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Sample records for agglutinin-conjugated horseradish peroxidase

  1. 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...

  2. 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

  3. Roles of horseradish peroxidase in response to terbium stress.

    PubMed

    Zhang, Xuanbo; Wang, Lihong; Zhou, Qing

    2014-10-01

    The pollution of the environment by rare earth elements (REEs) causes deleterious effects on plants. Peroxidase plays important roles in plant response to various environmental stresses. Here, to further understand the overall roles of peroxidase in response to REE stress, the effects of the REE terbium ion (Tb(3+)) on the peroxidase activity and H2O2 and lignin contents in the leaves and roots of horseradish during different growth stages were simultaneously investigated. The results showed that after 24 and 48 h of Tb(3+) treatment, the peroxidase activity in horseradish leaves decreased, while the H2O2 and lignin contents increased. After a long-term (8 and 16 days) treatment with Tb(3+), these effects were also observed in the roots. The analysis of the changes in peroxidase activity and H2O2 and lignin contents revealed that peroxidase plays important roles in not only reactive oxygen species scavenging but also cell wall lignification in horseradish under Tb(3+) stress. These roles were closely related to the dose of Tb(3+), duration of stress, and growth stages of horseradish.

  4. 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

  5. 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%.

  6. 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

  7. Relative binding affinities of monolignols to horseradish peroxidase

    DOE PAGES

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

    2016-07-22

    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 andmore » 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. Furthermore, 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.« less

  8. 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

  9. 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

  10. 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.

  11. 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

  12. Horseradish peroxidase. Complex formation with anions and hydrocyanic acid.

    PubMed

    Araiso, T; Dunford, H B

    1981-10-10

    Equilibrium binding experiments have been performed with perchlorate, chloride, and acetate in the presence of horseradish peroxidase. The binding of perchlorate and acetate appears to be like that of nitrate, at a site other than the sixth coordination position of the heme iron. Competitive experiments using both nitrate and cyanide demonstrate that two different binding sites are present on the enzyme. Chloride appears to bind at the sixth coordination position as do both fluoride and cyanide. Temperature jump experiments indicate that it is likely the nitrate anion and not undissociated nitric acid which is the binding species. Competitive stopped flow experiments indicate that the bound nitrate slows both the association rate and dissociation rate of cyanide, indicating that nitrate binds close to the sixth coordination position.

  13. 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.

  14. 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

  15. Kinetic modelling of phenol co-oxidation using horseradish peroxidase.

    PubMed

    Carvalho, R H; Lemos, F; Lemos, M A N D A; Vojinović, V; Fonseca, L P; Cabral, J M S

    2006-07-01

    Phenol is an industrial pollutant and its removal from industrial wastewaters is of great importance. In order to design optimised phenol removal procedures by using horseradish peroxidase-based systems, there are some points that have to be dealt with. One of the most important issues is the need for reliable kinetics as this is one of the difficulties found during process scale-up. Although simplified kinetics can be used for limited ranges of operating conditions, they are not usually reliable for the description of varying process conditions. The present work describes the implementation of a kinetic model, based on a mechanism, for the co-oxidation of phenol and 4-aminoantipyrine (Am-NH2), which is used as a chromogen agent, with hydrogen peroxide as the oxidant. The model covers not only the variation of the concentrations of all the species involved, but also the effect of temperature in the reaction. The estimation of kinetic rate constants and activation energies for the various steps in the mechanism is performed with a single optimisation procedure, and all the experimental results are described using a unique set of parameters, which, thus, is valid over an extended range of operating conditions. The mechanism allowed the determination of a reliable kinetic model which is appropriate for the range of experimental conditions used. The computational model was also tested with an independent set of experiments with different conditions from the ones for which the parameters were estimated. PMID:16612606

  16. 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

  17. 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

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

    PubMed

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

    2014-09-01

    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

  19. An updated view on horseradish peroxidases: recombinant production and biotechnological applications.

    PubMed

    Krainer, Florian W; Glieder, Anton

    2015-02-01

    Horseradish peroxidase has been the subject of scientific research for centuries. It has been used exhaustively as reporter enzyme in diagnostics and histochemistry and still plays a major role in these applications. Numerous studies have been conducted on the role of horseradish peroxidase in the plant and its catalytic mechanism. However, little progress has been made in its recombinant production. Until now, commercial preparations of horseradish peroxidase are still isolated from plant roots. These preparations are commonly mixtures of various isoenzymes of which only a small fraction has been described so far. The composition of isoenzymes in these mixed isolates is subjected to uncontrollable environmental conditions. Nowadays, horseradish peroxidase regains interest due to its broad applicability in the fields of medicine, life sciences, and biotechnology in cancer therapy, biosensor systems, bioremediation, and biocatalysis. These medically and commercially relevant applications, the recent discovery of new natural isoenzymes with different biochemical properties, as well as the challenges in recombinant production render this enzyme particularly interesting for future biotechnological solutions. Therefore, we reviewed previous studies as well as current developments with biotechnological emphasis on new applications and the major remaining biotechnological challenge-the efficient recombinant production of horseradish peroxidase enzymes.

  20. Subcellular location of horseradish peroxidase in horseradish leaves treated with La(III), Ce(III) and Tb(III).

    PubMed

    Ye, Yaxin; Wang, Lihong; Huang, Xiaohua; Lu, Tianhong; Ding, Xiaolan; Zhou, Qing; Guo, Shaofen

    2008-11-01

    The agricultural application of rare-earth elements (REEs) would promote REEs inevitably to enter in the environment and then to threaten the environmental safety and human health. Therefore, the distribution of the REEs ion, (141)Ce(III) and effects of La(III), Ce(III) and Tb(III) on the distribution of horseradish peroxidase (HRP) in horseradish mesophyll cells were investigated with electron microscopic radioautography and transmission electron microscopic cytochemistry. It was found for the first time that REEs ions can enter into the mesophyll cells, deposit in both extra and intra-cellular. Compared to the normal condition, after the horseradish leaves treated with La(III) or Tb(III), HRP located on the tonoplast is decreased and HRP is mainly located on the cell wall, while HRP is mainly located on the plasma membrane after the horseradish leaves were treated with Ce(III). This also indicated that REEs ions may regulate the plant growth through changing the distribution of enzymes.

  1. Signal amplification of streptavidin-horseradish peroxidase functionalized carbon nanotubes for amperometric detection of attomolar DNA.

    PubMed

    Gao, Wenchao; Dong, Haifeng; Lei, Jianping; Ji, Hanxu; Ju, Huangxian

    2011-05-14

    A novel biosensing strategy for selective electrochemical detection of DNA down to the attomolar level with a linear range of 5 orders of magnitude was developed by the specific recognitions of target DNA and streptavidin to biotin labelled molecular beacon and signal amplification of streptavidin-horseradish peroxidase functionalized carbon nanotubes.

  2. A comparison of horseradish peroxidase and manganese ions as catalysts for the oxidation of dihydroxyfumaric acid

    PubMed Central

    Hartree, E. F.

    1968-01-01

    With horseradish peroxidase as catalyst the main product was dihydroxytartrate, but small amounts of glycolaldehyde, mesoxalic semialdehyde, mesoxalate and possibly glyoxal were also formed. Mn2+ catalysis gave rise only to mesoxalate and oxalate. When oxygen uptake was followed by a manometric method the rate of the peroxidase-catalysed reaction was proportional to oxygen concentration and marked inhibition by cyanide was obtained only at low buffer concentration. The catalytic effects of peroxidase and Mn2+ were almost always additive. Chelating agents inhibited the Mn2+-catalysed reaction, but had either no effect or a slight accelerating effect on the peroxidase-catalysed reaction. It is concluded that Mn2+ does not function as cofactor in the peroxidase-catalysed oxidation. PMID:5660638

  3. Horseradish Peroxidase-Mediated, Iodide-Catalyzed Cascade Reaction for Plasmonic Immunoassays.

    PubMed

    Xianyu, Yunlei; Chen, Yiping; Jiang, Xingyu

    2015-11-01

    This report outlines an enzymatic cascade reaction for signal transduction and amplification for plasmonic immunoassays by using horseradish peroxidase (HRP)-mediated aggregation of gold nanoparticles (AuNPs). HRP-catalyzed oxidation of iodide and iodide-catalyzed oxidation of cysteine is employed to modulate the plasmonic signals of AuNPs. It agrees well with the current immunoassay platforms and allows naked-eye readout with enhanced sensitivity, which holds great promise for applications in resource-constrained settings. PMID:26460152

  4. Dynamic disorder in horseradish peroxidase observed with total internal reflection fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Hassler, Kai; Rigler, Per; Blom, Hans; Rigler, Rudolf; Widengren, Jerker; Lasser, Theo

    2007-04-01

    This paper discusses the application of objective-type total internal reflection fluorescence correlation spectroscopy (TIR-FCS) to the study of the kinetics of immobilized horseradish peroxidase on a single molecule level. Objective-type TIR-FCS combines the advantages of FCS with TIRF microscopy in a way that allows for simultaneous ultra-sensitive spectroscopic measurements using a single-point detector and convenient localization of single molecules on a surface by means of parallel imaging.

  5. 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

  6. Horseradish Peroxidase-Mediated, Iodide-Catalyzed Cascade Reaction for Plasmonic Immunoassays.

    PubMed

    Xianyu, Yunlei; Chen, Yiping; Jiang, Xingyu

    2015-11-01

    This report outlines an enzymatic cascade reaction for signal transduction and amplification for plasmonic immunoassays by using horseradish peroxidase (HRP)-mediated aggregation of gold nanoparticles (AuNPs). HRP-catalyzed oxidation of iodide and iodide-catalyzed oxidation of cysteine is employed to modulate the plasmonic signals of AuNPs. It agrees well with the current immunoassay platforms and allows naked-eye readout with enhanced sensitivity, which holds great promise for applications in resource-constrained settings.

