0.2 mM, which was about 20 times lower than that reported for acidic commercial horseradish peroxidase (HRP). Assessment of BP secondary structure showed 30% helical character, similar to HRP and cytochrome c peroxidase. BP lost only 25% activity after 10 min of heating at 55 degrees C and pH 6; it was stable in the pH range from 4 to 9 and showed an optimum pH of 4.6 using ABTS as substrate. BP was active on substrates normally involved in lignin biosynthesis, such as caffeic and ferulic acids, and also displayed good catechol oxidation activity in the presence of hydrogen peroxide. Reverse micellar extraction was successfully used as potential large-scale prepurification of broccoli peroxidase, achieving a purification factor of 7, with 60% activity yield. Stems from the broccoli processing industry have a high potential as an alternative for peroxidase purification. PMID:17997521

  19. Degradation of textile dyes mediated by plant peroxidases.

    PubMed

    Shaffiqu, T S; Roy, J Jegan; Nair, R Aswathi; Abraham, T Emilia

    2002-01-01

    The peroxidase enzyme from the plants Ipomea palmata (1.003 IU/g of leaf) and Saccharum spontaneum (3.6 IU/g of leaf) can be used as an alternative to the commercial source of horseradish and soybean peroxidase enzyme for the decolorization of textile dyes, mainly azo dyes. Eight textiles dyes currently used by the industry and seven other dyes were selected for decolorization studies at 25-200 mg/L levels using these plant enzymes. The enzymes were purified prior to use by ammonium sulfate precipitation, and ion exchange and gel permeation chromatographic techniques. Peroxidase of S. spontaneum leaf (specific activity of 0.23 IU/mg) could completely degrade Supranol Green and Procion Green HE-4BD (100%) dyes within 1 h, whereas Direct Blue, Procion Brilliant Blue H-7G and Chrysoidine were degraded >70% in 1 h. Peroxidase of Ipomea (I. palmata leaf; specific activity of 0.827 U/mg) degraded 50 mg/L of the dyes Methyl Orange (26%), Crystal Violet (36%), and Supranol Green (68%) in 2-4 h and Brilliant Green (54%), Direct Blue (15%), and Chrysoidine (44%) at the 25 mg/L level in 1 to 2 h of treatment. The Saccharum peroxidase was immobilized on a hydrophobic matrix. Four textile dyes, Procion Navy Blue HER, Procion Brilliant Blue H-7G, Procion Green HE-4BD, and Supranol Green, at an initial concentration of 50 mg/L were completely degraded within 8 h by the enzyme immobilized on the modified polyethylene matrix. The immobilized enzyme was used in a batch reactor for the degradation of Procion Green HE-4BD and the reusability was studied for 15 cycles, and the half-life was found to be 60 h. PMID:12396133

  20. Catalase-peroxidase (Mycobacterium tuberculosis KatG) catalysis and isoniazid activation.

    PubMed

    Chouchane, S; Lippai, I; Magliozzo, R S

    2000-08-15

    Resonance Raman spectra of native, overexpressed M. tuberculosis catalase-peroxidase (KatG), the enzyme responsible for activation of the antituberculosis antibiotic isoniazid (isonicotinic acid hydrazide), have confirmed that the heme iron in the resting (ferric) enzyme is high-spin five-coordinate. Difference Raman spectra did not reveal a change in coordination number upon binding of isoniazid to KatG. Stopped-flow spectrophotometric studies of the reaction of KatG with stoichiometric equivalents or small excesses of hydrogen peroxide revealed only the optical spectrum of the ferric enzyme with no hypervalent iron intermediates detected. Large excesses of hydrogen peroxide generated oxyferrous KatG, which was unstable and rapidly decayed to the ferric enzyme. Formation of a pseudo-stable intermediate sharing optical characteristics with the porphyrin pi-cation radical-ferryl iron species (Compound I) of horseradish peroxidase was observed upon reaction of KatG with excess 3-chloroperoxybenzoic acid, peroxyacetic acid, or tert-butylhydroperoxide (apparent second-order rate constants of 3.1 x 10(4), 1.2 x 10(4), and 25 M(-1) s(-1), respectively). Identification of the intermediate as KatG Compound I was confirmed using low-temperature electron paramagnetic resonance spectroscopy. Isoniazid, as well as ascorbate and potassium ferrocyanide, reduced KatG Compound I to the ferric enzyme without detectable formation of Compound II in stopped-flow measurements. This result differed from the reaction of horseradish peroxidase Compound I with isoniazid, during which Compound II was stably generated. These results demonstrate important mechanistic differences between a bacterial catalase-peroxidase and the homologous plant peroxidases and yeast cytochrome c peroxidase, in its reactions with peroxides as well as substrates. PMID:10933818

  1. Computer-controlled system for the study of oxidase reactions: application to the peroxidase-oxidase oscillator.

    PubMed

    McDonald, Andrew G; Tipton, Keith F

    2010-12-16

    An apparatus for the study of bisubstrate oxidase reactions at maintained steady-state substrate concentrations is described, and its specific application to the peroxidase-oxidase biochemical oscillator is reported. Instrument control and data acquisition are provided by custom software written in LabVIEW. The software allows measurement, recording, and control of dissolved oxygen through a Clark-type oxygen electrode, reaction monitoring by a UV/vis spectrophotometer, and controlled substrate delivery by a syringe infusion pump. For peroxidase from horseradish, the optimal pH for oscillatory behavior was found to be in the range 4.5-5.5. PMID:21049952

  2. Characterization of lignin and Mn peroxidases from Phanerochaete chrysosporium. Progress report

    SciTech Connect

    Not Available

    1991-12-31

    Long-term objectives are to elucidate the role and mechanism of the various isozymes in lignin biodegradation. Work is described on electrochemical studies on lignin and Mn peroxidases. This study was performed to investigate the structural aspects which confer the lignin and Mn peroxidases with their high reactivity. The experimentally determined redox potential of the Fe{sup 3+}/Fe{sup 2+} couple for the lignin peroxidase isozymes H1, H2, H8 and H10 are very similar, near-130 mV. The redox potential for the Mn peroxidase isozymes H3 and H4 are similar to each other ({minus}88 mV and {minus}95 mV, respectively) and are more positive than the lignin peroxidases. The higher redox potential for the Fe{sup 3+}/Fe{sup 2+} couple is consistent with the heme active site of these fungal peroxidases being more electron deficient. To investigate the accessibility of the heme active site to the substrate which is oxidized [veratryl alcohol and Mn (II)], we investigated whether these substrates had any affect on the redox potential of the heme. The E{sub m7} value for lignin and Mn peroxidases are not affected by their respective substrates, veratryl alcohol and Mn (II). These results suggest that substrates do not directly interact with the ferric heme-iron as axial ligands. This is consistent with the present model for peroxidase catalysis. Suicide inhibitor (1) and nmr studies (2) indicate that the heme-iron of horseradish peroxidase (HRP) is not fully accessible to bulky substrates occur at the periphery of the heme.

  3. Measurements of Heme Relaxation and Ligand Recombination in Strong Magnetic Fields

    PubMed Central

    Zhang, Zhenyu; Benabbas, Abdelkrim; Ye, Xiong; Yu, Anchi; Champion, Paul M.

    2009-01-01

    Heme cooling signals and diatomic ligand recombination kinetics are measured in strong magnetic fields (up to 10 Tesla). We examined diatomic ligand recombination to heme model compounds (NO and CO), myoglobin (NO and O2), and horseradish peroxidase (NO). No magnetic field induced rate changes in any of the samples were observed within the experimental detection limit. However, in the case of CO binding to heme in glycerol and O2 binding to myoglobin, we observe a small magnetic field dependent change in the early time amplitude of the optical response that is assigned to heme cooling. One possibility, consistent with this observation, is that there is a weak magnetic field dependence of the non-radiative branching ratio into the vibrationally hot electronic ground state during CO photolysis. Ancillary studies of the “spin-forbidden” CO binding reaction in a variety of heme compounds in the absence of magnetic field demonstrate a surprisingly wide range for the Arrhenius prefactor. We conclude that CO binding to heme is not always retarded by unfavorable spin selection rules involving a double spin-flip superexchange mechanism. In fact, it appears that the small prefactor (~109s−1) found for CO rebinding to Mb may be anomalous, rather than the general rule for heme-CO rebinding. These results point to unresolved fundamental issues that underlie the theory of heme-ligand photolysis and rebinding. PMID:19588986

  4. A new generation of flowerlike horseradish peroxides as a nanobiocatalyst for superior enzymatic activity.

    PubMed

    Ocsoy, Ismail; Dogru, Esra; Usta, Seyda

    2015-01-01

    Although various supports including nanomaterials have been widely utilized as platforms for enzymes immobilization in order to enhance their catalytic activities, most of immobilized enzymes exhibited reduced activities compared to free enzymes. In this study, for the first time, we used iron ions (Fe(2+)) and horseradish peroxidase (HRP) enzyme together to synthesize flowerlike hybrid nanostructures with greatly enhanced activity and stability and reported an explanation of the enhancements in both catalytic activity and stability. We demonstrated that Fe(2+)-HRP hybrid nanoflower (HNF) showed catalytic activity of ∼ 512% and ∼ 710%, respectively when stored at +4 °C and room temperature (RT = 20 °C) compared to free HRP. In addition, the HNF stored at +4 °C lost only 2.9% of its original activity within 30 days while the HNF stored at RT lost approximately 10% of its original activity. However, under the same conditions, free HRP enzymes stored at +4 °C and RT lost 68% and 91% of their activities, respectively. We claim that the drastic increases in activities of HNF are associated with to high local HRP concentration in nanoscale dimension, appropriate HRP conformation, less mass transfer limitations, and role of Fe(2+) ion as an activator for HRP. Further biosensors studies based on enhanced activity and stability of HNF are currently underway. PMID:26047912

  5. Antimicrobial activities of phenethyl isothiocyanate isolated from horseradish.

    PubMed

    Chen, Hongxia; Wang, Chengzhang; Ye, Jianzhong; Zhou, Hao; Chen, Xijuan

    2012-01-01

    Phenethyl isothiocyanate (PEITC) was obtained from horseradish. The preparation procedure was as follows: the horseradish powder was hydrolysed in the water first, and then, after filtration, the residue was extracted by petroleum ether; finally, PEITC was isolated by silica gel column. The structure of PEITC was identified by IR, MS, ¹H-NMR and ¹³C-NMR chromatography methods. The inhibitory activities of PEITC against Gibberella zeae, Xanthomonas axonopodis pv . citri, Cytospora sp . and Phytophthora capsisi showed that PEITC had good inhibition effects. The EC₅₀ values of G. zeae, X. axonopodis pv . citri, Cytospora sp . and P. capsisi were 13.92, 1.20, 0.73 and 3.69 µg mL⁻¹, respectively. PMID:21815843

  6. Molecular Phylogeny of Heme Peroxidases

    NASA Astrophysics Data System (ADS)

    Zámocký, Marcel; Obinger, Christian

    All currently available gene sequences of heme peroxidases can be phylogenetically divided in two superfamilies and three families. In this chapter, the phylogenetics and genomic distribution of each group are presented. Within the peroxidase-cyclooxygenase superfamily, the main evolutionary direction developed peroxidatic heme proteins involved in the innate immune defense system and in biosynthesis of (iodinated) hormones. The peroxidase-catalase superfamily is widely spread mainly among bacteria, fungi, and plants, and particularly in Class I led to the evolution of bifunctional catalase-peroxidases. Its numerous fungal representatives of Class II are involved in carbon recycling via lignin degradation, whereas Class III secretory peroxidases from algae and plants are included in various forms of secondary metabolism. The family of di-heme peroxidases are predominantly bacteria-inducible enzymes; however, a few corresponding genes were also detected in archaeal genomes. Four subfamilies of dyp-type peroxidases capable of degradation of various xenobiotics are abundant mainly among bacteria and fungi. Heme-haloperoxidase genes are widely spread among sac and club fungi, but corresponding genes were recently found also among oomycetes. All described families herein represent heme peroxidases of broad diversity in structure and function. Our accumulating knowledge about the evolution of various enzymatic functions and physiological roles can be exploited in future directed evolution approaches for engineering peroxidase genes de novo for various demands.

  7. Antioxidant Capacity of Poly(Ethylene Glycol) (PEG) as Protection Mechanism Against Hydrogen Peroxide Inactivation of Peroxidases.

    PubMed

    Juarez-Moreno, Karla; Ayala, Marcela; Vazquez-Duhalt, Rafael

    2015-11-01

    The ability of poly(ethylene glycol) (PEG) to protect enzymatic peroxidase activity was determined for horseradish peroxidase (HRP), versatile peroxidase (VP), commercial Coprinus peroxidase (BP), and chloroperoxidase (CPO). The operational stability measured as the total turnover number was determined for the four peroxidases. The presence of PEG significantly increased the operational stability of VP and HRP up to 123 and 195%, respectively, and dramatically increased the total turnover number of BP up to 597%. Chloroperoxidase was not protected by PEG, which may be due to the different oxidation mechanism, in which the oxidation is mediated by hypochlorous ion instead of free radicals as in the other peroxidases. The presence of PEG does not protect the enzyme when incubated only in the presence of H2O2 without reducing substrate. The catalytic constants (k cat) are insensitive to the presence of PEG, suggesting that the protection mechanism is not due to a competition between the PEG and the substrate as electron donors. On the other hand, PEG showed to have a significant antioxidant capacity. Thus, we conclude that the protection mechanism for peroxidases of PEG is based in its antioxidant capacity with which it is able scavenge or drain radicals that are harmful to the protein. PMID:26306530

  8. Impurity-induced peroxidase mimicry of nanoclay and its potential for the spectrophotometric determination of cholesterol.

    PubMed

    Aneesh, K; Vusa, Chiranjeevi Srinivasa Rao; Berchmans, Sheela

    2016-09-01

    A green version of the "Fe" impurity-induced peroxidase mimicry exhibited by simple and cheap substrate "nanoclay (NC)" along with the highly sensitive amperometric and spectrophotometric determination of cholesterol is demonstrated. The "Fe" impurity can act as the catalyst center for hydrogen peroxide reduction similar to the horseradish peroxidase (HRP)-catalyzed reaction. The Michaelis-Menten constant for the NC-catalyzed reaction is found to be lower than that of the HRP-catalyzed reaction indicating high affinity for the substrate. The NC-modulated peroxidase-like catalytic activity originates from the electron transfer between the reducing substrate in the catalyst center and H2O2 with the intermediate generation of hydroxyl radicals. The peroxidase mimicry is successfully applied for the low-potential electrochemical detection of H2O2 (linear detection range 1.96-10.71 mM, R (2) = 0.97). The H2O2 sensing platform is further modified with cholesterol oxidase (CHOx) for the spectrophotometric (linear detection range 50-244 μM, R (2) = 0.99) and amperometric detection of cholesterol (linear detection range 0.099-1.73 mM, R (2) = 0.998). Graphical abstract Peroxidase mimicry of nanoclay for the determination of cholesterol. PMID:27392749

  9. Bienzyme biosensors for glucose, ethanol and putrescine built on oxidase and sweet potato peroxidase.

    PubMed

    Castillo, Jaime; Gáspár, Szilveszter; Sakharov, Ivan; Csöregi, Elisabeth

    2003-05-01

    Amperometric biosensors for glucose, ethanol, and biogenic amines (putrescine) were constructed using oxidase/peroxidase bienzyme systems. The H(2)O(2) produced by the oxidase in reaction with its substrate is converted into a measurable signal via a novel peroxidase purified from sweet potato peels. All developed biosensors are based on redox hydrogels formed of oxidases (glucose oxidase, alcohol oxidase, or amine oxidase) and the newly purified sweet potato peroxidase (SPP) cross-linked to a redox polymer. The developed electrodes were characterized (sensitivity, stability, and performances in organic medium) and compared with similarly built ones using the 'classical' horseradish peroxidase (HRP). The SPP-based electrodes displayed higher sensitivity and better detection limit for putrescine than those using HRP and were also shown to retain their activity in organic phase much better than the HPR based ones. The importance of attractive or repulsive electrostatic interactions between the peroxidases and oxidases (determined by their isoelectric points) were found to play an important role in the sensitivity of the obtained sensors. PMID:12706582

  10. Understanding the formation of CuS concave superstructures with peroxidase-like activity

    NASA Astrophysics Data System (ADS)

    He, Weiwei; Jia, Huimin; Li, Xiaoxiao; Lei, Yan; Li, Jing; Zhao, Hongxiao; Mi, Liwei; Zhang, Lizhi; Zheng, Zhi

    2012-05-01

    Copper sulfide (CuS) concave polyhedral superstructures (CPSs) have been successfully prepared in an ethanolic solution by a simple solvothermal reaction without the use of surfactants or templates. Two typical well defined, high symmetry CuS concave polyhedrons, forming a concave truncated cuboctahedron and icosahedron were prepared. The effect of the reaction time, temperature and different Cu ion and sulfur sources on the formation of CuS CPSs were investigated and a possible formation mechanism was proposed and discussed based on gas chromatography-mass spectrometry. More importantly, we found, for the first time, that the CuS CPSs exhibit intrinsic peroxidase-like activity, as they can quickly catalyze the oxidation of typical horseradish peroxidase (HRP) substrates, 3,3',5,5'-tetramethylbenzidine (TMB) and o-phenylenediamine (OPD), in the presence of hydrogen peroxide. In addition to the recent discoveries regarding peroxidase mimetics on Fe3O4 NPs and carbon nanostructures, our findings suggest a new kind of candidate for peroxidase mimics. This may open up a new application field of CuS micro-nano structures in biodetection, biocatalysis and environmental monitoring.Copper sulfide (CuS) concave polyhedral superstructures (CPSs) have been successfully prepared in an ethanolic solution by a simple solvothermal reaction without the use of surfactants or templates. Two typical well defined, high symmetry CuS concave polyhedrons, forming a concave truncated cuboctahedron and icosahedron were prepared. The effect of the reaction time, temperature and different Cu ion and sulfur sources on the formation of CuS CPSs were investigated and a possible formation mechanism was proposed and discussed based on gas chromatography-mass spectrometry. More importantly, we found, for the first time, that the CuS CPSs exhibit intrinsic peroxidase-like activity, as they can quickly catalyze the oxidation of typical horseradish peroxidase (HRP) substrates, 3

  11. Carboxylic-group-functionalized single-walled carbon nanohorns as peroxidase mimetics and their application to glucose detection.

    PubMed

    Zhu, Shuyun; Zhao, Xian-En; You, Jinmao; Xu, Guobao; Wang, Hua

    2015-09-21

    Carboxylic-group-functionalized single-walled carbon nanohorns (SWCNHs-COOH) have been found to possess peroxidase-like activity for the first time. Similar to natural peroxidase, SWCNHs-COOH can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine by H2O2 to produce a blue color solution. Compared with horseradish peroxidase, SWCNHs-COOH exhibit higher activity and stability under harsh reaction conditions. The catalytic activity of SWCNHs-COOH depends on the concentration of H2O2. A colorimetric method for glucose detection was developed by combining the SWCNH-COOH catalytic reaction and the generation of H2O2 by the enzymatic oxidation of glucose with glucose oxidase. Taking into account the advantages of good stability, high biocompatibility in aqueous solutions, being metal-catalyst free, and high purity, SWCNHs-COOH are expected to have potential applications in biotechnology and clinical diagnostics as enzymatic mimics. PMID:26247806

  12. Chemoselective Oxidative Polymerization of m-Ethynylphenol by Peroxidase Catalyst to a New Reactive Polyphenol.

    PubMed

    Tonami, Hiroyuki; Uyama, Hiroshi; Kobayashi, Shiro; Fujita, Takayuki; Taguchi, Yoshihiro; Osada, Katsuhisa

    2000-06-13

    Enzymatic oxidative polymerization of m-ethynylphenol possessing two reactive groups, phenol and acetylene moieties, was carried out in aqueous methanol under air. Horseradish peroxidase and hydrogen peroxide were used as catalyst and oxidizing agent, respectively. (1)H NMR and IR analysis showed that only the phenolic moiety was polymerized to produce the polymer having the ethynyl group in the side chain. The reaction of the monomer using a copper/amine catalyst, a conventional catalyst for oxidative coupling, exclusively produced a diacetylene derivative. From these data, it was found that the peroxidase catalysis induced the chemoselective polymerization of the monomer. The resulting polymer was converted to carbonized polymer in a much higher yield than enzymatically synthesized poly(m-cresol) and is expected to have potential applications as a reactive starting polymer. PMID:11749146

  13. Chemoselective oxidative polymerization of m-ethynylphenol by peroxidase catalyst to a new reactive polyphenol.

    PubMed

    Tonami, H; Uyama, H; Kobayashi, S; Fujita, T; Taguchi, Y; Osada, K

    2000-01-01

    Enzymatic oxidative polymerization of m-ethynylphenol possessing two reactive groups, phenol and acetylene moieties, was carried out in aqueous methanol under air. Horseradish peroxidase and hydrogen peroxide were used as catalyst and oxidizing agent, respectively. 1H NMR and IR analysis showed that only the phenolic moiety was polymerized to produce the polymer having the ethynyl group in the side chain. The reaction of the monomer using a copper/amine catalyst, a conventional catalyst for oxidative coupling, exclusively produced a diacetylene derivative. From these data, it was found that the peroxidase catalysis induced the chemoselective polymerization of the monomer. The resulting polymer was converted to carbonized polymer in a much higher yield than enzymatically synthesized poly(m-cresol) and is expected to have potential applications as a reactive starting polymer. PMID:11710092

  14. Phenylbutazone Oxidation via Cu,Zn-SOD Peroxidase Activity: An EPR Study.

    PubMed

    Aljuhani, Naif; Whittal, Randy M; Khan, Saifur R; Siraki, Arno G

    2015-07-20

    We investigated the effect of Cu,Zn-superoxide dismutase (Cu,Zn-SOD)-peroxidase activity on the oxidation of the nonsteroidal anti-inflammatory drug phenylbutazone (PBZ). We utilized electron paramagnetic resonance (EPR) spectroscopy to detect free radical intermediates of PBZ, UV-vis spectrophotometry to monitor PBZ oxidation, oxygen analysis to determine the involvement of C-centered radicals, and LC/MS to determine the resulting metabolites. Using EPR spectroscopy and spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), we found that the spin adduct of CO3(•-) (DMPO/(•)OH) was attenuated with increasing PBZ concentrations. The resulting PBZ radical, which was assigned as a carbon-centered radical based on computer simulation of hyperfine splitting constants, was trapped by both DMPO and MNP spin traps. Similar to Cu,Zn-SOD-peroxidase activity, an identical PBZ carbon-centered radical was also detected with the presence of both myeloperoxidase (MPO/H2O2) and horseradish peroxidase (HRP/H2O2). Oxygen analysis revealed depletion in oxygen levels when PBZ was oxidized by SOD peroxidase-activity, further supporting carbon radical formation. In addition, UV-vis spectra showed that the λmax for PBZ (λ = 260 nm) declined in intensity and shifted to a new peak that was similar to the spectrum for 4-hydroxy-PBZ when oxidized by Cu,Zn-SOD-peroxidase activity. LC/MS evidence supported the formation of 4-hydroxy-PBZ when compared to that of a standard, and 4-hydroperoxy-PBZ was also detected in significant yield. These findings together indicate that the carbonate radical, a product of SOD peroxidase activity, appears to play a role in PBZ metabolism. Interestingly, these results are similar to findings from heme peroxidase enzymes, and the context of this metabolic pathway is discussed in terms of a mechanism for PBZ-induced toxicity. PMID:26090772

  15. Crystal structure and statistical coupling analysis of highly glycosylated peroxidase from royal palm tree (Roystonea regia).

    PubMed

    Watanabe, Leandra; de Moura, Patricia Ribeiro; Bleicher, Lucas; Nascimento, Alessandro S; Zamorano, Laura S; Calvete, Juan J; Sanz, Libia; Pérez, Alicia; Bursakov, Sergey; Roig, Manuel G; Shnyrov, Valery L; Polikarpov, Igor

    2010-02-01

    Royal palm tree peroxidase (RPTP) is a very stable enzyme in regards to acidity, temperature, H(2)O(2), and organic solvents. Thus, RPTP is a promising candidate for developing H(2)O(2)-sensitive biosensors for diverse applications in industry and analytical chemistry. RPTP belongs to the family of class III secretory plant peroxidases, which include horseradish peroxidase isozyme C, soybean and peanut peroxidases. Here we report the X-ray structure of native RPTP isolated from royal palm tree (Roystonea regia) refined to a resolution of 1.85A. RPTP has the same overall folding pattern of the plant peroxidase superfamily, and it contains one heme group and two calcium-binding sites in similar locations. The three-dimensional structure of RPTP was solved for a hydroperoxide complex state, and it revealed a bound 2-(N-morpholino) ethanesulfonic acid molecule (MES) positioned at a putative substrate-binding secondary site. Nine N-glycosylation sites are clearly defined in the RPTP electron-density maps, revealing for the first time conformations of the glycan chains of this highly glycosylated enzyme. Furthermore, statistical coupling analysis (SCA) of the plant peroxidase superfamily was performed. This sequence-based method identified a set of evolutionarily conserved sites that mapped to regions surrounding the heme prosthetic group. The SCA matrix also predicted a set of energetically coupled residues that are involved in the maintenance of the structural folding of plant peroxidases. The combination of crystallographic data and SCA analysis provides information about the key structural elements that could contribute to explaining the unique stability of RPTP. PMID:19854274

  16. Development of an amperometric biosensor based on peroxidases to quantify citrinin in rice samples.

    PubMed

    Zachetti, Vanesa Gimena Lourdes; Granero, Adrian Marcelo; Robledo, Sebastián Noel; Zon, María Alicia; Fernández, Héctor

    2013-06-01

    An amperometric biosensor based on horseradish peroxidase (EC1.11.1.7,H2O2-oxide-reductases) to determine the content of citrinin mycotoxin in rice samples is proposed by the first time. The method uses carbon paste electrodes filled up with multi-walled carbon nanotubes embedded in a mineral oil, horseradish peroxidase, and ferrocene as a redox mediator. The biosensor is covered externally with a dialysis membrane, which is fixed to the body side of the electrode with a Teflon laboratory film, and an O-ring. The reproducibility and the repeatability were of 7.0% and 3.0%, respectively, showing a very good biosensor performance. The calibration curve was linear in a concentration range from 1 to 11.6nM. The limits of detection and quantification were 0.25nM and 0.75nM, respectively. For comparison, the citrinin content in rice samples was also determined by fluorimetric measurements. A very good correlation was obtained between the electrochemical and spectrophotometric methods. PMID:23416359

  17. Enzymatic decolourisation of Methyl Orange and Bismarck Brown using crude peroxidase from Armoracia rusticana

    NASA Astrophysics Data System (ADS)

    Ambatkar, Mugdha; Mukundan, Usha

    2015-12-01

    The decolourisation of Methyl Orange (MO) and Bismarck Brown (BB) by crude peroxidase from Armoracia rusticana (Horseradish) was studied by varying different reaction parameters. The pH of the reaction mixture, initial dye concentration, amount of enzyme and hydrogen peroxide concentration were optimised for ambient temperatures (30 ± 2 °C). The optimum pH for decolourisation was 4.0 (72.95 %) and 3.0 (79.24 %) for MO and BB, respectively. Also it was found that the Chemical Oxygen Demand of the enzyme-treated sample was significantly lower than that of the untreated controls for both dyes. The addition of a complex iron salt like Ferric EDTA was found to enhance the decolourisation of both dyes at pH 6.0, showing an increase of 8.69 % and 14.17 % in the decolourisation of MO and of BB, respectively. The present study explores the potential of crude peroxidase from horseradish to decolourise representative monoazo and diazo dyes, MO and BB, respectively. An attempt has been made to utilise a crude enzyme with appreciable activity obtained after minimal processing for the decolourisation of the aforesaid dyes. The findings of this study would find application in the enzymatic treatment of wastewater containing azo dyes.

  18. Understanding the formation of CuS concave superstructures with peroxidase-like activity.

    PubMed

    He, Weiwei; Jia, Huimin; Li, Xiaoxiao; Lei, Yan; Li, Jing; Zhao, Hongxiao; Mi, Liwei; Zhang, Lizhi; Zheng, Zhi

    2012-06-01

    Copper sulfide (CuS) concave polyhedral superstructures (CPSs) have been successfully prepared in an ethanolic solution by a simple solvothermal reaction without the use of surfactants or templates. Two typical well defined, high symmetry CuS concave polyhedrons, forming a concave truncated cuboctahedron and icosahedron were prepared. The effect of the reaction time, temperature and different Cu ion and sulfur sources on the formation of CuS CPSs were investigated and a possible formation mechanism was proposed and discussed based on gas chromatography-mass spectrometry. More importantly, we found, for the first time, that the CuS CPSs exhibit intrinsic peroxidase-like activity, as they can quickly catalyze the oxidation of typical horseradish peroxidase (HRP) substrates, 3,3',5,5'-tetramethylbenzidine (TMB) and o-phenylenediamine (OPD), in the presence of hydrogen peroxide. In addition to the recent discoveries regarding peroxidase mimetics on Fe(3)O(4) NPs and carbon nanostructures, our findings suggest a new kind of candidate for peroxidase mimics. This may open up a new application field of CuS micro-nano structures in biodetection, biocatalysis and environmental monitoring. PMID:22552534

  19. Oxidase-peroxidase enzymes of Datura innoxia. Oxidation of formylphenylacetic acid ethyl ester.

    PubMed Central

    Kalyanaraman, V S; Mahadevan, S; Kumar, S A

    1975-01-01

    An enzyme system from Datura innoxia roots oxidizing formylphenylacetic acid ethyl ester was purified 38-fold by conventional methods such as (NH4)2SO4 fractionation, negative adsorption on alumina Cy gel and chromatography on DEAE-cellulose. The purified enzyme was shown to catalyse the stoicheiometric oxidation of formylphenylacetic acid ethyl ester to benzoylformic acid ethyl ester and formic acid, utilizing molecular O2. Substrate analogues such as phenylacetaldehyde and phenylpyruvate were oxidized at a very low rate, and formylphenylacetonitrile was an inhilating agents, cyanide, thiol compounds and ascorbic acid. This enzyme was identical with an oxidase-peroxidase isoenzyme. Another oxidase-peroxidase isoenzyme which separated on DEAE-chromatography also showed formylphenylacetic acid ethyl ester oxidase activity, albeit to a lesser extent. The properties of the two isoenzymes of the oxidase were compared and shown to differ in their oxidation and peroxidation properties. The oxidation of formylphenylacetic acid ethyl ester was also catalysed by horseradish peroxidase. The Datura isoenzymes exhibited typical haemoprotein spectra. The oxidation of formylphenylacetic acid ethyl ester was different from other peroxidase-catalysed reactions in not being activated by either Mn2+ or monophenols. The oxidation was inhibited by several mono- and poly-phenols and by catalase. A reaction mechanism for the oxidation is proposed. PMID:997

  20. Degradation of textile dyes using immobilized lignin peroxidase-like metalloporphines under mild experimental conditions

    PubMed Central

    2012-01-01

    Background Synthetic dyes represent a broad and heterogeneous class of durable pollutants, that are released in large amounts by the textile industry. The ability of two immobilized metalloporphines (structurally emulating the ligninolytic peroxidases) to bleach six chosen dyes (alizarin red S, phenosafranine, xylenol orange, methylene blue, methyl green, and methyl orange) was compared to enzymatic catalysts. To achieve a green and sustainable process, very mild conditions were chosen. Results IPS/MnTSPP was the most promising biomimetic catalyst as it was able to effectively and quickly bleach all tested dyes. Biomimetic catalysis was fully characterized: maximum activity was centered at neutral pH, in the absence of any organic solvent, using hydrogen peroxide as the oxidant. The immobilized metalloporphine kept a large part of its activity during multi-cycle use; however, well-known redox mediators were not able to increase its catalytic activity. IPS/MnTSPP was also more promising for use in industrial applications than its enzymatic counterparts (lignin peroxidase, laccase, manganese peroxidase, and horseradish peroxidase). Conclusions On the whole, the conditions were very mild (standard pressure, room temperature and neutral pH, using no organic solvents, and the most environmental-friendly oxidant) and a significant bleaching and partial mineralization of the dyes was achieved in approximately 1 h. Therefore, the process was consistent with large-scale applications. The biomimetic catalyst also had more promising features than the enzymatic catalysts. PMID:23256784

  1. Distribution of primary cochlear afferents in the bulbar nuclei of the rat: a horseradish peroxidase (HRP) study in parasagittal sections.

    PubMed Central

    Merchan, M A; Collia, F P; Merchan, J A; Ludeña, M D

    1986-01-01

    HRP was injected into the cochleae of 25 young albino rats in order to trace the primary afferents to the bulbar cochlear nuclei. Besides the classic V-shaped pattern and unconnected with it, HRP labelling revealed two plexuses stemming directly from the axons of the cochlear root. The plexuses cover the posterior area of the posteroventral cochlear nucleus (posterior plexus) and the anterolaterodorsal area of the anteroventral cochlear nucleus (anterior plexus). The fibres giving rise to these two plexuses were previously grouped in two bundles which have been called the posterior and anterior bundles, respectively. The origin of the anterior bundle is typically seen with the fibres stemming out at right angles; the origin and course of the posterior bundle, which characteristically cross over, is also a typical feature. Images Fig. 1 Figs. 2-3 (cont.) Figs. 2-3 Fig. 4 PMID:3319993

  2. Apoferritin Protein Nanoparticles Dually labeled with Aptamer and Horseradish Peroxidase as a Sensing Probe for Thrombin Detection

    SciTech Connect

    Zhao, Jie; Liu, Meiling; Zhang, Youyu; Li, Haitao; Lin, Yuehe; Yao, Shouzhuo

    2013-01-08

    A sandwich-type electrochemical aptasensor has been developed for the detection of thrombin, based on dual signal-amplification using HRP and apoferritin. Aptamer1 (Apt1) loaded on core/shell Fe3O4/Au magnetic nanoparticle (AuMNP) was used as recognition elements, and apoferritin dually labeled with Aptamer2 (Apt2) and HRP was used as a detection probe. Sandwich-type complex, Apt1/thrombin/Apt2–apoferritin NPs–HRP was formed by the affinity reactions between AuMNPs–Apt1, thrombin, and Apt2–apoferritin–HRP. The complex was anchored on a screen-printed carbon electrode (SPCE). Differential pulse voltammetry (DPV) was used to monitor the electrode response. The proposed aptasensor yielded a linear current response to thrombin concentrations over a broad range of 0.5 pM to 100 pM with a detection limit of 0.07 pM (S/N = 3). The detection signal was amplified by using apoferritin and HRP. This nanoparticle-based aptasensor offers a new method for rapid, sensitive, selective, and inexpensive quantification of thrombin, and offers a promising potential in biomarker detection and disease diagnosis. --------------------------------------------------------------------------------

  3. A Stable Bacterial Peroxidase with Novel Halogenating Activity and an Autocatalytically Linked Heme Prosthetic Group*

    PubMed Central

    Auer, Markus; Gruber, Clemens; Bellei, Marzia; Pirker, Katharina F.; Zamocky, Marcel; Kroiss, Daniela; Teufer, Stefan A.; Hofbauer, Stefan; Soudi, Monika; Battistuzzi, Gianantonio; Furtmüller, Paul G.; Obinger, Christian

    2013-01-01

    Reconstructing the phylogenetic relationships of the main evolutionary lines of the mammalian peroxidases lactoperoxidase and myeloperoxidase revealed the presence of novel bacterial heme peroxidase subfamilies. Here, for the first time, an ancestral bacterial heme peroxidase is shown to possess a very high bromide oxidation activity (besides conventional peroxidase activity). The recombinant protein allowed monitoring of the autocatalytic peroxide-driven formation of covalent heme to protein bonds. Thereby, the high spin ferric rhombic heme spectrum became similar to lactoperoxidase, the standard reduction potential of the Fe(III)/Fe(II) couple shifted to more positive values (−145 ± 10 mV at pH 7), and the conformational and thermal stability of the protein increased significantly. We discuss structure-function relationships of this new peroxidase in relation to its mammalian counterparts and ask for its putative physiological role. PMID:23918925

  4. Molecular characterization of the lignin-forming peroxidase: Role in growth, development and response to stress. Progress summary report, April 1, 1993--March 31, 1994

    SciTech Connect

    Lagrimini, L.M.

    1994-05-01

    Our group continues to focus on the characterization of the tobacco anionic peroxidase and its genes. Throughout this past year we have generated transgenic plants expressing {beta}-glucuronidase under control of the anionic peroxidase promoter, characterized effectors of peroxidase gene expression in transformed protoplasts, generated numerous transgenic plants which over- and under-express the anionic peroxidase in a tissue specific manner, characterized the role of the anionic peroxidase in the metabolism of auxin, introduced a marker (flag) into the anionic peroxidase primary protein sequence which will permit the identification of the recombinant protein in plant tissue, and described the enhancement of insect resistance as a result of over-expression of the anionic peroxidase. Although our research program has continued along the lines of the original proposal, we have redirected a significant effort to the role which this enzyme plays in the metabolism of auxin, and conversely, the role which auxin plays in regulating the expression of the anionic peroxidase gene.

  5. Phytochelatin homologs induced in hairy roots of horseradish.

    PubMed

    Kubota, H; Sato, K; Yamada, T; Maitani, T

    2000-01-01

    When exposed to excess heavy metals, plants induce phytochelatins and related peptides (all designated as PCAs). Thus, when hairy roots of horseradish (Armoracia rusticana) were exposed for 3 days to cadmium (1 mM) along with reduced glutathione (2 mM), PCA induction occurred. Moreover, a new family of thiol peptides was detected as well as the previously known PCAs, as revealed by postcolumn-derivatization HPLC. Two were isolated and their structures were identified as (gamma-Glu-Cys)n-Gln (n = 3 and 4) by electrospray ionization-mass spectrometer spectra, this being confirmed by chemical synthesis of the peptides. These new analogs constitute the sixth PCA family identified to date. PMID:10680177

  6. Model study of the enzymatic modification of natural extracts: peroxidase-based removal of eugenol from rose essential oil.

    PubMed

    Bouhlel, Charfeddine; Dolhem, Gwenn'Ann; Fernandez, Xavier; Antoniotti, Sylvain

    2012-02-01

    A protocol based on the use of horseradish peroxidase (HRP) is proposed for the removal of allergenic eugenol from rose essential oil without loss of the organoleptic quality and with a good conservation of the chemical composition. For the first time, an enzyme-based strategy is proposed for essential oils treatment and opens new opportunities in the detoxification of natural extracts used in perfumery and cosmetics. Our results on eugenol in rose essential oil constitute a first step toward the development of efficient and mild processes for the removal of more toxic compounds of natural extracts. PMID:22224510

  7. Structure and applications of a temperature responsive recombinant protein hydrogel based on silk- and elastin-like amino acid motifs

    NASA Astrophysics Data System (ADS)

    Drummy, Lawrence; Tomczak, Melanie; Macauliffe, Joseph; Vaia, Richard; Naik, Rajesh

    2008-03-01

    Proteins form the main components of many natural materials, and they can be designed to offer tailored functionality and material properties. Silk elastin-like proteins (SELP)s come from a family of repeat sequence protein polymers based on Bombyx mori silk and mammalian elastin that are recombinantly expressed in E. coli. SELP gels are formed by heating the protein solutions in order to induce physical crosslinking of the silk β-sheet regions, they contain approximately 80-90% water by weight and they can be used for encapsulation of enzymes or nanoparticles. For example, horseradish peroxidase demonstrates added resistance to drying and heat treatment when encapsulated in the gel matrix. During gel formation, small angle X-ray scattering shows intensity increases in two distinct regions of reciprocal space, one reversible with temperature and one irreversible. By fitting the scattering data to a unified power-law/Gunier model, morphological parameters are extracted. The thermally reversible intensity changes are attributed to a hydrophilic/hydrophobic transition in the elastin segments, while the irreversible intensity change is due to the crystalline regions formed by the silk blocks.

  8. Recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibody to turkey coronavirus.

    PubMed

    Abdelwahab, Mohamed; Loa, Chien Chang; Wu, Ching Ching; Lin, Tsang Long

    2015-06-01

    Nucleocapsid (N) protein gene of turkey coronavirus (TCoV) was expressed in a prokaryotic system and used to develop an enzyme-linked immunosorbent assay (ELISA) for detection of antibody to TCoV. Anti-TCoV hyperimmune turkey serum and normal turkey serum were used as positive or negative controls for optimization of the ELISA. Goat anti-turkey IgG (H+L) conjugated with horseradish peroxidase was used as detector antibody. Three hundred and twenty two turkey sera from the field were used to evaluate the performance of ELISA and determine the cut-off point of ELISA. The established ELISA was also examined with serum samples obtained from turkeys experimentally infected with TCoV. Those serum samples were collected at various time intervals from 1 to 63 days post-infection. The optimum conditions for differentiation between anti-TCoV hyperimmune serum and normal turkey serum were recombinant TCoV N protein concentration at 20 μg/ml, serum dilution at 1:800, and conjugate dilution at 1:10,000. Of the 322 sera from the field, 101 were positive for TCoV by immunofluorescent antibody assay (IFA). The sensitivity and specificity of the ELISA relative to IFA test were 86.0% and 96.8%, respectively, using the optimum cut-off point of 0.2 as determined by logistic regression method. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the recombinant N protein coated on the ELISA plates was not detected. These results indicated that the established antibody-capture ELISA in conjunction with recombinant TCoV N protein as the coating protein can be utilized for detection of antibodies to TCoV in turkey flocks. PMID:25745958

  9. Molecular Characterization of a Novel Peroxidase from the Cyanobacterium Anabaena sp. Strain PCC 7120 ▿

    PubMed Central

    Ogola, Henry Joseph Oduor; Kamiike, Takaaki; Hashimoto, Naoya; Ashida, Hiroyuki; Ishikawa, Takahiro; Shibata, Hitoshi; Sawa, Yoshihiro

    2009-01-01

    The open reading frame alr1585 of Anabaena sp. strain PCC 7120 encodes a heme-dependent peroxidase (Anabaena peroxidase [AnaPX]) belonging to the novel DyP-type peroxidase family (EC 1.11.1.X). We cloned and heterologously expressed the active form of the enzyme in Escherichia coli. The purified enzyme was a 53-kDa tetrameric protein with a pI of 3.68, a low pH optima (pH 4.0), and an optimum reaction temperature of 35°C. Biochemical characterization revealed an iron protoporphyrin-containing heme peroxidase with a broad specificity for aromatic substrates such as guaiacol, 4-aminoantipyrine and pyrogallol. The enzyme efficiently catalyzed the decolorization of anthraquinone dyes like Reactive Blue 5, Reactive Blue 4, Reactive Blue 114, Reactive Blue 119, and Acid Blue 45 with decolorization rates of 262, 167, 491, 401, and 256 μM·min−1, respectively. The apparent Km and kcat/Km values for Reactive Blue 5 were 3.6 μM and 1.2 × 107 M−1 s−1, respectively, while the apparent Km and kcat/Km values for H2O2 were 5.8 μM and 6.6 × 106 M−1 s−1, respectively. In contrast, the decolorization activity of AnaPX toward azo dyes was relatively low but was significantly enhanced 2- to ∼50-fold in the presence of the natural redox mediator syringaldehyde. The specificity and catalytic efficiency for hydrogen donors and synthetic dyes show the potential application of AnaPX as a useful alternative of horseradish peroxidase or fungal DyPs. To our knowledge, this study represents the only extensive report in which a bacterial DyP has been tested in the biotransformation of synthetic dyes. PMID:19801472

  10. Copper nanoclusters as peroxidase mimetics and their applications to H2O2 and glucose detection.

    PubMed

    Hu, Lianzhe; Yuan, Yali; Zhang, Ling; Zhao, Jianming; Majeed, Saadat; Xu, Guobao

    2013-01-31

    Copper nanoclusters (Cu NCs) are found to possess intrinsic peroxidase-like activity for the first time. Similar to nature peroxidase, they can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine by H(2)O(2) to produce a nice blue color reaction. Compared with horseradish peroxidase, Cu NCs exhibits higher activity near neutral pH, which is beneficial for biological applications. The increase in absorbance caused by the Cu NCs catalytic reaction allows the detection of H(2)O(2) in the range of 10 μM to 1 mM with a detection limit of 10 μM. A colorimetric method for glucose detection was also developed by combining the Cu NCs catalytic reaction and the enzymatic oxidation of glucose with glucose oxidase. Taking into account the advantages of ultra-small size, good stability, and high biocompatibility in aqueous solutions, Cu NCs are expected to have potential applications in biotechnology and clinical diagnosis as enzymatic mimics. PMID:23327949

  11. Anionic polythiophene derivative as peroxidase mimetics and their application for detection of hydrogen peroxide and glucose.