  7. 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).

  8. Effect of linking allyl and aromatic chains to histidine 170 in horseradish peroxidase.

    PubMed

    Urrutigoïty, M; Baboulène, M; Lattes, A; Souppe, J; Seris, J L

    1991-08-30

    Histidine residues in horseradish peroxidase (HRP) were modified chemically with diethyl pyrocarbonate, 4,omega-dibromoacetophenone or diallylpyrocarbonate. Histidines were chosen as His-170, the fifth ligand of the heme iron atom, forms part of the active site of this enzyme. Good yields of hemoprotein were obtained in all cases. Analysis by HPLC of peptides obtained after tryptic digestion showed that His-170 of HRP was in fact modified. The specific activity remained satisfactory after chemical modification of the histidine residues, and so the active site of HRP can thus be altered without a dramatic loss of hemoprotein or peroxidase activity. This may open routes to the preparation of novel biocatalysts.

  9. Oxidation of porphyrinogens by horseradish peroxidase and formation of a green pyrrole pigment.

    PubMed

    Jacobs, J M; Jacobs, N J; Kuhn, C B; Gorman, N; Dayan, F E; Duke, S O; Sinclair, J F; Sinclair, P R

    1996-10-01

    When humans or plants are exposed to certain chemicals which interfere with heme biosynthetic enzymes, porphyrinogen intermediates accumulate and are oxidized to cytotoxic porphyrins. Here we have investigated the role of peroxidases in porphyrinogen oxidation. Horseradish peroxidase (HRP) rapidly oxidizes uroporphyrinogen to uroporphyrin and this is inhibited by ascorbic acid. HRP also oxidizes deuteroporphyrinogen (a synthetic porphyrin similar to protoporphyrinogen), but the yield of porphyrin is lower than with uroporphyrinogen as substrate. This low yield is in part due to a rapid, HRP-dependent conversion of deuteroporphyrin (but not uroporphyrin) to a green compound with spectral characteristics of a chlorin with a large peak at 638 nm. This reaction requires addition of a sulfhydryl reductant such as glutathione and is inhibited by ascorbic acid. These findings suggest that cellular peroxidases and ascorbic acid levels may play a role in modifying the phototoxic tetrapyrroles which accumulate in plants and humans after certain environmental exposures.

  10. 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.

  11. 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.

  12. 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

  13. Temperature dependence of oxygen evolution through catalase-like activity of horseradish peroxidase

    NASA Astrophysics Data System (ADS)

    Popović-Bijelić, A.; Bijelić, G.; Kolar-Anić, Lj.; Vukojević, V.

    2007-09-01

    By experimental investigations of the temperature dependence of catalase-like activity of horseradish peroxidase in the temperature range 278 328 K, different kinetic profiles for oxygen evolution were found below and above 298 K. Extension of the model is proposed to account for these observations. By numeric simulations of the reaction kinetics at different temperatures, it was found that enhanced evaporation of molecular oxygen from the reaction solution is the main root through which oxygen is lost at elevated temperatures in laboratory conditions.

  14. 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

  15. HOW A SINGLE-POINT MUTATION IN HORSERADISH PEROXIDASE MARKEDLY ENHANCES ENANTIOSELECTIVITY

    PubMed Central

    Antipov, Eugene; Cho, Art E.; Klibanov, Alexander M.

    2009-01-01

    The effect of all possible mutations at position 178 on the enantioselectivity of yeast surface-bound horseradish peroxidase (HRP) toward chiral phenols has been investigated. In contrast to their wild-type predecessor, most HRP mutants are enantioselective, with the Arg178Glu variant exhibiting the greatest, 25-fold (S)/(R) preference. Using kinetic analysis of enzymatic oxidation of various substrate analogs and molecular modeling of enzyme-substrate complexes, this enantioselectivity enhancement is attributed to changes in the transition state energy due to electrostatic repulsion between the carboxylates of the enzyme's Glu178 and the substrate's (R)-enantiomer. PMID:19610634

  16. 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.

  17. Photophysics and Photochemistry of Horseradish Peroxidase A2 upon Ultraviolet Illumination

    PubMed Central

    Neves-Petersen, Maria Teresa; Klitgaard, Søren; Carvalho, Ana Sofia Leitão; Petersen, Steffen B.; Aires de Barros, Maria Raquel; e Melo, Eduardo Pinho

    2007-01-01

    Detailed analysis of the effects of ultraviolet (UV) and blue light illumination of horseradish peroxidase A2, a heme-containing enzyme that reduces H2O2 to oxidize organic and inorganic compounds, is presented. The effects of increasing illumination time on the protein's enzymatic activity, Reinheitzahl value, fluorescence emission, fluorescence lifetime distribution, fluorescence mean lifetime, and heme absorption are reported. UV illumination leads to an exponential decay of the enzyme activity followed by changes in heme group absorption. Longer UV illumination time leads to lower Tm values as well as helical content loss. Prolonged UV illumination and heme irradiation at 403 nm has a pronounced effect on the fluorescence quantum yield correlated with changes in the prosthetic group pocket, leading to a pronounced decrease in the heme's Soret absorbance band. Analysis of the picosecond-resolved fluorescence emission of horseradish peroxidase A2 with streak camera shows that UV illumination induces an exponential change in the preexponential factors distribution associated to the protein's fluorescence lifetimes, leading to an exponential increase of the mean fluorescence lifetime. Illumination of aromatic residues and of the heme group leads to changes indicative of heme leaving the molecule and/or that photoinduced chemical changes occur in the heme moiety. Our studies bring new insight into light-induced reactions in proteins. We show how streak camera technology can be of outstanding value to follow such ultrafast processes and how streak camera data can be correlated with protein structural changes. PMID:17189303

  18. Thyroid microsomal/thyroid peroxidase autoantibodies show discrete patterns of cross-reactivity to myeloperoxidase, lactoperoxidase and horseradish peroxidase.

    PubMed Central

    Banga, J P; Tomlinson, R W; Doble, N; Odell, E; McGregor, A M

    1989-01-01

    The recent cloning of the thyroid peroxidase (TPO) has shown that it is identical to the thyroid microsomal antigen (TMA), a potent antigen involved in autoimmune thyroid disease (ATD), which shares significant sequence homology with myeloperoxidase. The present study shows that autoantibodies (aAb) to the TMA/TPO antigen cross-react with human leucocyte myeloperoxidase, bovine lactoperoxidase and horseradish peroxidase. Cross-reactivity to myeloperoxidase was only apparent by ELISA using reduced and alkylated antigen preparations or by immunoblotting following denaturation with SDS. Sequential absorption of sera on SDS-denatured thyroid microsomes immobilized on Sepharose-4B followed by absorption on native microsomes removed all aAb specificities to TMA/TPO and the three peroxidase preparations, giving compelling evidence on the genuine cross-reactive nature of these aAbs. Sera from different patients contain different qualitative and quantitative specificities of aAb to the TMA/TPO antigen, confirming the polyclonal nature of this autoimmune response. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:2546881

  19. Direct interaction between terbium ion and peroxidase in horseradish at different pH values.

    PubMed

    Wang, Lihong; Zhou, Qing; Huang, Xiaohua

    2014-02-01

    Rare earth elements (REEs) entering plant cells can directly interact with peroxidase in plants, which is the structural basis for the decrease in the activity of peroxidase. Different cellular compartments have different pH values. However, little information is available regarding the direct interaction between REEs and peroxidase in plants at different pH values. Here, we investigated the charge distribution on the surface of horseradish peroxidase (HRP) molecule as well as the interaction of terbium ion (Tb(3+), one type of REEs) and HRP at different pH values. Using the molecular dynamics simulation, we found that when the pH value was from 4.0 to 8.0, a large amount of negative charges were intensively distributed on the surface of HRP molecule, and thus, we speculated that Tb(3+) with positive charges might directly interact with HRP at pH 4.0-8.0. Subsequently, using ultraviolet-visible spectroscopy, we demonstrated that Tb(3+) could directly interact with HRP in the simulated physiological solution at pH 7.0 and did not interact with HRP in other solutions at pH 5.0, pH 6.0 and pH 8.0. In conclusion, we showed that the direct interaction between Tb(3+) and HRP molecule depended on the pH value of cellular compartments.

  20. The mechanism of spontaneous heme release from horseradish peroxidase isoenzyme A2.

    PubMed

    Smith, M L; Hjortsberg, K; Ohlsson, P I; Paul, K G

    1983-01-01

    We have investigated the spontaneous release of heme from chromatographically homogeneous horseradish peroxidase A2 at varying temperature, pH and iron ligands. A "biphasic" rate of heme release was observed under all conditions. Upon further purification of HRP A2, a component was isolated that was homogeneous by polyacrylamide gel electrophoresis and exhibited a single first order rate of heme release. The rate of the release increased with pH and temperature, decreased in the presence of cyanide and increased slightly in the presence of fluoride. These results are consistent with the idea that the rate of heme release is a measure of the flexibility of the protein lining the heme "pocket" with iron bonding playing a secondary, though important role in heme-protein interactions.

  1. 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.

  2. 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.

  3. Increased ELISA sensitivity using a modified method for conjugating horseradish peroxidase to monoclonal antibodies.

    PubMed

    Madersbacher, S; Wolf, H; Gerth, R; Berger, P

    1992-07-31

    We investigated the importance of monoclonal antibody (MCA) purity and the input molar ratio of horseradish peroxidase (HRPO)/IgG used for MCA conjugation on various immunoenzymometric assay (IEMA) parameters. The sensitivity of IEMAs for human follicle stimulating hormone (hFSH), human chorionic gonadotropin (hCG) and the free alpha subunit of hCG (hCG alpha) could be increased up to 6-fold, whereas non-specific binding remained within tolerable limits (E less than 0.1), when MCAs purified by high performance liquid chromatography (HPLC) using a hydroxylapatite column (HPHT) were conjugated with an input molar HRPO/IgG ratio of four instead of the usual ratio of two.