    PubMed

    Liu, Mei; Li, Baoxin; Cui, Xiang

    2013-10-15

    In this paper, we discovered that the anionic polythiophenes derivative, poly[2-(3-thienyl)ethyloxy-4-butylsulfonate] (PTEBS), possesses intrinsic peroxidase-like activity that can catalyze the reaction of peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2 to produce a blue color reaction, which provides colorimetric detection of H2O2. PTEBS exhibits several advantages such as high catalytic efficiency, good stability, and rapid response over horseradish peroxidase (HRP). By coupling the oxidation of glucose catalyzed by glucose oxidase, a simple, inexpensive, highly sensitive and selective colorimetric method for glucose detection has been developed. The absorbance was proportional to the concentration of glucose in the range from 0.01 to 0.5mM with a detection limit of 0.004 mM. This work is not only of importance for a better understanding of the unique properties of polythiophenes derivative but also of great potential for medical diagnostics and biotechnology. PMID:24054671

  12. Purification and characterization of novel cationic peroxidases from Asparagus acutifolius L. with biotechnological applications.

    PubMed

    Guida, Vincenzo; Cantarella, Maria; Chambery, Angela; Mezzacapo, Maria C; Parente, Augusto; Landi, Nicola; Severino, Valeria; Di Maro, Antimo

    2014-08-01

    Four novel basic peroxidases, named AaP-1, AaP-2, AaP-3, and AaP-4, were purified from Asparagus acutifolius L. seeds by cation-exchange and gel filtration chromatographies. The four proteins showed a similar electrophoretic mobility of 46 kDa while, by MALDI-TOF MS, different Mr values of 42758.3, 41586.9, 42796.3, and 41595.5 were determined for AaP-1, AaP-2, AaP-3, and AaP-4, respectively. N-terminal sequences of AaPs 1-4 up to residue 20 showed a high percentage of identity with the peroxidase from Glycine max. In addition, AaP-1, AaP-2, AaP-3, and AaP-4 were found to be glycoproteins, containing 21.75, 22.27, 25.62, and 18.31 % of carbohydrates, respectively. Peptide mapping and MALDI-TOF MS analysis of AaPs 1-4 showed that the structural differences between AaP-1 and AaP-2 and AaP-3 and AaPs-4 were mainly due to their glycan content. We also demonstrate that AaPs were able to remove phenolic compounds from olive oil mill wastewaters with a higher catalytic efficiency with respect to horseradish peroxidase, thus representing candidate enzymes for potential biotechnological applications in the environmental field. PMID:24740695

  13. Regeneration of horseradish hairy roots incited by Agrobacterium rhizogenes infection.

    PubMed

    Noda, T; Tanaka, N; Mano, Y; Nabeshima, S; Ohkawa, H; Matsui, C

    1987-07-01

    Surface-sterilized leaf disks of horse-radish (Armoracia lapathifolia) were immersed in a suspension of Agrobacterium rhizogenes harboring the root-inducing plasmid (pRi) and cultured on a solid medium. Within about 10 days after inoculation, adventitious roots (hairy roots) emerged from the leaf disks. No roots emerged from the uninoculated leaf disks. The excised hairy roots grew vigorously in the dark and exhibited extensive lateral branches in the absence of phytohormones. When the hairy roots were moved into the light, numerous adventitious buds thrust out of the roots within about 10 days, and they developed into complete plants (R0 generation). R0 plants revealed leaf wrinkle. Root masses of cultured R0 plants were of two types. One had fibrous roots only and the other had both fibrous and tuberous roots Leaf disks of the R0 plants proliferated adventitious roots (R1 generation) on a solid medium after 1-2 weeks of culture. Phenotypical characters of the R1 roots were the same as those observed with the initial hairy roots. The T-DNA sequences of pRi were detected within DNA isolated from the hairy roots and their regenerants. PMID:24248760

  14. Peroxidase and peroxidase-oxidase activities of isolated human myeloperoxidases.

    PubMed Central

    Svensson, B E; Domeij, K; Lindvall, S; Rydell, G

    1987-01-01

    Isolated neutrophils from healthy donors were used for the isolation of four highly purified forms of myeloperoxidase as determined by spectral (A430/A280 ratio 0.80-0.87) and enzyme-activity measurements. Although the myeloperoxidases exhibited different elution profiles on cation-exchange chromatography, gel filtration indicated similar relative molecular masses. When these forms were assayed for peroxidase and peroxidase-oxidase activities with several substrates, they all exhibited virtually the same specific activities. These results suggest that possible functional differences between the enzymes may be related to differences in their sites of action rather than to differences in enzyme activity. Myeloperoxidase from a patient with chronic myeloid leukaemia also revealed a similar heterogeneity on cation-exchange chromatography. However, this myeloperoxidase contained in addition one form with a lower and one form with a higher relative molecular mass, as indicated by gel-filtration chromatography. PMID:3036098

  15. Peroxidase-encapsulated cyclodextrin nanosponge immunoconjugates as a signal enhancement tool in optical and electrochemical assays.

    PubMed

    Wajs, Ewelina; Caldera, Fabrizio; Trotta, Francesco; Fragoso, Alex

    2014-01-21

    Cyclodextrin nanosponges bearing carboxylate groups have been prepared by crosslinking β-cyclodextrin with pyromellitic dianhydride to form a carboxylic acid terminated nanoporous material. The surface of the particles was covalently modified with an anti-IgG antibody and then loaded with horseradish peroxidase. The structures of unmodified and protein modified nanosponge particles were investigated by Raman spectroscopy and imaging methods. Confocal microscopy indicates that the antibody is located in the outside of the particle while HRP is encapsulated in the inner part. The possibility to use these modified nanosponges as a signal enhancement tool in enzyme-linked colorimetric and electrochemical assays was evaluated using a sandwich format comprising immobilised gliadin as an antigen, a target anti-gliadin antibody and an anti-IgG antibody conjugated to the enzyme-loaded nanosponge immunoconjugates. PMID:24276364

  16. Bio-based Wrinkled Surfaces Harnessed from Biological Design Principles of Wood and Peroxidase Activity.

    PubMed

    Izawa, Hironori; Okuda, Noriko; Ifuku, Shinsuke; Morimoto, Minoru; Saimoto, Hiroyuki; Rojas, Orlando J

    2015-11-01

    A new and simple approach for surface wrinkling inspired by polymer assemblies in wood fibers is introduced. A hard skin is synthesized on a linear polysaccharide support that resembles the structural units of the cell wall. This skin, a wood mimetic layer, is produced through immersion in a solution containing phenolic precursor and subsequent surface reaction by horseradish peroxidase. A patterned surface with micron-scale wrinkles is formed upon drying and as a result of inhomogeneous shrinkage. We demonstrate that the design of the wrinkled surfaces can be controlled by the molecular structure of the phenolic precursor, temperature, and drying stress. It is noteworthy that this is a totally bio-based system involving green materials and processes. PMID:26489384

  17. Peroxidase induced oligo-tyrosine cross-links during polymerization of α-lactalbumin.

    PubMed

    Dhayal, Surender Kumar; Sforza, Stefano; Wierenga, Peter A; Gruppen, Harry

    2015-12-01

    Horseradish peroxidase (HRP) induced cross-linking of proteins has been reported to proceed through formation of di-tyrosine cross-links. In the case of low molar mass phenolic substrates, the enzymatic oxidation is reported to lead to polymerization of the phenols. The aim of this work was to investigate if during oxidative cross-linking of proteins oligo-tyrosine cross-links are formed in addition to dityrosine. To this end, α-lactalbumin (α-LA) was cross-linked using horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂). The reaction products were acid hydrolysed, after which the cross-linked amino acids were investigated by LC-MS and MALDI-MS. To test the effect of the size of the substrate, the cross-linking reaction was also performed with L-tyrosine, N-acetyl L-tyrosinamide and angiotensin. These products were analyzed by LC-MS directly, as well as after acid hydrolysis. In the acid hydrolysates of all samples oligo-tyrosine (Yn, n=3-8) was found in addition to di-tyrosine (Y2). Two stages of cross-linking of α-LA were identified: a) 1-2 cross-links were formed per monomer until the monomers were converted into oligomers, and b) subsequent cross-linking of oligomers formed in the first stage to form nanoparticles containing 3-4 cross-links per monomer. The transition from first stage to the second stage coincided with the point where di-tyrosine started to decrease and more oligo-tyrosines were formed. In conclusion, extensive polymerization of α-LA using HRP via oligo-tyrosine cross-links is possible, as is the case for low molar mass tyrosine containing substrates. PMID:26282909

  18. λ Recombination and Recombineering.

    PubMed

    Murphy, Kenan C

    2016-05-01

    The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics. PMID:27223821

  19. Structure-based discovery of the first non-covalent inhibitors of Leishmania major tryparedoxin peroxidase by high throughput docking

    PubMed Central

    Brindisi, Margherita; Brogi, Simone; Relitti, Nicola; Vallone, Alessandra; Butini, Stefania; Gemma, Sandra; Novellino, Ettore; Colotti, Gianni; Angiulli, Gabriella; Di Chiaro, Francesco; Fiorillo, Annarita; Ilari, Andrea; Campiani, Giuseppe

    2015-01-01

    Leishmaniasis is a neglected vector-born disease caused by a protozoan of the genus Leishmania and affecting more than 1.300.000 people worldwide. The couple tryparedoxin/tryparedoxin peroxidase is essential for parasite survival in the host since it neutralizes the hydrogen peroxide produced by macrophages during the infection. Herein we report a study aimed at discovering the first class of compounds able to non-covalently inhibit tryparedoxin peroxidase. We have solved the high-resolution structure of Tryparedoxin peroxidase I from Leishmania major (LmTXNPx) in the reduced state and in fully folded conformation. A first series of compounds able to inhibit LmTXNPx was identified by means of the high throughput docking technique. The inhibitory activity of these compounds was validated by a Horseradish peroxidase-based enzymatic assay and their affinity for LmTXNPx calculated by surface plasmon resonance experiments. On the basis of these results, the analysis of the enzyme-inhibitor docked models allowed us to rationally design and synthesize a series of N,N-disubstituted 3-aminomethyl quinolones. These compounds showed an inhibitory potency against LmTXNPx in the micromolar range. Among them, compound 12 represents the first non-covalent LmTXNPx inhibitor reported to date and could pave the way to the discovery of a new class of drugs against leishmaniasis. PMID:25951439

  20. Cationization of catalase, peroxidase, and superoxide dismutase. Effect of improved intraarticular retention on experimental arthritis in mice.

    PubMed Central

    Schalkwijk, J; van den Berg, W B; van de Putte, L B; Joosten, L A; van den Bersselaar, L

    1985-01-01

    Several enzymes and other proteins were made cationic either by coupling to polylysine or by shielding of anionic sites. These cationic proteins, all having an isoelectric point greater than 8.5 exhibited excellent retention in articular structures when injected in mouse knee joints. Autoradiography and histochemistry showed that cationic forms of catalase, superoxide dismutase, and horseradish peroxidase were firmly retained by synovial and cartilaginous tissues. The half-life of these enzymes in the joint is thus significantly extended compared with native enzymes. The native enzymes and their cationic derivatives were tested for antiinflammatory properties in mice, using antigen-induced arthritis and zymosan-induced arthritis. It was found that injection of cationic catalase or peroxidase induced a marked suppression of some parameters of the inflammatory response in both types of arthritis, as measured by 99m technetium pertechnetate uptake and leakage of 125I-labeled albumin. Native catalase and peroxidase were less, or not at all effective. Cationic superoxide dismutase or cationic nonenzyme proteins did not suppress inflammation. The observed suppression of two different types of inflammation (an immune and a nonimmune arthritis) by catalase and peroxidase suggests that elimination of peroxides contributes to the suppression of an inflammatory response. We would hypothesize that cationic enzymes offer the possibility for investigating the mechanisms of inflammation and, in addition, might be interesting from a therapeutical point of view. Images PMID:4019779

  1. Obtention of plant peroxidase and its potential for the decolorization of the reactive dye Remazol Turquoise G 133%.

    PubMed

    Silva, Maria Cristina; Torres, Juliana Arriel; Corrêa, Angelita Duarte; Junqueira, Allana Maria Bernardes; Amorim, Maria Teresa Pessoa; dos Santos, Custódio Donizete

    2012-01-01

    Peroxidases can be used in the decolorization process. There is a growing interest for new sources of this enzyme and for obtaining economically viable processes. In this work, a low-cost vegetable peroxidase extraction process is proposed; the resulting enzyme is characterized to determine its optimum pH, temperature, and stability conditions, and it is then applied in the decolorization of reactive dye Remazol Turquoise G 133%. The turnip peroxidase (TP) was utilized as an enzymatic source. This enzyme exhibited maximum activity at pH 7.0, and it was active in the temperature range of 30 to 50 °C, which favors its use in industrial processes. Acetone was the most efficient solvent to induce precipitation. The removal of Remazol Turquoise G 133% was 56.0% complete after 50 min, while 41.0% of the same dye was removed with the commercial horseradish peroxidase enzyme in 50 min. TP presents potential as a viable alternative in the decolorization of textile wastewaters. PMID:22277225

  2. Cyclometalated ruthenium(II) complexes as efficient redox mediators in peroxidase catalysis.

    PubMed

    Alpeeva, Inna S; Soukharev, Valentin S; Alexandrova, Larissa; Shilova, Nadezhda V; Bovin, Nicolai V; Csöregi, Elisabeth; Ryabov, Alexander D; Sakharov, Ivan Yu

    2003-07-01

    Cyclometalated ruthenium(II) complexes, [Ru(II)(C~N)(N~N)(2)]PF(6) [HC~N=2-phenylpyridine (Hphpy) or 2-(4'-tolyl)pyridine; N~N=2,2'-bipyridine, 1,10-phenanthroline, or 4,4'-dimethyl-2,2'-bipyridine], are rapidly oxidized by H(2)O(2) catalyzed by plant peroxidases to the corresponding Ru(III) species. The commercial isoenzyme C of horseradish peroxidase (HRP-C) and two recently purified peroxidases from sweet potato (SPP) and royal palm tree (RPTP) have been used. The most favorable conditions for the oxidation have been evaluated by varying the pH, buffer, and H(2)O(2) concentrations and the apparent second-order rate constants ( k(app)) have been measured. All the complexes studied are oxidized by HRP-C at similar rates and the rate constants k(app) are identical to those known for the best substrates of HRP-C (10(6)-10(7) M(-1) s(-1)). Both cationic (HRP-C) and anionic (SPP and RPTP) peroxidases show similar catalytic efficiency in the oxidation of the Ru(II) complexes. The mediating capacity of the complexes has been evaluated using the SPP-catalyzed co-oxidation of [Ru(II)(phpy)(bpy)(2)]PF(6) and catechol as a poor peroxidase substrate as an example. The rate of enzyme-catalyzed oxidation of catechol increases more than 10000-fold in the presence of the ruthenium complex. A simple routine for calculating the rate constant k(c) for the oxidation of catechol by the Ru(III) complex generated enzymatically from [Ru(II)(phpy)(bpy)(2)](+) is proposed. It is based on the accepted mechanism of peroxidase catalysis and involves spectrophotometric measurements of the limiting Ru(II) concentration at different concentrations of catechol. The calculated k(c) value of 0.75 M(-1) s(-1) shows that the cyclometalated Ru(II) complexes are efficient mediators in peroxidase catalysis. PMID:12774217

  3. Biosynthesis of N,N-dimethyltryptamine (DMT) in a melanoma cell line and its metabolization by peroxidases.

    PubMed

    Gomes, Melissa M; Coimbra, Janine B; Clara, Renan O; Dörr, Felipe A; Moreno, Ana Carolina R; Chagas, Jair R; Tufik, Sérgio; Pinto, Ernani; Catalani, Luiz H; Campa, Ana

    2014-04-01

    Tryptophan (TRP) is essential for many physiological processes, and its metabolism changes in some diseases such as infection and cancer. The most studied aspects of TRP metabolism are the kynurenine and serotonin pathways. A minor metabolic route, tryptamine and N,N-dimethyltryptamine (DMT) biosynthesis, has received far less attention, probably because of the very low amounts of these compounds detected only in some tissues, which has led them to be collectively considered as trace amines. In a previous study, we showed a metabolic interrelationship for TRP in melanoma cell lines. Here, we identified DMT and N,N-dimethyl-N-formyl-kynuramine (DMFK) in the supernatant of cultured SK-Mel-147 cells. Furthermore, when we added DMT to the cell culture, we found hydroxy-DMT (OH-DMT) and indole acetic acid (IAA) in the cell supernatant at 24 h. We found that SK-Mel-147 cells expressed mRNA for myeloperoxidase (MPO) and also had peroxidase activity. We further found that DMT oxidation was catalyzed by peroxidases. DMT oxidation by horseradish peroxidase, H2O2 and MPO from PMA-activated neutrophils produced DMFK, N,N-dimethyl-kynuramine (DMK) and OH-DMT. Oxidation of DMT by peroxidases apparently uses the common peroxidase cycle involving the native enzyme, compound I and compound II. In conclusion, this study describes a possible alternative metabolic pathway for DMT involving peroxidases that has not previously been described in humans and identifies DMT and metabolites in a melanoma cell line. The extension of these findings to other cell types and the biological effects of DMT and its metabolites on cell proliferation and function are key questions for future studies. PMID:24508833

  4. Mechanism and regulation of peroxidase-catalyzed nitric oxide consumption in physiological fluids: critical protective actions of ascorbate and thiocyanate.

    PubMed

    Rees, Martin D; Maiocchi, Sophie L; Kettle, Anthony J; Thomas, Shane R

    2014-07-01

    Catalytic consumption of nitric oxide (NO) by myeloperoxidase and related peroxidases is implicated as playing a key role in impairing NO bioavailability during inflammatory conditions. However, there are major gaps in our understanding of how peroxidases consume NO in physiological fluids, in which multiple reactive enzyme substrates and antioxidants are present. Notably, ascorbate has been proposed to enhance myeloperoxidase-catalyzed NO consumption by forming NO-consuming substrate radicals. However, we show that in complex biological fluids ascorbate instead plays a critical role in inhibiting NO consumption by myeloperoxidase and related peroxidases (lactoperoxidase, horseradish peroxidase) by acting as a competitive substrate for protein-bound redox intermediates and by efficiently scavenging peroxidase-derived radicals (e.g., urate radicals), yielding ascorbyl radicals that fail to consume NO. These data identify a novel mechanistic basis for how ascorbate preserves NO bioavailability during inflammation. We show that NO consumption by myeloperoxidase Compound I is significant in substrate-rich fluids and is resistant to competitive inhibition by ascorbate. However, thiocyanate effectively inhibits this process and yields hypothiocyanite at the expense of NO consumption. Hypothiocyanite can in turn form NO-consuming radicals, but thiols (albumin, glutathione) readily prevent this. Conversely, where ascorbate is absent, glutathione enhances NO consumption by urate radicals via pathways that yield S-nitrosoglutathione. Theoretical kinetic analyses provide detailed insights into the mechanisms by which ascorbate and thiocyanate exert their protective actions. We conclude that the local depletion of ascorbate and thiocyanate in inflammatory microenvironments (e.g., due to increased metabolism or dysregulated transport) will impair NO bioavailability by exacerbating peroxidase-catalyzed NO consumption. PMID:24704973

  5. Engineering Ascorbate Peroxidase Activity Into Cytochrome C Peroxidase

    SciTech Connect

    Meharenna, Y.T.; Oertel, P.; Bhaskar, B.; Poulos, T.L.

    2009-05-26

    Cytochrome c peroxidase (CCP) and ascorbate peroxidase (APX) have very similar structures, and yet neither CCP nor APX exhibits each others activities with respect to reducing substrates. APX has a unique substrate binding site near the heme propionates where ascorbate H-bonds with a surface Arg and one heme propionate (Sharp et al. (2003) Nat. Struct. Biol. 10, 303--307). The corresponding region in CCP has a much longer surface loop, and the critical Arg residue that is required for ascorbate binding in APX is Asn in CCP. In order to convert CCP into an APX, the ascorbate-binding loop and critical arginine were engineered into CCP to give the CCP2APX mutant. The mutant crystal structure shows that the engineered site is nearly identical to that found in APX. While wild-type CCP shows no APX activity, CCP2APX catalyzes the peroxidation of ascorbate at a rate of {approx}12 min{sup -1}, indicating that the engineered ascorbate-binding loop can bind ascorbate.

  6. Catalase and glutathione peroxidase mimics

    PubMed Central

    Day, Brian J.

    2009-01-01

    Overproduction of the reactive oxygen species (ROS) superoxide (O2−) and hydrogen peroxide (H2O2) are increasingly implicated in human disease and aging. ROS are also being explored as important modulating agents in a number of cell signaling pathways. Earlier work has focused on development of small catalytic scavengers of O2−, commonly referred to as superoxide dismutase (SOD) mimetics. Many of these compounds also have substantial abilities to catalytically scavenge H2O2 and peroxynitrite (ONOO−). Peroxides have been increasingly shown to disrupt cell signaling cascades associated with excessive inflammation associated with a wide variety of human diseases. Early studies with enzymatic scavengers like SOD frequently reported little or no beneficial effect in biologic models unless SOD was combined with catalase or a peroxidase. Increasing attention has been devoted to developing catalase or peroxidase mimetics as a way to treat overt inflammation associated with the pathophysiology of many human disorders. This review will focus on recent development of catalytic scavengers of peroxides and their potential use as therapeutic agents for pulmonary, cardiovascular, neurodegenerative and inflammatory disorders. PMID:18948086

  7. Carbodiimide or periodate method to prepare peroxidase hydrazide for its use in immunoassay.

    PubMed

    Shrivastav, Tulsidas G

    2004-01-01

    Peroxidase hydrazides were prepared by conjugating horseradish peroxidase (HRP) to adipic acid dihydrazide (ADH) by carbodiimide or periodate oxidation method. The resulting HRP hydrazides (ADH-HRP) were conjugated to cortisol-21-hemisuccinate (cortisol-21-HS) by forming diimide bonds using the N-hydroxysuccinimide (NHS) carbodiimide mediated reaction. The prepared cortisol-21-HS-ADH-HRP enzyme conjugates were utilized for the development of an enzyme linked immunosorbent assay (ELISA) for direct estimation of cortisol. To the cortisol antibody coated microtiter wells, standard or serum sample (50 microL), along with 100 microL of cortisol-21-HS-ADH-HRP enzyme conjugate (ADH-HRP used is prepared by either carbodiimide or periodate oxidation method), was incubated for 1 hr at 37 degrees C. Bound enzyme activity was measured by using tetramethyl benzidine/hydrogen peroxide (TMB/H202) as substrate. The sensitivity, specificity, and recovery of the assays were found to be identical when ELISAs were employed with cortisol enzyme conjugates prepared by conjugating cortisol-21-HS to HRP hydrazide, made either by the carbodiimide method or periodate oxidation method. PMID:15461389

  8. Peroxidase-mediated conjugation of corn fiber gum and bovine serum albumin to improve emulsifying properties.

    PubMed

    Liu, Yan; Qiu, Shuang; Li, Jinlong; Chen, Hao; Tatsumi, Eizo; Yadav, Madhav; Yin, Lijun

    2015-03-15

    The emulsifying properties of corn fiber gum (CFG), a naturally occurring polysaccharide-protein complex, was improved by kinetically controlled formation of hetero-covalent linkages with bovine serum albumin (BSA), using horseradish peroxidase (HRP). The formation of hetero-crosslinked CFG-BSA conjugates was confirmed using ultraviolet-visible and Fourier-transform infrared analyses. The optimum CFG-BSA conjugates were prepared at a CFG:BSA weight ratio of 10:1, and peroxidase:BSA weight ratio of 1:4000. Selected CFG-BSA conjugates were used to prepare oil-in-water emulsions; the emulsifying properties were better than those of emulsions stabilized with only CFG or BSA. Measurements of mean droplet sizes and zeta potentials showed that CFG-BSA-conjugate-stabilized emulsions were less susceptible to environmental stresses, such as pH changes, high K ionic strengths, and freeze-thaw treatments than CFG- or BSA-stabilized emulsions. These conjugates have potential applications as novel emulsifiers in food industry. PMID:25542109

  9. Independent evolution of four heme peroxidase superfamilies.

    PubMed

    Zámocký, Marcel; Hofbauer, Stefan; Schaffner, Irene; Gasselhuber, Bernhard; Nicolussi, Andrea; Soudi, Monika; Pirker, Katharina F; Furtmüller, Paul G; Obinger, Christian

    2015-05-15

    Four heme peroxidase superfamilies (peroxidase-catalase, peroxidase-cyclooxygenase, peroxidase-chlorite dismutase and peroxidase-peroxygenase superfamily) arose independently during evolution, which differ in overall fold, active site architecture and enzymatic activities. The redox cofactor is heme b or posttranslationally modified heme that is ligated by either histidine or cysteine. Heme peroxidases are found in all kingdoms of life and typically catalyze the one- and two-electron oxidation of a myriad of organic and inorganic substrates. In addition to this peroxidatic activity distinct (sub)families show pronounced catalase, cyclooxygenase, chlorite dismutase or peroxygenase activities. Here we describe the phylogeny of these four superfamilies and present the most important sequence signatures and active site architectures. The classification of families is described as well as important turning points in evolution. We show that at least three heme peroxidase superfamilies have ancient prokaryotic roots with several alternative ways of divergent evolution. In later evolutionary steps, they almost always produced highly evolved and specialized clades of peroxidases in eukaryotic kingdoms with a significant portion of such genes involved in coding various fusion proteins with novel physiological functions. PMID:25575902

  10. (Characterization of lignin peroxidases from Phanerochaete)

    SciTech Connect

    Not Available

    1990-11-14

    Work has continued on characterizing the kinetics of lignin peroxidases and has now expanded to include the chemistry of Mn peroxidases. Progress in these two area in addition to the authors work on the molecular biology of lignin biodegradation is briefly described below. Copies of two reprints and one preprint which have resulted from the work are attached.

  11. Biogenic magnetic nanoparticles from Burkholderia sp. YN01 exhibiting intrinsic peroxidase-like activity and their applications.

    PubMed

    Pan, Yu; Li, Na; Mu, Jianshuai; Zhou, Runhong; Xu, Yan; Cui, Daizong; Wang, Yan; Zhao, Min

    2015-01-01

    A novel bacterial strain containing biogenic magnetic nanoparticles (BMNPs) was isolated from the sediments of Songhua River in Harbin, China, and was identified as Burkholderia sp. YN01. Extracted BMNPs from YN01 were characterized as pure face-centered cubic Fe3O4 with an average size of 80 nm through transmission electron microscope (TEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). The hysteresis parameters of the BMNP samples such as Bc and Bcr and ratios Mrs/Ms were deduced as 35.6 mT, 43.2 mT, and 0.47, respectively, indicating that the BMNPs exhibit a ferromagnetic behavior. This is the first report concerning on biogenic Fe3O4 NPs produced in Burkholderia genus. Significantly, the BMNPs were proved to possess intrinsic peroxidase-like activity that could catalyze the oxidation of peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. Kinetic analysis indicates that the catalytic behavior is in accord with typical Michaelis-Menten kinetics and follows ping-pong mechanism. The catalytic constants (K cat) were 6.5 × 10(4) s(-1) and 0.78 × 10(4) s(-1) with H2O2 and TMB as substrate, respectively, which was higher than that of horseradish peroxidase (HRP). Electron spin resonance (ESR) spectroscopy experiments showed that the BMNPs could catalyze H2O2 to produce hydroxyl radicals. The origin of peroxidase-like activity is also associated with their ability to transfer electron between electrode and H2O2 according to an electrochemical study. As a novel peroxidase mimetic, the BMNPs were employed to offer a simple, sensitive, and selective colorimetric method for H2O2 and glucose determination, and the BMNPs could efficiently catalyze the degradation of phenol and Congo red dye. PMID:25030455

  12. Peroxidase-dependent metabolism of benzene's phenolic metabolites and its potential role in benzene toxicity and carcinogenicity.

    PubMed Central

    Smith, M T; Yager, J W; Steinmetz, K L; Eastmond, D A

    1989-01-01

    The metabolism of two of benzene's phenolic metabolites, phenol and hydroquinone, by peroxidase enzymes has been studied in detail. Studies employing horseradish peroxidase and human myeloperoxidase have shown that in the presence of hydrogen peroxide phenol is converted to 4,4'-diphenoquinone and other covalent binding metabolites, whereas hydroquinone is converted solely to 1,4-benzoquinone. Surprisingly, phenol stimulates the latter conversion rather than inhibiting it, an effect that may play a role in the in vivo myelotoxicity of benzene. Indeed, repeated coadministration of phenol and hydroquinone to B6C3F1 mice results in a dramatic and significant decrease in bone marrow cellularity similar to that observed following benzene exposure. A mechanism of benzene-induced myelotoxicity is therefore proposed in which the accumulation and interaction of phenol and hydroquinone in the bone marrow and the peroxidase-dependent formation of 1,4-benzoquinone are important components. This mechanism may also be responsible, at least in part, for benzene's genotoxic effects, as 1,4-benzoquinone has been shown to damage DNA and is shown here to induce multiple micronuclei in human lymphocytes. Secondary activation of benzene's phenol metabolites in the bone marrow may therefore play an important role in benzene's myelotoxic and carcinogenic effects. PMID:2551665

  13. Synthesis of hierarchical iron hydrogen phosphate crystal as a robust peroxidase mimic for stable H₂O₂ detection.

    PubMed

    Zhang, Tongbao; Lu, Yangcheng; Luo, Guangsheng

    2014-08-27

    To develop a green, cost-efficient and robust peroxidase mimic, micro/nano hierarchical morphology (for ease of separation and reuse), relative chemically stable composition (for ease of storage) and stable crystal structure (for long-term stability) are highly desired. Herein, using phosphoric acid as a chelating ligand to control the release of iron ions, hierarchical iron(III) hydrogen phosphate hydrate crystals are successfully prepared by nanosheets formation and following self-assembling in a facile low-temperature hydrothermal process. They are first found to have peroxidase-like activity and showed higher affinity for H2O2 and lower affinity for 3,3',5,5'-tetramethylbenzidine compared with horseradish peroxidase. The affinity feature is used for quantitative detection of H2O2 and shows a wide linear detection range from 57.4 to 525.8 μM (R(2) = 0.994) with a low detection limit of 1 μM. Benefited from chemical stability of hierarchical iron(III) salt crystals, they own good reproducibility (relative standard deviation = 1.95% for 10 independent measurements), long-term stability (no activity loss after 10 cycles), and ease of recovery (by simple centrifugation). Because the method is easily accessible, iron hydrogen phosphate hierarchical crystals have great potential for practical use of H2O2 sensing and detection under harsh conditions. PMID:25029358

  14. Combined experimental and theoretical study on the removal of pollutant compounds by peroxidases: affinity and reactivity toward a bioremediation catalyst.

    PubMed

    Silva, Maria Cristina; Torres, Juliana Arriel; Castro, Alexandre A; da Cunha, Elaine F F; Alves de Oliveira, Luiz Carlos; Corrêa, Angelita Duarte; Ramalho, Teodorico C

    2016-09-01

    Water pollution is a significant and growing problem throughout the world, especially in developing countries. In order to minimize environmental problems, catalysts have increasingly been designed to remove pollutants from the water. In an attempt to innovate by the creation of new low-cost alternatives to efficiently remove pollutants, the enzymatic treatment has been intensely studied for this purpose. Reactions catalyzed by enzymes are able to perform specific treatments, commonly with high rates of the final products. With this, the enzyme, peroxidase, is a promising candidate as a bioremediation catalyst. The efficiency of oxidoreductive enzymes, such as horseradish peroxidase (HRP) and soybean peroxidase (SP) have been studied, given that their performance depends on the substrate. In this investigation, experimental techniques and theoretical calculations have been employed in order to investigate the oxidative process for the ferulic acid and bromophenol blue dyes, performed by HRP and SP. Both enzymes showed a comparable behavior with respect to ferulic acid substrate. On the other hand, by utilizing bromophenol blue dye as a substrate, the behavior of the employed catalysts was significantly different. Experimental data have shown that HRP was more active toward bromophenol blue when compared to ferulic acid, being more rapidly degraded by the HRP enzyme. This tendency was confirmed by our theoretical docking, PM6 semi-empirical method, and DFT calculation results, in which the interaction, binding energies, and transition states were determined. PMID:26130457

  15. Use of crude extract of lentil plant (Lens culinaris Medikus) in peroxidase-based analyses: fast kinetic determination of hydrogen peroxide and sarcosine in urine.

    PubMed

    Pérez Galende, Patricia; Manzano Muñoz, Teresa; Roig, Manuel G; García de María, Cándido

    2012-11-01

    Peroxidase-catalysed reactions are used in a wide variety of analytical applications, most of them based on the final quantification of hydrogen peroxide. Clinical tests for glucose, cholesterol, creatine, creatinine or uric acid in blood or urine and enzyme-linked immunosorbent assays for pesticides, hepatitis or acquired immune deficiency syndrome are good examples of such applications. The most widely used and commercially available peroxidase for biotechnological processes and analytical applications is horseradish peroxidase followed, although in much lower proportion, by soybean peroxidase. The high commercial interest in peroxidases has led to the search for new sources of these enzymes. This work describes the analytical use of lentil plant peroxidase (LPP), which is a new peroxidase extracted from lentil plants (Lens culinaris Medikus); an abundant post-harvest agricultural waste in the area of Castilla y León (Spain). A procedure for the quantification of hydrogen peroxide in urine is first proposed using crude extract of lentil plant instead of the purified enzyme. This procedure is then applied to the determination of sarcosine; a natural amino acid that has attracted considerable interest in clinical diagnostics since urinary sarcosine was proposed and later questioned as a biomarker for prostate cancer. Under the action of sarcosine oxidase, sarcosine is oxidized by molecular oxygen to give glycine, formaldehyde and hydrogen peroxide that is quantified according to the previously proposed procedure. The limit of detection for both hydrogen peroxide and sarcosine is around 5 × 10(-7) M. In the determination of sarcosine, the high selectivity of the overall enzymatic reaction, the simple sample treatment and instrumentation, the high-sample throughput and the use of LPP in the plant extract instead of the purified enzyme provide a rapid and inexpensive procedure with characteristics very suitable for routine analysis in a clinical laboratory. PMID

  16. Selenium, glutathione peroxidase and other selenoproteins

    SciTech Connect

    Wilhelmsen, E.C.

    1983-01-01

    Selenium, as essential trace element, has long been associated with protein. The essentiality of selenium is partially understood as glutathione peroxidase contains an essential selenocysteine. Glutathione peroxidase has been purified from many tissues including rat liver. An estimated molecular weight of 105,000 was obtained for glutathione peroxidase by comparison to standards. A subunit size of 26,000 was obtained by SDS-gel electrophoresis. Glutathione peroxidase is not the only selenoprotein in the rat. In seven rat tissues examined, there were many different subunit sizes and change groups representing between 9 and 23 selenoproteins. Selenocysteine in glutathione peroxidase accounts for ca. 36% of the selenium in the rat. The mode of synthesis of glutathione peroxidase and the other selenoproteins is not understood. Glutathione peroxidase is strongly and reversibly inhibited by mercaptocarboxylic acids and other mercaptans, including some used as slow-acting drugs for the symtomatic treatment of rheumatoid arthritis. The mechanism and chemistry of this inhibition is discussed. This inhibition may provide a link between selenium and arthritis.

  17. Antisense RNA suppression of peroxidase gene expression

    SciTech Connect

    Lagrimini, L.M.; Bradford, S.; De Leon, F.D. )

    1989-04-01

    The 5{prime} half the anionic peroxidase cDNA of tobacco was inserted into a CaMV 35S promoter/terminator expression cassette in the antisense configuration. This was inserted into the Agrobacterium-mediated plant transformation vector pCIBIO which includes kanamycin selection, transformed into two species of tobacco (N. tabacum and M. sylvestris), and plants were subsequently regenerated on kanamycin. Transgenic plants were analyzed for peroxidase expression and found to have 3-5 fold lower levels of peroxidase than wild-type plants. Isoelectric focusing demonstrated that the antisense RNA only suppressed the anionic peroxidase. Wound-induced peroxidase expression was found not to be affected by the antisense RNA. Northern blots show a greater than 5 fold suppression of anionic peroxidase mRNA in leaf tissue, and the antisense RNA was expressed at a level 2 fold over the endogenous mRNA. Plants were self-pollinated and F1 plants showed normal segregation. N. sylvestris transgenic plants with the lowest level of peroxidase are epinastic, and preliminary results indicate elevated auxin levels. Excised pith tissue from both species of transgenic plants rapidly collapse when exposed to air, while pith tissue from wild-type plants showed little change when exposed to air. Further characterization of these phenotypes is currently being made.

  18. Removal of triclosan via peroxidases-mediated reactions in water: Reaction kinetics, products and detoxification.

    PubMed

    Li, Jianhua; Peng, Jianbiao; Zhang, Ya; Ji, Yuefei; Shi, Huanhuan; Mao, Liang; Gao, Shixiang

    2016-06-01

    This study investigated and compared reaction kinetics, product characterization, and toxicity variation of triclosan (TCS) removal mediated by soybean peroxidase (SBP), a recognized potential peroxidase for removing phenolic pollutants, and the commonly used horseradish peroxidase (HRP) with the goal of assessing the technical feasibility of SBP-catalyzed removal of TCS. Reaction conditions such as pH, H2O2 concentration and enzyme dosage were found to have a strong influence on the removal efficiency of TCS. SBP can retain its catalytic ability to remove TCS over broad ranges of pH and H2O2 concentration, while the optimal pH and H2O2 concentration were 7.0 and 8μM, respectively. 98% TCS was removed with only 0.1UmL(-1) SBP in 30min reaction time, while an HRP dose of 0.3UmL(-1) was required to achieve the similar conversion. The catalytic performance of SBP towards TCS was more efficient than that of HRP, which can be explained by catalytic rate constant (KCAT) and catalytic efficiency (KCAT/KM) for the two enzymes. MS analysis in combination with quantum chemistry computation showed that the polymerization products were generated via CC and CO coupling pathways. The polymers were proved to be nontoxic through growth inhibition of green alga (Scenedesmus obliquus). Taking into consideration of the enzymatic treatment cost, SBP may be a better alternative to HRP upon the removal and detoxification of TCS in water/wastewater treatment. PMID:26921508

  19. Recents patents in the use of peroxidases.

    PubMed

    Alvarado, Berenize; Torres, Eduardo

    2009-01-01

    Peroxidases are hemoenzymes with a wide range of applications, from fine chemical synthesis to environmental biocatalysis. These outstanding biocatalysts are able to catalyze reactions such as heteroatom oxidation (N- and S-oxidation), epoxidation, hydroxylation, and the oxidation of alcohols and indole, often giving high yields and enantiomeric excess values. This makes these biocatalysts very useful for application to several biotechnological processes. In this paper, recent advances and patents surrounding the use of peroxidases are reviewed, covering different aspects related to the applications of peroxidases and the modifications carried out to improve their functionality as biocatalysts. PMID:19519565

  20. Colorimetric detection of urea, urease, and urease inhibitor based on the peroxidase-like activity of gold nanoparticles.

    PubMed

    Deng, Hao-Hua; Hong, Guo-Lin; Lin, Feng-Lin; Liu, Ai-Lin; Xia, Xing-Hua; Chen, Wei

    2016-04-01

    Herein, we reported for the first time that gold nanoparticles-catalyzed 3,3',5,5'-tetramethylbenzidine-H2O2 system can serve as an ultrasensitive colorimetric pH indicator. Gold nanoparticles acted as a catalyst and imitated the function of horseradish peroxidase. The absorbance at 450 nm of the yellow-color product in the catalytic reaction exhibited a linear fashion over the pH range of 6.40-6.60. On the basis of this property, we constructed a novel sensing platform for the determination of urea, urease, and urease inhibitor. The limit of detection for urea and urease was 5 μM and 1.8 U/L, respectively. The half-maximal inhibition value IC50 of acetohydroxamic acid was found to be 0.05 mM. Urea in human urine and urease in soil were detected with satisfied results. PMID:26995642

  1. Bienzymatic glucose biosensor based on co-immobilization of peroxidase and glucose oxidase on a carbon nanotubes electrode.

    PubMed

    Zhu, Liande; Yang, Ruilan; Zhai, Jiangli; Tian, Chunyuan

    2007-11-30

    A bienzymatic glucose biosensor was proposed for selective and sensitive detection of glucose. This mediatorless biosensor was made by simultaneous immobilization of glucose oxidase (GOD) and horseradish peroxidase (HRP) in an electropolymerized pyrrole (PPy) film on a single-wall carbon nanotubes (SWNT) coated electrode. The amperometric detection of glucose was assayed by potentiostating the bienzymatic electrode at -0.1 versus Ag/AgCl to reduce the enzymatically produced H(2)O(2) with minimal interference from the coexisting electroactive compounds. The single-wall carbon nanotubes, sandwiched between the enzyme loading polypyrrole (PPy) layer and the conducting substrate (gold electrode), could efficiently promote the direct electron transfer of HRP. Operational characteristics of the bienzymatic sensor, in terms of linear range, detection limit, sensitivity, selectivity and stability, were presented in detail. PMID:17764922

  2. Self-enhanced N-(aminobutyl)-N-(ethylisoluminol) derivative-based electrochemiluminescence immunosensor for sensitive laminin detection using PdIr cubes as a mimic peroxidase

    NASA Astrophysics Data System (ADS)

    Jiang, Xinya; Wang, Huijun; Wang, Haijun; Zhuo, Ying; Yuan, Ruo; Chai, Yaqin

    2016-04-01

    Herein, a self-enhanced N-(aminobutyl)-N-(ethylisoluminol) (ABEI) derivative-based electrochemiluminescence (ECL) immunosensor was constructed for the determination of laminin (LN) using PdIr cubes as a mimic peroxidase for signal amplification. Initially, PdIr cubes with efficient peroxidase mimicking properties, large specific surface areas, and good stability and uniformity were synthesized. Then, l-cysteine (l-Cys) and ABEI were immobilized on the PdIr cubes to form the self-enhanced ECL nanocomplex (PdIr-l-Cys-ABEI). In this nanocomplex, PdIr cubes, whose catalytic constant is higher than that of horseradish peroxidase (HRP), could effectively catalyze H2O2 decomposition and thus enhance the ECL intensity of ABEI. Moreover, PdIr cubes can be easily modified with functional groups, which make them adaptable to desired supported platforms. On the other hand, l-Cys as a coreactant of ABEI could effectively enhance the luminous efficiency due to the intramolecular ECL reaction which could reduce the energy loss between l-Cys and ABEI by giving a shorter electron transfer distance. The developed strategy combined an ABEI derivative as a self-enhanced ECL luminophore and PdIr cubes as a mimic peroxidase, resulting in a significantly enhanced ECL signal output. Also, the strategy showed high sensitivity and selectivity for LN, which suggested that our new approach could be potentially applied in monitoring different proteins.

  3. 21 CFR 864.7675 - Leukocyte peroxidase test.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Leukocyte peroxidase test. 864.7675 Section 864... peroxidase test. (a) Identification. A leukocyte peroxidase test is a device used to distinguish certain... peroxidase activity as evidenced by staining. The results of this test are used in the differential...

  4. 21 CFR 864.7675 - Leukocyte peroxidase test.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Leukocyte peroxidase test. 864.7675 Section 864... peroxidase test. (a) Identification. A leukocyte peroxidase test is a device used to distinguish certain... peroxidase activity as evidenced by staining. The results of this test are used in the differential...

  5. 21 CFR 864.7675 - Leukocyte peroxidase test.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Leukocyte peroxidase test. 864.7675 Section 864... peroxidase test. (a) Identification. A leukocyte peroxidase test is a device used to distinguish certain... peroxidase activity as evidenced by staining. The results of this test are used in the differential...

  6. 21 CFR 864.7675 - Leukocyte peroxidase test.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Leukocyte peroxidase test. 864.7675 Section 864... peroxidase test. (a) Identification. A leukocyte peroxidase test is a device used to distinguish certain... peroxidase activity as evidenced by staining. The results of this test are used in the differential...

  7. 21 CFR 864.7675 - Leukocyte peroxidase test.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Leukocyte peroxidase test. 864.7675 Section 864... peroxidase test. (a) Identification. A leukocyte peroxidase test is a device used to distinguish certain... peroxidase activity as evidenced by staining. The results of this test are used in the differential...

  8. A heme peroxidase with a functional role as an L-tyrosine hydroxylase in the biosynthesis of anthramycin.

    PubMed

    Connor, Katherine L; Colabroy, Keri L; Gerratana, Barbara

    2011-10-18

    We report the first characterization and classification of Orf13 (S. refuineus) as a heme-dependent peroxidase catalyzing the ortho-hydroxylation of L-tyrosine to L-DOPA. The putative tyrosine hydroxylase coded by orf13 of the anthramycin biosynthesis gene cluster has been expressed and purified. Heme b has been identified as the required cofactor for catalysis, and maximal L-tyrosine conversion to L-DOPA is observed in the presence of hydrogen peroxide. Preincubation of L-tyrosine with Orf13 prior to the addition of hydrogen peroxide is required for L-DOPA production. However, the enzyme becomes inactivated by hydrogen peroxide during catalysis. Steady-state kinetic analysis of L-tyrosine hydroxylation revealed similar catalytic efficiency for both L-tyrosine and hydrogen peroxide. Spectroscopic data from a reduced-CO(g) UV-vis spectrum of Orf13 and electron paramagnetic resonance of ferric heme Orf13 are consistent with heme peroxidases that have a histidyl-ligated heme iron. Contrary to the classical heme peroxidase oxidation reaction with hydrogen peroxide that produces coupled aromatic products such as o,o'-dityrosine, Orf13 is novel in its ability to catalyze aromatic amino acid hydroxylation with hydrogen peroxide, in the substrate addition order and for its substrate specificity for L-tyrosine. Peroxygenase activity of Orf13 for the ortho-hydroxylation of L-tyrosine to L-DOPA by a molecular oxygen dependent pathway in the presence of dihydroxyfumaric acid is also observed. This reaction behavior is consistent with peroxygenase activity reported with horseradish peroxidase for the hydroxylation of phenol. Overall, the putative function of Orf13 as a tyrosine hydroxylase has been confirmed and establishes the first bacterial class of tyrosine hydroxylases. PMID:21919439

  9. A Recombinant Positive Control for Serology Diagnostic Tests Supporting Elimination of Onchocerca volvulus

    PubMed Central

    Golden, Allison; Stevens, Eric J.; Yokobe, Lindsay; Faulx, Dunia; Kalnoky, Michael; Peck, Roger; Valdez, Melissa; Steel, Cathy; Karabou, Potochoziou; Banla, Méba; Soboslay, Peter T.; Adade, Kangi; Tekle, Afework H.; Cama, Vitaliano A.; Fischer, Peter U.; Nutman, Thomas B.; Unnasch, Thomas R.; de los Santos, Tala; Domingo, Gonzalo J.

    2016-01-01

    Background Serological assays for human IgG4 to the Onchocerca volvulus antigen Ov16 have been used to confirm elimination of onchocerciasis in much of the Americas and parts of Africa. A standardized source of positive control antibody (human anti-Ov16 IgG4) will ensure the quality of surveillance data using these tests. Methodology/Principal Findings A recombinant human IgG4 antibody to Ov16 was identified by screening against a synthetic human Fab phage display library and converted into human IgG4. This antibody was developed into different positive control formulations for enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT) platforms. Variation in ELISA results and utility as a positive control of the antibody were assessed from multiple laboratories. Temperature and humidity conditions were collected across seven surveillance activities from 2011–2014 to inform stability requirements for RDTs and positive controls. The feasibility of the dried positive control for RDT was evaluated during onchocerciasis surveillance activity in Togo, in 2014. When the anti-Ov16 IgG4 antibody was used as a standard dilution in horseradish peroxidase (HRP) and alkaline phosphatase (AP) ELISAs, the detection limits were approximately 1ng/mL by HRP ELISA and 10ng/mL by AP ELISA. Positive control dilutions and spiked dried blood spots (DBS) produced similar ELISA results. Used as a simple plate normalization control, the positive control antibody may improve ELISA data comparison in the context of inter-laboratory variation. The aggregate temperature and humidity monitor data informed temperature parameters under which the dried positive control was tested and are applicable inputs for testing of diagnostics tools intended for sub-Saharan Africa. As a packaged positive control for Ov16 RDTs, stability of the antibody was demonstrated for over six months at relevant temperatures in the laboratory and for over 15 weeks under field conditions. Conclusions The

  10. (Molecular characteristics of the lignin forming peroxidase)

    SciTech Connect

    Lagrimini, L.M.

    1990-01-01

    Since this manuscript was submitted we have conducted a more thorough physiological analysis of water relations in wild-type and peroxidase overproducing plants. These experiments include pressure bomb, plasmolysis, and membrane integrity analysis. We are also in the process of analyzing other phenotypes in peroxidase overproducer plants such as excessive browning of tissue, the rapid death of tissue in culture, and poor germination of seed. Transformed plants of Nicotiana tabacum and Nicotiana sylvestris were obtained which have peroxidase activity 3--7 fold lower than wild-type plants. This was done by introducing a chimeric gene composed of the CaMV 35S promoter and the 5' half of the tobacco anionic peroxidase cDNA in the antisense RNA configuration. A manuscript which describes this work is being written, and will be submitted for publication in January 1990. The anionic peroxidase gene has been cloned by hybridization to the cloned cDNA. The entire gene is contained on an 8.7kb fragment within a lambda phage clone. Several smaller DNA fragments have been subcloned, and some have been sequenced. One exon within the coding sequence has been sequenced, along with the partial sequence of two introns. Further sequencing is being carried-out to identify the promoter, which will be later joined to a reporter gene. 6 figs.

  11. Vascular peroxidase-1 is rapidly secreted, circulates in plasma, and supports dityrosine cross-linking reactions.

    PubMed

    Cheng, Guangjie; Li, Hong; Cao, Zehong; Qiu, Xiaoyun; McCormick, Sally; Thannickal, Victor J; Nauseef, William M

    2011-10-01

    Members of the peroxidase-cyclooxygenase superfamily catalyze biochemical reactions essential to a broad spectrum of biological processes, including host defense, thyroid hormone biosynthesis, and modification of extracellular matrix, as well as contributing to the pathogenesis of chronic inflammatory diseases. We recently identified a novel member of this family, vascular peroxidase-1 (VPO1), that is highly expressed in the human cardiovascular system. Its biosynthesis and enzymatic properties are largely unknown. Here, we report that VPO1 was rapidly and efficiently secreted into the extracellular space when the gene was stably expressed in human embryonic kidney (HEK) cells. Secreted VPO1 is a monomer with complex N-linked oligosaccharides and exhibits peroxidase activity. Biosynthesis of endogenous VPO1 by cultured human umbilical vein endothelial cells (HUVECs) shares features exhibited by heterologous expression of recombinant VPO1 (rVPO1) in HEK cells. The proinflammatory agents lipopolysaccharide and tumor necrosis factor-α induce expression of VPO1 mRNA and protein in HUVECs. Furthermore, murine and bovine sera and human plasma contain enzymatically active VPO1. rVPO1 exhibits spectral and enzymatic properties characteristic of the peroxidase-cyclooxygenase family, except with regard to its heat stability. rVPO1 catalyzes tyrosyl radical formation and promotes dityrosine cross-linking. Taken together, these data demonstrate that VPO1 is a glycosylated heme peroxidase that is actively secreted into circulating plasma by vascular endothelial cells and shares several features with other members of the peroxidase-cyclooxygenase family, including the catalysis of dityrosine formation. PMID:21798344

  12. Genetic Recombination

    ERIC Educational Resources Information Center

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  13. A redox-dependent dimerization switch regulates activity and tolerance for reactive oxygen species of barley seed glutathione peroxidase.

    PubMed

    Navrot, Nicolas; Skjoldager, Nicklas; Bunkenborg, Jakob; Svensson, Birte; Hägglund, Per

    2015-05-01

    Monomeric and dimeric forms of recombinant barley (Hordeum vulgare subsp. vulgare) glutathione peroxidase 2 (HvGpx2) are demonstrated to display distinctly different functional properties in vitro. Monomeric HvGpx2 thus has five fold higher catalytic efficiency than the dimer towards tert-butyl hydroperoxide, but is more sensitive to inactivation by hydrogen peroxide. Treatment of the monomer with hydrogen peroxide results in dimer formation. This observed new behavior of a plant glutathione peroxidase suggests a mechanism involving a switch from a highly catalytically competent monomer to a less active, but more oxidation-resistant dimer. PMID:25796076

  14. Modelling a Peroxidase-based Optical Biosensor

    PubMed Central

    Baronas, Romas; Gaidamauskaite, Evelina; Kulys, Juozas

    2007-01-01

    The response of a peroxidase-based optical biosensor was modelled digitally. A mathematical model of the optical biosensor is based on a system of non-linear reaction-diffusion equations. The modelling biosensor comprises two compartments, an enzyme layer and an outer diffusion layer. The digital simulation was carried out using finite difference technique. The influence of the substrate concentration as well as of the thickness of both the enzyme and diffusion layers on the biosensor response was investigated. Calculations showed complex kinetics of the biosensor response, especially at low concentrations of the peroxidase and of the hydrogen peroxide.

  15. Kinetic study of the inactivation of ascorbate peroxidase by hydrogen peroxide.

    PubMed Central

    Hiner, A N; Rodríguez-López, J N; Arnao, M B; Lloyd Raven, E; García-Cánovas, F; Acosta, M

    2000-01-01

    The activity of ascorbate peroxidase (APX) has been studied with H(2)O(2) and various reducing substrates. The activity decreased in the order pyrogallol>ascorbate>guaiacol>2, 2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS). The inactivation of APX with H(2)O(2) as the sole substrate was studied. The number of H(2)O(2) molecules required for maximal inactivation of the enzyme was determined as approx. 2.5. Enzymic activity of approx. 20% of the original remained at the end of the inactivation process (i.e. approx. 20% resistance) when ascorbate or ABTS was used as the substrate in activity assays. With pyrogallol or guaiacol no resistance was seen. Inactivation by H(2)O(2) followed over time with ascorbate or pyrogallol assays exhibited single-exponential decreases in enzymic activity. Hyperbolic saturation kinetics were observed in both assay systems; a similar dissociation constant (0.8 microM) for H(2)O(2) was obtained in each case. However, the maximum rate constant (lambda(max)) obtained from the plots differed depending on the assay substrate. The presence of reducing substrate in addition to H(2)O(2) partly or completely protected the enzyme from inactivation, depending on how many molar equivalents of reducing substrate were added. An oxygen electrode system has been used to confirm that APX does not exhibit a catalase-like oxygen-releasing reaction. A kinetic model was developed to interpret the experimental results; both the results and the model are compared and contrasted with previously obtained results for horseradish peroxidase C. The kinetic model has led us to the conclusion that the inactivation of APX by H(2)O(2) represents an unusual situation in which no enzyme turnover occurs but there is a partition of the enzyme between two forms, one inactive and the other with activity towards reducing substrates such as ascorbate and ABTS only. The partition ratio is less than 1. PMID:10816425

  16. Modeling suberization with peroxidase-catalyzed polymerization of hydroxycinnamic acids: cross-coupling and dimerization reactions.

    PubMed

    Arrieta-Baez, Daniel; Stark, Ruth E

    2006-04-01

    An anionic potato peroxidase (EC 1.11.1.7, APP) thought to be involved in suberization after wounding was isolated from slices of Solanum tuberosum in order to elucidate the first steps of dehydrogenative polymerization between pairs of different hydroxycinnamic acids (FA, CafA, CA and SA) present in wound-healing plant tissues. Use of a commercial horseradish peroxidase (HRP)-H2O2 catalytic system gave the identical major products in these coupling reactions, providing sufficient quantities for purification and structural elucidation. Using an equimolar mixture of pairs of hydroxycinnamic acid suberin precursors, only caffeic acid is coupled to ferulic acid and sinapic acid in separate cross-coupling reactions. For the other systems, HRP and APP reacted as follows: (1) preferentially with ferulic acid in a reaction mixture that contained p-coumaric and ferulic acids; (2) with sinapic acid in a mixture of p-coumaric and sinapic acids; (3) with sinapic acid in a mixture of ferulic and sinapic acids; (4) with caffeic acid in a reaction mixture of p-coumaric and caffeic acids. The resulting products, isolated and identified by NMR and MS analysis, had predominantly beta-beta-gamma-lactone and beta-5 benzofuran molecular frameworks. Five cross-coupling products are described for the first time, whereas the beta-O-4 dehydrodimers identified from the caffeic acid and sinapic acid cross-coupling reaction are known materials that are highly abundant in plants. These reactivity trends lead to testable hypotheses regarding the molecular architecture of intractable suberin protective plant materials, complementing prior analysis of monomeric constituents by GC-MS and polymer functional group identification from solid-state NMR, respectively. PMID:16524605

  17. Enzymatic Degradation of Oxidized and Reduced Graphene Nanoribbons by Lignin Peroxidase

    PubMed Central

    Sitharaman, Balaji

    2014-01-01

    The expanding use of graphene for various industrial and biomedical applications requires efficient remediation strategies during their disposal into waste streams. Additionally, the interactions of graphene with the biota need thorough evaluation. In this study, we investigated the interactions of oxidized and reduced graphene oxide nanoribbons (GONRs and rGONRs) with lignin peroxidase (LiP), a ligninolytic enzyme released from white rot fungus. GONRs and rGONRs were treated with LiP in the presence and absence of veratryl alcohol (VA; an electron transfer mediator and secondary metabolite of white rot fungi). Transmission electron microscopy showed the formation of large defects (holes) in the graphene sheet, which increased in diameter with increased degradation time. Raman spectroscopic analysis indicated that, within 96 hours, in the presence of hydrogen peroxide and VA, the GONRs and rGONRs were completely and partially degraded by LiP, respectively. Comparisons between groups with or without VA showed that degradation of GONRs was accelerated in the presence of VA. These results indicated that LiP could efficiently degrade GONRs and rGONRs in the presence of VA, suggesting that VA may be an essential factor needed to degrade rGONRs via LiP treatment. Thus, the wide presence of white rot fungi, and thereby LiP, in nature, could lead to efficient degradation of graphene present in the environment. Additionally, LiP, which has a higher theoretical redox potential compared to horseradish peroxidases and myeloperoxidases, could be a better candidate for the environmental remediation of graphene. PMID:25215188

  18. INTERACTIONS OF HORSERADISH PEROXIDASE WITH MONTMORILLONITE HOMOIONIC TO NA+ AND CA2+: EFFECTS ON ENZYME ACTIVITY AND MICROBIAL DEGRADATION. (R826107)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  19. A Biosensor Based on Immobilization of Horseradish Peroxidase in Chitosan Matrix Cross-linked with Glyoxal for Amperometric Determination of Hydrogen Peroxide

    PubMed Central

    Wang, Huai-Sheng; Pan, Qian-Xiu; Wang, Gui-Xiang

    2005-01-01

    An amperometric biosensor for hydrogen peroxide (H2O2) was developed via an easy and effective enzyme immobilization method with the “sandwich” configuration: ferrocene-chitosan: HRP: chitosan-glyoxal using a glassy carbon electrode as the basic electrode. In order to prevent the loss of immobilized HRP under optimized conditions, the biosensor surface was cross-linked with glyoxal. Ferrocene was selected and immobilized on the glassy carbon electrode surface as a mediator. The fabrication procedure was systematically optimized to improve the biosensor performance. The biosensor had a fast response of less than 10 s to H2O2, with a linear range of 3.5×10-5 to 1.1×10-3 M, and a detection limit of 8.0×10-6 M based on S/N = 3.

  20. Controllable Biotinylated Poly(Ethylene-co-Glycidyl Methacrylate) (PE-co-GMA) Nanofibers to Bind Streptavidin-Horseradish Peroxidase (HRP) for Potential Biosensor Applications

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Poly(ethylene-co-glycidyl methacrylate) (PE-co-GMA) nanofibers with abundant active epoxy groups on surfaces were fabricated through a novel manufacturing process. The prepared PE-co-GMA nanofibers with different average diameters ranging from 100 to 400 nm were aminated by reacting the epoxy groups...

  1. Metal-organic framework-derived copper nanoparticle@carbon nanocomposites as peroxidase mimics for colorimetric sensing of ascorbic acid.

    PubMed

    Tan, Hongliang; Ma, Chanjiao; Gao, Li; Li, Qian; Song, Yonghai; Xu, Fugang; Wang, Tao; Wang, Li

    2014-12-01

    Metal-organic frameworks (MOFs) have emerged as very fascinating functional materials due to their diversity nature. A nanocomposite consisting of copper nanoparticles dispersed within a carbon matrix (Cu NPs@C) is prepared through a one-pot thermolysis of copper-based metal-organic framework precursors. Cu NPs@C can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to form a colored product in the presence of H2 O2 . As a peroxidase mimic, Cu NPs@C not only has the advantages of low cost, high stability, and easy preparation, but also follows Michaelis-Menten behaviors and shows strong affinity to H2 O2 . As the Cu NPs' surfaces are free from stabilizing agent, Cu NPs@C exhibited a higher affinity to H2 O2 than horseradish peroxidase. On the basis of the inhibitory effect of ascorbic acid (AA) on oxidation of TMB, this system serves as a colorimetric method for the detection of AA, suggesting that the present work would expand the potential applications of MOF-derived nanocomposites in biomedical fields. PMID:25332148

  2. Widespread occurrence of expressed fungal secretory peroxidases in forest soils.

    PubMed

    Kellner, Harald; Luis, Patricia; Pecyna, Marek J; Barbi, Florian; Kapturska, Danuta; Krüger, Dirk; Zak, Donald R; Marmeisse, Roland; Vandenbol, Micheline; Hofrichter, Martin

    2014-01-01

    Fungal secretory peroxidases mediate fundamental ecological functions in the conversion and degradation of plant biomass. Many of these enzymes have strong oxidizing activities towards aromatic compounds and are involved in the degradation of plant cell wall (lignin) and humus. They comprise three major groups: class II peroxidases (including lignin peroxidase, manganese peroxidase, versatile peroxidase and generic peroxidase), dye-decolorizing peroxidases, and heme-thiolate peroxidases (e.g. unspecific/aromatic peroxygenase, chloroperoxidase). Here, we have repeatedly observed a widespread expression of all major peroxidase groups in leaf and needle litter across a range of forest ecosystems (e.g. Fagus, Picea, Acer, Quercus, and Populus spp.), which are widespread in Europe and North America. Manganese peroxidases and unspecific peroxygenases were found expressed in all nine investigated forest sites, and dye-decolorizing peroxidases were observed in five of the nine sites, thereby indicating biological significance of these enzymes for fungal physiology and ecosystem processes. Transcripts of selected secretory peroxidase genes were also analyzed in pure cultures of several litter-decomposing species and other fungi. Using this information, we were able to match, in environmental litter samples, two manganese peroxidase sequences to Mycena galopus and Mycena epipterygia and one unspecific peroxygenase transcript to Mycena galopus, suggesting an important role of this litter- and coarse woody debris-dwelling genus in the disintegration and transformation of litter aromatics and organic matter formation. PMID:24763280

  3. Widespread Occurrence of Expressed Fungal Secretory Peroxidases in Forest Soils

    PubMed Central

    Kellner, Harald; Luis, Patricia; Pecyna, Marek J.; Barbi, Florian; Kapturska, Danuta; Krüger, Dirk; Zak, Donald R.; Marmeisse, Roland; Vandenbol, Micheline; Hofrichter, Martin

    2014-01-01

    Fungal secretory peroxidases mediate fundamental ecological functions in the conversion and degradation of plant biomass. Many of these enzymes have strong oxidizing activities towards aromatic compounds and are involved in the degradation of plant cell wall (lignin) and humus. They comprise three major groups: class II peroxidases (including lignin peroxidase, manganese peroxidase, versatile peroxidase and generic peroxidase), dye-decolorizing peroxidases, and heme-thiolate peroxidases (e.g. unspecific/aromatic peroxygenase, chloroperoxidase). Here, we have repeatedly observed a widespread expression of all major peroxidase groups in leaf and needle litter across a range of forest ecosystems (e.g. Fagus, Picea, Acer, Quercus, and Populus spp.), which are widespread in Europe and North America. Manganese peroxidases and unspecific peroxygenases were found expressed in all nine investigated forest sites, and dye-decolorizing peroxidases were observed in five of the nine sites, thereby indicating biological significance of these enzymes for fungal physiology and ecosystem processes. Transcripts of selected secretory peroxidase genes were also analyzed in pure cultures of several litter-decomposing species and other fungi. Using this information, we were able to match, in environmental litter samples, two manganese peroxidase sequences to Mycena galopus and Mycena epipterygia and one unspecific peroxygenase transcript to Mycena galopus, suggesting an important role of this litter- and coarse woody debris-dwelling genus in the disintegration and transformation of litter aromatics and organic matter formation. PMID:24763280

  4. Amperometric DNA-Peroxidase Sensor for the Detection of Pharmaceutical Preparations

    PubMed Central

    Evtugyn, G.A.; Goldfarb, O.E.; Budnikov, H.C.; Ivanov, A.N.; Vinter, V.G.

    2005-01-01

    Novel DNA-sensor with enzymatic amplification of the signal has been developed on the base of glassy carbon electrode modified with ds-DNA and horseradish peroxidase (HRP). Phenothiazine dyes Methylene Blue and Methylene Green were used as electrochemical markers for the detection of sulfonamide and anthracycline preparations able to interact with DNA. The biosensor signal related to HRP oxidation of the markers depends on the relation between their bonded and readily oxidized forms which depends on the nature and concentration of pharmaceuticals. Sulfonamides diminish surface concentration of MB accessible for HRP reaction whereas anthracyclines release intercalated marker and increase the signal. The DNA-HRP sensor developed makes it possible to detect down to 0.002 nmol L-1 of sulfamethoxazole, 0.1 nmol L-1 of sulfadiazine, 0.01 nmol L-1 of sulfamethazine, 0.1 nmol L-1 of sulfaguanine, 0.05 μmol L-1 of rubomycin and 0.08 μmol L-1 of doxorubicin.

  5. Sonoenzymatic decolourization of an azo dye employing immobilized horse radish peroxidase (HRP): a mechanistic study.

    PubMed

    Malani, Ritesh S; Khanna, Swati; Moholkar, Vijayanand S

    2013-07-15

    For degradation of biorefractory pollutants, enzymatic treatments and sonochemical treatments have shown high potential. A combined technique of sono-enzymatic treatment is of special interest as it has shown enhancement effect than the individual techniques. This work has attempted to give a mechanistic insight into the interaction of sonochemical and enzymatic treatments using immobilized horseradish peroxidase (HRP) enzyme on the decolourization of acid red dye (an azo dye). In order to segregate the effect of ultrasound and cavitation, experiments were conducted at elevated static pressure. The kinetic parameters of HRP, viz. Vmax and Km were marginally affected by immobilization. There was a minor change in pH optima and temperature optima for immobilized HRP (6.5, 25°C) from free HRP (7.0, 20-25°C). Though the specific activity of free enzyme (0.272U/mg) was found to be higher than the immobilized enzyme (0.104U/mg), immobilized enzyme exhibited higher stability (up to 3 cycles) and degradation potential than free enzyme in all experiments. The results revealed that the coupling of sonication and enzymatic treatment at high pressure in presence of polyethylene glycol (PEG) yielded the highest decolourization of acid red (61.2%). However, the total decolourization achieved with combined technique was lesser than the sum of individual techniques, indicating negative synergy between the sonochemical and enzymatic techniques. PMID:23708258

  6. Guaiacol Peroxidase Zymography for the Undergraduate Laboratory

    ERIC Educational Resources Information Center

    Wilkesman, Jeff; Castro, Diana; Contreras, Lellys M.; Kurz, Liliana

    2014-01-01

    This laboratory exercise presents a novel way to introduce undergraduate students to the specific detection of enzymatic activity by electrophoresis. First, students prepare a crude peroxidase extract and then analyze the homogenate via electrophoresis. Zymography, that is, a SDS-PAGE method to detect enzyme activity, is used to specifically…

  7. Microfluidic peroxidase biochip for polyphenol synthesis.

    PubMed

    Srinivasan, Aravind; Wu, Xiaoqiu; Lee, Moo-Yeal; Dordick, Jonathan S

    2003-03-01

    An enzyme-containing microfluidic biochip has been developed for the oxidative polymerization of phenols. The biochip consists of a simple T-junction with two feed reservoirs 20 mm apart and a microreaction channel 30 mm long. The channel is 15 microm deep and 200 microm wide at the center, giving a reaction volume of 90 nL. The biochip was fabricated using conventional photolithographic methods on a glass substrate etched using a HF-based solution. Fluid transport was enabled using electroosmotic flow. Soybean peroxidase was used as the phenol oxidizing catalyst, and in the presence of p-cresol and H(2)O(2), essentially complete conversion of the H(2)O(2) (the limiting substrate) occurred in the microchannel at a flow rate of ca. 290 nL/min. Thus, peroxidase was found to be intrinsically active even upon dramatic scale-down as achieved in microfluidic reactors. These results were extended to a series of phenols, thereby demonstrating that the microfluidic peroxidase reactor may have application in high-throughput screening of phenolic polymerization reactions for use in phenolic resin synthesis. Finally, rapid growth of poly(p-cresol) on the walls of the microreaction channel could be performed in the presence of higher H(2)O(2) concentrations. This finding suggests that solution-phase peroxidase catalysis can be used in the controlled deposition of polymers on the walls of microreactors. PMID:12514805

  8. Creation of a Thermally Tolerant Peroxidase.

    PubMed

    Watanabe, Y; Nakajima, H

    2016-01-01

    An artificial peroxidase with thermal tolerance and high catalytic activity has been successfully prepared by mutagenesis of an electron transfer protein, cytochrome c552 from Thermus thermophilus. The mutant enzymes were rationally designed based on the general peroxidase mechanism and spectroscopic analyses of an active intermediate formed in the catalytic reaction. Stopped flow UV-vis spectroscopy and EPR spectroscopy with a rapid freezing sample technique revealed that the initial double mutant, V49D/M69A, which was designed to reproduce the peroxidase mechanism, formed an active oxo-ferryl heme intermediate with a protein radical predominantly localized on Tyr45 during the catalytic reaction. The magnetic power saturation measurement obtained from EPR studies showed little interaction between the oxo-ferryl heme and the tyrosyl radical. Kinetics studies indicated that the isolated oxo-ferryl heme component in the active intermediate was a possible cause of heme degradation during the reaction with H2O2. Strong interaction between the oxo-ferryl heme and the radical was achieved by replacing Tyr45 with tryptophan (resulting in the Y45W/V49D/M69A mutant), which was similar to a tryptophanyl radical found in active intermediates of some catalase-peroxidases. Compared to the protein radical intermediates of V49D/M69A mutant, those of the Y45W/V49D/M69A mutant showed higher reactivity to an organic substrate than to H2O2. The Y45W/V49D/M69A mutant exhibited improved peroxidase activity and thermal tolerance. PMID:27586345

  9. Catalytic properties, localization, and in vivo role of Px IV, a novel tryparedoxin peroxidase of Trypanosoma brucei.

    PubMed

    Liu, Ilon; Bogacz, Marta; Schaffroth, Corinna; Dirdjaja, Natalie; Krauth-Siegel, R Luise

    2016-06-01

    Px IV is a distant relative of the known glutathione peroxidase-type enzymes of African trypanosomes. Immunofluorescence microscopy of bloodstream cells expressing C-terminally Myc6-tagged Px IV revealed a mitochondrial localization. Recombinant Px IV possesses very low activity as glutathione peroxidase but catalyzes the trypanothione/tryparedoxin-dependent reduction of hydrogen peroxide and, even more efficiently, of arachidonic acid hydroperoxide. Neither overexpression in bloodstream cells nor the deletion of both alleles in bloodstream or procyclic parasites affected the in vitro proliferation. Trypanosoma brucei Px IV shares 58% of all residues with TcGPXII. The orthologous enzymes have in common their substrate preference for fatty acid hydroperoxides. However, the T. cruzi protein has been reported to be localized in the endoplasmic reticulum and to be specific for glutathione as reducing agent. Taken together, our data show that Px IV is a low abundant tryparedoxin peroxidase of T. brucei that is not essential, at least under culture conditions. PMID:27262262

  10. Long-term chemiluminescence signal is produced in the course of luminol oxidation catalyzed by enhancer-independent peroxidase purified from Jatropha curcas leaves.

    PubMed

    Duan, Peipei; Cai, Feng; Luo, Yongting; Chen, Yangxi; Zou, Shujuan

    2015-09-01

    Isoenzyme c of horseradish peroxidase (HRP-C) is widely used in enzyme immunoassay combined with chemiluminescence (CL) detection. For this application, HRP-C activity measurement is usually based on luminol oxidation in the presence of hydrogen peroxide (H2O2). However, this catalysis reaction was enhancer dependent. In this study, we demonstrated that Jatropha curcas peroxidase (JcGP1) showed high efficiency in catalyzing luminol oxidation in the presence of H2O2. Compared with HRP-C, the JcGP1-induced reaction was enhancer independent, which made the enzyme-linked immunosorbent assay (ELISA) simpler. In addition, the JcGP1 catalyzed reaction showed a long-term stable CL signal. We optimized the conditions for JcGP1 catalysis and determined the favorable conditions as follows: 50 mM Tris buffer (pH 8.2) containing 10 mM H2 O2, 14 mM luminol and 0.75 M NaCl. The optimum catalysis temperature was 30°C. The detection limit of JcGP1 under optimum condition was 0.2 pM. Long-term stable CL signal combined with enhancer-independent property indicated that JcGP1 might be a valuable candidate peroxidase for clinical diagnosis and enzyme immunoassay with CL detection. PMID:25511847

  11. Axial histidyl imidazole non-exchangeable proton resonances as indicators of imidazole hydrogen bonding in ferric cyanide complexes of heme peroxidases.

    PubMed

    La Mar, G N; De Ropp, J S; Chacko, V P; Satterlee, J D; Erman, J E

    1982-11-19

    Proton NMR spectra of a model of low-spin cyanide complexes of ferric hemoproteins indicate that two broad single-protein resonances from the axial imidazole can be resolved outside the diamagnetic spectral region. Upon deprotonation of the imidazole in the model, the upfield resonance shifts dramatically to higher field, suggesting that its position may reflect the degree of hydrogen bonding or proton donation of the imidazole. Met-cyano myoglobin reveals a pair of such broad peaks in the regions expected for an essentially neutral axial imidazole. In the cyano complexes of horseradish peroxidase and cytochrome c peroxidase, a pair of single-proton resonances are located which are assigned to the same imidazole protons on the basis of their linewidth and shift changes upon altering the heme substituents. The upfiled proton, however, is found at much higher field than in metMbCN. The upfield bias of this resonance is taken as evidence for appreciable imidazolate character for the axial ligand in these heme peroxidases. PMID:6293582

  12. The interaction of diamines and polyamines with the peroxidase-catalyzed metabolism of aromatic amines: a potential mechanism for the modulation of aniline toxicity.

    PubMed

    Michail, Karim; Aljuhani, Naif; Siraki, Arno G

    2013-03-01

    Synthetic and biological amines such as ethylenediamine (EDA), spermine, and spermidine have not been previously investigated in free-radical biochemical systems involving aniline-based drugs or xenobiotics. We aimed to study the influence of polyamines in the modulation of aromatic amine radical metabolites in peroxidase-mediated free radical reactions. The aniline compounds tested caused a relatively low oxidation rate of glutathione in the presence of horseradish peroxidase (HRP), and H2O2; however, they demonstrated marked oxygen consumption when a polyamine molecule was present. Next, we characterized the free-radical products generated by these reactions using spin-trapping and electron paramagnetic resonance (EPR) spectrometry. Primary and secondary but not tertiary polyamines dose-dependently enhanced the N-centered radicals of different aniline compounds catalyzed by either HRP or myeloperoxidase, which we believe occurred via charge transfer intermediates and subsequent stabilization of aniline-derived radical species as suggested by isotopically labeled aniline. Aniline/peroxidase reaction product(s) were monitored at 435 nm by kinetic spectrophotometry in the presence and absence of a polyamine additive. Using gas chromatography-mass spectrometry, the dimerziation product of aniline, azobenzene, was significantly amplified when EDA was present. In conclusion, di- and poly-amines are capable of enhancing the formation of aromatic-amine-derived free radicals, a fact that is expected to have toxicological consequences. PMID:23537436

  13. [Characterization of lignin and Mn peroxidases from Phanerochaete chrysosporium

    SciTech Connect

    Not Available

    1992-01-01

    Lignin peroxidases were investigated with respect to enzyme kinetics and NMR spectroscopy of the heme domain. MN peroxidases were studied with respect to the role of oxalate in enzyme activity, the NMR spectroscopy of the heme domain. Gene expression of both lignin and MN peroxidases were examined as well as expression of site-directed mutants aimed at scale up production of these enzymes.

  14. PEROXIDASE GENE POLYMORPHISM IN BUFFALOGRASS AND OTHER GRASSES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant peroxidases are a family of related proteins possessing highly conserved domains. Degenerate oligonucleotide primers based on these conserved domains can be used to amplify DNA sequences coding for peroxidases from plants with unsequenced genomes. Polymorphisms in peroxidase genes among buffa...

  15. Cosmological Recombination

    NASA Astrophysics Data System (ADS)

    Wong, Wan Yan

    2008-11-01

    In this thesis we focus on studying the physics of cosmological recombination and how the details of recombination affect the Cosmic Microwave Background (CMB) anisotropies. We present a detailed calculation of the spectral line distortions on the CMB spectrum arising from the Lyman-alpha and the lowest two-photon transitions in the recombination of hydrogen (H), and the corresponding lines from helium (He). The peak of these distortions mainly comes from the Lyman-alpha transition and occurs at about 170 microns, which is the Wien part of the CMB. The major theoretical limitation for extracting cosmological parameters from the CMB sky lies in the precision with which we can calculate the cosmological recombination process. With this motivation, we perform a multi-level calculation of the recombination of H and He with the addition of the spin-forbidden transition for neutral helium (He I), plus the higher order two-photon transitions for H and among singlet states of He I. We find that the inclusion of the spin-forbidden transition results in more than a percent change in the ionization fraction, while the other transitions give much smaller effects. Last we modify RECFAST by introducing one more parameter to reproduce recent numerical results for the speed-up of helium recombination. Together with the existing hydrogen `fudge factor', we vary these two parameters to account for the remaining dominant uncertainties in cosmological recombination. By using a Markov Chain Monte Carlo method with Planck forecast data, we find that we need to determine the parameters to better than 10% for He I and 1% for H, in order to obtain negligible effects on the cosmological parameters.

  16. Nematicidal activity of allylisothiocyanate from horseradish (Armoracia rusticana) roots against Meloidogyne incognita.

    PubMed

    Aissani, Nadhem; Tedeschi, Paola; Maietti, Annalisa; Brandolini, Vincenzo; Garau, Vincenzo Luigi; Caboni, Pierluigi

    2013-05-22

    In recent years, there has been a great development in the search for new natural pesticides for crop protection aiming a partial or total replacement of currently used chemical nematicides. Glucosinolate breakdown products are volatile and are therefore good candidates for nematodes fumigants. In this article, the methanol-aqueous extract (1:1, w/v) of horseradish (Armoracia rusticana) fresh roots (MAH) was in vitro tested for nematicidal activity against second stage (J2) Meloidogyne incognita. The EC50 of MAH after 3 days of J2 immersion in test solutions was 251 ± 46 mg/L. The chemical composition analysis of the extract carried out by the GC-MS technique showed that allylisothicyanate was the most abundant compound. This pure compound induced J2 paralysis with an EC50 of 52.6 ± 45.6 and 6.6 ± 3.4 mg/L after 1 h and 3 days of incubation. The use of LC-MS/MS showed for the first time that horseradish root is rich in polyphenols. The study of isothiocyanate degradation in soil showed that allylisothiocyanate was the most quickly degradable compound (half-life <10 min), whereas no significant differences in half-life time were noted between degradation in regular and autoclaved soil. PMID:23627288

  17. Peroxidase Activity and Involvement in the Oxidative Stress Response of Roseobacter denitrificans Truncated Hemoglobin

    PubMed Central

    Wang, Yaya; Barbeau, Xavier; Bilimoria, Astha; Lagüe, Patrick; Couture, Manon; Tang, Joseph Kuo-Hsiang

    2015-01-01

    Roseobacter denitrificans is a member of the widespread marine Roseobacter genus. We report the first characterization of a truncated hemoglobin from R. denitrificans (Rd. trHb) that was purified in the heme-bound form from heterologous expression of the protein in Escherichia coli. Rd. trHb exhibits predominantly alpha-helical secondary structure and absorbs light at 412, 538 and 572 nm. The phylogenetic classification suggests that Rd. trHb falls into group II trHbs, whereas sequence alignments indicate that it shares certain important heme pocket residues with group I trHbs in addition to those of group II trHbs. The resonance Raman spectra indicate that the isolated Rd. trHb contains a ferric heme that is mostly 6-coordinate low-spin and that the heme of the ferrous form displays a mixture of 5- and 6-coordinate states. Two Fe-His stretching modes were detected, notably one at 248 cm-1, which has been reported in peroxidases and some flavohemoglobins that contain an Fe-His-Asp (or Glu) catalytic triad, but was never reported before in a trHb. We show that Rd. trHb exhibits a significant peroxidase activity with a (kcat/Km) value three orders of magnitude higher than that of bovine Hb and only one order lower than that of horseradish peroxidase. This enzymatic activity is pH-dependent with a pKa value ~6.8. Homology modeling suggests that residues known to be important for interactions with heme-bound ligands in group II trHbs from Mycobacterium tuberculosis and Bacillus subtilis are pointing toward to heme in Rd. trHb. Genomic organization and gene expression profiles imply possible functions for detoxification of reactive oxygen and nitrogen species in vivo. Altogether, Rd. trHb exhibits some distinctive features and appears equipped to help the bacterium to cope with reactive oxygen/nitrogen species and/or to operate redox biochemistry. PMID:25658318

  18. A highly sensitive flow-through amperometric immunosensor based on the Peroxidase chip and enzyme-channeling principle.

    PubMed

    Zeravik, J; Ruzgas, T; Fránek, M

    2003-10-01

    A concept based on the Peroxidase-chip (P-chip), antibody co-immobilization, competitive and enzyme-channeling principle was exploited to develop an integrated flow-through amperometric biosensor for detection of environmental pollutants such as s-triazine herbicides. In this concept, recombinant peroxidase is immobilized on the gold electrode (P-chip) in such a way that direct electron transfer is achieved. The recognition and quantitation the target analyte is realized through the competition between the simazine-glucose oxidase (GOD) conjugate and free simazine for the binding sites of the monoclonal antibody co-immobilized with peroxidase on the gold electrode. The arrangement allows to generate a specific signal in the presence of glucose through the channeling of H2O2 produced by GOD conjugate bound to the antibody. The immunosensor exhibited 50% signal decrease (IC50 value) at approximately 0.02 microg l(-1). A concentration of 0.1 ng l(-1) gave a signal clearly distinguishable from the blank whereas the ELISA using the same antibody had a typical detection limit of about 1 microg l(-1), which is four orders of magnitude higher compared to the presented biosensor system. The results demonstrated that gene engineering biomolecules, in this case recombinant peroxidase, might be attractive reagents for the development of electrochemical immunosensors. PMID:12896832

  19. Characterization and application of a novel class II thermophilic peroxidase from Myceliophthora thermophila in biosynthesis of polycatechol.