  4. Synthesis and characterization of starch-poly(methyl acrylate) graft copolymers using horseradish peroxidase.

    PubMed

    Wang, Su; Wang, Qiang; Fan, Xuerong; Xu, Jin; Zhang, Ying; Yuan, Jiugang; Jin, Heling; Cavaco-Paulo, Artur

    2016-01-20

    Horseradish peroxidase (HRP)-mediated graft polymerization in the presence of hydrogen peroxide (H2O2) and acetylacetone (Acac) has been successfully applied to the synthesis of starch-poly(methyl acrylate) (PMA). The graft copolymer was characterized by Fourier transform infrared (FT-IR), elemental analysis, nuclear magnetic resonance ((1)H NMR and (13)C NMR), and differential scanning calorimetry (DSC). FT-IR, elemental analysis and NMR confirmed that methyl acrylate (MA) was grafted onto starch successfully. DSC results showed the graft reaction had changed the crystalline regions of the gelatinized starch. The effects of pH, MA content, HRP dosage, incubation temperature and time on grafting percentage (GP) and grafting efficiency (GE) were also investigated. The GP and GE under optimal conditions reached 30.21% and 45.13%, respectively.

  5. 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

  6. Distribution of primary afferent fibres in the cochlear nuclei. A silver and horseradish peroxidase (HRP) study.

    PubMed Central

    Merchan, M A; Collia, F P; Merchan, J A; Saldana, E

    1985-01-01

    Horseradish peroxidase, when injected intracochlearly, is transported transganglionically to the brain stem cochlear nuclei, thus providing an excellent method for tracing the central projection of the spiral ganglion neurons. Silver impregnation using the Cajal-de Castro method, which stains axons even when inside the bone, was used as a reference technique. The combination of both procedures led to the following conclusions. Primary cochlear afferents are found only in the ventral zone of the dorsal cochlear nucleus. In this area they cover the deep and fusiform cell layers. The molecular layer shows no HRP label. The higher concentration of primary cochlear afferents in the ventral cochlear nucleus appears in its central zone; wide areas in this nucleus are not labelled at all. A thin bundle of primary cochlear afferents runs parallel to, and beneath, the granular region. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:4077711

  7. 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

  8. 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

  9. 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

  10. 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.

  11. A comparison between the oxidation with laccase and horseradish peroxidase for triclosan conversion.

    PubMed

    Melo, C F; Dezotti, M; Marques, M R C

    2016-01-01

    Triclosan is a broad-spectrum biocide used in personal-care products that is suspected to be linked to the emergence of antibiotic-resistant bacteria. In the present work, the enzymes horseradish peroxidase and laccase from Trametes versicolor were evaluated for the conversion of triclosan in an aqueous matrix. The removal of antibacterial activity by the enzymatic processes was evaluated by an assay based on the growth inhibition of Escherichia coli K12. The horseradish peroxidase (HRP) process appears more advantageous than the laccase process in removing triclosan from an aqueous matrix, considering the reaction parameters pH, temperature, catalytic efficiency, and enzyme concentration. The highest conversion of triclosan catalysed by laccase was observed at pH 5.0, that is, lower than the typical pH range (6.5-7.5) of sewage treatment plants' effluents. The efficiency of laccase process was much more impacted by variations in the temperature in the range of 10-40°C. Kinetic studies showed that triclosan is a substrate more specific for HRP than for laccase. The protein content for the HRP-catalysed process was 14 times lower than that for the laccase process. Decay kinetics suggest that reaction mechanisms depend on enzyme concentration and its concentration. Both processes were able to reduce the antibacterial activity, and the residual activity of the treated solution is probably due to non-converted triclosan and not due to the reaction products. The laccase-catalysed conversion of triclosan in an environmental relevant concentration required a higher amount of enzyme than that required in the HRP process.

  12. 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

  13. 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.

  14. 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.

  15. Functional expression of horseradish peroxidase in E. coli by directed evolution.

    PubMed

    Lin, Z; Thorsen, T; Arnold, F H

    1999-01-01

    In an effort to develop a bacterial expression system for horseradish peroxidase (HRP), we inserted the gene encoding HRP into the pET-22b(+) vector (Novagen) as a fusion to the signal peptide PelB. A similar construct for cytochrome c peroxidase (CcP) leads to high CcP activity in the supernatant. Expression of the wild-type HRP gene in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG) yielded no detectable activity against ABTS (azinobis(ethylbenzthiazoline sulfonate)). However, weak peroxidase activity was detected in the supernatant in the absence of IPTG. The HRP gene was subjected to directed evolution: random mutagenesis and gene recombination followed by screening in a 96-well microplate format. From 12 000 clones screened in the first generation, one was found that showed 14-fold higher HRP activity than wild-type, amounting to approximately 110 microg of HRP/L, which is similar to that reported from laborious in vitro refolding. No further improvement was obtained in subsequent generations of directed evolution. This level of expression has nonetheless enabled us to carry out further directed evolution to render the enzyme more thermostable and more resistant toward inactivation by H2O2. These results show that directed evolution can identify mutations that assist proteins to fold more efficiently in Escherichia coli. This approach will greatly facilitate efforts to "fine-tune" those many enzymes that are promising industrial biocatalysts, but for which suitable bacterial or yeast expression systems are currently lacking. PMID:10356264

  16. Effect of organic solvents on peroxidases from rice and horseradish: prospects for enzyme based applications.

    PubMed

    Singh, Priyanka; Prakash, Rajiv; Shah, Kavita

    2012-08-15

    A feasibility test for rice peroxidase (RP) enzyme as a substitute for horseradish peroxidase (HRP) was carried out. The activity of HRP was maximum at 30 °C with pH 6.0-7.0. The purified rice peroxidase showed optimum activity at 30 °C with pH 7-8 and was thermostable till 68 °C, which is higher than the temperature reported for HRP. RP obeyed Michaelis-Menten kinetics. With increasing substrate concentrations, RP and HRP had V(max) as 8.23 μM min(-1) and 4.21 μM min(-1) and K(m) as 5.585 and 3.662 mM, respectively. In 10% 1,4-dioxane and ethanol, RP exhibited 2 and 1.3 times higher activity, respectively than HRP. Shelf life studies show RP to be significantly stable till 60 h in 20% 1,4-dioxane and till 12 h in ethanol. The activity of RP/HRP increased gradually with 0%-40% ethanol or 0%-30% 1,4-dioxane till 20 h with a sharp decline thereafter. The stability of HRP and RP reduced with increasing storage period. Enzyme efficiencies compared as V(m)/K(m) showed water miscible organic solvents, viz.1,4-dioxane and ethanol, to exhibit a regular decrease in V(m)/K(m) with increase in organic solvent concentration whereas, a reverse trend was observed with water-immiscible solvent like chloroform. The relative activity of RP and HRP enzymes upon immobilization on poly-5-carboxy-indole shows increasing enzyme activity with time and with guaiacol/dopamine hydrochloride as substrates. Immobilized RP had a better relative activity with dopamine as substrate than immobilized HRP, whereas with guaiacol both RP and HRP had a comparable activity upon immobilization. Results suggest rice peroxidase to be a cheaper and convenient enzyme system for immobilization using organic solvents. The high thermal stability, more stability in organic solvents and longer shelf life of RP over the immobilizing matrix suggest conducting polyindole having carboxyl functional groups to be a suitable matrix for the covalent entrapment of rice peroxidase through amide linkage. Good

  17. 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

  18. Inactivation of Coprinus cinereus peroxidase by 4-chloroaniline during turnover: comparison with horseradish peroxidase and bovine lactoperoxidase.

    PubMed

    Chang, H C; Holland, R D; Bumpus, J A; Churchwell, M I; Doerge, D R

    1999-12-15

    The peroxidase from Coprinus cinereus (CPX) catalyzed oxidative oligomerization of 4-chloroaniline (4-CA) forming several products: N-(4-chlorophenyl)-benzoquinone monoamine (dimer D), 4,4'-dichloroazobenzene (dimer E); 2-(4-chloroanilino)-N-(4-chlorophenyl)-benzoquinone (trimer F); 2-amino-5-chlorobenzoquinone-di-4-chloroanil (trimer G); 2-(4-chloroanilino)-5-hydroxybenzoquinone-di-4-chloroanil (tetramer H) and 2-amino-5-(-4-chlroanilino)-benzoquinone-di-4-chloroanil (tetramer 1). In the presence of 4-CA and H2O2, CPX was irreversibly inactivated within 10 min. Inactivation of CPX in the presence of H2O2 was a time-dependent, first-order process when the concentration of 4-CA was varied between 0 and 2.5 mM. The apparent dissociation constant (Ki) for CPX and 4-CA was 0.71 mM. The pseudo-first order rate constant for inactivation (k(inact)), was 1.15 x 10(-2) s(-1). Covalent incorporation of 20 mole 14C-4-CA per mole of inactivated CPX was observed. The partition ratio was about 2200 when either 4-CA or H2O2 was used as the limiting substrate. These results show that 4-CA is a metabolically activated inactivator (i.e. a suicide substrate). Unmodified heme and hydroxymethyl heme were isolated from native, 4-CA-inactivated and H2O2-incubated CPX. Inactivation resulted in significant losses in both heme contents. Analysis of tryptic peptides from 4-CA-inactivated CPX by MALDI-TOF/ MS and UV-VIS spectrophotometry suggested that trimer G and tetramer H were the major 4-CA derivatives that were covalently bound, including to a peptide (MGDAGF-SPDEVVDLLAAHSLASQEGLNSAIFR) containing the heme binding site. These studies show that heme destruction and covalent modification of the polypeptide chain are both important for the inactivation of CPX. These results were compared with similar studies on 4-CA-inactivated horseradish peroxidase (HRP) and bovine lactoperoxidase (LPO) during the oxidation of 4-CA.

  19. Horseradish peroxidase-catalysed in situ-forming hydrogels for tissue-engineering applications.

    PubMed

    Bae, Jin Woo; Choi, Jong Hoon; Lee, Yunki; Park, Ki Dong

    2015-11-01

    In situ-forming hydrogels are an attractive class of implantable biomaterials that are used for biomedical applications. These injectable hydrogels are versatile and provide a convenient platform for delivering cells and drugs via minimally invasive surgery. Although several crosslinking methods for preparing in situ forming hydrogels have been developed over the past two decades, most hydrogels are not sufficiently versatile for use in a wide variety of tissue-engineering applications. In recent years, enzyme-catalysed crosslinking approaches have been emerged as a new approach for developing in situ-forming hydrogels. In particular, the horseradish peroxidase (HRP)-catalysed crosslinking approach has received increasing interest, due to its highly improved and tunable capacity to obtain hydrogels with desirable properties. The HRP-catalysed crosslinking reaction immediately occurs upon mixing phenol-rich polymers with HRP and hydrogen peroxide (H2O2) in aqueous media. Based on this unique gel-forming feature, recent studies have shown that various properties of formed hydrogels, such as gelation time, stiffness and degradation rate, can be easily manipulated by varying the concentrations of HRP and H2O2. In this review, we outline the versatile properties of HRP-catalysed in situ-forming hydrogels, with a brief introduction to the crosslinking mechanisms involved. In addition, the recent biomedical applications of HRP-catalysed in situ-forming hydrogels for tissue regeneration are described. PMID:24916126

  20. Formation of a porphyrin pi-cation radical in the fluoride complex of horseradish peroxidase.

    PubMed

    Farhangrazi, Z S; Sinclair, R; Powers, L; Yamazaki, I

    1995-11-21

    Horseradish peroxidase (HRP) was oxidized by IrCl6(2-) to a mixture of compounds I and II, the rate of oxidation and the ratio of the mixture being greatly affected by pH (Hayashi & Yamazaki, 1979). Oxidation of HRP by IrCl6(2-) in the presence of fluoride was significantly accelerated. This resulted in the formation of a new compound which is a ferric fluoride complex containing a porphyrin pi-cation radical. The spectrum of the new compound showed a decreased absorption band in the Soret region and a broad band at 570 nm; which was converted to that of the original ferric fluoride complex by addition of ascorbate or hydroquinone. Addition of cyanide slowed down the oxidation of HRP by IrCl6(2-), and the oxidation product was the same as that obtained in the absence of cyanide. Compound I was formed when H2O2 was added to HRP in the presence of fluoride or cyanide. The one-electron reduction potential (Eo') of the oxidized HRP-fluoride complex was measured at several pH values, the Eo' value at pH 7 being 861 +/- 4 mV. The ratio of delta Eo' to delta pH was 49 mV/pH unit.

  1. 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

  2. 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

  3. 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.

  4. Enzymatic decolorization of anthraquinone and diazo dyes using horseradish peroxidase enzyme immobilized onto various polysulfone supports.

    PubMed

    Celebi, Mithat; Kaya, Mehmet Arif; Altikatoglu, Melda; Yildirim, Huseyin

    2013-10-01

    In this study, covalent immobilization of the horseradish peroxidase (HRP) onto various polysulfone supports was investigated. For this purpose, different polysulfones were methacrylated with methacryloyl chloride, and then, nonwoven fabric samples were coated by using solutions of these methacrylated polysulfones. Finally, support materials were immersed into aquatic solution of HRP enzyme for covalent immobilization. Structural analysis of enzyme immobilization onto various polysulfones was confirmed with Fourier transform infrared spectroscopy, atomic force microscopy, and proton nuclear magnetic resonance spectroscopy. Decolorization of textile diazo (Acid Black 1) and anthraquinone (Reactive Blue 19) dyes was investigated by UV-visible spectrophotometer. Covalently immobilized enzyme has been used seven times in freshly prepared dye solutions through 63 days. Dye decolorization performance of the immobilized systems was observed that still remained high (70%) after reusing three times. Enzyme activities of immobilized systems were determined and compared to free enzyme activity at different conditions (pH, temperature, thermal stability, storage stability). Enzyme activities of immobilized systems and free enzyme were also investigated at the different temperatures and effects of temperature and thermal resistance for different incubation time at 50 °C. In addition, storage activity of free and immobilized enzymes was determined at 4 °C at different incubation days.

  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. Commercial horseradish peroxidase degrades myelin encephalitogenic protein during coupling for immunohistochemical studies.

    PubMed

    Johnson, A B; Cammer, W

    1977-05-01

    Conjugates of myelin encephalitogenic basic protein (EP) and commercial horseradish peroxidase (HRP) have been used for immunohistochemical demonstrations of anti-EP antibody in animals with experimental allergic encephalomyelitis. We performed gel electrophoresis studies on EP-HRP conjugates prepared with glutaraldehyde and on mixtures of EP and HRP incubated without glutaraldehyde. The results show that under conditions of one-and two-step coupling HRP causes rapid loss of the native EP band, apparently due to EP degradation. The EP-HRP mixtures are not encephalitogenic in rabbits, or encephalitogenic activity is lost during processing. The immunohistochemical reactivity of conjugates, however, signals some preservation of antibody-combining sites. The mechanism of the HRP effect on EP is unknown. The possibilities of a contaminating proteinase or direct peroxidatic attack are suggested. Until this action of HRP can be overcome, the effect of coupling procedures on the biological activities of EP will be difficult to assess, and EP-HRP conjugates cannot be expected to reveal sites that may bind encephalitogenic portions of the EP molecule.

  7. 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

  8. Refolding of horseradish peroxidase is enhanced in presence of metal cofactors and ionic liquids.

    PubMed

    Bae, Sang-Woo; Eom, Doyoung; Mai, Ngoc Lan; Koo, Yoon-Mo

    2016-03-01

    The effects of various refolding additives, including metal cofactors, organic co-solvents, and ionic liquids, on the refolding of horseradish peroxidase (HRP), a well-known hemoprotein containing four disulfide bonds and two different types of metal centers, a ferrous ion-containing heme group and two calcium atoms, which provide a stabilizing effect on protein structure and function, were investigated. Both metal cofactors (Ca(2+) and hemin) and ionic liquids have positive impact on the refolding of HRP. For instance, the HRP refolding yield remarkably increased by over 3-fold upon addition of hemin and calcium chloride to the refolding buffer as compared to that in the conventional urea-containing refolding buffer. Moreover, the addition of ionic liquids [EMIM][Cl] to the hemin and calcium cofactor-containing refolding buffer further enhanced the HRP refolding yield up to 80% as compared to 12% in conventional refolding buffer at relatively high initial protein concentration (5 mg/ml). These results indicated that refolding method utilizing metal cofactors and ionic liquids could enhance the yield and efficiency for metalloprotein.

  9. Immobilization of horseradish peroxidase on nonwoven polyester fabric coated with chitosan.

    PubMed

    Mohamed, Saleh A; Aly, A S; Mohamed, Tarek M; Salah, Hala A

    2008-02-01

    The immobilization of horseradish peroxidase (HRP) on composite membrane has been investigated. This membrane was prepared by coating nonwoven polyester fabric with chitosan glutamate in the presence of glutraldehyde as a crosslinking agent. The physicochemical properties of soluble and immobilized HRP were evaluated. The soluble HRP lost 90% of its activity after 4 weeks of storage at 4 degrees C, whereas the immobilized enzyme retained 85% of its original activity at the same time. A reusability study of immobilized HRP showed that the enzyme retained 54% of its activity after 10 cycles of reuse. Soluble and immobilized HRP showed the same pH optima at pH 5.5. The immobilized enzyme had significant stability at different pH values, where it had maximum stability at pH 3.0 and 6.0. The kinetic properties indicated that the immobilized enzyme had more affinity toward substrates than soluble enzyme. The soluble and immobilized enzymes had temperature optima at 30 and 40 degrees C and were stable up to 40 and 50 degrees C, respectively. The stability of HRP against metal ion inactivation was improved after immobilization. Immobilized HRP exhibited high resistance to proteolysis by trypsin. The immobilized HRP was more resistant to inactivation induced by urea, Triton X-100, and organic solvents compared to its soluble counterpart. The immobilized HRP showed very high yield of immobilization and markedly high stabilization against several forms of denaturants that offer potential for several applications. PMID:18456948

  10. A novel and efficient polymerization of lignosulfonates by horseradish peroxidase/H(2)O(2) incubation.

    PubMed

    Zhou, Haifeng; Yang, Dongjie; Qiu, Xueqing; Wu, Xiaolei; Li, Yuan

    2013-12-01

    Lignosulfonates(LSs), by-products from chemical pulping processes, are low-value products with limited dispersion properties. The ability of commercially available horseradish peroxidase (HRP) to polymerize LS macromolecules and improve the dispersion properties of LSs was investigated. The polymerization of LSs proceeded efficiently under mild reaction conditions in an aqueous solution with HRP/H2O2. Gel permeation chromatography showed a significant increase in weight-average molecular weight (M w ) of sulfonated kraft lignin and sodium lignosulfonate (NaLS) by 8.5-fold and 4.7-fold, respectively. The mechanism of polymerization was investigated by elemental analysis, surface charge measurement, headspace gas chromatography, infrared spectroscopy (IR), and hydrogen nuclear magnetic resonance spectrometry ((1)H-NMR). The functional group measurements indicated that HRP incubation did not reduce the sulfonic group content. However, it decreased the phenolic and methoxyl group contents. As the phenolic group content decreased, M w increased as a power function. The polymerization was proposed to involve the random coupling of phenoxy radical intermediates. The radicals coupled with each other to form different inter-unit linkages, most of which were the β-O-4' type, as the (1)H-NMR spectra indicated. Moreover, the HRP/H2O2 incubation induced a significant improvement in the adsorption and dispersion properties of LSs. Therefore, the HRP/H2O2 incubation is a promising approach for industrial applications of LSs. PMID:24196582

  11. Phosphate buffer effects on thermal stability and H2O2-resistance of horseradish peroxidase.

    PubMed

    Asad, Sedigheh; Torabi, Seyed-Fakhreddin; Fathi-Roudsari, Mehrnoosh; Ghaemi, Nasser; Khajeh, Khosro

    2011-05-01

    Horseradish peroxidase (HRP) has attracted intense research interest due to its potential applications in biotechnological fields. However, inadequate stability under prevalent conditions such as elevated temperatures and H(2)O(2) exposure, has limited its industrial application. In this study, stability of HRP was investigated in the presence of different buffer systems (potassium phosphate and Tris-HCl) and additives. It was shown that the concentration of phosphate buffer severely affects enzyme thermostability in a way that in diluted potassium phosphate buffer (10mM) half-life (from 13 to 35 min at 80 °C) and T(m) (from 73 to 77.5 °C) increased significantly. Among additives tested, trehalose had the most thermostabilizing effect. Exploring the role of glycosylation in stabilizing effect of phosphate buffer, non-glycosylated recombinant HRP was also examined for its thermal and H(2)O(2) stability in both diluted and concentrated phosphate buffers. The recombinant enzyme was more thermally stable in diluted buffer in accordance to glycosylated HRP; but interestingly recombinant HRP showed higher H(2)O(2) tolerance in concentrated buffer.