    PubMed

    Zerva, Anastasia; Christakopoulos, Paul; Topakas, Evangelos

    2015-01-01

    A peroxidase from the thermophilic fungus Myceliophthora thermophila that belongs to ascomycete Class II based on PeroxiBase classification was functionally expressed in methylotrophic yeast Pichia pastoris. The putative peroxidase from the genomic DNA was successfully cloned in P. pastoris X-33 under the transcriptional control of the alcohol oxidase (AOX1) promoter. The heterologous production was greatly enhanced by the addition of hemin with a titer of 0.41 U mL(-1) peroxidase activity at the second day of incubation. The recombinant enzyme was purified to homogeneity (50 kDa) and characterized using a series of phenolic substrates that indicated similar characteristics with those of generic peroxidases. In addition, the enzyme was found thermostable, retaining its activity for temperatures up to 60 °C after eight hours incubation. Moreover, the enzyme displayed remarkable H2O2 stability, retaining more than 80% of its initial activity after 24h incubation in 5000-fold molar excess of H2O2. The ability of the peroxidase to polymerize catechol at high superoxide concentrations, together with its high thermostability and substrate specificity, indicate a potential commercial significance of the enzyme. PMID:26047916

  20. Characterization and expression of the Schistosoma japonicum thioredoxin peroxidase-2 gene.

    PubMed

    Hong, Yang; Han, Yanhui; Fu, Zhiqiang; Han, Hongxiao; Qiu, Chunhui; Zhang, Min; Yang, Jianmei; Shi, Yaojun; Li, Xiangrui; Lin, Jiaojiao

    2013-02-01

    We analyzed proteins that were differentially expressed by 10-day-old schistosomula from 3 different hosts and determined that a functional thioredoxin peroxidase-2 gene has an important antioxidant role in Schistosoma japonicum , which we investigated further. A full-length cDNA encoding the S. japonicum thioredoxin peroxidase-2 (SjTPx-2) had an open reading frame of 681 bp that encoded 226 amino acids with a signal peptide of 24 amino acids. A cDNA encoding SjTPx-2 without the signal peptide sequence was isolated from 42-day-old schistosome cDNAs. Real-time quantitative RT-PCR analysis revealed that SjTPx-2 was upregulated in 7- and 13-day-old schistosomes, while the expression level in females was around 2-fold higher than that in male worms at 42 days. SjTPx was subcloned into pET28a(+) and expressed as both inclusion bodies and supernatant in Escherichia coli BL21 (DE3) cells. Western blotting showed that the recombinant SjTPx-2 (rSjTPx-2) was immunogenic. The purified recombinant protein could form disulfide-bonded dimers and it had peroxidase activity in vitro. An immunoprotection experiment in BALB/c mice showed that vaccination with recombinant SjTPx-2 could induce 31.2% and 34.0% reductions in the numbers of worms and eggs in the liver, respectively. This study suggests that SjTPx-2 may be an important antioxidative enzyme in scavenging ROS, and it may be a potential vaccine candidate or new drug target for schistosomiasis. PMID:22924918

  1. NADH peroxidase: kinetic mechanism and nucleotide specificity

    SciTech Connect

    Stoll, V.S.; Blanchard, J.S.

    1987-05-01

    NADH peroxidase is a flavoprotein reductase isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide dependent reduction of hydrogen peroxide to water. Initial velocity, product and dead-end inhibition studies have been performed and all support a ping-pong kinetic mechanism. Further support for the ping-pong nature of the kinetic mechanism are the hydrogen peroxide independent transhydrogenase activity of the enzyme, measured either with thio-NAD or with radiolabeled NAD (isotope exchange studies). Kinetic parameters will be presented for a number of reduced pyridine nucleotide analogs. Analogs which have been modified in the adenine ring exhibit much higher K/sub m/'s relative to their adenine analogs. NADH peroxidase catalyzes the stereo-specific removal of the 4S hydrogen of NADH and primary deuterium kinetic isotope effects have been determined for a number of these substrates with 4S-deuterated molecules. There is a strong correlation between their steady-state K/sub m/ and /sup D/V/K. Small values for /sup D/V are interpreted as supporting rate-limitation in the oxidative half-reaction. These data will be discussed in terms of a kinetic and chemical mechanism proposed for NADH peroxidase.

  2. Cytochrome bd Displays Significant Quinol Peroxidase Activity

    PubMed Central

    Al-Attar, Sinan; Yu, Yuanjie; Pinkse, Martijn; Hoeser, Jo; Friedrich, Thorsten; Bald, Dirk; de Vries, Simon

    2016-01-01

    Cytochrome bd is a prokaryotic terminal oxidase that catalyses the electrogenic reduction of oxygen to water using ubiquinol as electron donor. Cytochrome bd is a tri-haem integral membrane enzyme carrying a low-spin haem b558, and two high-spin haems: b595 and d. Here we show that besides its oxidase activity, cytochrome bd from Escherichia coli is a genuine quinol peroxidase (QPO) that reduces hydrogen peroxide to water. The highly active and pure enzyme preparation used in this study did not display the catalase activity recently reported for E. coli cytochrome bd. To our knowledge, cytochrome bd is the first membrane-bound quinol peroxidase detected in E. coli. The observation that cytochrome bd is a quinol peroxidase, can provide a biochemical basis for its role in detoxification of hydrogen peroxide and may explain the frequent findings reported in the literature that indicate increased sensitivity to hydrogen peroxide and decreased virulence in mutants that lack the enzyme. PMID:27279363

  3. Redox thermodynamics of lactoperoxidase and eosinophil peroxidase.

    PubMed

    Battistuzzi, Gianantonio; Bellei, Marzia; Vlasits, Jutta; Banerjee, Srijib; Furtmüller, Paul G; Sola, Marco; Obinger, Christian

    2010-02-01

    Eosinophil peroxidase (EPO) and lactoperoxidase (LPO) are important constituents of the innate immune system of mammals. These heme enzymes belong to the peroxidase-cyclooxygenase superfamily and catalyze the oxidation of thiocyanate, bromide and nitrite to hypothiocyanate, hypobromous acid and nitrogen dioxide that are toxic for invading pathogens. In order to gain a better understanding of the observed differences in substrate specificity and oxidation capacity in relation to heme and protein structure, a comprehensive spectro-electrochemical investigation was performed. The reduction potential (E degrees ') of the Fe(III)/Fe(II) couple of EPO and LPO was determined to be -126mV and -176mV, respectively (25 degrees C, pH 7.0). Variable temperature experiments show that EPO and LPO feature different reduction thermodynamics. In particular, reduction of ferric EPO is enthalpically and entropically disfavored, whereas in LPO the entropic term, which selectively stabilizes the oxidized form, prevails on the enthalpic term that favors reduction of Fe(III). The data are discussed with respect to the architecture of the heme cavity and the substrate channel. Comparison with published data for myeloperoxidase demonstrates the effect of heme to protein linkages and heme distortion on the redox chemistry of mammalian peroxidases and in consequence on the enzymatic properties of these physiologically important oxidoreductases. PMID:19944669

  4. Further characterization of human eosinophil peroxidase.

    PubMed Central

    Olsen, R L; Syse, K; Little, C; Christensen, T B

    1985-01-01

    The large and the small subunits (Mr 50 000 and 10 500 respectively) of human eosinophil peroxidase were isolated by gel filtration under reducing conditions. The subunits were very strongly associated but not apparently cross-linked by disulphide bridges. During storage, the large subunit tended to form aggregates, which required reduction to dissociate them. Amino acid analysis of the performic acid-treated large subunit showed the presence of 19 cysteic acid residues. The small subunit of eosinophil peroxidase had the same Mr value as the small subunit of myeloperoxidase. However, although these subunits have very similar amino acid compositions, they showed different patterns of peptide fragmentation after CNBr treatment. The carbohydrate of eosinophil peroxidase seemed associated exclusively with the large subunit and comprised mannose (4.5%, w/w) and N-acetylglucosamine (0.8%, w/w). The far-u.v.c.d. spectrum of the enzyme indicated the presence of relatively little ordered secondary structure. Images Fig. 3. PMID:4052025

  5. Specificity of an HPETE peroxidase from rat PMN

    SciTech Connect

    Skoog, M.T.; Nichols, J.S.; Harrison, B.L.; Wiseman, J.S.

    1988-09-01

    The 15,000xg supernatant of sonicated rat PMN contains 5-lipoxygenase that converts arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene A4 and an HPETE peroxidase that catalyzes reduction of the 5-HPETE. The specificity of this HPETE peroxidase for peroxides, reducing agents, and inhibitors has been characterized to distinguish this enzyme from other peroxidase activities. In addition to 5-HPETE, the HPETE peroxidase will catalyze reduction of 15-hydroperoxyeicosatetraenoic acid, 13-hydroperoxyoctadecadienoic acid, and 15-hydroperoxy-8,11,13-eicosatrienoic acid, but not cumene or t-butylhydroperoxides. The HPETE peroxidase accepted 5 of 11 thiols tested as reducing agents. However, glutathione is greater than 15 times more effective than any other thiol tested. Other reducing agents, ascorbate, NADH, NADPH, phenol, p-cresol, and homovanillic acid, were not accepted by HPETE peroxidase. This enzyme is not inhibited by 10 mM KCN, 2 mM aspirin, 2 mM salicylic acid, or 0.5 mM indomethacin. When 5-(14C)HPETE is generated from (14C)arachidonic acid in the presence of unlabeled 5-HPETE and the HPETE peroxidase, the 5-(14C)HETE produced is of much lower specific activity than the (14C)arachidonic acid. This indicates that the 5-(14C)HPETE leaves the active site of 5-lipoxygenase and mixes with the unlabeled 5-HPETE in solution prior to reduction and is a kinetic demonstration that 5-lipoxygenase has no peroxidase activity. Specificity for peroxides, reducing agents, and inhibitors differentiates HPETE peroxidase from glutathione peroxidase, phospholipid-hydroperoxide glutathione peroxidase, a 12-HPETE peroxidase, and heme peroxidases. The HPETE peroxidase could be a glutathione S-transferase selective for fatty acid hydroperoxides.

  6. Efficient expression of a Phanerochaete chrysosporium manganese peroxidase gene in Aspergillus oryzae

    SciTech Connect

    Stewart, P.; Whitwam, R.E.; Tien, Ming

    1996-03-01

    A manganese peroxidase (mnp1) from Phanerochaete chrysosporium was efficiently expressed in Aspergillus oryzae. Expression was achieved by fusing the mature cDNA of mnp1 with the A. oryzae Taka amylase promoter and secretion signal. The 3{prime} untranslated region of the glucoamylase gene of Asperigillus awamori provided the terminator. The recombinant protein (rMnP) was secreted in an active form, permitting rapid detection and purification. Physical and kinetic properties of rMnP were similar to those of the native protein. The A. oryzae expression system is well suited for both mechanistic and site-directed mutagenesis studies. 34 refs., 7 figs., 1 tab.

  7. Spectrum Recombination.

    ERIC Educational Resources Information Center

    Greenslade, Thomas B., Jr.

    1984-01-01

    Describes several methods of executing lecture demonstrations involving the recombination of the spectrum. Groups the techniques into two general classes: bringing selected portions of the spectrum together using lenses or mirrors and blurring the colors by rapid movement or foreshortening. (JM)

  8. Peroxidase-induced degradation of single-walled carbon nanotubes: hypochlorite is a major oxidant capable of in vivo degradation of carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Vlasova, I. I.; Vakhrusheva, T. V.; Sokolov, A. V.; Kostevich, V. A.; Ragimov, A. A.

    2011-04-01

    Due to their extraordinary properties, single-walled carbon nanotubes (SWNTs) have a tremendous potential for medical applications such as clinical diagnostics, targeted drug (or gene) delivery and cancer therapy. Hence, effects of SWNTs on living systems as well as mechanisms for biodegradation of SWTNs are of great importance and must be studied before starting to explore SWNTs for medical use. This study was undertaken to compare the potential of different peroxidases in degrading carboxylated SWNT (c-SWNT) and to elucidate the role of peroxidase-generated reactive products in this process. A detailed study showed that neither reactive intermediate products nor free radicals generated via peroxidase cycle can considerably oxidize c-SWNT. Biodegradation of c-SWNT in model system can be induced by free radicals generated as a result of heme degradation. The latter explains why hemoglobin, which is a pseudo-peroxidase possessing low peroxidase activity, is able to oxidize carbon nanotubes with a higher efficiency than horseradish peroxidase. However, c-SWNT in the presence of blood plasma (15 vol %) demonstrated no degradation even at high concentrations of hemoglobin and H2O2. The comparison of the ability of various peroxidases to degrade SWNTs in vitro revealed that MPO, due to its ability to produce hypochlorite, and lactoperoxidase, due to its ability to produce hypobromite, are extremely efficient in degrading carbon nanotubes. Since neutrophils are a main source of human MPO, we tested the effect of SWNTs on these cells. SWNTs were unable to stimulate neutrophils. On the other hand, they dose-dependently enhanced opsonized zymosan-induced cell stimulation as detected by measuring the amount of hypochlorite produced. This finding may be relevant to the in vivo situation, for example, at inflammatory sites. In order to imitate conditions characteristic of phagosomes and inflammatory sites, we titrated the suspension of c-SWNT in the presence of diluted blood

  9. A PI 4. 6 peroxidase that specifically crosslinks extensin precursors

    SciTech Connect

    Upham, B.L; Alizadeh, H.; Ryan, K.J.; Lamport, D.T.A. )

    1991-05-01

    The primary cell wall is a microcomposite of cellulose, pectin, hemicellulose and protein. The warp-weft model of the primary cell wall hypothesize that extensin monomers are intermolecularly crosslinked orthogonal to the cellulose microfibril thus mechanically coupling the major load-bearing polymer: cellulose. Media of tomato cell cultures contains heat labile, peroxide dependent crosslinking activity, as determined by the rate of decrease in monomer concentration analyzed via Superose-6. Isoelectric focusing of tomato cell culture media indicated crosslinking was predominantly in the acidic peroxidase fraction (pI4.6). This peroxidase was partially purified by ultracentrifugation, DEAE-Trisacryl and HPLC-DEAE chromatography techniques resulting in a 90 fold purification and 45% yield. A second acidic peroxidase eluted from the HPLC-DEAE column had 25% of the crosslinking activity of the pI 4.6 peroxidase. Purified basic peroxidase had only 0.7% of the activity of the pI 4.6 peroxidase. The specific activity of the pI 4.6 peroxidase was 5,473 mg extensin crosslinked/min/mg peroxidase. The pI 4.6 peroxidase crosslinked the following extensins: tomato I and II, carrot, Ginkgo II and did not crosslink Ginkgo I, Douglas Fir, Maize, Asparagus I and II, and sugarbeet extensins as well as bovine serum albumin. Comparison of motifs common to extensins that are crosslinked by the pI 4.6 peroxidase may help identify the crosslink domain(s) of extension.

  10. Peroxidase-induced wilting in transgenic tobacco plants

    SciTech Connect

    Lagrimini, L.M.; Bradford, S. ); Rothstein, S. )

    1990-01-01

    Peroxidases are a family of isoenzymes found in all higher plants. However, little is known concerning their role in growth, development or response to stress. Plant peroxidases are heme-containing monomeric glycoproteins that utilize either H{sub 2}O{sub 2} or O{sub 2} to oxidize a wide variety of molecules. To obtain more information on possible in planta functions of peroxidases, the authors have used a cDNA clone for the primary isoenzyme form of peroxidase to synthesize high levels of this enzyme in transgenic plants. They were able to obtain Nicotiana tabacum and N. sylvestris transformed plants with peroxidase activity that is 10-fold higher than in wild-type plants by introducing a chimeric gene composed of the cauliflower mosaic virus 35S promoter and the tobacco anionic peroxidase cDNA. The elevated peroxidase activity was a result of increased levels of two anionic peroxidases in N. tabacum, which apparently differ in post-translational modification. Transformed plants of both species have the unique phenotype of chronic severe wilting through loss of turgor in leaves, which was initiated a the time of flowering. The peroxidase-induced wilting was shown not to be an effect of diminished water uptake through the roots, decreased conductance of water through the xylem, or increased water loss through the leaf surface of stomata. Possible explanations for the loss of turgor, and the significance of these types of experiments in studying isoenzyme families, are discussed.

  11. Cytochrome c peroxidase from Methylococcus capsulatus Bath.

    PubMed

    Zahn, J A; Arciero, D M; Hooper, A B; Coats, J R; DiSpirito, A A

    1997-11-01

    A bacterial cytochrome c peroxidase was purified from the obligate methanotroph Methylococcus capsulatus Bath in either the fully oxidized or the half reduced form depending on the purification procedure. The cytochrome was a homo-dimer with a subunit mol mass of 35.8 kDa and an isoelectric point of 4.5. At physiological temperatures, the enzyme contained one high-spin, low-potential (Em7 = -254 mV) and one low-spin, high-potential (Em7 = +432 mM ) heme. The low-potential heme center exhibited a spin-state transition from the penta-coordinated, high-spin configuration to a low-spin configuration upon cooling the enzyme to cryogenic temperatures. Using M. capsulatus Bath ferrocytochrome c555 as the electron donor, the KM and Vmax for peroxide reduction were 510 +/- 100 nM and 425 +/- 22 mol ferrocytochrome c555 oxidized min-1 (mole cytochrome c peroxidase)-1, respectively. PMID:9325424

  12. Potent antioxidative and antigenotoxic activity in aqueous extract of Japanese rice bran--association with peroxidase activity.

    PubMed

    Higashi-Okai, Kiyoka; Kanbara, Keiko; Amano, Kanako; Hagiwara, Akiko; Sugita, Chie; Matsumoto, Norie; Okai, Yasuji

    2004-08-01

    To estimate the preventive potential of Japanese rice bran (Oryza sativa japonica) against the oxygen radical-related chronic diseases such as cardio-vascular diseases and cancer, antioxidative and antigenotoxic activities of the rice bran extracts were analyzed by using assay systems for lipid peroxidation and genotoxin-induced umu gene expression. When effects of the rice bran extracts under different extraction conditions on hydroperoxide generation from auto-oxidized linoleic acid were examined using aluminum chloride method, the water extract showed strong antioxidant activity, but the methanol and acetone extracts did not exhibit significant activity. The water extract of rice bran was divided into the ethanol-precipitable (EP) and supernatant fractions, and EP fraction showed the dominant antioxidant activity, but the supernatant fraction did not exhibit significant antioxidant activity. When the effect of EP fraction on umu C gene expression in SOS response associated with DNA damage in Salmonella typhimurium (TA 1535/pSK 1002) induced by 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was analyzed, it showed a dose-dependent suppressive activity against Trp-P-1-induced umu C gene expression. The bio-chemical analysis of EP fraction indicates that the major antioxidative and antigenotoxic activity of EP fraction is associated with a proteinous component with the molecular weight of more than 30 KDa. As a possible active principle for the antioxidative and antigenotoxic activity in EP fraction, the strong activity of an oxygen radical-scavenging enzyme, peroxidase was detected, and the purified horseradish peroxidase also caused the similar antioxidative and antigenotoxic activities. The significance of this finding is discussed from the viewpoint of the preventive role of rice bran against oxygen radical-related chronic diseases. PMID:15476307

  13. Site-directed mutagenesis of tobacco anionic peroxidase: Effect of additional aromatic amino acids on stability and activity.

    PubMed

    Poloznikov, A A; Zakharova, G S; Chubar, T A; Hushpulian, D M; Tishkov, V I; Gazaryan, I G

    2015-08-01

    Tobacco anionic peroxidase (TOP) is known to effectively catalyze luminol oxidation without enhancers, in contrast to horseradish peroxidase (HRP). To pursue structure-activity relationship studies for TOP, two amino acids have been chosen for mutation, namely Thr151, close to the heme plane, and Phe140 at the entrance to the active site pocket. Three mutant forms TOP F140Y, T151W and F140Y/T151W have been expressed in Escherichia coli, and reactivated to yield active enzymes. Single-point mutations introducing additional aromatic amino acid residues at the surface of TOP exhibit a significant effect on the enzyme catalytic activity and stability as judged by the results of steady-state and transient kinetics studies. TOP T151W is up to 4-fold more active towards a number of aromatic substrates including luminol, whereas TOP F140Y is 2-fold more stable against thermal inactivation and 8-fold more stable in the reaction course. These steady-state observations have been rationalized with the help of transient kinetic studies on the enzyme reaction with hydrogen peroxide in a single turnover regime. The stopped-flow data reveal (a) an increased stability of F140Y Compound I towards hydrogen peroxide, and thus, a higher operational stability as compared to the wild-type enzyme, and (b) a lesser leakage of oxidative equivalents from TOP T151W Compound I resulting in the increased catalytic activity. The results obtained show that TOP unique properties can be further improved for practical applications by site-directed mutagenesis. PMID:25957835

  14. Impact of aging on the formation of bound residues after peroxidase-mediated treatment of 2,4-DCP contaminated soils.

    PubMed

    Palomo, Mónica; Bhandari, Alok

    2006-05-15

    This study evaluated the impact of solute-soil contact time on the formation of "bound" residue in two surface soils exposed to solutions containing 2,4-dichlorophenol (DCP) or DCP polymerization products (DPP). DPP was generated by horseradish peroxidase (HRP) mediated oxidative polymerization of 14C-labeled DCP in the soil slurry. Soils were preloaded with DCP or DPP for durations ranging from 2 h to 84 days. Bound residue was described as solute that was resistant to methanol extraction. Alkali extractions were conducted to estimate the 14C-activity associated with the humic acid, fulvic acid, and humin/mineral components of the soil. Changes in the distribution of the preloaded 14C-DCP and 14C-DPP were observed as a function of the solute-soil contact time. Results suggest that an assumption of sorption equilibrium based solely on the achievement of constant aqueous- or solid-phase solute concentrations can lead to erroneous conclusions about the establishment of true thermodynamic sorption equilibrium. This work also illustrated that (i) significant "irreversible" binding of phenolic contaminants to soils can be achieved during peroxidase-mediated treatment; and (ii) the "aging" process can lead to greater bound-residue formation over time. PMID:16749713

  15. Peroxidase Isozymes from Meloidogyne spp. and Their Origin

    PubMed Central

    Starr, J. L.

    1979-01-01

    Two peroxidase isozymes (Ef 0.43 and 0.53) were detected by electrophoretic analysis in homogenates of Meloidogyne arenaria, M. hapla, M. javanica, and M. incognita females reared on tomato. No peroxidase isozymes were detected electrophoretically in homogenates of adult males, preparasitic larvae, or eggs. Peroxidase isozymes from females reared on bean, eggplant, or tobacco differed from those from females reared on tomato. Bean and eggplant populations had a single peroxidase isozyme each, respectively Ef 0.21 and 0.28. No peroxidase isozymes were detected in tobacco populations under the conditions used, although total activity assays did reveal low levels of peroxidase activity in homogenates of tobacco populations. The peroxidase isozymes detected in females reared on tomato or bean appear similar to the peroxidase isozymes present in root-knot galls, adjacent ungalled roots, and roots from uninoculated plants of the corresponding hosts. The probability is discussed that most of the peroxittase activity associated with Meloidogyne spp. females is of host origin. PMID:19305520

  16. Heterogeneity and regulation of manganese peroxidases from Phanerochaete chrysosporium.

    PubMed Central

    Pease, E A; Tien, M

    1992-01-01

    Lignin and Mn peroxidases are two families of isozymes produced by the lignin-degrading fungus Phanerochaete chrysosporium under nutrient nitrogen or carbon limitation. We purified to homogeneity the three major Mn peroxidase isozymes, H3 (pI = 4.9), H4 (pI = 4.5), and H5 (pI = 4.2). Amino-terminal sequencing of these isozymes demonstrates that they are encoded by different genes. We also analyzed the regulation of these isozymes in carbon- and nitrogen-limited cultures and found not only that the lignin and Mn peroxidases are differentially regulated but also that differential regulation occurs within the Mn peroxidase isozyme family. The isozyme profile and the time at which each isozyme appears in secondary metabolism differ in both nitrogen- and carbon-limited cultures. Each isozyme also responded differently to the addition of a putative inducer, divalent Mn. The stability of the Mn peroxidases in carbon- and nitrogen-limited cultures was also characterized after cycloheximide addition. The Mn peroxidases are more stable in carbon-limited cultures than in nitrogen-limited cultures. They are also more stable than the lignin peroxidases. These data collectively suggest that the Mn peroxidase isozymes serve different functions in lignin biodegradation. Images PMID:1592808

  17. Accelerating peroxidase mimicking nanozymes using DNA

    NASA Astrophysics Data System (ADS)

    Liu, Biwu; Liu, Juewen

    2015-08-01

    DNA-capped iron oxide nanoparticles are nearly 10-fold more active as a peroxidase mimic for TMB oxidation than naked nanoparticles. To understand the mechanism, the effect of DNA length and sequence is systematically studied, and other types of polymers are also compared. This rate enhancement is more obvious with longer DNA and, in particular, poly-cytosine. Among the various polymer coatings tested, DNA offers the highest rate enhancement. A similar acceleration is also observed for nanoceria. On the other hand, when the positively charged TMB substrate is replaced by the negatively charged ABTS, DNA inhibits oxidation. Therefore, the negatively charged phosphate backbone and bases of DNA can increase TMB binding by the iron oxide nanoparticles, thus facilitating the oxidation reaction in the presence of hydrogen peroxide.DNA-capped iron oxide nanoparticles are nearly 10-fold more active as a peroxidase mimic for TMB oxidation than naked nanoparticles. To understand the mechanism, the effect of DNA length and sequence is systematically studied, and other types of polymers are also compared. This rate enhancement is more obvious with longer DNA and, in particular, poly-cytosine. Among the various polymer coatings tested, DNA offers the highest rate enhancement. A similar acceleration is also observed for nanoceria. On the other hand, when the positively charged TMB substrate is replaced by the negatively charged ABTS, DNA inhibits oxidation. Therefore, the negatively charged phosphate backbone and bases of DNA can increase TMB binding by the iron oxide nanoparticles, thus facilitating the oxidation reaction in the presence of hydrogen peroxide. Electronic supplementary information (ESI) available: Methods, TEM, UV-vis and DLS data. See DOI: 10.1039/c5nr04176g

  18. Luffa aegyptiaca (Gourd) Fruit Juice as a Source of Peroxidase

    PubMed Central

    Yadav, R. S. S.; Yadav, K. S.; Yadav, H. S.

    2011-01-01

    Peroxidases have turned out to be potential biocatalyst for a variety of organic reactions. The research work reported in this communication was done with the objective of finding a convenient rich source of peroxidase which could be used as a biocatalyst for organic synthetic reactions. The studies made have shown that Luffa aegyptiaca (gourd) fruit juice contains peroxidase activity of the order of 180 enzyme unit/mL. The Km values of this peroxidase for the substrates guaiacol and hydrogen peroxide were 2.0 and 0.2 mM, respectively. The pH and temperature optima were 6.5 and 60°C, respectively. Like other peroxidases, it followed double displacement type mechanism. Sodium azide inhibited the enzyme competitively with Ki value of 3.35 mM. PMID:21804936

  19. Peroxidase-catalyzed oxidative coupling of phenols in the presence of geosorbents: rates of non-extractable product formation.

    PubMed

    Huang, Qingguo; Selig, Hildegarde; Weber, Walter J

    2002-02-15

    Oxidative coupling processes in subsurface systems comprise a form of natural contaminant attenuation in which hydroxylated aromatic compounds (HACs) are incorporated into soil/sediment organic matter matrices. Here we describe the oxidative coupling of phenol catalyzed by horseradish peroxidase (HRP) in systems containing two geosorbents having organic matter of different composition; specifically Chelsea soil, a near-surface geologically young soil having a predominantly humic-type soil/sediment organic matter (SOM) matrix, and Lachine shale, a diagenetically older natural material having a predominantly kerogen-type SOM matrix. It was found that each of these two different types of natural geosorbents increased the formation of non-extractable coupling products (NEPs) over that which occurred in solids-free systems. The extent of coupling was higher in the systems containing humic-type Chelsea SOM than in those containing kerogen-type Lachine SOM. It was observed that HRP inactivation by free radical attack was significantly reduced in the presence of each geosorbent. A rate model was developed to facilitate quantitative evaluation and mechanistic interpretation of such coupling processes. Experimental rate measurements revealed thatthe greater extent of reaction observed in the presence of Chelsea soil than in the presence of Lachine shale can be attributed to two factors: (i) more effective protection of HRP from inactivation by the Chelsea SOM and (ii) the greater reactivity of Chelsea SOM with respect to cross-coupling. Interrelationships among enzyme protection, cross-coupling reactivity, and SOM chemistry are discussed. PMID:11878372

  20. Development of porous alginate-based scaffolds covalently cross-linked through a peroxidase-catalyzed reaction.

    PubMed

    Sakai, Shinji; Kawakami, Koei

    2011-01-01

    Porous scaffolds are important in tissue engineering. We developed porous scaffolds from the hydrogels of an alginate derivative bearing phenolic hydroxyl groups. The hydrogels were prepared using horseradish peroxidase (HRP) to catalyze the cross-linking between the phenolic hydroxyl groups. A porous structure with a pore size of approx. 200 μm was developed through simultaneous water-extraction and ionic cross-linking by calcium ions by soaking frozen hydrogels in the mixture of ethanol and CaCl2 solution at -20°C. Due to the existence of the covalent cross-links developed through the enzymatic reaction, the porous form had a higher stability from a loss of cross-linked calcium ions than that obtained from non-modified sodium alginate (Na-Alg). The porous specimen developed from the hydrogel obtained with 10 U/ml HRP and 10 mM H2O2 showed about 1.5-times greater repulsion forces than those detected for the porous specimen obtained from Na-Alg toward compressions. No harmful effects of the enzymatically cross-linked specimens were detected on the growth and morphology of the entrapped cells: cells in the enzymatically cross-linked specimens showed almost the same growth profile and morphology with those in the porous specimen obtained from Na-Alg. PMID:21144141

  1. Changes in permeability of rabbit articular cartilage caused by joint contracture as revealed by the peroxidase method.

    PubMed

    Nakamura, K; Ohta, N; Kawaji, W; Takata, K; Hirano, H

    1984-11-01

    Changes in permeability of adult rabbit articular cartilage caused by joint contracture were studied by light and transmission electron microscopy, employing horseradish peroxidase (HRP) as an indicator. The knee joint was plaster-immobilized for 0, 2, 4, 6, or 8 weeks in the flexion position. One ml of 4% HRP was administered in the articular cavity of the knee joint and allowed to diffuse and permeate into the articular cartilage. Distribution of the permeated HRP was visualized in the cartilage taken from the lateral condyle of the femur, utilizing the DAB-H2O2 reaction. In the normal and the non-immobilized joints, the permeated HRP reached to the matrix and chondrocytes situated in the deep layer of the articular cartilage. HRP was heavily deposited in the intercellular matrices, particularly around the chondrocytes, and was actively endocytosed by these cells. In the plaster-immobilized joints, especially after 4 weeks or longer of immobilization, the administered HRP had not permeated well and was restricted to the surface (lamina splendens) and the superficial layer of the cartilage. These results show that administered HRP diffuses into the deep layer of the articular cartilage and is actively endocytosed by chondrocytes and that the permeability of articular cartilage is remarkably reduced by joint contracture. PMID:6532371

  2. The Quantum Mixed-Spin Heme State of Barley Peroxidase: A Paradigm for Class III Peroxidases

    SciTech Connect

    Howes, B.D.; Ma, J.; Marzocchi, M.P.; Schiodt, C.B.; Shelnutt, J.A.; Smulevich, G.; Welinder, K.G.; Zhang, J.

    1999-03-23

    Electronic absorption and resonance Raman (RR) spectra of the ferric form of barley grain peroxidase (BP 1) at various pH values both at room temperature and 20 K are . reported, together with EPR spectra at 10 K. The ferrous forms and the ferric complex with fluoride have also been studied. A quantum mechanically mixed-spin (QS) state has been identified. The QS heme species co-exists with 6- and 5-cHS heroes; the relative populations of these three spin states are found to be dependent on pH and temperature. However, the QS species remains in all cases the dominant heme spin species. Barley peroxidase appears to be further characterized by a splitting of the two vinyl stretching modes, indicating that the vinyl groups are differently conjugated with the porphyrin. An analysis of the presently available spectroscopic data for proteins from all three peroxidase classes suggests that the simultaneous occurrence of the QS heme state as well as the splitting of the two vinyl stretching modes is confined to class III enzymes. The former point is discussed in terms of the possible influences of heme deformations on heme spin state. It is found that moderate saddling alone is probably not enough to cause the QS state, although some saddling maybe necessary for the QS state.

  3. fPoxDB: fungal peroxidase database for comparative genomics

    PubMed Central

    2014-01-01

    Background Peroxidases are a group of oxidoreductases which mediate electron transfer from hydrogen peroxide (H2O2) and organic peroxide to various electron acceptors. They possess a broad spectrum of impact on industry and fungal biology. There are numerous industrial applications using peroxidases, such as to catalyse highly reactive pollutants and to breakdown lignin for recycling of carbon sources. Moreover, genes encoding peroxidases play important roles in fungal pathogenicity in both humans and plants. For better understanding of fungal peroxidases at the genome-level, a novel genomics platform is required. To this end, Fungal Peroxidase Database (fPoxDB; http://peroxidase.riceblast.snu.ac.kr/) has been developed to provide such a genomics platform for this important gene family. Description In order to identify and classify fungal peroxidases, 24 sequence profiles were built and applied on 331 genomes including 216 from fungi and Oomycetes. In addition, NoxR, which is known to regulate NADPH oxidases (NoxA and NoxB) in fungi, was also added to the pipeline. Collectively, 6,113 genes were predicted to encode 25 gene families, presenting well-separated distribution along the taxonomy. For instance, the genes encoding lignin peroxidase, manganese peroxidase, and versatile peroxidase were concentrated in the rot-causing basidiomycetes, reflecting their ligninolytic capability. As a genomics platform, fPoxDB provides diverse analysis resources, such as gene family predictions based on fungal sequence profiles, pre-computed results of eight bioinformatics programs, similarity search tools, a multiple sequence alignment tool, domain analysis functions, and taxonomic distribution summary, some of which are not available in the previously developed peroxidase resource. In addition, fPoxDB is interconnected with other family web systems, providing extended analysis opportunities. Conclusions fPoxDB is a fungi-oriented genomics platform for peroxidases. The sequence

  4. Molecular characterization of the lignin-forming peroxidase: Role in growth, development and response to stress

    SciTech Connect

    Lagrimini, L.M.

    1993-01-01

    This laboratory has continued its comprehensive study of the structure and function of plant peroxidases and their genes. Specifically, we are characterizing the anionic peroxidase of tobacco. During the past year we have completed the nucleotide sequence of the tobacco anionic peroxidase gene, joined the anionic peroxidase promoter to [Beta]-glucuronidase and demonstrated expression in transformed plants, measured lignin, auxin, and ethylene levels in transgenic tobacco plants over-expressing the anionic peroxidase, developed chimeric peroxidase genes to over-or under-express the anionic peroxidase in tissue specific manner in transgenic plants, and over-expressed the tobacco anionic peroxidase in transgenic tomato and sweetgum plants.

  5. Stabilization of lignin peroxidases in white rot fungi by tryptophan.

    PubMed Central

    Collins, P J; Field, J A; Teunissen, P; Dobson, A D

    1997-01-01

    Supplementation of various cultures of white rot fungi with tryptophan was found to have a large stimulatory effect on lignin peroxidase activity levels. This enhancement was greater than that observed in the presence of the lignin peroxidase recycling agent veratryl alcohol. Using reverse transcription-PCR, we found that tryptophan does not act to induce lignin peroxidase expression at the level of gene transcription. Instead, the activity enhancement observed is likely to result from the protective effect of tryptophan against H2O2 inactivation. In experiments using a partially purified lignin peroxidase preparation, tryptophan and its derivative indole were determined to function in the same way as veratryl alcohol in converting compound II, an oxidized form of lignin peroxidase, to ferric enzyme, thereby completing the catalytic cycle. Furthermore, tryptophan was found to be a better substrate for lignin peroxidase than veratryl alcohol. Inclusion of either tryptophan or indole enhanced the oxidation of the azo dyes methyl orange and Eriochrome blue black. Stimulation of azo dye oxidations by veratryl alcohol has previously been shown to be due to its enzyme recycling function. Our data allow us to propose that tryptophan stabilizes lignin peroxidase by acting as a reductant for the enzyme. PMID:9212404

  6. The molecular characterization of the lignin-forming peroxidase

    SciTech Connect

    Lagrimini, L.M.

    1992-01-01

    This laboratory is committed to understanding the function of plant peroxidases via a multi-disciplinary approach. We have chosen the lignin-forming peroxidase from tobacco as the first isoenzyme to be subjected to this comprehensive approach. The goals which were set out upon the initiation of this project were as follows: (1) utilize a cDNA clone to the tobacco anionic peroxidase to generate transgenic plants which either over-produced this isoenzyme or specifically under-produced this isoenzyme via antisense RNA, (2) describe any phenotypic changes resulting from altered peroxidase expression, (3) perform morphological, physiological, and biochemical analysis of the above mentioned plants to help in determining the in planta function for this enzyme, and (4) clone and characterize the gene for the tobacco anionic peroxidase. A summary of progress thus far which includes both published and unpublished work will be presented in three sections: generation and characterization of transgenic plants, description of phenotypes, and biochemical and physiological analysis of peroxidase function, and cloning and characterization of the tobacco anionic peroxidase gene.

  7. Application of peroxidase labelled antibody assays for detection of porcine IgG antibodies to hog cholera and bovine viral diarrhea viruses.

    PubMed

    Afshar, A; Dulac, G C; Bouffard, A

    1989-03-01

    Rapid, sensitive peroxidase labelled antibody (PLA) assays using microtiter systems, were developed for detection of hog cholera virus (HCV) and cross-reacting bovine viral diarrhea virus (BVDV) antibodies in pig sera. HCV-infected pig kidney cell line (PK 15) prepared in microtiter plates were fixed and used in PLA assays. After inoculation with test serum, bound antibodies (HCV/BVDV) were reacted with either horseradish peroxidase (HRP) conjugated anti-porcine immunoglobulin (H & L) or biotinylated protein A (BPA) and subsequent HRP labelled avidin (A). Positive reactions were easily visualized under an inverted light microscope as foci of brown colored cells after enzyme degradation of hydrogen peroxidase in the presence of amino-ethylcarbazole (AEC). The PLA assays were superior to the indirect fluorescent antibody (IFA) test in detecting anti-HCV antibodies in porcine sera collected early after inoculation of pigs with a lapinized HCV vaccine. The performances of the PLA, IFA and FA neutralization (FAN) tests in measuring the immune response in the vaccinated pigs were comparable. Cross-reacting anti-BVDV antibody, as measured by a microtiter serum neutralization (MTSN) test, was not demonstrable in vaccinated pigs until they were challenged with a virulent HCV, 13 weeks later. The PLA assays relative to the IFA test detected more reactive samples among porcine field sera collected from HC-free pigs in Canada. Of 795 samples, 24 (3.01%) were reactive in the PLA employing HRP anti-porcine IgG, and 21 (2.6%) in the PLA, using BPA-HRP-A. When 324 of these sera were screened by the IFA test (using HC antigen), only one sample (0.30%) was found reactive.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2541152

  8. Horseradish curly top virus is a distinct subgroup II geminivirus species with rep and C4 genes derived from a subgroup III ancestor.

    PubMed

    Klute, K A; Nadler, S A; Stenger, D C

    1996-07-01

    The complete nucleotide sequence (3080 nt) of an infectious DNA clone derived from the geminivirus horseradish curly top virus (HrCTV) has been determined. The relationship of HrCTV to other geminiviruses was examined using dot matrix plots of nucleotide sequence similarities, and by phylogeny of predicted amino acid sequences of individual ORFs based upon parsimony or neighbour-joining methods. These analyses indicate that the V1 and V2 virion sense ORFs of HrCTV are most closely related to, yet distinct from, the corresponding ORFs of the subgroup II geminivirus beet curly top virus (BCTV). HrCTV also encodes a third virion sense ORF (V3) which is similar (72-74 percent amino acid identity) to the BCTV V3 ORF; however, the HrCTV V3 ORF has diverged in sequence to a greater extent relative to that observed among isolates of BCTV (98-100% amino acid identity). The HrCTV genome encodes only three complementary sense ORFs (Cl, C2 and C4) and lacks a C3 ORF which is conserved among all other subgroup II and III geminiviruses characterized to date. Although the neighbour-joining analysis indicated that the HrCTV C2 ORF was distantly related to the C2 ORF of BCTV, the predicted amino acid sequence deduced from the HrCTV C2 ORF lacks the characteristic zinc-finger domain present in the transcriptional activating protein (TrAP) encoded by the subgroup III ORF AC2, which is also retained within the TrAP-related product of the BCTV C2 ORF. Surprisingly, the rep and C4 proteins encoded by HrCTV share a closer phylogenetic relationship to the corresponding proteins of the subgroup III geminivirus squash leaf curl virus (SLCV) than to BCTV. These results suggest that the HrCTV genome may have arisen by a recombination event between a BCTV-like subgroup II virus ancestor and an SLCV-like subgroup III virus ancestor. Possible mechanisms that may explain recombination events among geminiviruses are discussed. PMID:8757976

  9. Redundancy among manganese peroxidases in Pleurotus ostreatus.

    PubMed

    Salame, Tomer M; Knop, Doriv; Levinson, Dana; Yarden, Oded; Hadar, Yitzhak

    2013-04-01

    Manganese peroxidases (MnPs) are key players in the ligninolytic system of white rot fungi. In Pleurotus ostreatus (the oyster mushroom) these enzymes are encoded by a gene family comprising nine members, mnp1 to -9 (mnp genes). Mn(2+) amendment to P. ostreatus cultures results in enhanced degradation of recalcitrant compounds (such as the azo dye orange II) and lignin. In Mn(2+)-amended glucose-peptone medium, mnp3, mnp4, and mnp9 were the most highly expressed mnp genes. After 7 days of incubation, the time point at which the greatest capacity for orange II decolorization was observed, mnp3 expression and the presence of MnP3 in the extracellular culture fluids were predominant. To determine the significance of MnP3 for ligninolytic functionality in Mn(2+)-sufficient cultures, mnp3 was inactivated via the Δku80 strain-based P. ostreatus gene-targeting system. In Mn(2+)-sufficient medium, inactivation of mnp3 did not significantly affect expression of nontargeted MnPs or their genes, nor did it considerably diminish the fungal Mn(2+)-mediated orange II decolorization capacity, despite the significant reduction in total MnP activity. Similarly, inactivation of either mnp4 or mnp9 did not affect orange II decolorization ability. These results indicate functional redundancy within the P. ostreatus MnP gene family, enabling compensation upon deficiency of one of its members. PMID:23377936

  10. Redundancy among Manganese Peroxidases in Pleurotus ostreatus

    PubMed Central

    Salame, Tomer M.; Knop, Doriv; Levinson, Dana; Yarden, Oded

    2013-01-01

    Manganese peroxidases (MnPs) are key players in the ligninolytic system of white rot fungi. In Pleurotus ostreatus (the oyster mushroom) these enzymes are encoded by a gene family comprising nine members, mnp1 to -9 (mnp genes). Mn2+ amendment to P. ostreatus cultures results in enhanced degradation of recalcitrant compounds (such as the azo dye orange II) and lignin. In Mn2+-amended glucose-peptone medium, mnp3, mnp4, and mnp9 were the most highly expressed mnp genes. After 7 days of incubation, the time point at which the greatest capacity for orange II decolorization was observed, mnp3 expression and the presence of MnP3 in the extracellular culture fluids were predominant. To determine the significance of MnP3 for ligninolytic functionality in Mn2+-sufficient cultures, mnp3 was inactivated via the Δku80 strain-based P. ostreatus gene-targeting system. In Mn2+-sufficient medium, inactivation of mnp3 did not significantly affect expression of nontargeted MnPs or their genes, nor did it considerably diminish the fungal Mn2+-mediated orange II decolorization capacity, despite the significant reduction in total MnP activity. Similarly, inactivation of either mnp4 or mnp9 did not affect orange II decolorization ability. These results indicate functional redundancy within the P. ostreatus MnP gene family, enabling compensation upon deficiency of one of its members. PMID:23377936

  11. Peroxidase gene expression during tomato fruit ripening

    SciTech Connect

    Biggs, M.S.; Flurkey, W.H.; Handa, A.K.