  12. 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.

  13. Electrochemically deposited chitosan hydrogel for horseradish peroxidase immobilization through gold nanoparticles self-assembly.

    PubMed

    Luo, Xi-Liang; Xu, Jing-Juan; Zhang, Qing; Yang, Gong-Jun; Chen, Hong-Yuan

    2005-07-15

    A new strategy for immobilization of horseradish peroxidase (HRP) has been presented by self-assembling gold nanoparticles on chitosan hydrogel modified Au electrode. From a mildly acidic chitosan solution, a chitosan film is electrochemically deposited on Au electrode surface via a negative voltage bias. This process is accompanied by the hydrogen evolution reaction, and the released hydrogen gas made the deposited chitosan film with porous structure, which facilitates the assembly of gold nanoparticles and HRP. The resulting substrates were characterized by atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). The immobilized HRP displayed an excellent catalytic property to the reduction of H2O2 in the presence of methylene blue mediator. The resulting biosensor (HRP-modified electrode) showed a wide dynamic range of 8.0 microM-15 mM H2O2, and the linear ranges were 8.0 microM-0.12 mM and 0.50-12 mM, with a detection limit of 2.4 microM estimated at a signal-to-noise ratio of 3. Moreover, the biosensor remained about 85% of its original sensitivity after four weeks' storage.

  14. Horseradish peroxidase-catalysed in situ-forming hydrogels for tissue-engineering applications.

    PubMed

    Bae, Jin Woo; Choi, Jong Hoon; Lee, Yunki; Park, Ki Dong

    2015-11-01

    In situ-forming hydrogels are an attractive class of implantable biomaterials that are used for biomedical applications. These injectable hydrogels are versatile and provide a convenient platform for delivering cells and drugs via minimally invasive surgery. Although several crosslinking methods for preparing in situ forming hydrogels have been developed over the past two decades, most hydrogels are not sufficiently versatile for use in a wide variety of tissue-engineering applications. In recent years, enzyme-catalysed crosslinking approaches have been emerged as a new approach for developing in situ-forming hydrogels. In particular, the horseradish peroxidase (HRP)-catalysed crosslinking approach has received increasing interest, due to its highly improved and tunable capacity to obtain hydrogels with desirable properties. The HRP-catalysed crosslinking reaction immediately occurs upon mixing phenol-rich polymers with HRP and hydrogen peroxide (H2O2) in aqueous media. Based on this unique gel-forming feature, recent studies have shown that various properties of formed hydrogels, such as gelation time, stiffness and degradation rate, can be easily manipulated by varying the concentrations of HRP and H2O2. In this review, we outline the versatile properties of HRP-catalysed in situ-forming hydrogels, with a brief introduction to the crosslinking mechanisms involved. In addition, the recent biomedical applications of HRP-catalysed in situ-forming hydrogels for tissue regeneration are described.

  15. 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

  16. 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

  17. Nano reengineering of horseradish peroxidase with dendritic macromolecules for stability enhancement.

    PubMed

    Khosravi, Arezoo; Vossoughi, Manouchehr; Shahrokhian, Saeed; Alemzadeh, Iran

    2012-01-01

    A simple bio-conjugation procedure to surround a single horseradish peroxidase (HRP) enzyme molecule with dendritic polyester macromolecules (polyester-32-hydroxyl-1-carboxyl bis-MPA dendron, generation 5) was proposed. The characterization of resultant nanoparticles entitled HRP dendrozyme, was performed by transmission electron microscopy, dynamic light scattering, gel permeation chromatography and Fourier transform infrared spectroscopy. The results showed that HRP nanoparticles were spherical in shape and have an average size of 14±2 nm in diameter. Furthermore, bio-conformational characterization of HRP dendrozyme was performed by means of circular dichroism and fluorescence spectroscopy to evaluate the secondary and tertiary structure changes after enzyme modification. These investigations revealed that protein conformation had small changes (in secondary and tertiary structures) after bio-conjugation. We also reported here that dendritic modification did not significantly affect the kinetic parameters of free HRP. The stabilization of HRP with dendron macromolecules as single enzyme nanoparticles resulted in improvement of half-life over 70 days storage at 4 °C as well as its tolerance under different elevated temperatures up to 80 °C and in the presence of organic solvents for 15 min. These significant results promise extensive applications of HRP particularly in harsh environmental conditions. PMID:22133434

  18. 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

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

    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.

  20. Bioelectrochemical systems with oleylamine-stabilized gold nanostructures and horseradish peroxidase for hydrogen peroxide sensor.

    PubMed

    Koposova, Ekaterina; Liu, Xiao; Kisner, Alexandre; Ermolenko, Yury; Shumilova, Galina; Offenhäusser, Andreas; Mourzina, Yulia

    2014-07-15

    This paper describes ultrathin gold nanowires (NWs) and nanoparticles (NPs) prepared by oleylamine (OA) synthesis and their assembly with horseradish peroxidase enzyme (HRP) for bioelectrochemical sensing of hydrogen peroxide for the first time. The immobilization of oxidoreductase enzyme HRP on the electrodes modified with OA gold nanostructures (OANSs) is discussed. The HRP-sensor characteristics, namely sensitivity, working concentration range, sensor-to-sensor and measurement-to-measurement reproducibility as well as long-term stability, are improved significantly compared to the planar thin-film sensors by using OANSs. The thin-film gold electrodes modified with OANWs and OANPs exhibit a catalytic activity towards oxidation of hydrogen peroxide with a working concentration range from 20 µM to 500 µM, a sensitivity of 0.031 A M(-1) cm(-2) (RSD 0.046) and 0.027 A M(-1) cm(-2) (RSD 0.045), and a detection limit of 5 µM and 8 µM, respectively (RSD near the detection limits was 9-12%). Our study shows that ultrathin gold nanowires and nanoparticles prepared by oleylamine synthesis are prospective materials to assemble biomolecules into functional nanoarchitectures for enzyme-based bioelectrochemical sensors, metalloprotein bioelectronics, and energy research.

  1. 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

  2. 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.

  3. Characterization of an organic phase peroxide biosensor based on horseradish peroxidase immobilized in Eastman AQ.

    PubMed

    Konash, Anastassija; Magner, Edmond

    2006-07-15

    Due to their frequent occurrence in food, cosmetics and pharmaceutical products, and their poor solubility in water, the detection of peroxides in organic solvents has aroused significant interest. For diagnostics or on-site testing, a fast and specific experimental approach is required. Although aqueous peroxide biosensors are well known, they are usually not suitable for nonaqueous applications due to their instability. Here we describe an organic phase biosensor for hydrogen peroxide based on horseradish peroxidase immobilized in an Eastman AQ 55 polymer matrix. Rotating disc amperometry was used to examine the effect of the solvent properties, the amount and pH of added buffer, the concentration of peroxide and ferrocene dimethanol, and the amount of Eastman AQ 55 and of enzyme on the response of the biosensor to hydrogen peroxide. The response of the biosensor was limited by diffusion. Linear responses (with detection limits to hydrogen peroxide given in parentheses) were obtained in methanol (1.2 microM), ethanol (0.6 microM), 1-propanol (2.8 microM), acetone (1.4 microM), acetonitrile (2.6 microM), and ethylene glycol (13.6 microM). The rate of diffusion of ferrocene dimethanol was more constrained than the rate of diffusion of hydrogen peroxide, resulting in a comparatively narrow linear range. The main advantages of the sensor are its ease of use and a high degree of reproducibility, together with good operational and storage stability.

  4. 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

  5. Two-dimensional 1H-NMR studies of horseradish peroxidase C and its interaction with indole-3-propionic acid.

    PubMed

    Veitch, N C; Williams, R J

    1990-04-30

    The binding of aromatic donor molecules to plant peroxidases has been investigated by examining the complex formed between horseradish peroxidase isoenzyme C and indole-3-propionic acid using two-dimensional 1H-NMR spectroscopy. Despite the relatively high molecular mass and paramagnetism of the protein, this technique can be successfully applied to provide new information on the structure of the complex. A number of relatively well-resolved resonances in certain regions of the one-dimensional spectrum are assigned to amino acid type on the basis of the two-dimensional experiments. Two phenylalanine side chains are found to interact at positions close to the haem group as shown by nuclear Overhauser effect spectroscopy (NOESY). Furthermore, the NOESY spectrum of the complex reveals distinct interactions between these phenylalanine residues and the indole ring of the donor molecule. The binding site is found to comprise of these phenylalanine side chains and also the methyl group of a leucine or valine residue. On the basis of the model structure of horseradish peroxidase isoenzyme C proposed by Welinder and Nørskov-Lauritsen and information from previous studies of the related turnip peroxidases, possible locations for this binding site are discussed. The NMR methods adopted here may be generally applicable to the study of peroxidase--aromatic-donor interactions. PMID:2338080

  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. Preliminary studies on long distance, retrograde transport of horseradish peroxidase in equine peripheral nerves.