    1987-04-01

    Auxin oxidation has been reported to play a critical role in the initiation of pear fruit ripening and a tomato fruit peroxidase (POD) has been shown to have IAA-oxidase activity. However, little is known about changes in the expression of POD mRNA in tomato fruit development. They are investigating the expression of POD mRNA during tomato fruit maturation. Fruit pericarp tissues from six stages of fruit development and ripening (immature green, mature green, breaker, turning, ripe, and red ripe fruits) were used to extract poly (A)/sup +/ RNAs. These RNAs were translated in vitro in a rabbit reticulocyte lysate system using L-/sup 35/S-methionine. The /sup 35/S-labeled products were immunoprecipitated with POD antibodies to determine the relative proportions of POD mRNA. High levels of POD mRNA were present in immature green and mature green pericarp, but declined greatly by the turning stage of fruit ripening. In addition, the distribution of POD mRNA on free vs bound polyribosomes will be presented, as well as the presence or absence of POD mRNA in other tomato tissues.

  12. Expression of a Phanerochaete chrysosporium manganese peroxidase gene in the yeast Pichia pastoris.

    PubMed

    Gu, Lina; Lajoie, Curtis; Kelly, Christine

    2003-01-01

    A gene encoding manganese peroxidase (mnp1) from Phanerochaete chrysosporium was cloned downstream of a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter in the methylotrophic yeast Pichia pastoris. Three different expression vectors were constructed: pZBMNP contains the native P. chrysosporium fungal secretion signal, palphaAMNP contains an alpha-factor secretion signal derived from Saccharomyces cerevisiae, and pZBIMNP has no secretion signal and was used for intracellular expression. Both the native fungal secretion signal sequence and alpha-factor secretion signal sequence directed the secretion of active recombinant manganese peroxidase (rMnP) from P. pastoris transformants. The majority of the rMnP produced by P. pastoris exhibited a molecular mass (55-100 kDa) considerably larger than that of the wild-type manganese peroxidase (wtMnP, 46 kDa). Deletion of the native fungal secretion signal yielded a molecular mass of 39 kDa for intracellular rMnP in P. pastoris. Treatment of the secreted rMnP with endoglycosidase H (Endo H) resulted in a considerable decrease in the mass of rMnP, indicating N-linked hyperglycosylation. Partially purified rMnP showed kinetic characteristics similar to those of wtMnP. Both enzymes also had similar pH stability profiles. Addition of exogenous Mn(II), Ca(II), and Fe(III) conferred additional thermal stability to both enzymes. However, rMnP was slightly less thermostable than wtMnP, which demonstrated an extended half-life at 55 degrees C. PMID:14524699

  13. Crystallographic, kinetic, and spectroscopic study of the first ligninolytic peroxidase presenting a catalytic tyrosine.

    PubMed

    Miki, Yuta; Calviño, Fabiola R; Pogni, Rebecca; Giansanti, Stefania; Ruiz-Dueñas, Francisco J; Martínez, María Jesús; Basosi, Riccardo; Romero, Antonio; Martínez, Angel T

    2011-04-29

    Trametes cervina lignin peroxidase (LiP) is a unique enzyme lacking the catalytic tryptophan strictly conserved in all other LiPs and versatile peroxidases (more than 30 sequences available). Recombinant T. cervina LiP and site-directed variants were investigated by crystallographic, kinetic, and spectroscopic techniques. The crystal structure shows three substrate oxidation site candidates involving His-170, Asp-146, and Tyr-181. Steady-state kinetics for oxidation of veratryl alcohol (the typical LiP substrate) by variants at the above three residues reveals a crucial role of Tyr-181 in LiP activity. Moreover, assays with ferrocytochrome c show that its ability to oxidize large molecules (a requisite property for oxidation of the lignin polymer) originates in Tyr-181. This residue is also involved in the oxidation of 1,4-dimethoxybenzene, a reaction initiated by the one-electron abstraction with formation of substrate cation radical, as described for the well known Phanerochaete chrysosporium LiP. Detailed spectroscopic and kinetic investigations, including low temperature EPR, show that the porphyrin radical in the two-electron activated T. cervina LiP is unstable and rapidly receives one electron from Tyr-181, forming a catalytic protein radical, which is identified as an H-bonded neutral tyrosyl radical. The crystal structure reveals a partially exposed location of Tyr-181, compatible with its catalytic role, and several neighbor residues probably contributing to catalysis: (i) by enabling substrate recognition by aromatic interactions; (ii) by acting as proton acceptor/donor from Tyr-181 or H-bonding the radical form; and (iii) by providing the acidic environment that would facilitate oxidation. This is the first structure-function study of the only ligninolytic peroxidase described to date that has a catalytic tyrosine. PMID:21367853

  14. Thiol Peroxidase Is an Important Component of Streptococcus pneumoniae in Oxygenated Environments

    PubMed Central

    Hajaj, Barak; Yesilkaya, Hasan; Benisty, Rachel; David, Maayan; Andrew, Peter W.

    2012-01-01

    Streptococcus pneumoniae is an aerotolerant Gram-positive bacterium that causes an array of diseases, including pneumonia, otitis media, and meningitis. During aerobic growth, S. pneumoniae produces high levels of H2O2. Since S. pneumoniae lacks catalase, the question of how it controls H2O2 levels is of critical importance. The psa locus encodes an ABC Mn2+-permease complex (psaBCA) and a putative thiol peroxidase, tpxD. This study shows that tpxD encodes a functional thiol peroxidase involved in the adjustment of H2O2 homeostasis in the cell. Kinetic experiments showed that recombinant TpxD removed H2O2 efficiently. However, in vivo experiments revealed that TpxD detoxifies only a fraction of the H2O2 generated by the pneumococcus. Mass spectrometry analysis demonstrated that TpxD Cys58 undergoes selective oxidation in vivo, under conditions where H2O2 is formed, confirming the thiol peroxidase activity. Levels of TpxD expression and synthesis in vitro were significantly increased in cells grown under aerobic versus anaerobic conditions. The challenge with D39 and TIGR4 with H2O2 resulted in tpxD upregulation, while psaBCA expression was oppositely affected. However, the challenge of ΔtpxD mutants with H2O2 did not affect psaBCA, implying that TpxD is involved in the regulation of the psa operon, in addition to its scavenging activity. Virulence studies demonstrated a notable difference in the survival time of mice infected intranasally with D39 compared to that of mice infected intranasally with D39ΔtpxD. However, when bacteria were administered directly into the blood, this difference disappeared. The findings of this study suggest that TpxD constitutes a component of the organism's fundamental strategy to fine-tune cellular processes in response to H2O2. PMID:23027531

  15. Altered phenotypes in plants transformed with chimeric tobacco peroxidase genes

    SciTech Connect

    Lagrimini, L.M.

    1990-01-01

    Peroxidases have been implicated in a variety of secondary metabolic reactions including lignification, cross-linking of cell wall polysaccharides, oxidation of indole-3-acetic acid, regulation of cell elongation, wound-healing, phenol oxidation, and pathogen defense. However, due to the many different isoenzymes and even more potential substrates, it has proven difficult to verify actual physiological roles for peroxidase. We are studying the molecular biology of the tobacco peroxidase genes, and have utilized genetic engineering techniques to produce transgenic plants which differ only in their expression of an individual peroxidase isoenzyme. Many of the in planta functions for any individual isoenzyme may be predicted through the morphological and physiological analysis of transformed plants.

  16. Altered phenotypes in plants transformed with chimeric tobacco peroxidase genes

    SciTech Connect

    Lagrimini, L.M.

    1990-12-31

    Peroxidases have been implicated in a variety of secondary metabolic reactions including lignification, cross-linking of cell wall polysaccharides, oxidation of indole-3-acetic acid, regulation of cell elongation, wound-healing, phenol oxidation, and pathogen defense. However, due to the many different isoenzymes and even more potential substrates, it has proven difficult to verify actual physiological roles for peroxidase. We are studying the molecular biology of the tobacco peroxidase genes, and have utilized genetic engineering techniques to produce transgenic plants which differ only in their expression of an individual peroxidase isoenzyme. Many of the in planta functions for any individual isoenzyme may be predicted through the morphological and physiological analysis of transformed plants.

  17. Stimuli-responsive peroxidase mimicking at a smart graphene interface.

    PubMed

    Liu, Meng; Zhao, Huimin; Chen, Shuo; Yu, Hongtao; Quan, Xie

    2012-07-18

    A synergistic graphene-based catalyst was engineered by the in situ growth of "naked" Au-nanoparticles (NPs) on graphene sheets. The catalyst exhibits excellent switchable peroxidase-like activity in response to specific DNA. PMID:22673613

  18. Cell wall bound anionic peroxidases from asparagus byproducts.

    PubMed

    Jaramillo-Carmona, Sara; López, Sergio; Vazquez-Castilla, Sara; Jimenez-Araujo, Ana; Rodriguez-Arcos, Rocio; Guillen-Bejarano, Rafael

    2014-10-01

    Asparagus byproducts are a good source of cationic soluble peroxidases (CAP) useful for the bioremediation of phenol-contaminated wastewaters. In this study, cell wall bound peroxidases (POD) from the same byproducts have been purified and characterized. The covalent forms of POD represent >90% of the total cell wall bound POD. Isoelectric focusing showed that whereas the covalent fraction is constituted primarily by anionic isoenzymes, the ionic fraction is a mixture of anionic, neutral, and cationic isoenzymes. Covalently bound peroxidases were purified by means of ion exchange chromatography and affinity chromatography. In vitro detoxification studies showed that although CAP are more effective for the removal of 4-CP and 2,4-DCP, anionic asparagus peroxidase (AAP) is a better option for the removal of hydroxytyrosol (HT), the main phenol present in olive mill wastewaters. PMID:25195693

  19. Multidomain Human Peroxidasin 1 Is a Highly Glycosylated and Stable Homotrimeric High Spin Ferric Peroxidase*

    PubMed Central

    Soudi, Monika; Paumann-Page, Martina; Delporte, Cedric; Pirker, Katharina F.; Bellei, Marzia; Edenhofer, Eva; Stadlmayr, Gerhard; Battistuzzi, Gianantonio; Boudjeltia, Karim Zouaoui; Furtmüller, Paul G.; Van Antwerpen, Pierre; Obinger, Christian

    2015-01-01

    Human peroxidasin 1 (hsPxd01) is a multidomain heme peroxidase that uses bromide as a cofactor for the formation of sulfilimine cross-links. The latter confers critical structural reinforcement to collagen IV scaffolds. Here, hsPxd01 and various truncated variants lacking nonenzymatic domains were recombinantly expressed in HEK cell lines. The N-glycosylation site occupancy and disulfide pattern, the oligomeric structure, and unfolding pathway are reported. The homotrimeric iron protein contains a covalently bound ferric high spin heme per subunit with a standard reduction potential of the Fe(III)/Fe(II) couple of −233 ± 5 mV at pH 7.0. Despite sequence homology at the active site and biophysical properties similar to human peroxidases, the catalytic efficiency of bromide oxidation (kcat/KMapp) of full-length hsPxd01 is rather low but increased upon truncation. This is discussed with respect to its structure and proposed biosynthetic function in collagen IV cross-linking. PMID:25713063

  20. Grafting of Functional Molecules: Insights into Peroxidase-Derived Materials

    NASA Astrophysics Data System (ADS)

    Nyanhongo, Gibson S.; Prasetyo, Endry Nugroho; Kudanga, Tukayi; Guebitz, Georg

    An insight into the progress made in applying heme peroxidases in grafting processes, starting from the production of simple resins to more complex polymers, is presented. The refinement of the different reaction conditions (solvents, concentrations of the reactants) and careful study of the reaction mechanisms have been instrumental in advancing enzymatic grafting processes. A number of processes described here show how peroxidase mediated catalysis could provide a new strategy as an alternative to conventional energy intensive procedures mediated by chemical catalysts.

  1. Heterologous Expression of Phanerochaete chrysoporium Glyoxal Oxidase and its Application for the Coupled Reaction with Manganese Peroxidase to Decolorize Malachite Green

    PubMed Central

    Son, Yu-Lim; Kim, Hyoun-Young; Thiyagarajan, Saravanakumar; Xu, Jing Jing

    2012-01-01

    cDNA of the glx1 gene encoding glyoxal oxidase (GLX) from Phanerochaete chrysosporium was isolated and expressed in Pichia pastoris. The recombinant GLX (rGLX) produces H2O2 over 7.0 nmol/min/mL using methyl glyoxal as a substrate. Use of rGLX as a generator of H2O2 improved the coupled reaction with recombinant manganese peroxidase resulting in decolorization of malachite green up to 150 µM within 90 min. PMID:23323052

  2. The impact of thiol peroxidases on redox regulation.

    PubMed

    Flohé, Leopold

    2016-01-01

    The biology of glutathione peroxidases and peroxiredoxins is reviewed with emphasis on their role in metabolic regulation. Apart from their obvious function in balancing oxidative challenge, these thiol peroxidases are not only implicated in orchestrating the adaptive response to oxidative stress, but also in regulating signaling triggered by hormones, growth factors and cytokines. The mechanisms presently discussed comprise dampening of redox-sensitive regulatory processes by elimination of hydroperoxides, suppression of lipoxygenase activity, committing suicide to save H2O2 for signaling, direct binding to receptors or regulatory proteins in a peroxidase activity-independent manner, or acting as sensors for hydroperoxides and as transducers of oxidant signals. The various mechanistic proposals are discussed in the light of kinetic data, which unfortunately are scarce. Taking into account pivotal criteria of a meaningful regulatory circuit, kinetic plausibility and specificity, the mechanistic concepts implying a direct sensor/transducer function of the thiol peroxidases appear most appealing. With rate constants for the reaction with hydroperoxide of 10(5)-10(8) M(-1) s(-1), thiol peroxidases are qualified as kinetically preferred hydroperoxide sensors, and the ability of the oxidized enzymes to react with defined protein thiols lends specificity to the transduction process. The versatility of thiol peroxidases, however, allows multiple ways of interaction with regulatory pathways. PMID:26291534

  3. Serine incorporation into the selenocysteine moiety of glutathione peroxidase

    SciTech Connect

    Sunde, R.A.; Evenson, J.K.

    1987-01-15

    The selenium in mammalian glutathione peroxidase is present as a selenocysteine ((Se)Cys) moiety incorporated into the peptide backbone 41-47 residues from the N-terminal end. To study the origin of the skeleton of the (Se)Cys moiety, we perfused isolated rat liver with /sup 14/C- or /sup 3/H-labeled amino acids for 4 h, purified the GSH peroxidase, derivatized the (Se)Cys in GSH peroxidase to carboxymethylselenocysteine ((Se)Cys(Cm)), and determined the amino acid specific activity. Perfusion with (/sup 14/C)cystine resulted in (/sup 14/C)cystine incorporation into GSH peroxidase without labeling (Se)Cys(Cm), indicating that cysteine is not a direct precursor for (Se)Cys. (/sup 14/C)Serine perfusion labeled serine, glycine (the serine hydroxymethyltransferase product), and (Se)Cys(Cm) in purified GSH peroxidase, whereas (3-3H)serine perfusion only labeled serine and (Se)Cys(Cm), thus demonstrating that the (Se)Cys in GSH peroxidase is derived from serine. The similar specific activities of serine and (Se)Cys(Cm) strongly suggest that the precursor pool of serine used for (Se) Cys synthesis is the same or similar to the serine pool used for acylation of seryl-tRNAs.

  4. Study of a reagent- and mediator-less biosensor for D-amino acids based on co-immobilized D-amino acid oxidase and peroxidase in carbon paste electrodes.

    PubMed

    Johansson, E; Marko-Varga, G; Gorton, L

    1993-10-01

    A biosensor for the analysis of D-amino acids is described. Carbon paste (graphite/paraffin oil) was chemically modified with immobilized D-amino acid oxidase and either horse-radish peroxidase or fungal peroxidase from Arthromyces ramosus. The two enzymes dissolved in buffer, together with an amine containing oligomer or polymer, were adsorbed on dry graphite. Prior to immobilization, the graphite was heat treated at 700 degrees C for 15 s to promote an efficient electron transfer between graphite and the peroxidase. The mixture was dried before addition of the pasting liquid. The sensor is based on the fact that the hydrogen peroxide produced by the action of D-amino acid oxidase is electrocatalytically reduced through the action of the peroxidase. The amine containing compound acted as a stabilizer and activator of the enzymes in the paste. The enzyme electrode was investigated as a sensor for D-phenylalanine and hydrogen peroxide in a flow through electrochemical cell connected to a single line flow injection system. The influences on the response by different additives to the paste and pH are reported. Linear calibration curves were obtained between 0.1 and 1.4 mM for D-phenylalanine and 5 and 1000 microM for hydrogen peroxide at an applied potential of -50 mV vs. Ag/AgCl. The sensor was also active for the following D-amino acids: D-alanine, D-valine, D-leucine, D-isoleucine, D-serine, D-aspartic acid, D-glutamic acid, D-lysine, D-histidine, D-arginine, D-tryptophan, D-methionine, and D-proline. PMID:7902430

  5. Engineering the proximal heme cavity of catalase-peroxidase.

    PubMed

    Jakopitsch, Christa; Regelsberger, Günther; Furtmüller, Paul Georg; Rüker, Florian; Peschek, Günter A; Obinger, Christian

    2002-07-25

    Catalase-peroxidases (KatGs) are prokaryotic heme peroxidases with homology to yeast cytochrome c peroxidase (CCP) and plant ascorbate peroxidases (APXs). KatGs, CCP and APXs contain identical amino acid triads in the heme pocket (distal Arg/Trp/His and proximal His/Trp/Asp), but differ dramatically in their reactivities towards hydrogen peroxide and various one-electron donors. Only KatGs have high catalase activity in addition to a peroxidase activity of broad specificity. Here, we investigated the effect of mutating the conserved proximal triad on KatG catalysis. With the exception of W341F, all variants (H290Q, W341A, D402N, D402E) exhibited a catalase activity <1% of wild-type KatG and spectral properties indicating alterations in heme coordination and spin states. Generally, the peroxidase activity was much less effected by these mutations. Compared with wild-type KatG the W341F variant had a catalase and halogenation activity of about 40% and an even increased overall peroxidase activity. This variant, for the first time, allowed to monitor the hydrogen peroxide mediated transitions of ferric KatG to compound I and back to the resting enzyme. Compound I reduction by aromatic one-electron donors (o-dianisidine, pyrogallol, aniline) was not influenced by exchanging Trp by Phe. The findings are discussed in comparison with the data known from CCP and APX and a reaction mechanism for the multifunctional activity of the W341F variant is suggested. PMID:12121764

  6. Purification and characterization of an intracellular peroxidase from Streptomyces cyaneus

    SciTech Connect

    Mliki, A.; Zimmermann, W. )

    1992-03-01

    Peroxidases play an important role in the oxidation of a large number of aromatic compounds, including recalcitrant substances. An intracellular peroxidase (EC 1.11.1.7) from Streptomyces cyaneus was purified to homogeneity. The enzyme had a molecular weight of 185,000 and was composed of two subunits of equal size. It had an isoelectric point of 6.1. The enzyme had a peroxidase activity toward o-dianisidine with a K{sub m} of 17.8 {mu}M and a pH optimum of 5.0. It also showed catalase activity with a K{sub m} of 2.07 mM H{sub 2}O{sub 2} and a pH optimum of 8.0. The purified enzyme did not catalyze C{alpha}-C{beta} bond cleavage of 1,3-dihydroxy-2-(2-methoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl) propane, a nonphenolic dimeric lignin model compound. The spectrum of te peroxidase showed a soret band at 405 nm, which disappeared after reduction with sodium dithionite, indicating that the enzyme is a hemoprotein. Testing the effects of various inhibitors on the enzyme activity showed that it is a bifunctional enzyme having catalase and peroxidase activities.

  7. Peroxidase activation of cytoglobin by anionic phospholipids: Mechanisms and consequences.

    PubMed

    Tejero, Jesús; Kapralov, Alexandr A; Baumgartner, Matthew P; Sparacino-Watkins, Courtney E; Anthonymutu, Tamil S; Vlasova, Irina I; Camacho, Carlos J; Gladwin, Mark T; Bayir, Hülya; Kagan, Valerian E

    2016-05-01

    Cytoglobin (Cygb) is a hexa-coordinated hemoprotein with yet to be defined physiological functions. The iron coordination and spin state of the Cygb heme group are sensitive to oxidation of two cysteine residues (Cys38/Cys83) and/or the binding of free fatty acids. However, the roles of redox vs lipid regulators of Cygb's structural rearrangements in the context of the protein peroxidase competence are not known. Searching for physiologically relevant lipid regulators of Cygb, here we report that anionic phospholipids, particularly phosphatidylinositolphosphates, affect structural organization of the protein and modulate its iron state and peroxidase activity both conjointly and/or independently of cysteine oxidation. Thus, different anionic lipids can operate in cysteine-dependent and cysteine-independent ways as inducers of the peroxidase activity. We establish that Cygb's peroxidase activity can be utilized for the catalysis of peroxidation of anionic phospholipids (including phosphatidylinositolphosphates) yielding mono-oxygenated molecular species. Combined with the computational simulations we propose a bipartite lipid binding model that rationalizes the modes of interactions with phospholipids, the effects on structural re-arrangements and the peroxidase activity of the hemoprotein. PMID:26928591

  8. Peroxidase extraction from jicama skin peels for phenol removal

    NASA Astrophysics Data System (ADS)

    Chiong, T.; Lau, S. Y.; Khor, E. H.; Danquah, M. K.

    2016-06-01

    Phenol and its derivatives exist in various types of industrial effluents, and are known to be harmful to aquatic lives even at low concentrations. Conventional treatment technologies for phenol removal are challenged with long retention time, high energy consumption and process cost. Enzymatic treatment has emerged as an alternative technology for phenol removal from wastewater. These enzymes interact with aromatic compounds including phenols in the presence of hydrogen peroxide, forming free radicals which polymerize spontaneously to produce insoluble phenolic polymers. This work aims to extract peroxidase from agricultural wastes materials and establish its application for phenol removal. Peroxidase was extracted from jicama skin peels under varying extraction conditions of pH, sample-to-buffer ratio (w/v %) and temperature. Experimental results showed that extraction process conducted at pH 10, 40% w/v and 25oC demonstrated a peroxidase activity of 0.79 U/mL. Elevated temperatures slightly enhanced the peroxidase activities. Jicama peroxidase extracted at optimum extraction conditions demonstrated a phenol removal efficiency of 87.5% at pH 7. Phenol removal efficiency was ∼ 97% in the range of 30 - 40oC, and H2O2 dosage has to be kept below 100 mM for maximum removal under phenol concentration tested.

  9. Identification and molecular characterization of a novel DyP-type peroxidase from Pseudomonas aeruginosa PKE117.

    PubMed

    Li, Jing; Liu, Chen; Li, Baozhen; Yuan, Hongli; Yang, Jinshui; Zheng, Beiwen

    2012-02-01

    A new DyP-type peroxidase from Pseudomonas aeruginosa PKE117 was identified and characterized. The dypPa was first identified via sequence analysis and then cloned in Escherichia coli. Subsequently, the recombinant protein DyPPa was expressed and purified. Its DNA sequence analysis revealed an open reading frame of 897 bp, encoding a protein monomer of 299 amino acid residues with isoelectric point 4.62. According to SDS-PAGE analysis and FPLC result, DyPPa mainly existed as homodimer (64 kDa). DyPPa displayed typical heme absorbance of Soret band, with an Rz value of 1.18. Inductively coupled plasma-atomic absorption spectrum data also indicated DyPPa contained iron. Multiple amino acid sequence alignment of DyPPa with other members of the DyP-type peroxidases family showed the presence of conserved D139, H210, and R227 amino acids and GXXDG motifs, which were commonly shared by the DyP-type peroxidase family. Although the primary structure homology between DyPPa and other family members was very low, their secondary and tertiary structure displayed high homology, which explained the high decolorizing activity of DyPPa. Specifically, DyPPa displayed a good thermal stability and maximal activity on Reactive blue 5 under pH 3.5. Therefore, it was proposed that DyPPa, with a wide range of substrate specificity, was a novel member of the DyP-type peroxidases family. PMID:22161141

  10. Recombinant protein production technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant protein production is an important technology for antibody production, biochemical activity study, and structural determination during the post-genomic era. Limiting factors in recombinant protein production include low-level protein expression, protein precipitation, and loss of protein...

  11. Diverse functions and reactions of class III peroxidases.

    PubMed

    Shigeto, Jun; Tsutsumi, Yuji

    2016-03-01

    Higher plants contain plant-specific peroxidases (class III peroxidase; Prxs) that exist as large multigene families. Reverse genetic studies to characterize the function of each Prx have revealed that Prxs are involved in lignification, cell elongation, stress defense and seed germination. However, the underlying mechanisms associated with plant phenotypes following genetic engineering of Prx genes are not fully understood. This is because Prxs can function as catalytic enzymes that oxidize phenolic compounds while consuming hydrogen peroxide and/or as generators of reactive oxygen species. Moreover, biochemical efforts to characterize Prxs responsible for lignin polymerization have revealed specialized activities of Prxs. In conclusion, not only spatiotemporal regulation of gene expression and protein distribution, but also differentiated oxidation properties of each Prx define the function of this class of peroxidases. PMID:26542837

  12. Purification, crystallization and preliminary crystallographic analysis of banyan peroxidase

    PubMed Central

    Sharma, Anurag; Palm, Gottfried J.; Kumari, Moni; Panjikar, Santosh; Jagannadham, M. V.; Hinrichs, Winfried

    2012-01-01

    Plant peroxidases are extensively used in a wide range of biotechnological applications owing to their high environmental and thermal stability. A new peroxidase, named banyan peroxidase, was purified from the latex of Ficus benghalensis and crystallized. X-ray diffraction data were collected from native crystals and from bromide and xenon derivatives to resolutions of up to 1.66 Å in the trigonal space group P3221, with unit-cell parameters a = b = 73.1, c = 164.6 Å. The anomalous signal of the intrinsic iron and calcium ions was sufficient for structure solution by SAD, although the sequence is not yet known. PMID:22869125

  13. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  14. Nucleotide sequence of the tobacco (Nicotiana tabacum) anionic peroxidase gene

    SciTech Connect

    Diaz-De-Leon, F.; Klotz, K.L.; Lagrimini, L.M. )

    1993-03-01

    Peroxidases have been implicated in numerous physiological processes including lignification (Grisebach, 1981), wound-healing (Espelie et al., 1986), phenol oxidation (Lagrimini, 1991), pathogen defense (Ye et al., 1990), and the regulation of cell elongation through the formation of interchain covalent bonds between various cell wall polymers (Fry, 1986; Goldberg et al., 1986; Bradley et al., 1992). However, a complete description of peroxidase action in vivo is not available because of the vast number of potential substrates and the existence of multiple isoenzymes. The tobacco anionic peroxidase is one of the better-characterized isoenzymes. This enzyme has been shown to oxidize a number of significant plant secondary compounds in vitro including cinnamyl alcohols, phenolic acids, and indole-3-acetic acid (Maeder, 1980; Lagrimini, 1991). A cDNA encoding the enzyme has been obtained, and this enzyme was shown to be expressed at the highest levels in lignifying tissues (xylem and tracheary elements) and also in epidermal tissue (Lagrimini et al., 1987). It was shown at this time that there were four distinct copies of the anionic peroxidase gene in tobacco (Nicotiana tabacum). A tobacco genomic DNA library was constructed in the [lambda]-phase EMBL3, from which two unique peroxidase genes were sequenced. One of these clones, [lambda]POD1, was designated as a pseudogene when the exonic sequences were found to differ from the cDNA sequences by 1%, and several frame shifts in the coding sequences indicated a dysfunctional gene (the authors' unpublished results). The other clone, [lambda]POD3, described in this manuscript, was designated as the functional tobacco anionic peroxidase gene because of 100% homology with the cDNA. Significant structural elements include an AS-2 box indicated in shoot-specific expression (Lam and Chua, 1989), a TATA box, and two intervening sequences. 10 refs., 1 tab.

  15. Recombineering homologous recombination constructs in Drosophila.

    PubMed

    Carreira-Rosario, Arnaldo; Scoggin, Shane; Shalaby, Nevine A; Williams, Nathan David; Hiesinger, P Robin; Buszczak, Michael

    2013-01-01

    The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner. PMID:23893070

  16. Suicide inactivation of peroxidase from Chamaerops excelsa palm tree leaves.

    PubMed

    Cuadrado, Nazaret Hidalgo; Zhadan, Galina G; Roig, Manuel G; Shnyrov, Valery L

    2011-12-01

    The concentration and time-dependences and the mechanism of the inactivation of Chamaerops excelsa peroxidase (CEP) by hydrogen peroxide were studied kinetically with four co-substrates (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), guaiacol, o-dianisidine and o-phenylenediamine). The turnover number (r) of H(2)O(2) required to complete the inactivation of the enzyme varied for the different substrates, the enzyme most resistant to inactivation (r=4844) with ABTS being the most useful substrate for biotechnological applications, opening a new avenue of enquiry with this peroxidase. PMID:21925205

  17. Genome Sequence of Pectobacterium carotovorum Phage PPWS1, Isolated from Japanese Horseradish [Eutrema japonicum (Miq.) Koidz] Showing Soft-Rot Symptoms

    PubMed Central

    Kashihara, Misako; Horiike, Tokumasa; Suzuki, Tomohiro; Dohra, Hideo; Netsu, Osamu; Tsuyumu, Shinji

    2016-01-01

    Pectobacterium carotovorum subsp. carotovorum and its lytic bacteriophage PPWS1 were isolated from a Japanese horseradish rhizome with soft rot. Sequencing of the phage genomic DNA suggested that PPWS1 is a new species of the family Podoviridae and has high similarity to the bacteriophage Peat1 infectious to P. atrosepticum. PMID:27103734

  18. Fumigant activity of plant essential oils and components from horseradish (Armoracia rusticana), anise (Pimpinella anisum) and garlic (Allium sativum) oils against Lycoriella ingenua (Diptera: Sciaridae).

    PubMed

    Park, Ii-Kwon; Choi, Kwang-Sik; Kim, Do-Hyung; Choi, In-Ho; Kim, Lee-Sun; Bak, Won-Chull; Choi, Joon-Weon; Shin, Sang-Chul

    2006-08-01

    Plant essential oils from 40 plant species were tested for their insecticidal activities against larvae of Lycoriella ingénue (Dufour) using a fumigation bioassay. Good insecticidal activity against larvae of L. ingenua was achieved with essential oils of Chenopodium ambrosioides L., Eucalyptus globulus Labill, Eucalyptus smithii RT Baker, horseradish, anise and garlic at 10 and 5 microL L(-1) air. Horseradish, anise and garlic oils showed the most potent insecticidal activities among the plant essential oils. At 1.25 microL L(-1), horseradish, anise and garlic oils caused 100, 93.3 and 13.3% mortality, but at 0.625 microL L(-1) air this decreased to 3.3, 0 and 0% respectively. Analysis by gas chromatography-mass spectrometry led to the identification of one major compound from horseradish, and three each from anise and garlic oils. These seven compounds and m-anisaldehyde and o-anisaldehyde, two positional isomers of p-anisaldehyde, were tested individually for their insecticidal activities against larvae of L. ingenua. Allyl isothiocyanate was the most toxic, followed by trans-anethole, diallyl disulfide and p-anisaldehyde with LC(50) values of 0.15, 0.20, 0.87 and 1.47 microL L(-1) respectively. PMID:16786497

  19. A novel 1-Cys thioredoxin peroxidase gene in Apis cerana cerana: characterization of AccTpx4 and its role in oxidative stresses.

    PubMed

    Huaxia, Yifeng; Wang, Fang; Yan, Yan; Liu, Feng; Wang, Hongfang; Guo, Xingqi; Xu, Baohua

    2015-07-01

    Thioredoxin peroxidase (Tpx), also named peroxiredoxin (Prx), is an important peroxidase that can protect organisms against stressful environments. AccTpx4, a 1-Cys thioredoxin peroxidase gene from the Chinese honey bee Apis cerana cerana, was cloned and characterized. The AccTpx4 gene encodes a protein that is predicted to contain the conserved PVCTTE motif from 1-Cys peroxiredoxin. Quantitative real-time PCR (Q-PCR) and Western blotting revealed that AccTpx4 was induced by various oxidative stresses, such as cold, heat, insecticides, H(2)O(2), and HgCl(2). The in vivo peroxidase activity assay showed that recombinant AccTpx4 protein could efficiently degrade H(2)O(2) in the presence of DL-dithiothreitol (DTT). In addition, disc fusion assays revealed that AccTpx4 could function to protect cells against oxidative stresses. These results indicate that AccTpx4 plays an important role in oxidative stress responses and may contribute to the conservation of honeybees. PMID:25971604

  20. Photoionization and Recombination

    NASA Technical Reports Server (NTRS)

    Nahar, Sultana N.

    2000-01-01

    Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

  1. Crystallization and preliminary X-ray analysis of a decameric form of cytosolic thioredoxin peroxidase 1 (Tsa1), C47S mutant, from Saccharomyces cerevisiae

    SciTech Connect

    Oliveira, Marcos Antonio de Genu, Victor; Discola, Karen Fulan; Alves, Simone Vidigal; Netto, Luis Eduardo Soares; Guimarães, Beatriz Gomes

    2007-08-01

    A recombinant mutant (C47S) of cytosolic thioredoxin peroxidase 1 from S. cerevisiae was expressed, purified and crystallized by the hanging-drop vapour-diffusion method from protein previously treated with 1,4-dithiothreitol. The crystals belong to the monoclinic space group C2 and diffraction data were collected to 2.8 Å resolution using a synchrotron-radiation source. Saccharomyces cerevisiae cytosolic thioredoxin peroxidase 1 (cTPxI or Tsa1) is a bifunctional enzyme with protective roles in cellular defence against oxidative and thermal stress that exhibits both peroxidase and chaperone activities. Protein overoxidation and/or high temperatures induce great changes in its quaternary structure and lead to its assembly into large complexes that possess chaperone activity. A recombinant mutant of Tsa1 from S. cerevisiae, with Cys47 substituted by serine, was overexpressed in Escherichia coli as a His{sub 6}-tagged fusion protein and purified by nickel-affinity chromatography. Crystals were obtained from protein previously treated with 1,4-dithiothreitol by the hanging-drop vapour-diffusion method using PEG 3000 as precipitant and sodium fluoride as an additive. Diffraction data were collected to 2.8 Å resolution using a synchrotron-radiation source. The crystal structure was solved by molecular-replacement methods and structure refinement is currently in progress.

  2. Comparison of peroxidase-labeled DNA probes with radioactive RNA probes for detection of human papillomaviruses by in situ hybridization in paraffin sections

    SciTech Connect

    Park, J.S.; Kurman, R.J.; Kessis, T.D.; Shah, K.V. )

    1991-01-01

    A study comparing in situ hybridization using nonradioactive DNA probes directly conjugated with horseradish peroxidase (HRP), and {sup 35}S-labeled antisense RNA probes for human papillomavirus (HPV) types 6/11, 16, and 18 was performed on formalin-fixed, paraffin-embedded tissue from 34 lesions of the cervix and vulva. These lesions included exophytic condylomas and intraepithelial and invasive neoplasms. HPV 6/11 was detected in two of four condylomata acuminata by both in situ techniques. HPV 16 was detected in 13 of 30 cases of intraepithelial and invasive neoplasms by both methods. Discordance between the two methods occurred in two instances. The radiolabeled probe but not the HRP probe detected HPV 16 in one case of cervical intraepithelial neoplasia (CIN 3), whereas the converse occurred in one case of vulvar intraepithelial neoplasia (VIN 3). HPV 18 was not detected in any of the specimens by either method. This study demonstrates that nonradioactive HRP-labeled probes for the detection of specific HPV types are as sensitive as the more laborious and potentially hazardous radioactive probes.

  3. Investigating the effect of gallium curcumin and gallium diacetylcurcumin complexes on the structure, function and oxidative stability of the peroxidase enzyme and their anticancer and antibacterial activities.

    PubMed

    Jahangoshaei, Parisa; Hassani, Leila; Mohammadi, Fakhrossadat; Hamidi, Akram; Mohammadi, Khosro

    2015-10-01

    Curcumin has a wide spectrum of biological and pharmacological activities including anti-inflammatory, antioxidant, antiproliferative, antimicrobial and anticancer activities. Complexation of curcumin with metals has gained attention in recent years for improvement of its stability. In this study, the effect of gallium curcumin and gallium diacetylcurcumin on the structure, function and oxidative stability of horseradish peroxidase (HRP) enzyme were evaluated by spectroscopic techniques. In addition to the enzymatic investigation, the cytotoxic effect of the complexes was assessed on bladder, MCF-7 breast cancer and LNCaP prostate carcinoma cell lines by MTT assay. Furthermore, antibacterial activity of the complexes against S. aureus and E. coli was explored by dilution test method. The results showed that the complexes improve activity of HRP and also increase its tolerance against the oxidative condition. After addition of the complexes, affinity of HRP for hydrogen peroxide substrate decreases, while the affinity increases for phenol substrate. Circular dichroism, intrinsic and synchronous fluorescence spectra showed that the enzyme structure around the catalytic heme group becomes less compact and also the distance between the heme group and tryptophan residues increases due to binding of the complexes to HRP. On the whole, it can be concluded that the change in the enzyme structure upon binding to the gallium curcumin and gallium diacetylcurcumin complexes results in an increase in the antioxidant efficiency and activity of the peroxidise enzyme. The result of anticancer and antibacterial activities suggested that the complexes exhibit the potential for cancer treatment, but they have no significant antibacterial activity. PMID:26369539

  4. Detection of Semliki Forest virus in cell culture by use of an enzyme immunoassay with peroxidase-labeled monoclonal antibodies specific for glycoproteins E1 and E2.

    PubMed Central

    van Tiel, F H; Boere, W A; Vinjé, J; Harmsen, T; Benaissa-Trouw, B J; Kraaijeveld, C A; Snippe, H

    1984-01-01

    Four noncompeting monoclonal antibodies (MA) directed against either the E1 (UM 8.64 and 8.139) or E2 (UM 8.55 and 8.73) glycoprotein of Semliki Forest virus were purified and labeled with horseradish peroxidase. Each enzyme-labeled MA was tested alone and in combination with others for its sensitivity to detect virus-infected cells. Semliki Forest virus-infected L cells seeded as monolayers in 96-well plates were screened for the virus after incubation with enzyme-labeled MA and a substrate. In this system single enzyme-labeled MA even at high dilution (10(3.0) to 10(4.5] were able to detect virus-infected cells. The sensitivity of the test could be enhanced by combining two noncompeting MA (10(4.5) to 10(5.0]. Combinations of three and four MA were less effective, due to high absorbance values for noninfected cells. The threshold of virus defection was between 10(5) and 10(6) PFU/ml. This test is sensitive and specific and therefore may be useful for diagnostic purposes. PMID:6386855

  5. Direct Electron Transfer Kinetics of Peroxidase at Edge Plane Sites of Cup-Stacked Carbon Nanofibers and Their Comparison with Single-Walled Carbon Nanotubes.

    PubMed

    Komori, Kikuo; Tatsuma, Tetsu; Sakai, Yasuyuki

    2016-09-13

    Electron transfer kinetics at the graphene edge site is of great interest from the viewpoints of application to sensing and energy conversion and storage. Here we analyzed kinetics of direct electron transfer of horseradish peroxidase (HRP) adsorbed through surfactant sodium dodecyl sulfate at cup-stacked carbon nanofibers (CSCNFs), which provide highly ordered graphene edges, and compared it with that at single-walled carbon nanotubes (SWCNTs), which consist of a rolled-up basal plane graphene. The heterogeneous electron transfer rate constant of the Fe(2+/3+) couple of the HRP reaction center at CSCNFs (ca. 34.8 s(-1)) was an order of magnitude larger than that at SWCNTs (ca. 4.7 s(-1)). In addition, the overall rate constant of the electron transfer reaction from CSCNFs to HRP oxidized by H2O2 was higher than that from SWCNTs by a factor of 3. CSCNFs also allowed enhancement of the complex-formation reaction rate of HRP with H2O2, in comparison with that at SWCNTs. CSCNFs would therefore be applied to not only biosensors but also biofuel cells with enhanced performance. PMID:27529505

  6. Immobilization of peroxidase on SPEU film via radiation grafting

    NASA Astrophysics Data System (ADS)

    Hongfei, Ha; Guanghui, Wang; Jilan, Wu

    The acrylic acid or acrylamide were grafted via radiation onto segmented polyetherurethane (SPEU) film which is a kind of biocompatible material. Then the Horse radish peroxidase was immobilized on the grafted SPEU film through chemical binding. Some quantitative relationships between the percent graft and the activity, amount of immobilized enzyme were given. The properties and application of obtained biomaterial was studied as well.