    PubMed

    Fubini, S L; Cummings, J F; Todhunter, R J

    1985-11-01

    As a prelude to studies on retrograde axonal transport of neurotoxin (ie, so-called suicide transport) as a means to prevent post neurectomy neuroma formation, preliminary studies were conducted with an innocuous enzymatic marker, horseradish peroxidase (HRP). The proximal stumps of resected medial and lateral palmar digital nerves in six ponies were injected via a tuberculin syringe and needle with 50 micron 1 of a 30 per cent solution of HRP in order to assess long distance retrograde axonal transport. The dorsal root ganglion of the cervical spinal enlargement (ie, C6, C7, C8, T1, T2) were removed at post injection intervals of two, four, six, eight, 10 and 12 days. These were sectioned serially and reacted by the tetramethylbenzidine method to demonstrate transported enzyme in the ganglionic cell bodies which give rise to sensory fibres of the palmar digital nerves. Enzyme, retrogradely transported over axon lengths of 115 cm, was first demonstrated in spinal ganglia four days after injections of the palmar digital nerves. The calculated transport velocity of 287 mm/day, although almost certainly an underestimate, greatly exceeded rates of 72 to 120 mm/day recorded previously with HRP in the peripheral nerves of small laboratory animals. The intensity of the HRP reaction product in ganglionic neurons was strong at four days and it remained unabated in ganglia examined at six, eight, 10 and 12 days post injection. The major sources of the sensory fibres of the palmar digital nerves appeared to be the ganglia of the C8 and T1 spinal segments which contained more than 90 per cent of all labelled neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. 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

  9. The pontocerebellar system in the opossum, Didelphis virginiana. A horseradish peroxidase study.

    PubMed

    Mihailoff, G A; Martin, G F; Linauts, M

    1980-01-01

    The method of retrograde axonal transport of horseradish peroxidase was employed to demonstrate certain organizational features of the pontocerebellar system in adult opossums. The spinal cerebellum (anterior lobe, pyramis and paramedian lobule) receives input from neurons situated in ventral and lateral regions throughout the rostro-caudal extent of the pons. The projection to the anterior lobe and pyramis was primarily contralateral, whereas the projection to the paramedian lobule included a substantial ipsilateral contribution. The pontine projection to the vermal visual-auditory area was also found to include significant bilateral components which were observed to be organized in mirror image locations in medial, ventral and lateral regions. The paraflocculus was found to receive input from a relatively large number of pontine neurons, the medial injection producing a more bilateral distribution of labeled neurons while the lateral injection resulted in primarily contralateral labeling. Crus I and crus II of the cerebellar hemispheres received a bilateral projection which also included neurons distributed in mirror image locations in both halves of the pontine gray. Taken together such observations indicate that the pontocerebellar system includes a more substantial ipsilateral contribution than has heretofore been recognized. In addition, comparison of the locations of various groups of labeled neurons resulting from spatially separate injection sites suggests the possibility that some pontocerebellar neurons might diverge to reach more than one cerebellar zone, i.e. cells in a similar ventrolateral pontine region were labeled following injections in the anterior lobe, pyramis and crus I. Evidence for convergence of multiple pontine areas to single cerebellar foci was not as compelling.

  10. [Investigation of micro-aqueous covalent immobilization of horseradish peroxidase by "conformation memory"].

    PubMed

    Cai, Yixuan; Chen, Junhua; Yao, Dongsheng; Liu, Daling

    2009-12-01

    We has studied the feasibility of preventing protein from denature during covalent immobilization by "conformation memory", which was achieved by freeze-drying under enzyme active conformation and cross-linked with carrier under micro-aqueous media (MAM). Horseradish peroxidase (HRP) and chitosan beads have been used as the model enzyme and carrier. The MAM consisted of 99% dioxane and 1% water. We compared the immobilized HRP under MAM with that under traditional aqueous solvent, found that the optimum temperature of both was raised to 60 degrees C, and the optimum pH was 6.5. However, the MAM-immobilized HRP had shown less activity loss during usage and six times higher activity than that immobilized under aqueous solvent. After 30 min incubation at 70 degrees C, the MAM-immobilized HRP remained 75.42% activity while the aqueous-media-immobilized enzyme only 15.4%. The MAM-immobilized HRP has shown a better operation stability with 77.69% residue activity after 5 times of repeat operation while the aqueous-media-immobilized enzyme only 16.67%. In addition, the MAM-immobilized HRP had also shown more advantages when used in phenol removal. We constructed enzyme electrodes (CS-HRP-SWCNTs/Au) to further display the different properties of the two immobilized HRP. MAM-immobilized HRP-electrode has shown two times stronger response signal to H2O2 than that immobilized under aqueous media, which indicated a better enzyme activity of MAM-immobilized HRP. Our research demonstrated that the conformation memory, to some extent, did contribute to preventing protein from denaturing when use HRP as a model, and it is feasible to immobilize enzyme by covalent cross-linking method under micro-aqueous media.

  11. 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

  12. Poly(2-hydroxyethyl methacrylate) for enzyme immobilization: impact on activity and stability of horseradish peroxidase.

    PubMed

    Lane, Sarah M; Kuang, Zhifeng; Yom, Jeannie; Arifuzzaman, Shafi; Genzer, Jan; Farmer, Barry; Naik, Rajesh; Vaia, Richard A

    2011-05-01

    On the basis of their versatile structure and chemistry as well as tunable mechanical properties, polymer brushes are well-suited as supports for enzyme immobilization. However, a robust surface design is hindered by an inadequate understanding of the impact on activity from the coupling motif and enzyme distribution within the brush. Herein, horseradish peroxidase C (HRP C, 44 kDa), chosen as a model enzyme, was immobilized covalently through its lysine residues on a N-hydroxysuccinimidyl carbonate-activated poly(2-hydroxyethyl methacrylate) (PHEMA) brush grafted chemically onto a flat impenetrable surface. Up to a monolayer coverage of HRP C is achieved, where most of the HRP C resides at or near the brush-air interface. Molecular modeling shows that lysines 232 and 241 are the most probable binding sites, leading to an orientation of the immobilized HRP C that does not block the active pocket of the enzyme. Michaelis-Menten kinetics of the immobilized HRP C indicated little change in the K(m) (Michaelis constant) but a large decrease in the V(max) (maximum substrate conversion rate) and a correspondingly large decrease in the k(cat) (overall catalytic rate). This indicates a loss in the percentage of active enzymes. Given the relatively ideal geometry of the HRPC-PHEMA brush, the loss of activity is most likely due to structural changes in the enzyme arising from either secondary constraints imposed by the connectivity of the N-hydroxysuccinimidyl carbonate linking moiety or nonspecific interactions between HRP C and DSC-PHEMA. Therefore, a general enzyme-brush coupling motif must optimize reactive group density to balance binding with neutrality of surroundings. PMID:21438540

  13. Influence of Cu(II) on the interaction between sulfite and horseradish peroxidase in vitro

    NASA Astrophysics Data System (ADS)

    Lan, Jie; Guo, Dong-Sheng; Yuan, Xiao-Ying

    2007-06-01

    This paper discussed the quantitative influence of Cu(II) on the interaction between horseradish peroxidase (HRP) and sulfite (SO 32-), which is a derivate of sulfite dioxide in human bodies, by using fluorescence spectrum and ultraviolet (UV) absorption spectrometry in vitro. The results show that under the conditions of physiological pH and room-temperature, Cu(II) can bind strongly with both the protein part and the ferroporphyrin part in HRP at a low concentration (10 -4 mol L -1), and the combination constants are 2.047 × 10 3 and 7.66 × 10 2 L mol -1, respectively. Under the same conditions, SO 32- at low concentrations (<0.15 mol L -1) has little quenching for the fluorescence of HRP at 330 nm, and the combination constant is 0.108 L mol -1. While the fluorescence intensity at 440 nm enhance gradually with the increased concentration of SO 32- (<0.1 mol L -1), and the combination constant is 8.219 L mol -1. These indicate that SO 32- at low concentration has little reaction with the enzyme protein part in HRP but obvious reaction with the ferroporphyrin part in HRP. After SO 32- at low concentrations is added into the HRP-Cu(II) binary system, the reaction constants between SO 32- and the enzyme protein part in HRP increase rapidly. Compared with the absence of Cu(II), the combination constant of SO 32- with the enzyme protein part in HRP increases nearly 70 times with a certain Cu(II) concentration (5.0 × 10 -4 mol L -1) in the system. However, the presence of Cu(II) in the system has little effect on the reaction constants between SO 32- and the ferroporphyrin part in HRP.