  7. Peroxidase activity in cotton cell culture infected with Verticillium dahliae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In our studies with cotton, we have shown that the plant’s induced anionic peroxidases bind to chitin, which is a component of the cell wall of the plant pathogenic fungus Verticillium dahliae. In binding to the cell wall surface, they disrupt the integrity of the pathogen’s cell wall. Thus, these...

  8. TiO(2) nanotube arrays: intrinsic peroxidase mimetics.

    PubMed

    Zhang, Lingling; Han, Lei; Hu, Peng; Wang, Li; Dong, Shaojun

    2013-11-18

    TiO2 nanotube arrays (NTA), prepared by potentiostatic anodization, were discovered to possess an intrinsic peroxidase-like activity. The colorimetric and electrochemical assays both demonstrated their excellent catalytic activity towards H2O2 reduction. On this basis, a simple and inexpensive electrochemical biosensor for glucose detection was developed. PMID:24084751

  9. Structural and spectroscopic characterisation of a heme peroxidase from sorghum.

    PubMed

    Nnamchi, Chukwudi I; Parkin, Gary; Efimov, Igor; Basran, Jaswir; Kwon, Hanna; Svistunenko, Dimitri A; Agirre, Jon; Okolo, Bartholomew N; Moneke, Anene; Nwanguma, Bennett C; Moody, Peter C E; Raven, Emma L

    2016-03-01

    A cationic class III peroxidase from Sorghum bicolor was purified to homogeneity. The enzyme contains a high-spin heme, as evidenced by UV-visible spectroscopy and EPR. Steady state oxidation of guaiacol was demonstrated and the enzyme was shown to have higher activity in the presence of calcium ions. A Fe(III)/Fe(II) reduction potential of -266 mV vs NHE was determined. Stopped-flow experiments with H2O2 showed formation of a typical peroxidase Compound I species, which converts to Compound II in the presence of calcium. A crystal structure of the enzyme is reported, the first for a sorghum peroxidase. The structure reveals an active site that is analogous to those for other class I heme peroxidase, and a substrate binding site (assigned as arising from binding of indole-3-acetic acid) at the γ-heme edge. Metal binding sites are observed in the structure on the distal (assigned as a Na(+) ion) and proximal (assigned as a Ca(2+)) sides of the heme, which is consistent with the Ca(2+)-dependence of the steady state and pre-steady state kinetics. It is probably the case that the structural integrity (and, thus, the catalytic activity) of the sorghum enzyme is dependent on metal ion incorporation at these positions. PMID:26666777

  10. Towards uncovering the roles of switchgrass peroxidases in plant processes

    PubMed Central

    Saathoff, Aaron J.; Donze, Teresa; Palmer, Nathan A.; Bradshaw, Jeff; Heng-Moss, Tiffany; Twigg, Paul; Tobias, Christian M.; Lagrimini, Mark; Sarath, Gautam

    2013-01-01

    Herbaceous perennial plants selected as potential biofuel feedstocks had been understudied at the genomic and functional genomic levels. Recent investments, primarily by the U.S. Department of Energy, have led to the development of a number of molecular resources for bioenergy grasses, such as the partially annotated genome for switchgrass (Panicum virgatum L.), and some related diploid species. In its current version, the switchgrass genome contains 65,878 gene models arising from the A and B genomes of this tetraploid grass. The availability of these gene sequences provides a framework to exploit transcriptomic data obtained from next-generation sequencing platforms to address questions of biological importance. One such question pertains to discovery of genes and proteins important for biotic and abiotic stress responses, and how these components might affect biomass quality and stress response in plants engineered for a specific end purpose. It can be expected that production of switchgrass on marginal lands will expose plants to diverse stresses, including herbivory by insects. Class III plant peroxidases have been implicated in many developmental responses such as lignification and in the adaptive responses of plants to insect feeding. Here, we have analyzed the class III peroxidases encoded by the switchgrass genome, and have mined available transcriptomic datasets to develop a first understanding of the expression profiles of the class III peroxidases in different plant tissues. Lastly, we have identified switchgrass peroxidases that appear to be orthologs of enzymes shown to play key roles in lignification and plant defense responses to hemipterans. PMID:23802005

  11. MacA is a Second Cytochrome c Peroxidase of Geobacter sulfurreducens

    PubMed Central

    Seidel, Julian; Hoffmann, Maren; Ellis, Katie E.; Seidel, Antonia; Spatzal, Thomas; Gerhardt, Stefan; Elliott, Sean J.

    2012-01-01

    The metal-reducing δ-proteobacterium Geobacter sulfurreducens produces a large number of c-type cytochromes, many of which have been implicated in the transfer of electrons to insoluble metal oxides. Among these, the dihemic MacA was assigned a central role. Here we have produced G. sulfurreducens MacA by recombinant expression in Escherichia coli and have solved its three-dimensional structure in three different oxidation states. Sequence comparisons group MacA into the family of diheme cytochrome c peroxidases, and the protein indeed showed hydrogen peroxide reductase activity with ABTS2– as an electron donor. The observed KM was 38.5 ± 3.7 μM H2O2 and vmax was 0.78 ± 0.03 μmol H2O2·min–1·mg–1, resulting in a turnover number kcat = 0.46 · s–1. In contrast, no Fe(III) reductase activity was observed. MacA was found to display similar electrochemical properties to other bacterial diheme peroxidases, in additional to the ability to electrochemically mediate electron transfer to the soluble cytochrome PpcA. Differences in activity between CcpA and MacA can be rationalized with structural variations in one of the three loop regions, loop 2, that undergo conformational changes during reductive activation of the enzyme. This loop is adjacent to the active site heme and forms an open loop structure rather than a more rigid helix as in CcpA. For the activation of the protein the loop has to displace the distal ligand to the active site heme, H93, in loop 1. A H93G variant showed an unexpected formation of a helix in loop 2 and disorder in loop 1, while a M297H variant that altered the properties of the electron transfer heme abolished reductive activation. PMID:22417533

  12. New dye-decolorizing peroxidases from Bacillus subtilis and Pseudomonas putida MET94: towards biotechnological applications.

    PubMed

    Santos, Ana; Mendes, Sónia; Brissos, Vânia; Martins, Lígia O

    2014-03-01

    This work provides spectroscopic, catalytic, and stability fingerprints of two new bacterial dye-decolorizing peroxidases (DyPs) from Bacillus subtilis (BsDyP) and Pseudomonas putida MET94 (PpDyP). DyPs are a family of microbial heme-containing peroxidases with wide substrate specificity, including high redox potential aromatic compounds such as synthetic dyes or phenolic and nonphenolic lignin units. The genes encoding BsDyP and PpDyP, belonging to subfamilies A and B, respectively, were cloned and heterologously expressed in Escherichia coli. The recombinant PpDyP is a 120-kDa homotetramer while BsDyP enzyme consists of a single 48-kDa monomer. The optimal pH of both enzymes is in the acidic range (pH 4-5). BsDyP has a bell-shape profile with optimum between 20 and 30 °C whereas PpDyP shows a peculiar flat and broad (10-30 °C) temperature profile. Anthraquinonic or azo dyes, phenolics, methoxylated aromatics, and also manganese and ferrous ions are substrates used by the enzymes. In general, PpDyP exhibits higher activities and accepts a wider scope of substrates than BsDyP; the spectroscopic data suggest distinct heme microenvironments in the two enzymes that might account for the distinctive catalytic behavior. However, the Bs enzyme with activity lasting for up to 53 h at 40 °C is more stable towards temperature or chemical denaturation than the PpDyP. The results of this work will guide future optimization of the biocatalytis towards their utilization in the fields of environmental or industrial biotechnology. PMID:23820555

  13. Heterologous expression and physicochemical characterization of a fungal dye-decolorizing peroxidase from Auricularia auricula-judae.

    PubMed

    Linde, Dolores; Coscolín, Cristina; Liers, Christiane; Hofrichter, Martin; Martínez, Angel T; Ruiz-Dueñas, Francisco J

    2014-11-01

    An efficient heterologous expression system for Auricularia auricula-judae dye-decolorizing peroxidase (DyP) has been constructed. DNA coding for the mature protein sequence was cloned into the pET23a vector and expressed in Escherichia coli BL21(DE3)pLysS. Recombinant DyP was obtained in high yield as inclusion bodies, and different parameters for its in vitro activation were optimized with a refolding yield of ∼8.5% of the E. coli-expressed DyP. Then, a single chromatographic step allowed the recovery of 17% of the refolded DyP as pure enzyme (1.5mg per liter of culture). The thermal stabilities of wild DyP from A. auricula-judae and recombinant DyP from E. coli expression were similar up to 60°C, but the former was more stable in the 62-70°C range. Stabilities against pH and H2O2 were also measured, and a remarkably high stability at extreme pH values (from pH 2 to 12) was observed. The kinetic constants of recombinant DyP for the oxidation of different substrates were determined and, when compared with those of wild DyP, no important differences were ascertained. Both enzymes showed high affinity for Reactive Blue 19 (anthraquinone dye), Reactive Black 5 (azo dye), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and 2,6-dimethoxyphenol, with similar acidic pH optima and oxidative stabilities. Oxidation of veratryl alcohol and a nonphenolic lignin model dimer were confirmed, although as minor enzymatic activities. Interestingly, two sets of kinetic constants could be obtained for the oxidation of Reactive Blue 19 and other substrates, suggesting the existence of more than one oxidation site in this new peroxidase family. PMID:25153532

  14. Phenol removal by peroxidases extracted from Chinese cabbage root

    SciTech Connect

    Rhee, H.I.; Jeong, Y.H.

    1995-12-31

    More than four million tons of Chinese cabbages are produced in Korea. Most of them are used as raw materials for Kimchi, but root parts of them are discarded as agricultural wastes. A trial for the application of agricultural waste to industrial waste water treatment was made as an effort to the efficient use of natural resources and to reduce water pollution problem simultaneously. Peroxidases of both solid and liquid phases were obtained from Chinese cabbage roots by using commercial juicer. The differences in peroxidase activity among the various cultivars of Chinese cabbages in Korea were little and electrophoretic patterns of various peroxidases will be discussed. The optimum pH and temperature for enzyme activity will be discussed also. Since peroxidases are distributed into 66% in liquid (juice) and 34% in solid phase (pulp), enzymes from both phases were applied to investigate the enzymatic removal of phenol from waste water. After phenol solution at 150 ppm being reacted with liquid phase enzyme (1,800 unit/1) for 3 hours in a batch stirred reactor, 96% of phenol could be removed through polymerization and precipitation. Also, phenol could be removed from initial 120 ppm to final 5 ppm by applying solid phase enzyme in an air lift reactor (600 unit/1). Almost equivalent efficiencies of phenol removal were observed between two systems, even though only one third of the enzymes in batch stirred reactor was applied in air lift reactor. The possible reason for this phenomenon is because peroxidases exist as immobilized forms in solid phase.

  15. Pt74Ag26 nanoparticle-decorated ultrathin MoS2 nanosheets as novel peroxidase mimics for highly selective colorimetric detection of H2O2 and glucose

    NASA Astrophysics Data System (ADS)

    Cai, Shuangfei; Han, Qiusen; Qi, Cui; Lian, Zheng; Jia, Xinghang; Yang, Rong; Wang, Chen

    2016-02-01

    To extend the functionalities of two-dimensional graphene-like layered compounds as versatile materials, the modification of transition metal dichalcogenide nanosheets such as MoS2 with metal nanoparticles is of great and widespread interest. However, few studies are available on the preparation of bimetallic nanoparticles supported on MoS2. Herein, a facile and efficient method to synthesize MoS2-PtAg nanohybrids by decorating ultrathin MoS2 nanosheets with octahedral Pt74Ag26 alloy nanoparticles has been reported. The as-prepared MoS2-Pt74Ag26 nanohybrids were investigated as novel peroxidase mimics to catalyze the oxidation of classical peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2, producing a blue colored reaction and exhibiting typical Michaelis-Menten kinetics. MoS2-Pt74Ag26 has a higher affinity for H2O2 than horseradish peroxidase (HRP) and a higher vmax value with TMB as the substrate than MoS2. The improved catalytic activity of hybrids for colorimetric reactions could be attributed to the synergistic effects of octahedral Pt74Ag26 nanoparticles and ultrathin MoS2 nanosheets as supports. Meanwhile, the generation of active oxygen species (&z.rad;OH) by H2O2 decomposition with MoS2-Pt74Ag26 was responsible for the oxidation of TMB. On the basis of these findings, a colorimetric method based on MoS2-Pt74Ag26 nanohybrids that is highly sensitive and selective was developed for glucose detection. Lower values of the limit of detection (LOD) were obtained, which is more sensitive than MoS2 nanosheets.To extend the functionalities of two-dimensional graphene-like layered compounds as versatile materials, the modification of transition metal dichalcogenide nanosheets such as MoS2 with metal nanoparticles is of great and widespread interest. However, few studies are available on the preparation of bimetallic nanoparticles supported on MoS2. Herein, a facile and efficient method to synthesize MoS2-PtAg nanohybrids by decorating

  16. Variation in Yield of Near-isogenic Soybean Lines for High and Low Seed Coat Peroxidase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Peroxidase is an enzyme present in soybean [Glycine max (L.) Merrill] seed coats and is characterized as either high (dominant allele) or low (recessive allele) activity. Cultivar Cutler 71 is a mixture of high and low seed coat peroxidase genotypes. Mechanical mixtures of 1 high: 1 low peroxidase...

  17. Identification of IAA-oxidase in peroxidase isozymes from cotton plant leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    One of the functions of plant peroxidase is to regulate the indole acetic acid (IAA) hormonal level by oxidizing it to inactive 3-methyleneoxyindole. IAA-binding proteins and plant peroxidase revealed five structurally similar fragments. We have isolated peroxidase isozymes with IAA-oxidase activit...

  18. Lignin-degrading Peroxidases from Genome of Selective Ligninolytic Fungus Ceriporiopsis subvermispora*

    PubMed Central

    Fernández-Fueyo, Elena; Ruiz-Dueñas, Francisco J.; Miki, Yuta; Martínez, María Jesús; Hammel, Kenneth E.; Martínez, Angel T.

    2012-01-01

    The white-rot fungus Ceriporiopsis subvermispora delignifies lignocellulose with high selectivity, but until now it has appeared to lack the specialized peroxidases, termed lignin peroxidases (LiPs) and versatile peroxidases (VPs), that are generally thought important for ligninolysis. We screened the recently sequenced C. subvermispora genome for genes that encode peroxidases with a potential ligninolytic role. A total of 26 peroxidase genes was apparent after a structural-functional classification based on homology modeling and a search for diagnostic catalytic amino acid residues. In addition to revealing the presence of nine heme-thiolate peroxidase superfamily members and the unexpected absence of the dye-decolorizing peroxidase superfamily, the search showed that the C. subvermispora genome encodes 16 class II enzymes in the plant-fungal-bacterial peroxidase superfamily, where LiPs and VPs are classified. The 16 encoded enzymes include 13 putative manganese peroxidases and one generic peroxidase but most notably two peroxidases containing the catalytic tryptophan characteristic of LiPs and VPs. We expressed these two enzymes in Escherichia coli and determined their substrate specificities on typical LiP/VP substrates, including nonphenolic lignin model monomers and dimers, as well as synthetic lignin. The results show that the two newly discovered C. subvermispora peroxidases are functionally competent LiPs and also suggest that they are phylogenetically and catalytically intermediate between classical LiPs and VPs. These results offer new insight into selective lignin degradation by C. subvermispora. PMID:22437835

  19. [Characterization of lignin and Mn peroxidases from Phanerochaete chrysosporium]. Progress report

    SciTech Connect

    Not Available

    1992-12-31

    Lignin peroxidases were investigated with respect to enzyme kinetics and NMR spectroscopy of the heme domain. MN peroxidases were studied with respect to the role of oxalate in enzyme activity, the NMR spectroscopy of the heme domain. Gene expression of both lignin and MN peroxidases were examined as well as expression of site-directed mutants aimed at scale up production of these enzymes.

  20. Recombination of cluster ions

    NASA Technical Reports Server (NTRS)

    Johnsen, Rainer

    1993-01-01

    Some of our recent work on molecular band emissions from recombination of molecular dimer ions (N4(+) and CO(+) CO) is discussed. Much of the experimental work was done by Y. S. Cao; the results on N4(+) recombination have been published. A brief progress report is given on our ongoing measurements of neutral products of recombination using the flowing-afterglow Langmuir-probe technique in conjunction with laser-induced fluorescence.

  1. Recombination in electron coolers

    NASA Astrophysics Data System (ADS)

    Wolf, A.; Gwinner, G.; Linkemann, J.; Saghiri, A. A.; Schmitt, M.; Schwalm, D.; Grieser, M.; Beutelspacher, M.; Bartsch, T.; Brandau, C.; Hoffknecht, A.; Müller, A.; Schippers, S.; Uwira, O.; Savin, D. W.

    2000-02-01

    An introduction to electron-ion recombination processes is given and recent measurements are described as examples, focusing on low collision energies. Discussed in particular are fine-structure-mediated dielectronic recombination of fluorine-like ions, the moderate recombination enhancement by factors of typically 1.5-4 found for most ion species at relative electron-ion energies below about 10 meV, and the much larger enhancement occurring for specific highly charged ions of complex electronic structure, apparently caused by low-energy dielectronic recombination resonances. Recent experiments revealing dielectronic resonances with very large natural width are also described.

  2. Data on the catalytic mechanism of thiol peroxidase mimics.

    PubMed

    Zadehvakili, B; Giles, N M; Fawcett, J P; Giles, G I

    2016-09-01

    We have recently reported SAR data describing the pharmacological activity of a series of phenyl alkyl selenides and tellurides which catalyse the oxidation of thiols by hydrogen peroxide (H2O2), "The design of redox active thiol peroxidase mimics: dihydrolipoic acid recognition correlates with cytotoxicity and prooxidant action" B. Zadehvakili, S.M. McNeill, J.P. Fawcett, G.I. Giles (2016) [1]. This thiol peroxidase (TPx) activity is potentially useful for a number of therapeutic applications, as it can alter the outcome of oxidative stress related pathologies and modify redox signalling. This article presents data describing the molecular changes that occur to a TPx mimic upon exposure to H2O2, and then the thiol mercaptoethanol, as characterised by UV-vis spectroscopy and HPLC retention time. PMID:27331089

  3. Genetic recombination. [Escherichia coli

    SciTech Connect

    Stahl, F.W.

    1987-02-01

    The molecular pathways of gene recombination are explored and compared in studies of the model organisms, Escherichia coli and phase lambda. In the discussion of data from these studies it seems that recombination varies with the genetic idiosyncrasies of the organism and may also vary within a single organism.

  4. Thermal denaturation and regeneration of japanese-radish peroxidase.

    PubMed

    Tamura, Y; Morita, Y

    1975-09-01

    Thermal denaturation of Japanese-radish peroxidase [EC 1.11.1.7] was investigated with respect to its spectrophotometric properties and effect on the enzymatic activity. Inactivation of the peroxidase occurred at temperatures higher than 60degrees and involved three processes, i.e., dissociation of protohemin from the holoperoxidase, a conformation change in the apperoxidase, and the modification or degradation of protohemin. The splitting process of protohemin from holoperoxidase as followed by the change in the absorption spectrum at high temperatures coincided with the degrease in the activity, and it was found to be at least biphasic. The regeneration of peroxidase on cooling to room temperature was essentially reversible at neutral pH, while at pH 5 and pH 9 these processes were irreversible. The irreversibility at acidic pH was mainly due to an irreversible change in the conformation of the apoenzyme. The difference spectrum of heat-treated apoperoxidase exhibited a denaturation blueshift with negative maxima at 287 and 294 nm, and the total protein fluorescence quantum yield. qprotein, increased by 20% compared to that of the untreated apoenzyme. On the other hand, the irreversibility at alkaline pH was largely attributable to the modification of protohemin. Apoperoxidase was more resistnat to heat denaturation but the modification or degradation of protohemin in heated enzyme was greater at alkaline pH than at acidic pH. The pyridine-ferrohemochrome spectrum of peroxidase exhibited slight shifts of the maxima of the alpha-band to shorter wavelength on heat treatment, and the paper chromatogram showed the presence of a new derivative other than protohemin. The modified product is probably (2(4)-vinyl-4(2)-hydroxyethyldeuterohemin. PMID:5412

  5. Prokaryotic origins of the non-animal peroxidase superfamily and organelle-mediated transmission to eukaryotes.

    PubMed

    Passardi, Filippo; Bakalovic, Nenad; Teixeira, Felipe Karam; Margis-Pinheiro, Marcia; Penel, Claude; Dunand, Christophe

    2007-05-01

    Members of the superfamily of plant, fungal, and bacterial peroxidases are known to be present in a wide variety of living organisms. Extensive searching within sequencing projects identified organisms containing sequences of this superfamily. Class I peroxidases, cytochrome c peroxidase (CcP), ascorbate peroxidase (APx), and catalase peroxidase (CP), are known to be present in bacteria, fungi, and plants, but have now been found in various protists. CcP sequences were detected in most mitochondria-possessing organisms except for green plants, which possess only ascorbate peroxidases. APx sequences had previously been observed only in green plants but were also found in chloroplastic protists, which acquired chloroplasts by secondary endosymbiosis. CP sequences that are known to be present in prokaryotes and in Ascomycetes were also detected in some Basidiomycetes and occasionally in some protists. Class II peroxidases are involved in lignin biodegradation and are found only in the Homobasidiomycetes. In fact class II peroxidases were identified in only three orders, although degenerate forms were found in different Pezizomycota orders. Class III peroxidases are specific for higher plants, and their evolution is thought to be related to the emergence of the land plants. We have found, however, that class III peroxidases are present in some green algae, which predate land colonization. The presence of peroxidases in all major phyla (except vertebrates) makes them powerful marker genes for understanding the early evolutionary events that led to the appearance of the ancestors of each eukaryotic group. PMID:17355904

  6. Mechanistic study of a diazo dye degradation by Soybean Peroxidase

    PubMed Central

    2013-01-01

    Background Enzyme based remediation of wastewater is emerging as a novel, efficient and environmentally-friendlier approach. However, studies showing detailed mechanisms of enzyme mediated degradation of organic pollutants are not widely published. Results The present report describes a detailed study on the use of Soybean Peroxidase to efficiently degrade Trypan Blue, a diazo dye. In addition to examining various parameters that can affect the dye degradation ability of the enzyme, such as enzyme and H2O2 concentration, reaction pH and temperature, we carried out a detailed mechanistic study of Trypan Blue degradation. HPLC-DAD and LC-MS/MS studies were carried out to confirm dye degradation and analyze the intermediate metabolites and develop a detailed mechanistic dye degradation pathway. Conclusion We report that Soybean peroxidase causes Trypan Blue degradation via symmetrical azo bond cleavage and subsequent radical-initiated ring opening of the metabolites. Interestingly, our results also show that no high molecular weight polymers were produced during the peroxidase-H2O2 mediated degradation of the phenolic Trypan Blue. PMID:23711110

  7. Kinetic mechanism and nucleotide specificity of NADH peroxidase

    SciTech Connect

    Stoll, V.S.; Blanchard, J.S.

    1988-02-01

    NADH peroxidase is a flavoprotein isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide-dependent reduction of hydrogen peroxide to water. Initial velocity, product, and dead-end inhibition studies have been performed at pH 7.5 and support a ping-pong kinetic mechanism. In the absence of hydrogen peroxide, both transhydrogenation between NADH and thioNAD, and isotope exchange between (/sup 14/C)NADH and NAD, have been demonstrated, although in both these experiments, the maximal velocity of nucleotide exchange was less than 1.5% the maximal velocity of the peroxidatic reaction. We propose that NADH binds tightly to both oxidized and two-electron reduced enzyme. NADH oxidation proceeds stereospecifically with the transfer of the 4S hydrogen to enzyme, and then, via exchange, to water. No primary tritium kinetic isotope effect was observed, and no statistically significant primary deuterium kinetic isotope effects on V/K were determined, although primary deuterium kinetic isotope effects on V were observed in the presence and absence of sodium acetate. NADH peroxidase thus shares with other flavoprotein reductases striking kinetic, spectroscopic, and stereochemical similarities. On this basis, we propose a chemical mechanism for the peroxide cleaving reaction catalyzed by NADH peroxidase which involves the obligate formation of a flavinperoxide, and peroxo bond cleavage by nucleophilic attack by enzymatic dithiols.

  8. Production and characterization of monoclonal antibodies to wall-localized peroxidases from corn seedlings

    NASA Technical Reports Server (NTRS)

    Kim, S. H.; Terry, M. E.; Hoops, P.; Dauwalder, M.; Roux, S. J.

    1988-01-01

    A library of 22 hybridomas, which make antibodies to soluble wall antigens from the coleoptiles and primary leaves of etiolated corn (Zea mays L.) seedlings, was raised and cloned three times by limit dilution to assure monoclonal growth and stability. Two of these hybridomas made immunoglobulin G antibodies, designated mWP3 and mWP19, which both effectively immunoprecipitated peroxidase activity from crude and partially purified preparations of wall peroxidases. Direct peroxidase-binding assays revealed that both antibodies bound enzymes with peroxidase activity. As judged by immunoblot analyses, mWP3 recognized a Mr 98,000 wall peroxidase with an isoelectric point near 4.2, and mWP19 recognized a Mr 58,000 wall peroxidase. Immunogold localization studies showed both peroxidases are predominately in cell walls.

  9. [Enhancement of Coprinus cinereus peroxidase in Pichia pastoris by co-expression chaperone PDI and Ero1].

    PubMed

    Chen, Fei; Hu, Meirong; Jiang, Xianzhang; Tao, Yong; Huang, Jianzhong

    2015-12-01

    The 1,095 bp gene encoding peroxidase from Coprinus cinereus was synthesized and integrated into the genome of Pichia pastoris with a highly inducible alcohol oxidase. The recombinant CiP (rCiP) fused with the a-mating factor per-pro leader sequence derived from Saccharomyces cerevisiae was secreted into the culture medium and identified as the target protein by mass spectrometry, confirming that a C. cinereus peroxidase (CiP) was successfully expressed in P. pastoris. The endoplasmic reticulum oxidoreductase 1 (Ero1) and protein disulfide isomerase (PDI) were co-expressed with rCiP separately and simultaneously. Compared with the wild type, overexpression of PDI and Erol-PDI increaseed Cip activity in 2.43 and 2.6 fold and their activity reached 316 U/mL and 340 U/mL respectively. The strains co-expressed with Erol-PDI was used to high density fermentation, and their activity reached 3,379 U/mL, which was higher than previously reported of 1,200 U/mL. PMID:27093831

  10. The role of ascorbate peroxidase, guaiacol peroxidase, and polysaccharides in cassava (Manihot esculenta Crantz) roots under postharvest physiological deterioration.

    PubMed

    Uarrota, Virgílio Gavicho; Moresco, Rodolfo; Schmidt, Eder Carlos; Bouzon, Zenilda Laurita; Nunes, Eduardo da Costa; Neubert, Enilto de Oliveira; Peruch, Luiz Augusto Martins; Rocha, Miguel; Maraschin, Marcelo

    2016-04-15

    This study aimed to investigate the role of ascorbate peroxidase (APX), guaiacol peroxidase (GPX), polysaccharides, and protein contents associated with the early events of postharvest physiological deterioration (PPD) in cassava roots. Increases in APX and GPX activity, as well as total protein contents occurred from 3 to 5 days of storage and were correlated with the delay of PPD. Cassava samples stained with Periodic Acid-Schiff (PAS) highlighted the presence of starch and cellulose. Degradation of starch granules during PPD was also detected. Slight metachromatic reaction with toluidine blue is indicative of increasing of acidic polysaccharides and may play an important role in PPD delay. Principal component analysis (PCA) classified samples according to their levels of enzymatic activity based on the decision tree model which showed GPX and total protein amounts to be correlated with PPD. The Oriental (ORI) cultivar was more susceptible to PPD. PMID:26617011

  11. Comparative analysis of lignin peroxidase and manganese peroxidase activity on coniferous and deciduous wood using ToF-SIMS.

    PubMed

    MacDonald, Jacqueline; Goacher, Robyn E; Abou-Zaid, Mamdouh; Master, Emma R

    2016-09-01

    White-rot fungi are distinguished by their ability to efficiently degrade lignin via lignin-modifying type II peroxidases, including manganese peroxidase (MnP) and lignin peroxidase (LiP). In the present study, time-of flight secondary ion mass spectrometry (ToF-SIMS) was used to evaluate lignin modification in three coniferous and three deciduous wood preparations following treatment with commercial preparations of LiP and MnP from two different white-rot fungi. Percent modification of lignin was calculated as a loss of intact methoxylated lignin over nonfunctionalized aromatic rings, which is consistent with oxidative cleavage of methoxy moieties within the lignin structure. Exposure to MnP resulted in greater modification of lignin in coniferous compared to deciduous wood (28 vs. 18 % modification of lignin); and greater modification of G-lignin compared to S-lignin within the deciduous wood samples (21 vs. 12 %). In contrast, exposure to LiP resulted in similar percent modification of lignin in all wood samples (21 vs 22 %), and of G- and S-lignin within the deciduous wood (22 vs. 23 %). These findings suggest that the selected MnP and LiP may particularly benefit delignification of coniferous and deciduous wood, respectively. Moreover, the current analysis further demonstrates the utility of ToF-SIMS for characterizing enzymatic modification of lignin in wood fibre along with potential advantages over UV and HPCL-MS detection of solubilized delignification products. PMID:27138198

  12. Does the autoantibody immunodominant region on thyroid peroxidase include amino acid residues 742-771?

    PubMed

    Xiong, Z; Farilla, L; Guo, J; McLachlan, S; Rapoport, B

    2001-03-01

    Identification of the thyroid peroxidase (TPO) amino acid residues that comprise the autoantibody immunodominant region is an important goal that has proven difficult because of the conformational nature of the epitopes involved. Recent data suggest that the immunodominant region has been located. Thus, by autoantibody recognition of tryptic fragments of native TPO, as well as of conformational portions of TPO expressed as cell-free translates, the autoantibody immunodominant region appears to include amino acid residues 742-771, near the C terminus of the ectodomain. To evaluate this deduction, we expressed as cell-free translates the full TPO ectodomain, as well as TPO truncated after residues 741 and 771. The epitopic integrity of these molecules was first confirmed by immunoprecipitation by patient sera containing TPO autoantibodies. However, autoantibody recognition could involve a minority of TPO autoantibodies with the individual sera, not fulfilling the strict criteria for immunodominance. In order to obtain definitive data, we performed immunoprecipitations on these TPO variants with four recombinant human monoclonal autoantibodies that define the immunodominant region. All four monoclonal autoantibodies immunoprecipitated TPO 1-741 to the same extent as they did TPO 1-771 and the full TPO ectodomain, indicating that the immunodominant region comprises (at least in large part) amino acid residues upstream of residue 741. PMID:11327613

  13. Heterologous Expression of Pleurotus eryngii Peroxidase Confirms Its Ability To Oxidize Mn2+ and Different Aromatic Substrates

    PubMed Central

    Ruiz-Dueñas, Francisco Javier; Martínez, María Jesús; Martínez, Ángel T.

    1999-01-01

    A versatile ligninolytic peroxidase has been cloned from Pleurotus eryngii and its allelic variant MnPL2 expressed in Aspergillus nidulans, with properties similar to those of the mature enzyme from P. eryngii. These include the ability to oxidize Mn2+ and aromatic substrates, confirming that this is a new peroxidase type sharing catalytic properties of lignin peroxidase and manganese peroxidase. PMID:10508113

  14. Identification of an orthologous clade of peroxidases that respond to feeding by greenbugs (Schizaphis graminum) in c4 grasses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Scully et al. FY15 – C4 grass peroxidases and greenbug herbivory TECHNICAL ABSTRACT The role of peroxidases in response to green bug-feeding has been well characterized in several plant species, but information linking specific peroxidases to resistance and the identification of peroxidases that re...

  15. Alterations in cytosol free calcium in horseradish roots simultaneously exposed to lanthanum(III) and acid rain.

    PubMed

    Zhang, Xuanbo; Wang, Lihong; Zhou, Anhua; Zhou, Qing; Huang, Xiaohua

    2016-04-01

    The extensive use of rare earth elements (REEs) has increased their environmental levels. REE pollution concomitant with acid rain in many agricultural regions can affect crop growth. Cytosol free calcium ions (Ca(2+)) play an important role in almost all cellular activities. However, no data have been reported regarding the role of cytosol free Ca(2+) in plant roots simultaneously exposed to REE and acid rain. In this study, the effects of exposures to lanthanum(III) and acid rain, independently and in combination, on cytosol free Ca(2+) levels, root activity, metal contents, biomass, cytosol pH and La contents in horseradish roots were investigated. The simultaneous exposures to La(III) and acid rain increased or decreased the cytosol free Ca(2+) levels, depending on the concentration of La(III), and these effects were more evident than independent exposure to La(III) or acid rain. In combined exposures, cytosol free Ca(2+) played an important role in the regulation of root activity, metal contents and biomass. These roles were closely related to La(III) dose, acid rain strength and treatment mode (independent exposure or simultaneous exposure). A low concentration of La(III) (20 mg L(-1)) could alleviate the adverse effects on the roots caused by acid rain, and the combined exposures at higher concentrations of La(III) and acid rain had synergic effects on the roots. PMID:26720810

  16. Broad spectrum antibacterial activity of a mixture of isothiocyanates from nasturtium (Tropaeoli majoris herba) and horseradish (Armoraciae rusticanae radix).

    PubMed

    Conrad, A; Biehler, D; Nobis, T; Richter, H; Engels, I; Biehler, K; Frank, U

    2013-02-01

    Isothiocyanates have been reported to exert antimicrobial activity. These compounds are found in a licensed native preparation of nasturtium (Tropaeoli majoris herba) and horseradish (Armoraciae rusticanae radix) which is used for treatment of upper respiratory and urinary tract infections. The aim of our investigation was to assess the antimicrobial activity of a mixture of the contained benzyl-, allyl-, and phenylethyl- isothiocyanates against clinically important bacterial and fungal pathogens including antimicrobial resistant isolates. Susceptibility testing was performed by agar-dilution technique. Isothiocyanates were mixed in proportions identical to the licensed drug. Minimum inhibitory- and minimum bactericidal concentrations were assessed. The Minimum inhibitory concentration90 was defined as the concentration which inhibited 90% of the microbial species tested. H. influenzae, M. catarrhalis, S. marcescens, P. vulgaris, and Candida spp. were found to be highly susceptible, with minimum inhibitory concentration90 -values ranging between ≤0.0005% and 0.004% (v/v) of total ITC. Intermediate susceptibilities were observed for S. aureus, S. pyogenes, S. pneumoniae, K. pneumoniae, E. coli and P. aeruginosa, with Minimum inhibitory concentration90 -values ranging between 0.004% and 0.125% (v/v), but with elevated Minimum bactericidal concentrations90-values (2-7 dilution steps above Minimum inhibitory concentration90). Low susceptibilities were determined for viridans streptococci and enterococci. Interestingly, both resistant and non-resistant bacteria were similarly susceptible to the test preparation. PMID:23447075

  17. Food as a Source for Quorum Sensing Inhibitors: Iberin from Horseradish Revealed as a Quorum Sensing Inhibitor of Pseudomonas aeruginosa

    PubMed Central

    Jakobsen, Tim Holm; Bragason, Steinn Kristinn; Phipps, Richard Kerry; Christensen, Louise Dahl; van Gennip, Maria; Alhede, Morten; Skindersoe, Mette; Larsen, Thomas Ostenfeld; Høiby, Niels; Bjarnsholt, Thomas

    2012-01-01

    Foods with health-promoting effects beyond nutritional values have been gaining increasing research focus in recent years, although not much has been published on this subject in relation to bacterial infections. With respect to treatment, a novel antimicrobial strategy, which is expected to transcend problems with selective pressures for antibiotic resistance, is to interrupt bacterial communication, also known as quorum sensing (QS), by means of signal antagonists, the so-called QS inhibitors (QSIs). Furthermore, QSI agents offer a potential solution to the deficiencies associated with use of traditional antibiotics to treat infections caused by bacterial biofilms and multidrug-resistant bacteria. Several QSIs of natural origin have been identified, and in this study, several common food products and plants were extracted and screened for QSI activity in an attempt to isolate and characterize previously unknown QSI compounds active against the common opportunistic pathogen Pseudomonas aeruginosa. Several extracts displayed activity, but horseradish exhibited the highest activity. Chromatographic separation led to the isolation of a potent QSI compound that was identified by liquid chromatography-diode array detector-mass spectrometry (LC-DAD-MS) and nuclear magnetic resonance (NMR) spectroscopy as iberin—an isothiocyanate produced by many members of the Brassicaceae family. Real-time PCR (RT-PCR) and DNA microarray studies showed that iberin specifically blocks expression of QS-regulated genes in P. aeruginosa. PMID:22286987

  18. Peroxidase synthesis and activity in the interaction of soybean with Phytophthora megasperma f. sp. glycinea (Pmg)

    SciTech Connect

    Chibbar, R.N.; Esnault, R.; Lee, D.; van Huystee, R.B.; Ward, E.W.B.

    1986-04-01

    Changes, in peroxidase (EC1.11.1.7) have been reported following infection. However, determinations of biosynthesis of quantities of the peroxidase protein molecule have not been madeexclamation In this study hypocotyl of soybean seedlings (Glycine max; cv Harosoy, susceptible; cv Harosoy 63, resistant) were inoculated with zoospores of Pmg. Incorporation of /sup 35/S-methionine (supplied with inoculum) in TCA precipitates was measured. Peroxidase synthesis was measured by immuno precipitation using antibodies against a cationic and an anionic peroxidase derived from peanut cells. Specific peroxidase activity increased rapidly from 5 to 9 h following infection in the resistant reaction but not in the susceptible reaction or the water controls. There was increased synthesis of the anionic peroxidase but not of the cationic peroxidase in the resistant reaction. The anionic peroxidase did not increase in the susceptible until 15 h. The ratio of peroxidase synthesis to total protein synthesis decreased in inoculated tissues compared to control. Peroxidase synthesis is, therefore, a relative minor host response to infection.

  19. [Recombinant antibodies against bioweapons].

    PubMed

    Thullier, Philippe; Pelat, Thibaut; Vidal, Dominique

    2009-12-01

    The threat posed by bioweapons (BW) could lead to the re-emergence of such deadly diseases as plague or smallpox, now eradicated from industrialized countries. The development of recombinant antibodies allows tackling this risk because these recombinant molecules are generally well tolerated in human medicine, may be utilized for prophylaxis and treatment, and because antibodies neutralize many BW. Recombinant antibodies neutralizing the lethal toxin of anthrax, botulinum toxins and the smallpox virus have in particular been isolated recently, with different technologies. Our approach, which uses phage-displayed immune libraries built from non-human primates (M. fascicularis) to obtain recombinant antibodies, which may later be super-humanized (germlinized), has allowed us to obtain such BWs-neutralizing antibodies. PMID:20035695

  20. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  1. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  2. Demonstration of Lignin-to-Peroxidase Direct Electron Transfer

    PubMed Central

    Sáez-Jiménez, Verónica; Baratto, Maria Camilla; Pogni, Rebecca; Rencoret, Jorge; Gutiérrez, Ana; Santos, José Ignacio; Martínez, Angel T.; Ruiz-Dueñas, Francisco Javier

    2015-01-01

    Versatile peroxidase (VP) is a high redox-potential peroxidase of biotechnological interest that is able to oxidize phenolic and non-phenolic aromatics, Mn2+, and different dyes. The ability of VP from Pleurotus eryngii to oxidize water-soluble lignins (softwood and hardwood lignosulfonates) is demonstrated here by a combination of directed mutagenesis and spectroscopic techniques, among others. In addition, direct electron transfer between the peroxidase and the lignin macromolecule was kinetically characterized using stopped-flow spectrophotometry. VP variants were used to show that this reaction strongly depends on the presence of a solvent-exposed tryptophan residue (Trp-164). Moreover, the tryptophanyl radical detected by EPR spectroscopy of H2O2-activated VP (being absent from the W164S variant) was identified as catalytically active because it was reduced during lignosulfonate oxidation, resulting in the appearance of a lignin radical. The decrease of lignin fluorescence (excitation at 355 nm/emission at 400 nm) during VP treatment under steady-state conditions was accompanied by a decrease of the lignin (aromatic nuclei and side chains) signals in one-dimensional and two-dimensional NMR spectra, confirming the ligninolytic capabilities of the enzyme. Simultaneously, size-exclusion chromatography showed an increase of the molecular mass of the modified residual lignin, especially for the (low molecular mass) hardwood lignosulfonate, revealing that the oxidation products tend to recondense during the VP treatment. Finally, mutagenesis of selected residues neighboring Trp-164 resulted in improved apparent second-order rate constants for lignosulfonate reactions, revealing that changes in its protein environment (modifying the net negative charge and/or substrate accessibility/binding) can modulate the reactivity of the catalytic tryptophan. PMID:26240145

  3. Fluoride inhibits the antimicrobial peroxidase systems in human whole saliva.

    PubMed

    Hannuksela, S; Tenovuo, J; Roger, V; Lenander-Lumikari, M; Ekstrand, J

    1994-01-01

    Fluoride (F-) ions at concentrations present in vivo at the plaque/enamel interface (0.05-10 mM) inhibited the activities of lactoperoxidase (LP), myeloperoxidase (MP) and total salivary peroxidase (TSP) in a pH- and dose-dependent way. The inhibition was observed only at pH < or = 6.5 and with F- concentrations > or = 0.1 mM. At pH 5.5 LP activity was inhibited by 85% and MP by 34% with 10 mM F-. TSP activity was also inhibited only at low pH (5.5) by approximately 25%. Furthermore, the generation of the actual antimicrobial agent in vivo, hypothiocyanite (HOSCN/OSCN-), of the oral peroxidase systems was inhibited by F-, again at low pH (5.0-5.5) both in buffer (by 45%) and in saliva (by 15%). This inhibition was observed only with the highest F- concentrations studied (5-10 mM). Fluoridated toothpaste (with 0.10 or 0.14% F) mixed with saliva did not inhibit TSP or HOSCN/OSCN- generation. This may have been due to the 'buffering' effect of toothpaste which did not allow salivary pH to drop below 5.9. We conclude that the F- ions in acidic fluoride products, e.g. in gels or varnishes (but not in toothpastes), may have the potential to locally inhibit the generation of a nonimmune host defense factor, HOSCN/OSCN/SCN-, produced by oral peroxidase systems. The possible clinical significance of this finding remains to be shown. PMID:7850846

  4. Antifungal Properties of Haem Peroxidase from Acorus calamus

    PubMed Central

    GHOSH, MODHUMITA

    2006-01-01

    • Background and Aims Plants have evolved a number of inducible defence mechanisms against pathogen attack, including synthesis of pathogenesis-related proteins. The aim of the study was to purify and characterize antifungal protein from leaves of Acorus calamus. • Methods Leaf proteins from A. calamus were fractionated by cation exchange chromatography and gel filtration and the fraction inhibiting the hyphal extension of phytopathogens was characterized. The temperature stability and pH optima of the protein were determined and its presence was localized in the leaf tissues. • Key Results The purified protein was identified as a class III haem peroxidase with a molecular weight of approx. 32 kDa and pI of 7·93. The temperature stability of the enzyme was observed from 5 °C to 60 °C with a temperature optimum of 36 °C. Maximum enzyme activity was registered at pH 5·5. The pH and temperature optima were corroborated with the antifungal activity of the enzyme. The enzyme was localized in the leaf epidermal cells and lumen tissues of xylem, characteristic of class III peroxidases. The toxic nature of the enzyme which inhibited hyphal growth was demonstrated against phytopathogens such as Macrophomina phaseolina, Fusarium moniliforme and Trichosporium vesiculosum. Microscopic observations revealed distortion in the hyphal structure with stunted growth, increased volume and extensive hyphal branching. • Conclusions This study indicates that peroxidases may have a role to play in host defence by inhibiting the hyphal extension of invading pathogens. PMID:17056613

  5. Synthesis and properties of lignin peroxidase from Streptomyces viridosporus T7A

    SciTech Connect

    Lodha, S.J.; Korus, R.A.; Crawford, D.L.

    1991-12-31

    The production of lignin peroxidase by Streptomyces viridosporus T7A was studied in shake flasks and under aerobic conditions in a 7.5-L batch fermentor. Lignin peroxidase synthesis was found to be strongly affected by catabolite repression. Lignin peroxidase was a non-growth-associated, secondary metabolite. The maximum lignin peroxidase activity was 0.064 U/mL at 36 h. In order to maximize lignin peroxidase activity, optimal conditions were determined. The optimal incubation temperature, pH, and substrate (2,4-dichlorophenol) concentration for the enzyme assays were 45{degrees}C, 6, and 3 m-M, respectively. Stability of lignin peroxidase was determined at 37, 45, and 60{degrees}C, and over the pH range 4-9.

  6. Purification and characterization of a cationic peroxidase Cs in Raphanus sativus.

    PubMed

    Kim, Soung Soo; Lee, Dong Ju

    2005-06-01

    A short distance migrating cationic peroxidase from Korean radish seeds (Raphanus sativus) was detected. Cationic peroxidase Cs was purified to apparent homogeneity and characterized. The molecular mass of the purified cationic peroxidase Cs was estimated to be about 44 kDa on SDS-PAGE. After reconstitution of apoperoxidase Cs with protohemin, the absorption spectra revealed a new peak in the Soret region around 400 nm, which is typical in a classical type III peroxidase family. The optimum pH of peroxidase activity for o-dianisidine oxidation was observed at pH 7.0. Kinetic studies revealed that the reconstituted cationic peroxidase Cs has Km values of 1.18 mM and of 1.27 mM for o-dianisidine and H2O2, respectively. The cationic peroxidase Cs showed the peroxidase activities for native substrates, such as coumaric acid, ferulic acid, and scopoletin. This result suggested that cationic peroxidase Cs plays an important role in plant cell wall formation during seed germination. PMID:16008083

  7. A structural and functional perspective of DyP-type peroxidase family.

    PubMed

    Yoshida, Toru; Sugano, Yasushi

    2015-05-15

    Dye-decolorizing peroxidase from the basidiomycete Bjerkandera adusta Dec 1 (DyP) is a heme peroxidase. This name reflects its ability to degrade several anthraquinone dyes. The substrate specificity, the amino acid sequence, and the tertiary structure of DyP are different from those of the other heme peroxidase (super)families. Therefore, many proteins showing the similar amino acid sequences to that of DyP are called DyP-type peroxidase which is a new family of heme peroxidase identified in 2007. In fact, all structures of this family show a similar structure fold. However, this family includes many proteins whose amino acid sequence identity to DyP is lower than 15% and/or whose catalytic efficiency (kcat/Km) is a few orders of magnitude less than that of DyP. A protein showing an activity different from peroxidase activity (dechelatase activity) has been also reported. In addition, the precise physiological roles of DyP-type peroxidases are unknown. These facts raise a question of whether calling this family DyP-type peroxidase is suitable. Here, we review the differences and similarities of structure and function among this family and propose the reasonable new classification of DyP-type peroxidase family, that is, class P, I and V. In this contribution, we discuss the adequacy of this family name. PMID:25655348

  8. Wound-induced deposition of polyphenols in transgenic plants overexpressing peroxidase

    SciTech Connect

    Lagrimini, L.M. )

    1991-06-01

    Tobacco (Nicotiana tabacum) plants transformed with a chimeric tobacco anionic peroxidase gene have previously been shown to synthesize high levels of peroxidase in all tissues throughout the plant. One of several distinguishable phenotypes of transformed plants is the rapid browning of pith tissue upon wounding. Pith tissue from plants expressing high levels of peroxidase browned within 24 hours of wounding, while tissue from control plants did not brown as late as 7 days after wounding. A correlation between peroxidase activity and wound-induced browning was observed, whereas no relationship between polyphenol oxidase activity and browning was found. The purified tobacco anionic peroxidase was subjected to kinetic analysis with substrates which resemble the precursors of lignin or polyphenolic acid. The purified enzyme was found to readily polymerize phenolic acids in the presence of H{sub 2}O{sub 2} via a modified ping-pong mechanism. The percentage of lignin and lignin-related polymers in cell walls was nearly twofold greater in pith tissue isolated from peroxidase-overproducer plants compared to control plants. Lignin deposition in wounded pith tissue from control plants closely followed the induction of peroxidase activity. However, wound-induced lignification occurred 24 to 48 hours sooner in plants overexpressing the anionic peroxidase. This suggests that the availability of peroxidase rather than substrate may delay polyphenol deposition in wounded tissue.

  9. Application of a Novel Alkali-Tolerant Thermostable DyP-Type Peroxidase from Saccharomonospora viridis DSM 43017 in Biobleaching of Eucalyptus Kraft Pulp

    PubMed Central

    Huang, Huoqing; Zheng, Fei; Wang, Xiaoyu; Wu, Yuying; Li, Kangjia; Xie, Xiangming; Jin, Yi

    2014-01-01

    Saccharomonospora viridis is a thermophilic actinomycete that may have biotechnological applications because of its dye decolorizing activity, though the enzymatic oxidative system responsible for this activity remains elusive. Bioinformatic analysis revealed a DyP-type peroxidase gene in the genome of S. viridis DSM 43017 with sequence similarity to peroxidase from dye-decolorizing microbes. This gene, svidyp, consists of 1,215 bp encoding a polypeptide of 404 amino acids. The gene encoding SviDyP was cloned, heterologously expressed in Escherichia coli, and then purified. The recombinant protein could efficiently decolorize several triarylmethane dyes, anthraquinonic and azo dyes under neutral to alkaline conditions. The optimum pH and temperature for SviDyP was pH 7.0 and 70°C, respectively. Compared with other DyP-type peroxidases, SviDyP was more active at high temperatures, retaining>63% of its maximum activity at 50–80°C. It also showed broad pH adaptability (>35% activity at pH 4.0–9.0) and alkali-tolerance (>80% activity after incubation at pH 5–10 for 1 h at 37°C), and was highly thermostable (>60% activity after incubation at 70°C for 2 h at pH 7.0). SviDyP had an accelerated action during the biobleaching of eucalyptus kraft pulp, resulting in a 21.8% reduction in kappa number and an increase of 2.98% (ISO) in brightness. These favorable properties make SviDyP peroxidase a promising enzyme for use in the pulp and paper industries. PMID:25333297

  10. Improving the pH-stability of Versatile Peroxidase by Comparative Structural Analysis with a Naturally-Stable Manganese Peroxidase

    PubMed Central

    Sáez-Jiménez, Verónica; Fernández-Fueyo, Elena; Medrano, Francisco Javier; Romero, Antonio; Martínez, Angel T.; Ruiz-Dueñas, Francisco J.

    2015-01-01

    Versatile peroxidase (VP) from the white-rot fungus Pleurotus eryngii is a high redox potential peroxidase of biotechnological interest able to oxidize a wide range of recalcitrant substrates including lignin, phenolic and non-phenolic aromatic compounds and dyes. However, the relatively low stability towards pH of this and other fungal peroxidases is a drawback for their industrial application. A strategy based on the comparative analysis of the crystal structures of VP and the highly pH-stable manganese peroxidase (MnP4) from Pleurotus ostreatus was followed to improve the VP pH stability. Several interactions, including hydrogen bonds and salt bridges, and charged residues exposed to the solvent were identified as putatively contributing to the pH stability of MnP4. The eight amino acid residues responsible for these interactions and seven surface basic residues were introduced into VP by directed mutagenesis. Furthermore, two cysteines were also included to explore the effect of an extra disulfide bond stabilizing the distal Ca2+ region. Three of the four designed variants were crystallized and new interactions were confirmed, being correlated with the observed improvement in pH stability. The extra hydrogen bonds and salt bridges stabilized the heme pocket at acidic and neutral pH as revealed by UV-visible spectroscopy. They led to a VP variant that retained a significant percentage of the initial activity at both pH 3.5 (61% after 24 h) and pH 7 (55% after 120 h) compared with the native enzyme, which was almost completely inactivated. The introduction of extra solvent-exposed basic residues and an additional disulfide bond into the above variant further improved the stability at acidic pH (85% residual activity at pH 3.5 after 24 h when introduced separately, and 64% at pH 3 when introduced together). The analysis of the results provides a rational explanation to the pH stability improvement achieved. PMID:26496708

  11. Improving the pH-stability of Versatile Peroxidase by Comparative Structural Analysis with a Naturally-Stable Manganese Peroxidase.

    PubMed

    Sáez-Jiménez, Verónica; Fernández-Fueyo, Elena; Medrano, Francisco Javier; Romero, Antonio; Martínez, Angel T; Ruiz-Dueñas, Francisco J

    2015-01-01

    Versatile peroxidase (VP) from the white-rot fungus Pleurotus eryngii is a high redox potential peroxidase of biotechnological interest able to oxidize a wide range of recalcitrant substrates including lignin, phenolic and non-phenolic aromatic compounds and dyes. However, the relatively low stability towards pH of this and other fungal peroxidases is a drawback for their industrial application. A strategy based on the comparative analysis of the crystal structures of VP and the highly pH-stable manganese peroxidase (MnP4) from Pleurotus ostreatus was followed to improve the VP pH stability. Several interactions, including hydrogen bonds and salt bridges, and charged residues exposed to the solvent were identified as putatively contributing to the pH stability of MnP4. The eight amino acid residues responsible for these interactions and seven surface basic residues were introduced into VP by directed mutagenesis. Furthermore, two cysteines were also included to explore the effect of an extra disulfide bond stabilizing the distal Ca2+ region. Three of the four designed variants were crystallized and new interactions were confirmed, being correlated with the observed improvement in pH stability. The extra hydrogen bonds and salt bridges stabilized the heme pocket at acidic and neutral pH as revealed by UV-visible spectroscopy. They led to a VP variant that retained a significant percentage of the initial activity at both pH 3.5 (61% after 24 h) and pH 7 (55% after 120 h) compared with the native enzyme, which was almost completely inactivated. The introduction of extra solvent-exposed basic residues and an additional disulfide bond into the above variant further improved the stability at acidic pH (85% residual activity at pH 3.5 after 24 h when introduced separately, and 64% at pH 3 when introduced together). The analysis of the results provides a rational explanation to the pH stability improvement achieved. PMID:26496708

  12. Manganese peroxidase gene transcription in Phanerochaete chrysosporium: Activation by manganese

    SciTech Connect

    Brown, J.A.; Alic, M. Gold, M.H. )

    1991-07-01

    The expression of manganese peroxidase in nitrogen-limited cultures of Phanerochaete chrysosporium is dependent on Mn, and initial work suggested that Mn regulates transcription of the mnp gene. In this study, using Northern (RNA) blot analysis of kinetic, dose-response, and inhibitor experiments, the authors demonstrate unequivocally that Mn regulates mnp gene transcription. The amount of mnp mRNA in cells of 4-day-old nitrogen-limited cultures is a direct function of the concentration of Mn in the culture medium up to a maximum of 180 {mu}M. Addition of Mn to nitrogen-limited Mn-deficient secondary metabolic (4-, 5-, and 6-day-old) cultures results in the appearance of mnp mRNA within 40 min. The appearance of this message is completely inhibited by the RNA synthesis inhibitor dactinomycin but not by the protein synthesis inhibitor cycloheximide. Furthermore, the amount of mnp mRNA produced is a direct function of the concentration of added Mn. In contrast, addition of Mn to low-nitrogen Mn-deficient 2- or 3-day-old cultures does not result in the appearance of mnp mRNA. Manganese peroxidase protein is detected by specific immunoprecipitation of the in vitro translation products of poly(A) RNA isolated from Mn-supplemented (but nor from Mn-deficient) cells. All of these results demonstrate that Mn, the substrate for the enzyme, regulates mnp gene transcription via a growth-stage-specific and concentration-dependent mechanism.

  13. High Conformational Stability of Secreted Eukaryotic Catalase-peroxidases

    PubMed Central

    Zámocký, Marcel; García-Fernández, Queralt; Gasselhuber, Bernhard; Jakopitsch, Christa; Furtmüller, Paul G.; Loewen, Peter C.; Fita, Ignacio; Obinger, Christian; Carpena, Xavi

    2012-01-01

    Catalase-peroxidases (KatGs) are bifunctional heme enzymes widely spread in archaea, bacteria, and lower eukaryotes. Here we present the first crystal structure (1.55 Å resolution) of an eukaryotic KatG, the extracellular or secreted enzyme from the phytopathogenic fungus Magnaporthe grisea. The heme cavity of the homodimeric enzyme is similar to prokaryotic KatGs including the unique distal +Met-Tyr-Trp adduct (where the Trp is further modified by peroxidation) and its associated mobile arginine. The structure also revealed several conspicuous peculiarities that are fully conserved in all secreted eukaryotic KatGs. Peculiarities include the wrapping at the dimer interface of the N-terminal elongations from the two subunits and cysteine residues that cross-link the two subunits. Differential scanning calorimetry and temperature- and urea-mediated unfolding followed by UV-visible, circular dichroism, and fluorescence spectroscopy combined with site-directed mutagenesis demonstrated that secreted eukaryotic KatGs have a significantly higher conformational stability as well as a different unfolding pattern when compared with intracellular eukaryotic and prokaryotic catalase-peroxidases. We discuss these properties with respect to the structure as well as the postulated roles of this metalloenzyme in host-pathogen interactions. PMID:22822072

  14. Peroxidase-catalysed interfacial adhesion of aquatic caddisworm silk.

    PubMed

    Wang, Ching-Shuen; Pan, Huaizhong; Weerasekare, G Mahika; Stewart, Russell J

    2015-11-01

    Casemaker caddisfly (Hesperophylax occidentalis) larvae use adhesive silk fibres to construct protective shelters under water. The silk comprises a distinct peripheral coating on a viscoelastic fibre core. Caddisworm silk peroxinectin (csPxt), a haem-peroxidase, was shown to be glycosylated by lectin affinity chromatography and tandem mass spectrometry. Using high-resolution H2O2 and peroxidase-dependent silver ion reduction and nanoparticle deposition, imaged by electron microscopy, csPxt activity was shown to be localized in the peripheral layer of drawn silk fibres. CsPxt catalyses dityrosine cross-linking within the adhesive peripheral layer post-draw, initiated perhaps by H2O2 generated by a silk gland-specific superoxide dismutase 3 (csSOD3) from environmental reactive oxygen species present in natural water. CsSOD3 was also shown to be a glycoprotein and is likely localized in the peripheral layer. Using a synthetic fluorescent phenolic copolymer and confocal microscopy, it was shown that csPxt catalyses oxidative cross-linking to external polyphenolic compounds capable of diffusive interpenetration into the fuzzy peripheral coating, including humic acid, a natural surface-active polyphenol. The results provide evidence of enzyme-mediated covalent cross-linking of a natural bioadhesive to polyphenol conditioned interfaces as a mechanism of permanent adhesion underwater. PMID:26490632

  15. Enzymatic degradation of Congo Red by turnip (Brassica rapa) peroxidase.

    PubMed

    Ahmedi, Afaf; Abouseoud, Mahmoud; Couvert, Annabelle; Amrane, Abdeltif

    2012-01-01

    The enzyme peroxidase is known for its capacity to remove phenolic compounds and aromatic amines from aqueous solutions and also to decolourize textile effluents. This study aims at evaluating the potential of a turnip (Brassica rapa) peroxidase (TP) preparation in the discolouration of textile azo dyes and effluents. An azo dye, Congo Red (CR), was used as a model pollutant for treatment by the enzyme. The effects of various operating conditions like pH value, temperature, initial dye and hydrogen peroxide concentrations, contact time, and enzyme concentration were evaluated. The optimal conditions for maximal colour removal were at pH 2.0, 40 degrees C, 50 mM hydrogen peroxide, 50 mg/l CR dye, and TP activity of 0.45 U/ml within 10 min of incubation time. Analysis of the by-products from the enzymatic treatment by UV-Vis and IR spectroscopy showed no residual compounds in the aqueous phase and a precipitate of polymeric nature. PMID:23016283

  16. Regulation of Meiotic Recombination

    SciTech Connect

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  17. A novel amperometric alcohol biosensor developed in a 3rd generation bioelectrode platform using peroxidase coupled ferrocene activated alcohol oxidase as biorecognition system.

    PubMed

    Chinnadayyala, Somasekhar R; Kakoti, Ankana; Santhosh, Mallesh; Goswami, Pranab

    2014-05-15

    Alcohol oxidase (AOx) with a two-fold increase in efficiency (Kcat/Km) was achieved by physical entrapment of the activator ferrocene in the protein matrix through a simple microwave based partial unfolding technique and was used to develop a 3rd generation biosensor for improved detection of alcohol in liquid samples. The ferrocene molecules were stably entrapped in the AOx protein matrix in a molar ratio of ~3:1 through electrostatic interaction with the Trp residues involved in the functional activity of the enzyme as demonstrated by advanced analytical techniques. The sensor was fabricated by immobilizing ferrocene entrapped alcohol oxidase (FcAOx) and sol-gel chitosan film coated horseradish peroxidase (HRP) on a multi-walled carbon nanotube (MWCNT) modified glassy carbon electrode through layer-by-layer technique. The bioelectrode reactions involved the formation of H2O2 by FcAOx biocatalysis of substrate alcohol followed by HRP-catalyzed reduction of the liberated H2O2 through MWCNT supported direct electron transfer mechanism. The amperometric biosensor exhibited a linear response to alcohol in the range of 5.0 × 10(-6) to 30 × 10(-4)mol L(-1) with a detection limit of 2.3 × 10(-6) mol L(-1), and a sensitivity of 150 µA mM(-1) cm(-2). The biosensor response was steady for 28 successive measurements completed in a period of 5h and retained ~90% of the original response even after four weeks when stored at 4 °C. The biosensor was successfully applied for the determination of alcohol in commercial samples and its performance was validated by comparing with the data obtained by GC analyses of the samples. PMID:24368229

  18. Roles of Lignin Peroxidase and Manganese Peroxidase from Phanerochaete chrysosporium in the Decolorization of Olive Mill Wastewaters

    PubMed Central

    Sayadi, S.; Ellouz, R.

    1995-01-01

    The relative contributions of lignin peroxidase (LiP) and manganese peroxidase (MnP) to the decolorization of olive mill wastewaters (OMW) by Phanerochaete chrysosporium were investigated. A relatively low level (25%) of OMW decolorization was found with P. chrysosporium which was grown in a medium with a high Mn(II) concentration and in which a high level of MnP (0.65 (mu)M) was produced. In contrast, a high degree of OMW decolorization (more than 70%) was observed with P. chrysosporium which was grown in a medium with a low Mn(II) concentration but which resulted in a high level of LiP activity (0.3 (mu)M). In this culture medium, increasing the Mn(II) concentration resulted in decreased levels of OMW decolorization and LiP activity. Decolorization by reconstituted cultures of P. chrysosporium was found to be more enhanced by the addition of isolated LiP than by the addition of isolated MnP. The highest OMW decolorization levels were obtained at low initial chemical oxygen demands combined with high levels of extracellular LiP. These data, plus the positive effect of veratryl alcohol on OMW decolorization and LiP activity, indicate that culture conditions which yield high levels of LiP activity lead to high levels of OMW decolorization. PMID:16534959

  19. Role of manganese peroxidases and lignin peroxidases of Phanerochaete chrysosporium in the decolorization of kraft bleach plant effluent.

    PubMed Central

    Michel, F C; Dass, S B; Grulke, E A; Reddy, C A

    1991-01-01

    The role of lignin peroxidases (LIPs) and manganese peroxidases (MNPs) of Phanerochaete chrysosporium in decolorizing kraft bleach plant effluent (BPE) was investigated. Negligible BPE decolorization was exhibited by a per mutant, which lacks the ability to produce both the LIPs and the MNPs. Also, little decolorization was seen when the wild type was grown in high-nitrogen medium, in which the production of LIPs and MNPs is blocked. A lip mutant of P. chrysosporium, which produces MNPs but not LIPs, showed about 80% of the activity exhibited by the wild type, indicating that the MNPs play an important role in BPE decolorization. When P. chrysosporium was grown in a medium with 100 ppm of Mn(II), high levels of MNPs but no LIPs were produced, and this culture also exhibited high rates of BPE decolorization, lending further support to the idea that MNPs play a key role in BPE decolorization. When P. chrysosporium was grown in a medium with no Mn(II), high levels of LIPs but negligible levels of MNPs were produced and the rate and extent of BPE decolorization by such cultures were quite low, indicating that LIPs play a relatively minor role in BPE decolorization. Furthermore, high rates of BPE decolorization were seen on days 3 and 4 of incubation, when the cultures exhibit high levels of MNP activity but little or no LIP activity. These results indicate that MNPs play a relatively more important role than LIPs in BPE decolorization by P. chrysosporium. Images PMID:1768105

  20. Meiotic recombination mechanisms.

    PubMed

    Grelon, Mathilde

    2016-01-01

    Meiosis is a specialized cell division at the origin of the haploid cells that eventually develop into the gametes. It therefore lies at the heart of Mendelian heredity. Recombination and redistribution of the homologous chromosomes arising during meiosis constitute an important source of genetic diversity, conferring to meiosis a particularly important place in the evolution and the diversification of the species. Our understanding of the molecular mechanisms governing meiotic recombination has considerably progressed these last decades, benefiting from complementary approaches led on various model species. An overview of these mechanisms will be provided as well as a discussion on the implications of these recent discoveries. PMID:27180110

  1. Recombinant expression of four oxidoreductases in Phanerochaete chrysosporium improves degradation of phenolic and non-phenolic substrates.

    PubMed

    Coconi-Linares, Nancy; Ortiz-Vázquez, Elizabeth; Fernández, Francisco; Loske, Achim M; Gómez-Lim, Miguel A

    2015-09-10

    Phanerochaete chrysosporium belongs to a group of lignin-degrading fungi that secretes various oxidoreductive enzymes, including lignin peroxidase (LiP) and manganese peroxidase (MnP). Previously, we demonstrated that the heterologous expression of a versatile peroxidase (VP) in P. chrysosporium recombinant strains is possible. However, the production of laccases (Lac) in this fungus has not been completely demonstrated and remains controversial. In order to investigate if the co-expression of Lac and VP in P. chrysosporium would improve the degradation of phenolic and non-phenolic substrates, we tested the constitutive co-expression of the lacIIIb gene from Trametes versicolor and the vpl2 gene from Pleurotus eryngii, and also the endogenous genes mnp1 and lipH8 by shock wave mediated transformation. The co-overexpression of peroxidases and laccases was improved up to five-fold as compared with wild type species. Transformant strains showed a broad spectrum in phenolic/non-phenolic biotransformation and a high percentage in synthetic dye decolorization in comparison with the parental strain. Our results show that the four enzymes can be constitutively expressed in a single transformant of P. chrysosporium in minimal medium. These data offer new possibilities for an easy and efficient co-expression of laccases and peroxidases in suitable basidiomycete species. PMID:26113215

  2. Effects of elevated peroxidase levels and corn earworm feeding on gene expression in tomato

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tomato gene arrays were used to investigate how high levels of transgenic peroxidase expression and feeding by the corn earworm, Helicoverpa zea, affected expression of defensive and other genes. High peroxidase activity significantly upregulated proteinase inhibitors and a few other defensive gene...

  3. Structural and Functional Features of Peroxidases with a Potential as Industrial Biocatalysts

    NASA Astrophysics Data System (ADS)

    Ruiz-Dueñas, Francisco J.; Martínez, Angel T.

    This chapter begins with a description of the main structural features of heme peroxidases representative of the two large superfamilies of plant-fungal-bacterial and animal peroxidases, and the four additional (super)families described to date. Then, we focus on several fungal peroxidases of high biotechnological potential as industrial biocatalysts. These include (1) ligninolytic peroxidases from white-rot basidiomycetes being able to oxidize high redox-potential substrates at an exposed protein radical; (2) heme-thiolate peroxidases that are structural hybrids of typical peroxidases and cytochrome P450 enzymes and, after their discovery in sooty molds, are being described in basidiomycetes with even more interesting catalytic properties, such as selective aromatic oxygenation; and (3) the so-called dye-decolorizing peroxidases that are still to be thoroughly investigated but have been identified in different basidiomycete genomes. The structural-functional description of these peroxidases includes an analysis of the heme environment and a description of their substrate oxidation sites, with the purpose of understanding their interesting catalytic properties and biotechnological potential.

  4. Selective oxidation of enzyme extracts for improved quantification of peroxidase activity.

    PubMed

    Jiang, Shu; Penner, Michael H

    2015-05-01

    Natural components endogenous to plant material extracts often interfere with traditional peroxidase assays by reducing the oxidized product generated as a result of the peroxidase-catalyzed reaction. This leads to an underestimation of peroxidase activity when the oxidized product provides the signal for enzyme activity quantification. This article describes a relatively simple way to alleviate complications arising due to the presence of such confounding compounds. The method is based on using 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as the reducing substrate. The oxidized product of the reaction is ABTS(+), the accumulation of which can be followed spectrophotometrically. It is shown here that one can selectively inactivate the endogenous compounds that confound the peroxidase assay by treating the enzyme preparation with the oxidized product itself, ABTS(+), prior to initiating the quantification assay. This approach is selective for those compounds likely to interfere with peroxidase quantification. The presented method is shown to alleviate the complications associated with lag phases typical of plant extract peroxidase assays and, thus, to more accurately reflect total peroxidase activity. The presented assay is expected to be applicable to the wide range of biological systems for which the determination of peroxidase activity is desired. PMID:25640588

  5. Characterization of Peroxidase Changes in Resistant and Susceptible Warm- Season Turfgrasses Challenged by Blissus Occiduus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Peroxidases play an important role in plant stress related interactions. This research assessed the role of peroxidases in the defense response of resistant and susceptible buffalograsses [Buchloe dactyloides (Nutt.) Engelm] and zoysiagrasses (Zoysia japonica Steudel) to the western chinch bug, Bli...

  6. Interference by morpholine ethanesulfonic acid (MES) and related buffers in phenolic oxidation by peroxidase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    While characterizing the kinetic parameters of apoplastic phenolic oxidation by peroxidase, we found anomalies caused by the 4-morpholine ethanesulfonic acid (MES) buffer being used. In the presence of MES, certain phenolics appeared not to be oxidized by peroxidase, yet the oxidant, H2O2, was uti...

  7. Recombinant renewable polyclonal antibodies

    PubMed Central

    Ferrara, Fortunato; D’Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew RM

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products. PMID:25530082

  8. Recombinant DNA for Teachers.

    ERIC Educational Resources Information Center

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  9. Recombineering Pseudomonas syringae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we report the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecE...

  10. Oligonucleotide recombination in bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Today, there are more than 1,500 completed or draft bacterial genome sequences available for public access. To functionally analyze these genomes and to test the hypotheses that are generated from the sequence information we require new and generically useful tools. Recombineering (genetic engineer...

  11. Intrinsic peroxidase-like activity of ferromagnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Gao, Lizeng; Zhuang, Jie; Nie, Leng; Zhang, Jinbin; Zhang, Yu; Gu, Ning; Wang, Taihong; Feng, Jing; Yang, Dongling; Perrett, Sarah; Yan, Xiyun

    2007-09-01

    Nanoparticles containing magnetic materials, such as magnetite (Fe3O4), are particularly useful for imaging and separation techniques. As these nanoparticles are generally considered to be biologically and chemically inert, they are typically coated with metal catalysts, antibodies or enzymes to increase their functionality as separation agents. Here, we report that magnetite nanoparticles in fact possess an intrinsic enzyme mimetic activity similar to that found in natural peroxidases, which are widely used to oxidize organic substrates in the treatment of wastewater or as detection tools. Based on this finding, we have developed a novel immunoassay in which antibody-modified magnetite nanoparticles provide three functions: capture, separation and detection. The stability, ease of production and versatility of these nanoparticles makes them a powerful tool for a wide range of potential applications in medicine, biotechnology and environmental chemistry.

  12. Glutathione peroxidase 4 prevents necroptosis in mouse erythroid precursors

    PubMed Central

    Canli, Özge; Alankuş, Yasemin B.; Grootjans, Sasker; Vegi, Naidu; Hültner, Lothar; Hoppe, Philipp S.; Schroeder, Timm; Vandenabeele, Peter; Bornkamm, Georg W.

    2016-01-01

    Maintaining cellular redox balance is vital for cell survival and tissue homoeostasis because imbalanced production of reactive oxygen species (ROS) may lead to oxidative stress and cell death. The antioxidant enzyme glutathione peroxidase 4 (Gpx4) is a key regulator of oxidative stress–induced cell death. We show that mice with deletion of Gpx4 in hematopoietic cells develop anemia and that Gpx4 is essential for preventing receptor-interacting protein 3 (RIP3)-dependent necroptosis in erythroid precursor cells. Absence of Gpx4 leads to functional inactivation of caspase 8 by glutathionylation, resulting in necroptosis, which occurs independently of tumor necrosis factor α activation. Although genetic ablation of Rip3 normalizes reticulocyte maturation and prevents anemia, ROS accumulation and lipid peroxidation in Gpx4-deficient cells remain high. Our results demonstrate that ROS and lipid hydroperoxides function as not-yet-recognized unconventional upstream signaling activators of RIP3-dependent necroptosis. PMID:26463424

  13. Cytochrome c peroxidase activity of heme bound amyloid β peptides.

    PubMed

    Seal, Manas; Ghosh, Chandradeep; Basu, Olivia; Dey, Somdatta Ghosh

    2016-09-01

    Heme bound amyloid β (Aβ) peptides, which have been associated with Alzheimer's disease (AD), can catalytically oxidize ferrocytochrome c (Cyt c(II)) in the presence of hydrogen peroxide (H2O2). The rate of catalytic oxidation of Cyt(II) c has been found to be dependent on several factors, such as concentration of heme(III)-Aβ, Cyt(II) c, H2O2, pH, ionic strength of the solution, and peptide chain length of Aβ. The above features resemble the naturally occurring enzyme cytochrome c peroxidase (CCP) which is known to catalytically oxidize Cyt(II) c in the presence of H2O2. In the absence of heme(III)-Aβ, the oxidation of Cyt(II) c is not catalytic. Thus, heme-Aβ complex behaves as CCP. PMID:27270708

  14. Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria.

    PubMed

    Kathiresan, Meena; Martins, Dorival; English, Ann M

    2014-12-01

    In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H2O2 levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H2O2 because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1's heme is labilized by hyperoxidation of the protein during the burst in H2O2 production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H2O2 in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼ 85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H2O2 via a previously unidentified mechanism involving H2O2-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H2O2 detoxification. PMID:25422453

  15. A Redundant Role of Human Thyroid Peroxidase Propeptide for Cellular, Enzymatic, and Immunological Activity

    PubMed Central

    Góra, Monika; Buckle, Ashley M.; Porebski, Benjamin T.; Kemp, E. Helen; Sutton, Brian J.; Czarnocka, Barbara; Banga, J. Paul

    2014-01-01

    Background: Thyroid peroxidase (TPO) is a dimeric membrane-bound enzyme of thyroid follicular cells, responsible for thyroid hormone biosynthesis. TPO is also a common target antigen in autoimmune thyroid disease (AITD). With two active sites, TPO is an unusual enzyme, and thus there is much interest in understanding its structure and role in AITD. Homology modeling has shown TPO to be composed of different structural modules, as well as a propeptide sequence. During the course of studies to obtain homogeneous preparations of recombinant TPO for structural studies, we investigated the role of the large propeptide sequence in TPO. Methods: An engineered recombinant human TPO preparation expressed in Chinese hamster ovary (CHO) cells lacking the propeptide (TPOΔpro; amino acid residues 21–108) was characterized and its properties compared to wild-type TPO. Plasma membrane localization was determined by cell surface protein biotinylation, and biochemical studies were performed to evaluate enzymatic activity and the effect of deglycosylation. Immunological investigations using autoantibodies from AITD patients and other epitope-specific antibodies that recognize conformational determinants on TPO were evaluated for binding to TPOΔpro by flow cytometry, immunocytochemistry, and capture enzyme-linked immunosorbent assay. Molecular modeling and dynamics simulation of TPOΔpro comprising a dimer of myeloperoxidase-like domains was performed in order to investigate the impact of propeptide removal and the role of glycosylation. Results: The TPOΔpro was expressed on the cell surface at comparable levels to wild-type TPO. The TPOΔpro was enzymatically active and recognized by patients' autoantibodies and a panel of epitope-specific antibodies, confirming structural integrity of the two major conformational determinants recognized by autoantibodies. Faithful intracellular trafficking and N-glycosylation of TPOΔpro was also maintained. Molecular modeling and dynamics

  16. Recombinant Human Lactoferrin as a biomaterial for Bone Tissue Engineering: Mechanism of Antiapoptotic and Osteogenic Activity

    PubMed Central

    Amini, Ashley A.

    2014-01-01

    Lactoferrin is a bioactive globular protein with unique properties towards musculo-skeletal cells and anabolic to bone in vivo. Even though the potent anti-apoptotic and osteogenic activity of lactoferrin has been reported, the mechanism of action has not been fully elucidated. The study demonstrated that the anti-apoptotic effect of rhLF towards MC3T3 pre-osteoblast cells is mediated by Wnt5a/PKA pathway and the stabilization of β-catenin by rhLF is dependent on PKA/LRP6 signaling pathway. The study also investigated the feasibility of developing rhLF as a biomaterial for cell delivery. The injectable rhLF cell delivery vehicles were prepared by enzymatic crosslinking of tyramine-modified rhLF in the presence of hydrogen peroxide and horseradish peroxidase. The modified rhLF showed bioactivity similar to unmodified rhLF. The rhLF gels supported encapsulated MC3T3 cell viability, proliferation and differentation, as well as phosphorylation of signaling proteins. In conclusion, the study demonstrated the involvement of Wnt5a, LRP6 and PKA signaling in rhLF mediated bioactivity towards MC3T3 cells and the feasibility of developing an injectable cell delivery vehicle from rhLF. PMID:24352833

  17. Recombinant human lactoferrin as a biomaterial for bone tissue engineering: mechanism of antiapoptotic and osteogenic activity.

    PubMed

    Amini, Ashley A; Nair, Lakshmi S

    2014-06-01

    Lactoferrin is a bioactive globular protein with unique properties towards musculo-skeletal cells and anabolic to bone in vivo. Even though the potent anti-apoptotic and osteogenic activity of lactoferrin has been reported, the mechanism of action has not been fully elucidated. The study demonstrates that the anti-apoptotic effect of rhLF towards MC3T3 pre-osteoblast cells is mediated by Wnt5a/PKA pathway and the stabilization of β-catenin by rhLF is dependent on PKA/LRP6 signaling pathway. The study also investigates the feasibility of developing rhLF as a biomaterial for cell delivery. The injectable rhLF cell delivery vehicles are prepared by enzymatic crosslinking of tyramine-modified rhLF in the presence of hydrogen peroxide and horseradish peroxidase. The modified rhLF shows bioactivity similar to unmodified rhLF. The rhLF gels support encapsulated MC3T3 cell viability, proliferation, and differentiation, as well as phosphorylation of signaling proteins. In conclusion, the study demonstrates the involvement of Wnt5a, LRP6, and PKA signaling in rhLF-mediated bioactivity towards MC3T3 cells and the feasibility of developing an injectable cell delivery vehicle from rhLF. PMID:24352833

  18. Storage of Heparinised Canine Whole Blood for the Measurement of Glutathione Peroxidase Activity.

    PubMed

    van Zelst, Mariëlle; Hesta, Myriam; Gray, Kerry; Janssens, Geert P J

    2016-08-01

    Glutathione peroxidase activity is used as a biomarker of selenium status in dogs. Freshly collected blood samples are usually measured, due to the lack of knowledge on the effect of storing the samples. This study investigated if the analysis of glutathione peroxidase activity in whole blood collected from dogs was affected by storage of between 5 and 164 days. Results indicated that glutathione peroxidase activity was more variable in the freshly analysed samples compared to the stored samples. Although the mean differences between fresh and stored samples were not always equal to zero, this is thought to be caused by the variability of reagent preparation rather than by storage, as no consistent increase or decrease in glutathione peroxidase activity was found. Therefore, it can be concluded that heparinised dog blood samples can be successfully stored up to 164 days before analysis of glutathione peroxidase activity. PMID:26701335

  19. Effect of methylmercury on the activity of glutathione peroxidase in rat liver

    SciTech Connect

    Hirota, Y.

    1986-09-01

    The effect of methylmercury on the activity of glutathione peroxidase in rat liver was studied in vivo. A daily dose of 10mg methylmercuric chloride/kg body weight was administered subcutaneously to 15 male Wistar rats for 10 days, and the glutathione peroxidase activity in the liver was measured to compare with the control activity. A marked decrease was observed in the glutathione peroxidase activity in the experimental animals, which measured as low as 40% in comparison to that in the control animals. It can be speculated that the inhibition of glutathione peroxidase activity plays a significant role in the development of mercury toxicity and that the protective effect of selenium and vitamin E on the mercury intoxication might be partly due to preserving the glutathione peroxidase activity in the antioxidative defense mechanisms.

  20. Genomic homologous recombination in planta.

    PubMed Central

    Gal, S; Pisan, B; Hohn, T; Grimsley, N; Hohn, B

    1991-01-01

    A system for monitoring intrachromosomal homologous recombination in whole plants is described. A multimer of cauliflower mosaic virus (CaMV) sequences, arranged such that CaMV could only be produced by recombination, was integrated into Brassica napus nuclear DNA. This set-up allowed scoring of recombination events by the appearance of viral symptoms. The repeated homologous regions were derived from two different strains of CaMV so that different recombinant viruses (i.e. different recombination events) could be distinguished. In most of the transgenic plants, a single major virus species was detected. About half of the transgenic plants contained viruses of the same type, suggesting a hotspot for recombination. The remainder of the plants contained viruses with cross-over sites distributed throughout the rest of the homologous sequence. Sequence analysis of two recombinant molecules suggest that mismatch repair is linked to the recombination process. Images PMID:2026150

  1. Proton NMR investigation of the heme active site structure of an engineered cytochrome c peroxidase that mimics manganese peroxidase.

    PubMed

    Wang, X; Lu, Y

    1999-07-13

    The heme active site structure of an engineered cytochrome c peroxidase [MnCcP; see Yeung, B. K., et al. (1997) Chem. Biol. 4, 215-221] that closely mimics manganese peroxidase (MnP) has been characterized by both one- and two-dimensional NMR spectroscopy. All hyperfine-shifted resonances from the heme pocket as well as resonances from catalytically relevant amino acid residues in the congested diamagnetic envelope have been assigned. From the NMR spectral assignment and the line broadening pattern of specific protons in NOESY spectra of MnCcP, the location of the engineered Mn(II) center is firmly identified. Furthermore, we found that the creation of the Mn(II)-binding site in CcP resulted in no detectable structural changes on the distal heme pocket of the protein. However, notable structural changes are observed at the proximal side of the heme cavity. Both CepsilonH shift of the proximal histidine and (15)N shift of the bound C(15)N(-) suggest a weaker heme Fe(III)-N(His) bond in MnCcP compared to WtCcP. Our results indicate that the engineered Mn(II)-binding site in CcP resulted in not only a similar Mn(II)-binding affinity and improved MnP activity, but also weakened the Fe(III)-N(His) bond strength of the template protein CcP so that its bond strength is similar to that of the target protein MnP. The results presented here help elucidate the impact of designing a metal-binding site on both the local and global structure of the enzyme, and provide a structural basis for engineering the next generation of MnCcP that mimics MnP more closely. PMID:10413489

  2. Lignin-degrading peroxidases in Polyporales: an evolutionary survey based on 10 sequenced genomes.

    PubMed

    Ruiz-Dueñas, Francisco J; Lundell, Taina; Floudas, Dimitrios; Nagy, Laszlo G; Barrasa, José M; Hibbett, David S; Martínez, Angel T

    2013-01-01

    The genomes of three representative Polyporales (Bjerkandera adusta, Phlebia brevispora and a member of the Ganoderma lucidum complex) were sequenced to expand our knowledge on the diversity of ligninolytic and related peroxidase genes in this Basidiomycota order that includes most wood-rotting fungi. The survey was completed by analyzing the heme-peroxidase genes in the already available genomes of seven more Polyporales species representing the antrodia, gelatoporia, core polyporoid and phlebioid clades. The study confirms the absence of ligninolytic peroxidase genes from the manganese peroxidase (MnP), lignin peroxidase (LiP) and versatile peroxidase (VP) families, in the brown-rot fungal genomes (all of them from the antrodia clade), which include only a limited number of predicted low redox-potential generic peroxidase (GP) genes. When members of the heme-thiolate peroxidase (HTP) and dye-decolorizing peroxidase (DyP) superfamilies (up to a total of 64 genes) also are considered, the newly sequenced B. adusta appears as the Polyporales species with the highest number of peroxidase genes due to the high expansion of both the ligninolytic peroxidase and DyP (super)families. The evolutionary relationships of the 111 genes for class-II peroxidases (from the GP, MnP, VP, LiP families) in the 10 Polyporales genomes is discussed including the existence of different MnP subfamilies and of a large and homogeneous LiP cluster, while different VPs mainly cluster with short MnPs. Finally, ancestral state reconstructions showed that a putative MnP gene, derived from a primitive GP that incorporated the Mn(II)-oxidation site, is the precursor of all the class-II ligninolytic peroxidases. Incorporation of an exposed tryptophan residue involved in oxidative degradation of lignin in a short MnP apparently resulted in evolution of the first VP. One of these ancient VPs might have lost the Mn(II)-oxidation site being at the origin of all the LiP enzymes, which are found only in

  3. Recombinant vaccines against leptospirosis.

    PubMed

    Dellagostin, Odir A; Grassmann, André A; Hartwig, Daiane D; Félix, Samuel R; da Silva, Éverton F; McBride, Alan J A

    2011-11-01

    Leptospirosis is an important neglected infectious disease that occurs in urban environments, as well as in rural regions worldwide. Rodents, the principal reservoir hosts of pathogenic Leptospira spp., and other infected animals shed the bacteria in their urine. During occupational or even recreational activities, humans that come into direct contact with infected animals or with a contaminated environment, particularly water, are at risk of infection. Prevention of urban leptospirosis is largely dependent on sanitation measures that are often difficult to implement, especially in developing countries. Vaccination with inactivated whole-cell preparations (bacterins) has limited efficacy due to the wide antigenic variation of the pathogen. Intensive efforts towards developing improved recombinant vaccines are ongoing. During the last decade, many reports on the evaluation of recombinant vaccines have been published. Partial success has been obtained with some surface-exposed protein antigens. The combination of protective antigens and new adjuvants or delivery systems may result in the much-needed effective vaccine. PMID:22048111

  4. Recombinant influenza vaccines.

    PubMed

    Sedova, E S; Shcherbinin, D N; Migunov, A I; Smirnov, Iu A; Logunov, D Iu; Shmarov, M M; Tsybalova, L M; Naroditskiĭ, B S; Kiselev, O I; Gintsburg, A L

    2012-10-01

    This review covers the problems encountered in the construction and production of new recombinant influenza vaccines. New approaches to the development of influenza vaccines are investigated; they include reverse genetics methods, production of virus-like particles, and DNA- and viral vector-based vaccines. Such approaches as the delivery of foreign genes by DNA- and viral vector-based vaccines can preserve the native structure of antigens. Adenoviral vectors are a promising gene-delivery platform for a variety of genetic vaccines. Adenoviruses can efficiently penetrate the human organism through mucosal epithelium, thus providing long-term antigen persistence and induction of the innate immune response. This review provides an overview of the practicability of the production of new recombinant influenza cross-protective vaccines on the basis of adenoviral vectors expressing hemagglutinin genes of different influenza strains. PMID:23346377

  5. Site directed recombination

    DOEpatents

    Jurka, Jerzy W.