  14. Organization within the cranial IX-X complex in ranid frogs: a horseradish peroxidase transport study.

    PubMed

    Stuesse, S L; Cruce, W L; Powell, K S

    1984-01-20

    Cranial nerves IX and X in frogs have been described as originating from a nuclear group referred to as the IX-X complex. We studied the central nervous system components of this complex in Rana pipiens and R. catesbiana by labeling peripheral branches of cranial nerves IX and X and identifying the central nervous system contributions of these branches. Various peripheral nerves (IX and the cardiac, gastric, pulmonary, and laryngeal branches of X) were identified and soaked in horseradish peroxidase (HRP). One to 2 weeks later, the frogs were killed and processed for HRP by the tetramethylbenzidine method. Glossopharyngeal efferents originated from a small ventrolateral cell group found at the level of IX root exit. Vagal efferents formed a single column of cells in a ventrolateral position from the level of the brainstem exist of the vagus nerve (approximately 2,000 micrometers above the obex) to 200 micrometers below the obex (values given are for an 80-g frog). This cell group was separate from and just caudal to efferent cells of the glossopharyngeal nerve. Within the vagal portion of the column, cells projecting through the gastric branch were found throughout the rostral-caudal extent of the nucleus. "Cardiac" cells tended to be more rostral than "pulmonary" cells, and both groups of cells were located in the middle of the nucleus. "Laryngeal" cells were located more caudally in the nucleus. This peripheral representation within the vagal nucleus corresponds more closely to the organization found in the mammalian nucleus ambiguus, rather than to the apparent lack of organization found in the mammalian dorsal motor nucleus. Afferents of IX and X entered slightly rostral to the ventral roots of their respective nerves and descended in two tracts. The majority entered the tractus solitarius and descended in a medial position to cervical spinal cord. A portion of the afferents from the vagus nerve crossed the midline in the lower myelencephalon just dorsal to the

  15. Arginine-to-lysine substitutions influence recombinant horseradish peroxidase stability and immobilisation effectiveness

    PubMed Central

    Ryan, Barry J; Ó'Fágáin, Ciarán

    2007-01-01

    Background Horseradish Peroxidase (HRP) plays important roles in many biotechnological fields, including diagnostics, biosensors and biocatalysis. Often, it is used in immobilised form. With conventional immobilisation techniques, the enzyme adheres in random orientation: the active site may face the solid phase rather than bulk medium, impeding substrate access and leading to sub-optimal catalytic performance. The ability to immobilise HRP in a directional manner, such that the active site would always face outwards from the insoluble matrix, would maximise the immobilised enzyme's catalytic potential and could increase HRP's range of actual and potential applications. Results We have replaced arginine residues on the face of glycan-free recombinant HRP opposite to the active site by lysines. Our strategy differs from previous reports of specific HRP immobilisation via an engineered affinity tag or single reactive residue. These conservative Arg-to-Lys substitutions provide a means of multipoint covalent immobilisation such that the active site will always face away from the immobilisation matrix. One triple and one pentuple mutant were generated by substitution of solvent-exposed arginines on the "back" of the polypeptide (R118, R159 and R283) and of residues known to influence stability (K232 and K241). Orientated HRP immobilisation was demonstrated using a modified polyethersulfone (PES) membrane; the protein was forced to orientate its active site away from the membrane and towards the bulk solution phase. Mutant properties and bioinformatic analysis suggested the reversion of K283R to improve stability, thus generating two additional mutants (K118/R159K and R118K/K232N/K241F/R283K). While most mutants were less stable in free solution than wild type rHRP, the quadruple revertant regained some stability over its mutant counterparts. A greater degree of immobilisation on CNBr-activated Sepharose™ was noted with increased lysine content; however, only marginal

  16. 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.

  17. Direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized in sol-gel-derived ceramic-carbon nanotube nanocomposite film.

    PubMed

    Chen, Hongjun; Dong, Shaojun

    2007-03-15

    The sol-gel-derived ceramic-carbon nanotube (SGCCN) nanocomposite film fabricated by doping multiwall carbon nanotubes (MWNTs) into a silicate gel matrix was used to immobilize protein. The SGCCN film can provide a favorable microenvironment for horseradish peroxidase (HRP) to perform direct electron transfer (DET) at glassy carbon electrode. The HRP immobilized in the SGCCN film shows a pair of well-defined redox waves and retains its bioelectrocatalytic activity to the reduction of O2 and H2O2, which is superior to that immobilized in silica sol-gel film.

  18. 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

  19. An ultrasensitive electrochemical aptasensor for thrombin based on the triplex-amplification of hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme and horseradish peroxidase decorated FeTe nanorods.

    PubMed

    Jiang, Liping; Yuan, Ruo; Chai, Yaqin; Yuan, Yali; Bai, Lijuan; Wang, Yan

    2013-03-01

    In the present study, we fabricated an ultrasensitive sandwich-type electrochemical aptasensor for thrombin (TB) based on a triplex signal amplification strategy. The hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme (HRP-DNAzyme) as well as blocking reagent-horseradish peroxidase (HRP) and iron telluride nanorods (FeTe NRs) could simultaneously amplify the electrochemical signal of thionine (Thi) in the presence of H(2)O(2). Herein, FeTe NRs, as a newly discovered HRP-mimicking enzyme, were employed to construct an aptasensor for the first time. And, the FeTe NRs decorated by gold nanoparticles (abbreviated as AuNPs@FeTe NRs), were not only used as carriers of secondary thrombin aptamer (TBA 2), electron mediator thionine (Thi) and HRP, but also catalyzed the electrochemical reaction of Thi in the presence of H(2)O(2). As can be seen from experiment results, with the triplex signal amplification strategy, the reduction peak current of the fabricated aptasensor was logarithmically related to the concentration of thrombin (TB) over a wide range from 1 pM to 20 nM, and a detection limit of 0.5 pM was obtained. Hence, the proposed aptamer-based sandwich sensing approach for amplified detection of TB could provide a promising way for highly sensitive determination of other analytes.

  20. 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

  1. An amperometric biosensor utilizing a ferrocene-mediated horseradish peroxidase reaction for the determination of capsaicin (chili hotness).

    PubMed

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

    2013-08-05

    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.

  2. Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics.

    PubMed

    Toledo, José Carlos; Audi, Renata; Ogusucu, Renata; Monteiro, Gisele; Netto, Luis Eduardo Soares; Augusto, Ohara

    2011-05-01

    Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol-disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV-Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pK(a) of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4 ± 0.2) × 10⁷M⁻¹ s⁻¹) and peroxynitrite ((3.7 ± 0.4) × 10⁵ M⁻¹ s⁻¹) at pH 7.4 and 25°C.

  3. 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

  4. 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.

  5. 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.

  6. Colorimetric detection of copper(II) ion using click chemistry and hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme.

    PubMed

    Ge, Chenchen; Luo, Quan; Wang, Dou; Zhao, Shiming; Liang, Xiaoling; Yu, Luxin; Xing, Xuerong; Zeng, Lingwen

    2014-07-01

    G-quadruplex-forming sequence can be formed through a copper(I) ion (Cu(+))-catalyzed click chemistry between azide- and alkyne-modified short G-rich sequences in aqueous solution, eliminating immobilization and washing steps of conventional assays. The source for Cu(+) was generated from the reduction of Cu(2+) with the reductant of sodium ascorbate. In the presence of hemin and K(+), the self-assembly of hemin/G-quadruplex structure has the activity of horseradish peroxidase (HRP), which can catalyze its colorless substrate tetrazmethyl benzidine (TMB) into a colored product. Hence, the concentration of Cu(2+) can be evaluated visually for qualitative analysis according to the color change of the solution, and the optical density (OD) value of the resulting solution at 450 nm was also recorded using a microplate reader for quantitative analysis. PMID:24950121

  7. 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

  8. Probing the aromatic-donor-binding site of horseradish peroxidase using site-directed mutagenesis and the suicide substrate phenylhydrazine.

    PubMed

    Gilfoyle, D J; Rodriguez-Lopez, J N; Smith, A T

    1996-03-01

    The haem groups from two classes of site-directed mutants of horseradish peroxidase isoenzyme C (HRP-C) (distal haem pocket mutants, [H42L]HRP-C* and [R38K]-HRP-C* and peripheral-haem-access-channel mutants, [F142A]HRP-C* and [F143A]HRP-C*) were extracted and analysed by reverse-phase HPLC after phenylhydrazine-induced suicide inactivation. The relative abundance of the two covalently modified haems, C20-phenyl (delta-meso phenyl) and C18-hydroxymethyl haem, provided a sensitive topological probe for changes induced in the protein architecture in the vicinity of the haem active site and substrate-access channel. Although differing considerably in their efficiency as peroxidases ([H42L]HRP-C* exhibited only approximately 0.03% of the peroxidase activity of wild type), the variants studied gave rise to a modification pattern typical of an exposed haem edge thereby strengthening the argument that it is the overall protein topology rather than the intrinsic catalytic activity of the active site that determines the sites of covalent haem modification. Mutants which showed impaired ability to bind the aromatic donor benzhydroxamic acid were less readily modified by the phenyl radical at the haem C18-methyl position although the level of arylation at the haem C20 position remained remarkable constant. Our findings suggest that the overall efficacy of haem modification catalysed by HRP-C during turnover with phenylhydrazine and its vulnerability towards inactivation are related to its general ability to bind aromatic donor molecules. Results from phenylhydrazine treatment of HRP-C wild-type and mutant variants were compared with those obtained for Coprinus cinereus peroxidase, an enzyme which from its structure is known to have a remarkably open access channel to the haem edge. We show evidence that C. cinereus peroxidase is able to bind benzhydroxamic acid, albeit with a relatively high Kd (Kd 3.7 mM), a probe for aromatic-donor binding. We suggest reasons why

  9. 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

  10. Simultaneous visualization of cortical barrels and horseradish peroxidase-injected layer 5b vibrissa neurones in the rat.