    1997-01-01

    Enhanced homologous recombination is obtained by employing a consensus sequence which has been found to be associated with integration of repeat sequences, such as Alu and ID. The consensus sequence or sequence having a single transition mutation determines one site of a double break which allows for high efficiency of integration at the site. By introducing single or double stranded DNA having the consensus sequence flanking region joined to a sequence of interest, one can reproducibly direct integration of the sequence of interest at one or a limited number of sites. In this way, specific sites can be identified and homologous recombination achieved at the site by employing a second flanking sequence associated with a sequence proximal to the 3'-nick.

  6. Measurement of tomato plant gene expression on a genomic scale for tomato plants that over express peroxidase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We examined the gene expression of transgenic tomato plants that over-express the plant defense peroxidase in comparison to the control tomato plants with normal levels of peroxidase. In general, jasmonate-related plant defenses such as putative protease inhibitors were suppressed in peroxidase-rel...

  7. Molecular characterization of the lignin-forming peroxidase: Role in growth, development and response to stress. Progress summary report, April 1, 1992--March 31, 1993

    SciTech Connect

    Lagrimini, L.M.

    1993-03-01

    This laboratory has continued its comprehensive study of the structure and function of plant peroxidases and their genes. Specifically, we are characterizing the anionic peroxidase of tobacco. During the past year we have completed the nucleotide sequence of the tobacco anionic peroxidase gene, joined the anionic peroxidase promoter to {Beta}-glucuronidase and demonstrated expression in transformed plants, measured lignin, auxin, and ethylene levels in transgenic tobacco plants over-expressing the anionic peroxidase, developed chimeric peroxidase genes to over-or under-express the anionic peroxidase in tissue specific manner in transgenic plants, and over-expressed the tobacco anionic peroxidase in transgenic tomato and sweetgum plants.

  8. The recombination epoch revisited

    NASA Technical Reports Server (NTRS)

    Krolik, Julian H.

    1989-01-01

    Previous studies of cosmological recombination have shown that this process produces as a by-product a highly superthermal population of Ly-alpha photons which retard completion of recombination. Cosmological redshifting was thought to determine the frequency distribution of the photons, while two-photon decay of hydrogen's 2s state was thought to control their numbers. It is shown here that frequency diffusion due to photon scattering dominate the cosmological redshift in the frequency range near line center which fixes the ratio of ground state to excited state population, while incoherent scattering into the far-red damping wing effectively destroys Ly-alpha photons as a rate which is competitive with two-photon decay. The former effect tends to hold back recombination, while the latter tends to accelerate it; the net results depends on cosmological parameters, particularly the combination Omega(b) h/sq rt (2q0), where Omega(b) is the fraction of the critical density provided by baryons.

  9. Hevea brasiliensis cell suspension peroxidase: purification, characterization and application for dye decolorization

    PubMed Central

    2013-01-01

    Peroxidases are oxidoreductase enzymes produced by most organisms. In this study, a peroxidase was purified from Hevea brasiliensis cell suspension by using anion exchange chromatography (DEAE-Sepharose), affinity chromatography (Con A-agarose) and preparative SDS-PAGE. The obtained enzyme appeared as a single band on SDS-PAGE with molecular mass of 70 kDa. Surprisingly, this purified peroxidase also had polyphenol oxidase activity. However, the biochemical characteristics were only studied in term of peroxidase because similar experiments in term of polyphenol oxidase have been reported in our pervious publication. The optimal pH of the purified peroxidase was 5.0 and its activity was retained at pH values between 5.0–10.0. The enzyme was heat stable over a wide range of temperatures (0–60°C), and less than 50% of its activity was lost at 70°C after incubation for 30 min. The enzyme was completely inhibited by β-mercaptoethanol and strongly inhibited by NaN3; in addition, its properties indicated that it was a heme containing glycoprotein. This peroxidase could decolorize many dyes; aniline blue, bromocresol purple, brilliant green, crystal violet, fuchsin, malachite green, methyl green, methyl violet and water blue. The stability against high temperature and extreme pH supported that the enzyme could be a potential peroxidase source for special industrial applications. PMID:23402438

  10. Site-specific Effects of DUOX1-Related Peroxidase on Intercellular Apoptosis Signaling.

    PubMed

    Heinzelmann, Sonja; Bauer, Georg

    2015-11-01

    Intercellular apoptosis-inducing HOCl signaling is known as an interplay between superoxide anions/H₂O₂ of transformed target cells and dual oxidase 1 (DUOX1)-related peroxidase that is released from neighboring non-transformed or transformed effector cells. Effector cells are dispensable when the release of the peroxidase domain of DUOX1 from target cells is prevented through inhibition of matrix metalloproteinase (MMP) activity. Membrane-associated peroxidase is then co-localized to NADPH oxidase 1 (NOX1) and establishes HOCl signaling specifically in transformed cells, using the same biochemical pathways as classical intercellular HOCl signaling. Membrane-associated peroxidase protects against exogenous HOCl through reversal of the peroxidase reaction. In addition, membrane-associated peroxidase protects against NO/peroxynitrite signaling as it oxidates NO and decomposes peroxynitrite. The protective function of membrane-associated peroxidase (in the absence of MMP) is analogous to that of catalase, whereas the destructive effect of the enzyme, i.e. the synthesis of HOCl, is independent of its localization and of MMP activity. PMID:26504019

  11. Uncovering a new role for peroxidase enzymes as drivers of angiogenesis.

    PubMed

    Panagopoulos, Vasilios; Zinonos, Irene; Leach, Damien A; Hay, Shelley J; Liapis, Vasilios; Zysk, Aneta; Ingman, Wendy V; DeNichilo, Mark O; Evdokiou, Andreas

    2015-11-01

    Peroxidases are heme-containing enzymes released by activated immune cells at sites of inflammation. To-date their functional role in human health has mainly been limited to providing a mechanism for oxidative defence against invading bacteria and other pathogenic microorganisms. Our laboratory has recently identified a new functional role for peroxidase enzymes in stimulating fibroblast migration and collagen biosynthesis, offering a new insight into the causative association between inflammation and the pro-fibrogenic events that mediate tissue repair and regeneration. Peroxidases are found at elevated levels within and near blood vessels however, their direct involvement in angiogenesis has never been reported. Here we report for the first time that myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are readily internalised by human umbilical vein endothelial cells (HUVEC) where they promote cellular proliferation, migration, invasion, and stimulate angiogenesis both in vitro and in vivo. These pro-angiogenic effects were attenuated using the specific peroxidase inhibitor 4-ABAH, indicating the enzyme's catalytic activity is essential in mediating this response. Mechanistically, we provide evidence that MPO and EPO regulate endothelial FAK, Akt, p38 MAPK, ERK1/2 phosphorylation and stabilisation of HIF-2α, culminating in transcriptional regulation of key angiogenesis pathways. These findings uncover for the first time an important and previously unsuspected role for peroxidases as drivers of angiogenesis, and suggest that peroxidase inhibitors may have therapeutic potential for the treatment of angiogenesis related diseases driven by inflammation. PMID:26386352

  12. Is Peroxiredoxin II's peroxidase activity strongly inhibited in human erythrocytes?

    PubMed

    Benfeitas, Rui; Selvaggio, Gianluca; Antunes, Fernando; Coelho, Pedro; Salvador, Armindo

    2014-10-01

    H2O2 elimination in human erythrocytes is mainly carried out by catalase (Cat), glutathione peroxidase (GPx1) and the more recently discovered peroxiredoxin 2 (Prx2). However, the contribution of Prx2 to H2O2 consumption is still unclear. Prx2's high reactivity with H2O2 (kPrx2=10×10(7) M(-1)s(-1), kCat =7×10(7) M(-1)s(-1), kGPx1 =4×10(7) M(-1)s(-1)) and high abundance ([Prx2]= 570µM, [Cat]= 32µM, [GPx1]= 1µM) suggest that under low H2O2 supply rates it should consume >99% of the H2O2. However, extensive evidence indicates that in intact erythrocytes Prx2 contributes no more than Cat to H2O2 consumption. In order for this to be attained, Prx2's effective rate constant with H2O2would have to be just ~10(5) M(-1)s(-1), much lower than that determined in multiple experiments with the purified proteins. Nevertheless, nearly all Prx2 is oxidized within 1min of exposing erythrocytes to a H2O2 bolus, which is inconsistent with an irreversible inhibition. A mathematical model of the H2O2 metabolism in human erythrocytes [Benfeitas et al. (2014) Free Radic. Biol. Med.] where Prx2 either has a low kPrx2 or is subject to a strong (>99%) but readily reversible inhibition achieves quantitative agreement with detailed experimental observations of the responses of the redox status of Prx2 in human erythrocytes and suggests functional advantages of this design (see companion abstract). By contrast, a variant where Prx2 is fully active with kPrx2=10(8) M(-1)s(-1) shows important qualitative discrepancies. Altogether, these results suggest that Prx2's peroxidase activity is strongly inhibited in human erythrocytes. We acknowledge fellowship SFRH/BD/51199/2010, grants PEst-C/SAU/LA0001/2013-2014, PEst-OE/QUI/UI0612/2013, PEst-OE/QUI/UI0313/2014, and FCOMP-01-0124-FEDER-020978 (PTDC/QUI-BIQ/119657/2010) co-financed by FEDER through the COMPETE program and by FCT. PMID:26461310

  13. Phenolic mediators enhance the manganese peroxidase catalyzed oxidation of recalcitrant lignin model compounds and synthetic lignin.

    PubMed

    Nousiainen, Paula; Kontro, Jussi; Manner, Helmiina; Hatakka, Annele; Sipilä, Jussi

    2014-11-01

    Fungal oxidative enzymes, such as peroxidases and laccases, are the key catalysts in lignin biodegradation in vivo, and consequently provide an important source for industrial ligninolytic biocatalysts. Recently, it has been shown that some syringyl-type phenolics have potential as industrial co-oxidants or mediators, in laccase-catalyzed modification of lignocellulosic material. We have now studied the effect of such mediators with ligninolytic peroxidases on oxidation of the most recalcitrant lignin model compounds. We found that they are able to enhance the manganese peroxidase (MnP) catalyzed oxidation reactions of small non-phenolic compounds, veratryl alcohol and veratrylglycerol β-guaiacyl ether (adlerol), which are not usually oxidized by manganese peroxidases alone. In these experiments we compared two peroxidases from white-rot fungi, MnP from Phlebia sp. Nf b19 and versatile peroxidase (VP) from Bjerkandera adusta under two oxidation conditions: (i) the Mn(III) initiated mediated oxidation by syringyl compounds and (ii) the system involving MnP-dependent lipid peroxidation, both with production of (hydrogen) peroxides in situ to maintain the peroxidase catalytic cycle. It was found that both peroxidases produced α-carbonyl oxidation product of veratryl alcohol in clearly higher yields in reactions mediated by phenoxy radicals than in lipid-peroxyl radical system. The oxidation of adlerol, on the other hand, was more efficient in lipid-peroxidation-system. VP was more efficient than MnP in the oxidation of veratryl alcohol and showed its lignin peroxidase type activity in the reaction conditions indicated by some cleavage of Cα-Cβ-bond of adlerol. Finally, the mediator assisted oxidation conditions were applied in the oxidation of synthetic lignin (DHP) and the structural analysis of the oxidized polymers showed clear modifications in the polymer outcome, e.g. the oxidation resulted in reduced amount of aliphatic hydroxyls indicated by (31)P NMR. PMID

  14. Peroxidase activity in Aloe barbadensis commercial gel: probable role in skin protection.

    PubMed

    Esteban, A; Zapata, J M; Casano, L; Martín, M; Sabater, B

    2000-12-01

    A basic peroxidase (EC 1.11.1.7) (pl around 9.0) has been identified in commercial gel of Aloe barbadensis. In vivo, the activity is localised in the vascular system of inner aqueous leaf parenchyma. Some relevant properties of this basic peroxidase of Aloe have been investigated in leaf extract and in commercial gel where it is notably stable. The acid optimum pH (5.0) for activity and the low KM for H2O2 (0.14 mM) suggest that, when topically applied, Aloe peroxidase may scavenge H2O2 in skin surface. PMID:11199129

  15. Identification of a copper-sensitive ascorbate peroxidase in the unicellular green alga Selenastrum capricornutum.

    PubMed

    Sauser, K R; Liu, J K; Wong, T Y

    1997-07-01

    Extracts from the unicellular green alga Selenastrum capricornutum exhibit high superoxide dismutase activity, but only traces of catalase activity. The excess hydrogen peroxide (H2O2) generated by the superoxide dismutase in S. capricornutum may be degraded by a unique peroxidase. This peroxidase has a high specificity for ascorbate as its electron donor. The enzyme has an optimum pH at 8, is insensitive to cyanide and is inhibited by oxine. Addition of low concentrations of copper to algal cultures stimulates the peroxidase activity threefold. This enzymatic system could be used as a sensitive bioindicator for copper in fresh water. PMID:9243795

  16. The multihued palette of dye-decolorizing peroxidases.

    PubMed

    Singh, Rahul; Eltis, Lindsay D

    2015-05-15

    Dye-decolorizing peroxidases (DyPs; EC 1.11.1.19) are heme enzymes that comprise a family of the dimeric α+β barrel structural superfamily of proteins. The first DyP, identified relatively recently in the fungus Bjerkandera adusta, was characterized for its ability to catalyze the decolorization of anthraquinone-based industrial dyes. These enzymes are now known to be present in all three domains of life, but do not appear to occur in plants or animals. They are involved in a range of physiological processes, although in many cases their roles remain unknown. This has not prevented the development of their biocatalytic potential, which includes the transformation of lignin. This review highlights the functional diversity of DyPs in the light of phylogenetic, structural and biochemical data. The phylogenetic analysis reveals the existence of at least five classes of DyPs. Their potential physiological roles are discussed based in part on synteny analyses. Finally, the considerable biotechnological potential of DyPs is summarized. PMID:25743546

  17. Identification of S-RNase and peroxidase in petunia nectar.

    PubMed

    Hillwig, Melissa S; Kanobe, Charles; Thornburg, Robert W; Macintosh, Gustavo C

    2011-05-01

    Previous SDS PAGE gel analysis of the floral nectars from petunia and tobacco plants revealed significant differences in the protein patterns. Petunia floral nectar was shown to contain a number of RNase activities by in gel RNase activity assay. To identify these proteins in more detail, the bands with RNase activity were excised from gel and subjected to trypsin digestion followed by LC-MS/MS analysis. This analysis revealed that S-RNases accumulate in nectar from Petunia hybrida, where they should carry out a biological function different from self-pollen rejection. In addition, other proteins were identified by the LC-MS/MS analysis. These proteins include a peroxidase, an endochitinase, and a putative fructokinase. Each of these proteins contained a secretory signal sequence that marked them as potential nectar proteins. We developed RT-PCR assays for each of these five proteins and demonstrated that each of these proteins was expressed in the petunia floral nectary. A discussion of the role of these proteins in antimicrobial activity in nectar is presented. PMID:21093100

  18. Biodegradation of Single-Walled Carbon Nanotubes by Eosinophil Peroxidase

    PubMed Central

    Andón, Fernando T.; Kapralov, Alexandr A.; Yanamala, Naveena; Feng, Weihong; Baygan, Arjang; Chambers, Benedict J.; Hultenby, Kjell; Ye, Fei; Toprak, Muhammet S.; Brandner, Birgit D.; Fornara, Andrea; Klein-Seetharaman, Judith; Kotchey, Gregg P.; Star, Alexander; Shvedova, Anna A.

    2014-01-01

    Eosinophil peroxidase (EPO) is one of the major oxidant-producing enzymes during inflammatory states in the human lung. The degradation of single-walled carbon nanotubes (SWCNTs) upon incubation with human EPO and H2O2 is reported. Biodegradation of SWCNTs is higher in the presence of NaBr, but neither EPO alone nor H2O2 alone caused the degradation of nanotubes. Molecular modeling reveals two binding sites for SWCNTs on EPO, one located at the proximal side (same side as the catalytic site) and the other on the distal side of EPO. The oxidized groups on SWCNTs in both cases are stabilized by electrostatic interactions with positively charged residues. Biodegradation of SWCNTs can also be executed in an ex vivo culture system using primary murine eosinophils stimulated to undergo degranulation. Biodegradation is proven by a range of methods including transmission electron microscopy, UV-visible-NIR spectroscopy, Raman spectroscopy, and confocal Raman imaging. Thus, human EPO (in vitro) and ex vivo activated eosinophils mediate biodegradation of SWCNTs: an observation that is relevant to pulmonary responses to these materials. PMID:23447468

  19. Aflatoxin detoxification by manganese peroxidase purified from Pleurotus ostreatus

    PubMed Central

    Yehia, Ramy Sayed

    2014-01-01

    Manganese peroxidase (MnP) was produced from white rot edible mushroom Pleurotus ostreatus on the culture filtrate. The enzyme was purified to homogeneity using (NH4)2SO4 precipitation, DEAE-Sepharose and Sephadex G-100 column chromatography. The final enzyme activity achieved 81 U mL−1, specific activity 78 U mg−1 with purification fold of 130 and recovery 1.2% of the crude enzyme. SDS-PAGE indicated that the pure enzyme have a molecular mass of approximately 42 kDa. The optimum pH was between 4–5 and the optimum temperature was 25 °C. The pure MnP activity was enhanced by Mn2+, Cu2+, Ca2+ and K+ and inhibited by Hg+2 and Cd+2. H2O2 at 5 mM enhanced MnP activity while at 10 mM inhibited it significantly. The MnP-cDNA encoding gene was sequenced and determined (GenBank accession no. AB698450.1). The MnP-cDNA was found to consist of 497 bp in an Open Reading Frame (ORF) encoding 165 amino acids. MnP from P. ostreatus could detoxify aflatoxin B1 (AFB1) depending on enzyme concentration and incubation period. The highest detoxification power (90%) was observed after 48 h incubation at 1.5 U mL−1 enzyme activities. PMID:24948923

  20. Colorimetric peroxidase mimetic assay for uranyl detection in sea water.

    PubMed

    Zhang, Dingyuan; Chen, Zhuo; Omar, Haneen; Deng, Lin; Khashab, Niveen M

    2015-03-01

    Uranyl (UO2(2+)) is a form of uranium in aqueous solution that represents the greatest risk to human health because of its bioavailability. Different sensing techniques have been used with very sensitive detection limits especially the recently reported uranyl-specific DNAzymes systems. However, to the best of our knowledge, few efficient detection methods have been reported for uranyl sensing in seawater. Herein, gold nanoclusters (AuNCs) are employed in an efficient spectroscopic method to detect uranyl ion (UO2(2+)) with a detection limit of 1.86 μM. In the absence of UO2(2+), the BSA-stabilized AuNCs (BSA-AuNCs) showed an intrinsic peroxidase-like activity. In the presence of UO2(2+), this activity can be efficiently restrained. The preliminary quenching mechanism and selectivity of UO2(2+) was also investigated and compared with other ions. This design strategy could be useful in understanding the binding affinity of protein-stabilized AuNCs to UO2(2+) and consequently prompt the recycling of UO2(2+) from seawater. PMID:25658750

  1. Calcium promotes activity and confers heat stability on plant peroxidases

    PubMed Central

    Plieth, Christoph; Vollbehr, Sonja

    2012-01-01

    In this paper we demonstrate how peroxidase (PO) activities and their heat stability correlate with the availability of free Ca2+ ions. Calcium ions work as a molecular switch for PO activity and exert a protective function, rendering POs heat stable. The concentration ranges of these two activities differ markedly. POs are activated by µM Ca2+ concentration ranges, whereas heat stabilization is observed in the nM range. This suggests the existence of different Ca2+ binding sites. The heat stability of POs depends on the source plant species. Terrestrial plants have POs that exhibit higher temperature stability than those POs from limnic and marine plants. Different POs from a single species can differ in terms of heat stability. The abundance of different POs within a plant is dependent on age and developmental stage. The heat stability of a PO does not necessarily correlate with the maximum temperature the source species is usually exposed to in its natural habitat. This raises questions on the role of POs in the heat tolerance of plants. Consequently, detailed investigations are needed to identify and characterize individual POs, with regard to their genetic origin, subcellular expression, tissue abundance, developmental emergence and their functions in innate and acquired heat tolerance. PMID:22580695

  2. Atherogenic diets exacerbate colitis in mice deficient in glutathione peroxidase

    PubMed Central

    Gao, Qiang; Esworthy, R. Steven; Kim, Byung-Wook; Synold, Timothy W.; Smith, David D.; Chu, Fong-Fong

    2010-01-01

    The pro-inflammatory effect of high-fat diet has been observed beyond the cardiovascular system, but there is little evidence to support its role in triggering inflammatory bowel disease. GPx1/2-double knockout (DKO) mice deficient in two intracellular glutathione peroxidases, GPx1 and GPx2, on a C57BL/6 (B6) background, have mild ileocolitis on a conventional chow. We fed B6 DKO mice two atherogenic diets to test the dietary effect on atherosclerosis and ileocolitis. Both atherogenic diets have high cholesterol, the Chol+/CA diet has cholic acid (CA) and the Chol+ diet has no CA. The Chol+/CA diet induced severe colitis, but not ileitis, in the DKO mice compared with Chol+ and a Chol- control diet. On the Chol+/CA diet, the wild-type (WT) mice had similar levels of aortic lesions and hypercholesterolemia as DKO mice did, but had no intestinal pathology. The diet-associated inflammatory responses in the DKO mice included increase of colonic pro-inflammatory serum amyloid A 3 expression, plasma lipopolysaccharide and TNF-α levels. The Chol+/CA diet has lowered the expression of unfolded protein response genes, ATF6, CHOP, unspliced XbpU and Grp78/Bip, in WT and DKO mice on the Chol- diet. Thus, we conclude that cholesterol diet weakens colon unfolded protein response, which can aggravate spontaneous colitis leading to gut barrier breakdown. GPx has no impact on atherosclerosis without ultra-hypercholesterolemia. PMID:20848490

  3. Eosinophil peroxidase-dependent hydroxyl radical generation by human eosinophils.

    PubMed

    McCormick, M L; Roeder, T L; Railsback, M A; Britigan, B E

    1994-11-11

    Eosinophil production of superoxide (O2-.) and hydrogen peroxide (H2O2) is important in host defense. The present study assessed the potential of eosinophils to generate another potent cytotoxic species, the hydroxyl radical (.OH). .OH formation by phorbol myristate acetate (PMA)-stimulated eosinophils was demonstrated using an alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone/ethanol spin trapping system. Additionally, .OH was spin trapped following the addition of purified eosinophil peroxidase (EPO) to a cell-free O2-./H2O2 generating systems. Effects of superoxide dismutase, catalase, azide, aminotriazole, chloride-depleted buffer, and extensive metal chelation were consistent with .OH formation via the reaction of O2-. and EPO-generated hypohalous acid. Under chloride-depleted conditions, physiologic concentrations of Br- increased .OH formation by both PMA-stimulated eosinophils and the cell-free EPO system. Physiologic concentrations of SCN-, however, did not increase .OH formation, and in the presence of both Br- and SCN-, .OH formation was similar to SCN- only. Eosinophils appear to form .OH via an EPO-dependent mechanism, the magnitude of which varies with the availability of various EPO substrates. Given the highly reactive nature of this radical and the ability of EPO to adhere to cell membranes, even small amounts of .OH formed at such sites could contribute to eosinophil-mediated cytotoxicity. PMID:7961724

  4. Activation of lignin peroxidase in organic media by reversed micelles.

    PubMed

    Kimura, Masayuki; Michizoe, Junji; Oakazaki, Shin-Ya; Furusaki, Shintaro; Goto, Masahiro; Tanaka, Hiroo; Wariishi, Hiroyuki

    2004-11-20

    Activation of lignin peroxidase (LIP) in an organic solvent by reversed micelles was investigated. Bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT) was used as a surfactant to form a reversed micelle. Lyophilized LIP from an optimized aqueous solution exhibited no enzymatic activity in any organic solvents examined in this study; however, LIP was catalytically active by being entrapped in the AOT reversed micellar solution. LIP activity in the reversed micelle was enhanced by optimizing either the preparation or the operation conditions, such as water content and pH in water pools of the reversed micelle and the reaction temperature. Stable activity was obtained in isooctane because of the stability of the reversed micelle. The optimal pH was 5 in the reversed micellar system, which shifted from pH 3 in the aqueous solution. The degradation reaction of several environmental pollutants was attempted using LIP hosted in the AOT reversed micelle. Degradation achieved after a 1-h reaction reached 81%, 50%, and 22% for p-nonylphenol, bisphenol A, and 2,4-dichlorophenol, respectively. This is the first report on the utilization of LIP in organic media. PMID:15459910

  5. Oxidation of pharmaceutically active compounds by a ligninolytic fungal peroxidase.

    PubMed

    Eibes, Gemma; Debernardi, Gianfranco; Feijoo, Gumersindo; Moreira, M Teresa; Lema, Juan M

    2011-06-01

    Pharmaceuticals are an important group of emerging pollutants with increasing interest due to their rising consumption and the evidence for ecotoxicological effects associated to trace amounts in aquatic environments. In this paper, we assessed the potential degradation of a series of pharmaceuticals: antibiotics (sulfamethoxazole), antidepressives (citalopram hydrobromide and fluoxetine hydrochloride), antiepileptics (carbamazepine), anti-inflammatory drugs (diclofenac and naproxen) and estrogen hormones (estrone, 17β-estradiol, 17α-ethinylestradiol) by means of a versatile peroxidase (VP) from the ligninolytic fungus Bjerkandera adusta. The effects of the reaction conditions: VP activity, organic acid concentration and H(2)O(2) addition rate, on the kinetics of the VP based oxidation system were evaluated. Diclofenac and estrogens were completely degraded after only 5-25 min even with a very low VP activity (10 U l(-1)). High degradation percentages (80%) were achieved for sulfamethoxazole and naproxen. Low or undetectable removal yields were observed for citalopram (up to 18%), fluoxetine (lower than 10%) and carbamazepine (not degraded). PMID:20972884

  6. Bacterial Recombineering: Genome Engineering via Phage-Based Homologous Recombination.

    PubMed

    Pines, Gur; Freed, Emily F; Winkler, James D; Gill, Ryan T

    2015-11-20

    The ability to specifically modify bacterial genomes in a precise and efficient manner is highly desired in various fields, ranging from molecular genetics to metabolic engineering and synthetic biology. Much has changed from the initial realization that phage-derived genes may be employed for such tasks to today, where recombineering enables complex genetic edits within a genome or a population. Here, we review the major developments leading to recombineering becoming the method of choice for in situ bacterial genome editing while highlighting the various applications of recombineering in pushing the boundaries of synthetic biology. We also present the current understanding of the mechanism of recombineering. Finally, we discuss in detail issues surrounding recombineering efficiency and future directions for recombineering-based genome editing. PMID:25856528

  7. Peroxiredoxin 6 homodimerization and heterodimerization with glutathione S-transferase pi are required for its peroxidase but not phospholipase A2 activity.

    PubMed

    Zhou, Suiping; Sorokina, Elena M; Harper, Sandra; Li, Haitao; Ralat, Luis; Dodia, Chandra; Speicher, David W; Feinstein, Sheldon I; Fisher, Aron B

    2016-05-01

    Peroxiredoxin 6 (Prdx6) is a unique 1-Cys member of the peroxiredoxin family with both GSH peroxidase and phospholipase A2 (PLA2) activities. It is highly expressed in the lung where it plays an important role in antioxidant defense and lung surfactant metabolism. Glutathionylation of Prdx6 mediated by its heterodimerization with GSH S-transferase π (πGST) is required for its peroxidatic catalytic cycle. Recombinant human Prdx6 crystallizes as a homodimer and sedimentation equilibrium analysis confirmed that this protein exists as a high affinity dimer in solution. Based on measurement of molecular mass, dimeric Prdx6 that was oxidized to the sulfenic acid formed a sulfenylamide during storage. After examination of the dimer interface in the crystal structure, we postulated that the hydrophobic amino acids L145 and L148 play an important role in homodimerization of Prdx6 as well as in its heterodimerization with πGST. Oxidation of Prdx6 also was required for its heterodimerization. Sedimentation equilibrium analysis and the Duolink proximity ligation assay following mutation of the L145 and L148 residues of Prdx6 to Glu indicated greatly decreased dimerization propensity reflecting the loss of hydrophobic interactions between the protein monomers. Peroxidase activity was markedly reduced by mutation at either of the Leu sites and was essentially abolished by the double mutation, while PLA2 activity was unaffected. Decreased peroxidase activity following mutation of the interfacial leucines presumably is mediated via impaired heterodimerization of Prdx6 with πGST that is required for reduction and re-activation of the oxidized enzyme. PMID:26891882

  8. Predominance of a Versatile-Peroxidase-Encoding Gene, mnp4, as Demonstrated by Gene Replacement via a Gene Targeting System for Pleurotus ostreatus

    PubMed Central

    Salame, Tomer M.; Knop, Doriv; Tal, Dana; Levinson, Dana; Yarden, Oded

    2012-01-01

    Pleurotus ostreatus (the oyster mushroom) and other white rot filamentous basidiomycetes are key players in the global carbon cycle. P. ostreatus is also a commercially important edible fungus with medicinal properties and is important for biotechnological and environmental applications. Efficient gene targeting via homologous recombination (HR) is a fundamental tool for facilitating comprehensive gene function studies. Since the natural HR frequency in Pleurotus transformations is low (2.3%), transformed DNA is predominantly integrated ectopically. To overcome this limitation, a general gene targeting system was developed by producing a P. ostreatus PC9 homokaryon Δku80 strain, using carboxin resistance complemented by the development of a protocol for hygromycin B resistance protoplast-based DNA transformation and homokaryon isolation. The Δku80 strain exhibited exclusive (100%) HR in the integration of transforming DNA, providing a high efficiency of gene targeting. Furthermore, the Δku80 strains produced showed a phenotype similar to that of the wild-type PC9 strain, with similar growth fitness, ligninolytic functionality, and capability of mating with the incompatible strain PC15 to produce a dikaryon which retained its resistance to the corresponding selection and was capable of producing typical fruiting bodies. The applicability of this system is demonstrated by inactivation of the versatile peroxidase (VP) encoded by mnp4. This enzyme is part of the ligninolytic system of P. ostreatus, being one of the nine members of the manganese-peroxidase (MnP) gene family, and is the predominantly expressed VP in Mn2+-deficient media. mnp4 inactivation provided a direct proof that mnp4 encodes a key VP responsible for the Mn2+-dependent and Mn2+-independent peroxidase activity under Mn2+-deficient culture conditions. PMID:22636004

  9. Dielectronic recombination theory

    SciTech Connect

    LaGattuta, K.J.

    1991-12-31

    A theory now in wide use for the calculation of dielectronic recombination cross sections ({sigma}{sup DR}) and rate coefficients ({alpha}{sup DR}) was one introduced originally by Feshbach for nuclear physics applications, and then later adapted for atomic scattering problems by Hahn. In the following, we briefly review this theory in a very general form, which allows one to account for the effects of overlapping and interacting resonances, as well as continuum-continuum coupling. An extension of our notation will then also allow for the inclusion of the effects of direct radiative recombination, along with a treatment of the interference between radiative and dielectronic recombination. Other approaches to the calculation of {sigma}{sup DR} have been described by Fano and by Seaton. We will not consider those theories here. Calculations of {alpha}{sup DR} have progressed considerably over the last 25 years, since the early work of Burgess. Advances in the reliability of theoretical predictions have also been promoted recently b a variety of direct laboratory measurements of {sigma}{sup DR}. While the measurements of {sigma}{sup DR} for {delta}n {ne} 0 excitations have tended to agree very well with calculations, the case of {delta}n = 0 has been much problematic. However, by invoking a mechanism originally proposed by Jacobs, which takes into account the effect of stray electric fields on high Rydberg states (HRS) participating in the DR process, new calculations have improved the agreement between theory and experiment for these cases. Nevertheless, certain discrepancies still remain.

  10. Recombinant Collagenlike Proteins

    NASA Technical Reports Server (NTRS)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  11. Did the universe recombine?

    NASA Technical Reports Server (NTRS)

    Bartlett, James G.; Stebbins, Albert

    1991-01-01

    The Zel'dovich-Sunyaev model-independent arguments for the existence of a neutral hydrogen phase is reviewed in light of new limits on the Compton y parameter from COBE. It is concluded that with baryon densities compatible with standard cosmological nucleosynthesis, the universe could have remained fully ionized throughout its history without producing a detectable spectral distortion. It is argued that it is unlikely that spectral observations of the cosmic microwave background will ever require the universe to have recombined for flat cosmologies.

  12. Research Advances: Horseradish Peroxide Mixture May End War of the Noses-- Even Low Levels of Benzene Are Hemotoxic--New "Nuclear Battery" Runs 10 Years. 10 Times More Powerful

    ERIC Educational Resources Information Center

    King, Angela G.

    2005-01-01

    Experts have mixed horseradish root with hydrogen peroxide or calcium peroxide in laboratory studies to get rid of the problem of odors from farm manure. Researchers evaluated how well the system reduced the concentration of volatile fatty acids, indole and skatole, compounds that are also associated with the stink of animal manure.

  13. A catalytic approach to estimate the redox potential of heme-peroxidases

    SciTech Connect

    Ayala, Marcela . E-mail: maa@ibt.unam.mx; Roman, Rosa; Vazquez-Duhalt, Rafael

    2007-06-08

    The redox potential of heme-peroxidases varies according to a combination of structural components within the active site and its vicinities. For each peroxidase, this redox potential imposes a thermodynamic threshold to the range of oxidizable substrates. However, the instability of enzymatic intermediates during the catalytic cycle precludes the use of direct voltammetry to measure the redox potential of most peroxidases. Here we describe a novel approach to estimate the redox potential of peroxidases, which directly depends on the catalytic performance of the activated enzyme. Selected p-substituted phenols are used as substrates for the estimations. The results obtained with this catalytic approach correlate well with the oxidative capacity predicted by the redox potential of the Fe(III)/Fe(II) couple.

  14. Inhibition of Peroxidase Activity of Cytochrome c: De Novo Compound Discovery and Validation

    PubMed Central

    Bakan, Ahmet; Kapralov, Alexandr A.; Bayir, Hulya; Hu, Feizhou; Kagan, Valerian E.

    2015-01-01

    Cytochrome c (cyt c) release from mitochondria is accepted to be the point of no return for eliciting a cascade of interactions that lead to apoptosis. A strategy for containing sustained apoptosis is to reduce the mitochondrial permeability pore opening. Pore opening is enhanced by peroxidase activity of cyt c gained upon its complexation with cardiolipin in the presence of reactive oxygen species. Blocking access to the heme group has been proposed as an effective intervention method for reducing, if not eliminating, the peroxidase activity of cyt c. In the present study, using a combination of druggability simulations, pharmacophore modeling, virtual screening, and in vitro fluorescence measurements to probe peroxidase activity, we identified three repurposable drugs and seven compounds that are validated to effectively inhibit the peroxidase activity of cyt c. PMID:26078313

  15. IMPROVED TOLERANCE TO ENVIRONMENTALLY INDUCED OXIDATIVE STRESSES IN TRANSGENIC TOMATO OVEREXPRESSING ASCORBATE PEROXIDASE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We studied the effect on oxidative stress resistance of overexpressing, in transgenic tomato (Lycopersicon esculentum) plants, a cytosolic ascorbate peroxidase (APX) gene derived from pea (Pisum sativum). Transformants were selected using Kanamycin resistance and confirmed by PCR, Southern and Nort...

  16. Degradation of disperse dye from textile effluent by free and immobilized Cucurbita pepo peroxidase

    NASA Astrophysics Data System (ADS)

    Boucherit, N.; Abouseoud, M.; Adour, L.

    2012-06-01

    Disperse dyes constitute the largest group of dyes used in local textile industry. This work evaluates the potential of the Cucurbita peroxidase(C-peroxidase) extracted from courgette in the decolourization of disperse dye in free and immobilized form. The optimal conditions for immobilization of C-peroxidase in Ca-alginate were identified. The immobilization was optimized at 2%(w/v) of sodium alginate and 0.2 M of calcium chloride. After optimization of treatment parameters, the results indicate that at pH 2, dye concentration: 80 mg/L(for FCP) and 180 mg/L(for ICP), H2O2 dose: 0,02M (for FCP) and 0,12M(for ICP), the decolourization by free and immobilized C-peroxidase were 72.02% and 69.71 % respectively. The degradation pathway and the metabolic products formed after the degradation were also predicted using UV-vis spectroscopy analysis.

  17. Amino Acid Sequence of Anionic Peroxidase from the Windmill Palm Tree Trachycarpus fortunei

    PubMed Central

    2015-01-01

    Palm peroxidases are extremely stable and have uncommon substrate specificity. This study was designed to fill in the knowledge gap about the structures of a peroxidase from the windmill palm tree Trachycarpus fortunei. The complete amino acid sequence and partial glycosylation were determined by MALDI-top-down sequencing of native windmill palm tree peroxidase (WPTP), MALDI-TOF/TOF MS/MS of WPTP tryptic peptides, and cDNA sequencing. The propeptide of WPTP contained N- and C-terminal signal sequences which contained 21 and 17 amino acid residues, respectively. Mature WPTP was 306 amino acids in length, and its carbohydrate content ranged from 21% to 29%. Comparison to closely related royal palm tree peroxidase revealed structural features that may explain differences in their substrate specificity. The results can be used to guide engineering of WPTP and its novel applications. PMID:25383699

  18. Influence of organophosphorus pesticides on peroxidase and chlorination activity of human myeloperoxidase.

    PubMed

    Lazarević-Pašti, Tamara; Momić, Tatjana; Radojević, Miloš M; Vasić, Vesna

    2013-09-01

    Inhibitory effects of five organophosphorus pesticides (diazinon, malathion, chlorpyrifos, azinphos-methyl and phorate) and their oxo-analogs on human myeloperoxidase (MPO) activity were investigated. While inspecting separately peroxidase and chlorination activity, it was observed that investigated OPs affect peroxidase activity, but not chlorination activity. Among investigated pesticides, malathion and malaoxon have showed the highest power to inhibit MPO peroxidase activity with IC50 values of the order of 3×10(-7) and 5×10(-9) M, respectively. It was proposed that inhibition trend is rendered by molecular structure which invokes steric hindrance for OPs interaction with MPO active center responsible for peroxidase activity. In addition, it was concluded that physiological function of MPO is not affected by any of the investigated OPs. PMID:25149236

  19. Manganese regulation of manganese peroxidase expression and lignin degradation by the white rot fungus Dichomitus squalens

    SciTech Connect

    Perie, F.; Gold, M.H. )

    1991-08-01

    Extracellular manganese peroxidase and laccase activities were detected in cultures of Dichomitus squalens (Polyporus anceps) under conditions favoring lignin degradation. In contrast, neither extracellular lignin peroxidase nor aryl alcohol oxidase activity was detected in cultures grown under a wide variety of conditions. The mineralization of {sup 14}C-ring-, -side chain-, and -methoxy-labeled synthetic guaiacyl lignins by D. squalens and the expression of extracellular manganese peroxidase were dependent on the presence of Mn(II), suggesting that manganese peroxidase is an important component of this organism's lignin degradation system. The expression of laccase activity was independent of manganese. In contrast to previous findings with Phanero-chaete chrysosporium, lignin degradation by D. squalens proceeded in the cultures containing excess carbon and nitrogen.

  20. Determination of estrogenic/antiestrogenic potential of antifertility substances using rat uterine peroxidase assay.

    PubMed

    Johri, R K; Pahwa, G S; Sharma, S C; Zutshi, U

    1991-11-01

    The effect of three compounds (clomiphene citrate, centchroman, embelin) and plant-derived methanolic extracts (Abutilon indicum and Butea monosperma) was studied on uterotropic and uterine peroxidase activities in ovariectomized rats. It was observed that these two parameters were highly correlated in response to treatment with these test materials and also to estradiol. It was suggested that the uterine peroxidase assay could be utilized as a biochemical parameter in the screening of new antifertility agents for their estrogenic/antiestrogenic properties. PMID:1665776