    PubMed Central

    Ito, M

    1992-01-01

    1. Using diaminobenzidine (DAB) as a chromagen, horseradish peroxidase-injected neurones and cytochrome oxidase-stained barrels were visualized simultaneously in the rat vibrissa cortex. Neurones were initially tested during extracellular recording for responses to whisker deflections. This was followed by intracellular injection of the soma with horseradish peroxidase (HRP) and histological processing to visualize the HRP-stained neurone in an incubation solution which contained, in addition to DAB, cytochrome C for cytochrome oxidase (CO) reaction of the barrels. 2. Recording and intracellular staining were made in layer 5b under urethane anaesthesia. CO-stained barrels were observed in layer 4. Physiologically and morphologically characterized neurones were mostly large pyramidal neurones that responded to more than one whisker and displayed transient-type responses. 3. In tangential sections, the apical dendrite of the HRP-filled neurone was followed from the soma level upward as it ascended through the barrelfield in layer 4. The cross-section of the apical dendrite was found in the periphery of the CO-stained barrel. Using the apical dendrite as a guide, the basal dendritic field of the layer 5b pyramidal neurone was aligned on the pattern of layer 4 barrels. The soma was seen to project basal dendrites in all directions, involving one or two neighbouring barrels/columns. 4. In sixteen neurones examined in tangential sections, a complete spatial tuning map constructed by measuring sensitivity of the neurone to different whiskers could be compared to the basal dendritic field in relation to the pattern of overlying layer 4 barrels. The mean receptive field size in terms of the number of effective whiskers was 5.8 whereas the mean dendritic field size in terms of the number of barrels/columns involved was 2.2. In addition to the well-documented role of intracortical connectivity in elaboration of multi-whisker receptor fields in the cortical neurones, the role

  11. pH effects on the haem iron co-ordination state in the nitric oxide and deoxy derivatives of ferrous horseradish peroxidase and cytochrome c peroxidase.

    PubMed Central

    Ascenzi, P; Brunori, M; Coletta, M; Desideri, A

    1989-01-01

    The spectral (e.p.r. and absorbance) properties of the NO and deoxy derivatives of ferrous horseradish peroxidase (HRP; EC 1.11.1.7) and baker's-yeast cytochrome c peroxidase (CCP; EC 1.11.1.5) were investigated between pH 7 and pH 2; over the same pH range the kinetics for CO binding were also determined. At neutral pH the e.p.r. and absorption spectra of the NO and deoxy derivatives of HRP and CCP are typical of systems in which the haem iron is in the hexaco-ordinated state and the pentaco-ordinated state respectively. By lowering pH, the e.p.r. and absorption spectra of HRP and CCP undergo reversible transitions, with pKa values of 4.1 for the NO derivatives and less than or equal to 3 for the deoxy derivatives of the ferrous forms. By analogy with O2-carrying proteins and haem model compounds, the pH-dependent spectral changes of HRP and CCP were interpreted as indicative of the protonation of the N(epsilon) atom of the proximal histidine residue and of the cleavage of the Fe-N(epsilon) bond. However, the slow second-order rate constant (0.003 microM-1.s-1) for CO binding to deoxy ferrous HRP and CCP does not increase substantially even at pH 2.6, suggesting that changes in the Fe-haem plane geometry, presumably associated with the cleavage of the Fe-N(epsilon) bond, do not affect appreciably the observed ligand association rate constant. PMID:2539809

  12. How modification of accessible lysines to phenylalanine modulates the structural and functional properties of horseradish peroxidase: a simulation study.

    PubMed

    Navapour, Leila; Mogharrab, Navid; Amininasab, Mehriar

    2014-01-01

    Horseradish Peroxidase (HRP) is one of the most studied peroxidases and a great number of chemical modifications and genetic manipulations have been carried out on its surface accessible residues to improve its stability and catalytic efficiency necessary for biotechnological applications. Most of the stabilized derivatives of HRP reported to date have involved chemical or genetic modifications of three surface-exposed lysines (K174, K232 and K241). In this computational study, we altered these lysines to phenylalanine residues to model those chemical modifications or genetic manipulations in which these positively charged lysines are converted to aromatic hydrophobic residues. Simulation results implied that upon these substitutions, the protein structure becomes less flexible. Stability gains are likely to be achieved due to the increased number of stable hydrogen bonds, improved heme-protein interactions and more integrated proximal Ca2+ binding pocket. We also found a new persistent hydrogen bond between the protein moiety (F174) and the heme prosthetic group as well as two stitching hydrogen bonds between the connecting loops GH and F'F″ in mutated HRP. However, detailed analysis of functionally related structural properties and dynamical features suggests reduced reactivity of the enzyme toward its substrates. Molecular dynamics simulations showed that substitutions narrow the bottle neck entry of peroxide substrate access channel and reduce the surface accessibility of the distal histidine (H42) and heme prosthetic group to the peroxide and aromatic substrates, respectively. Results also demonstrated that the area and volume of the aromatic-substrate binding pocket are significantly decreased upon modifications. Moreover, the hydrophobic patch functioning as a binding site or trap for reducing aromatic substrates is shrunk in mutated enzyme. Together, the results of this simulation study could provide possible structural clues to explain those experimental

  13. 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

  14. An adhesive conducting electrode material based on commercial mesoporous titanium dioxide as a support for Horseradish peroxidase for bioelectrochemical applications.

    PubMed

    Rahemi, Vanoushe; Trashin, Stanislav; Meynen, Vera; De Wael, Karolien

    2016-01-01

    An adhesive conducting electrode material containing of graphite, biocompatible ion exchange polymer nafion(®) and commercial mesoporous TiO2 impregnated with horseradish peroxidase (HRP) is prepared and characterized by amperometric, UV-vis and N2 sorption methods. The factors influencing the performance of the resulting biosensor are studied in detail. The optimal electrode material consists of 45% graphite, 50% impregnated HRP-TiO2 and 5% nafion(®). The optimum conditions for H2O2 reduction are an applied potential of -0.3 V and 0.1 mM hydroquinone. Sensitivity and limit of detection in the optimum conditions are 1 A M(-1) cm(-2) and 1 µM correspondingly. The N2 sorption results show that the pore volume of TiO2 decreases sharply upon adsorption of HRP. The preparation process of the proposed enzyme electrode is straightforward and potentially can be used for preparation of carbon paste electrodes for bioelectrochemical detections. PMID:26695318

  15. 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

  16. 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

  17. 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.

  18. High resolution autoantibody detection by optimized protein G-horseradish peroxidase immunostaining in a western blotting assay.

    PubMed

    Hu, G R; Harrop, P; Warlow, R S; Gacis, M L; Walls, R S

    1997-03-28

    We report a procedure for optimisation of Western blotting using protein G-horseradish peroxidase (protein G-HRP) which avoids the false positive reactions often caused by second antibodies and increases the detection of autoantibodies by protein G conjugate. A number of modifications were investigated. Higher concentrations of serum and protein G-HRP at 1:5 to 1:10 and 1:100, respectively, increased the detection to the same order as that obtained with second antibody systems and gold staining with silver enhancement. The role of various detergents in the procedure was established. 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) in incubation with protein G-HRP increased the binding between protein G and immunoglobulin G. Addition of Tween-20 for blocking produced little background so that protein blockers could be avoided. Prolonged incubation with serum increased markedly the sensitivity of the procedure when compared with the recommended 2 h incubation period. Polyvinylidene difluoride membrane provided better transfer effect, lower background and higher mechanical strength than nitrocellulose membrane. The utilization of only one antibody-specific ligand increased the simplicity, reliability, economy, efficiency and specificity of the method. These modifications make this method significantly better for detection and screening for autoantibodies.

  19. 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.

  20. Nanosheet-based titania microspheres with hollow core-shell structure encapsulating horseradish peroxidase for a mediator-free biosensor.

    PubMed

    Xie, Qing; Zhao, Yingying; Chen, Xu; Liu, Haimei; Evans, David G; Yang, Wensheng

    2011-09-01

    Nanosheet-based titania (TiO(2)) microspheres with a hollow core-shell structure have been synthesized and employed to immobilize horseradish peroxidase (HRP) in order to fabricate a mediator-free biosensor. The morphology and structure of the TiO(2) microspheres were characterized by X-ray diffraction, scanning electron microscopy and transmission electronic microscopy. A possible growth mechanism has been proposed. Spectroscopic and electrochemical measurements revealed that the TiO(2) microspheres are an immobilization support with biocompatibility for enzymes, affording good enzyme stability and bioactivity. Due to the nanosheet-based hollow core-shell structure of the TiO(2) microspheres, the direct electron transfer of HRP is facilitated and the resulting biosensor displayed good performance for the detection of H(2)O(2), with both a low detection limit of 0.05 μM and a wide linear range of 0.4-140 μM, as well as a fast response and excellent long-term stability. The nanosheet-based TiO(2) microspheres with hollow core-shell structure, can be used for the efficient entrapment of other redox-active proteins and have wide potential applications in biosensors, biocatalysis, biomedical devices and bioelectronics. PMID:21663956

  1. 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.

  2. 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.

  3. Amperometric inhibition biosensors based on horseradish peroxidase and gold sononanoparticles immobilized onto different electrodes for cyanide measurements.

    PubMed

    Attar, Aisha; Cubillana-Aguilera, Laura; Naranjo-Rodríguez, Ignacio; de Cisneros, José Luis Hidalgo-Hidalgo; Palacios-Santander, José María; Amine, Aziz

    2015-02-01

    New biosensors based on inhibition for the detection of cyanide and the comparison of the analytical performances of nine enzyme biosensor designs by using three different electrodes: Sonogel-Carbon, glassy carbon and gold electrodes were discussed. Three different horseradish peroxidase immobilization procedures with and without gold sononanoparticles were studied. The amperometric measurements were performed at an applied potential of -0.15V vs. Ag/AgCl in 50mM sodium acetate buffer solution pH=5.0. The apparent kinetic parameters (Kmapp, Vmaxapp) of immobilized HRP were calculated in the absence of inhibitor (cyanide) by using caffeic acid, hydroquinone, and catechol as substrates. The presence of gold sononanoparticles enhanced the electron transfer reaction and improved the analytical performance of the biosensors. The HRP kinetic interactions reveal non-competitive binding of cyanide with an apparent inhibition constant (Ki) of 2.7μM and I50 of 1.3μM. The determination of cyanide can be achieved in a dynamic range of 0.1-58.6μM with a detection limit of 0.03μM which is lower than those reported by previous studies. Hence this biosensing methodology can be used as a new promising approach for detecting cyanide.

  4. 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

  5. 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.

  6. Horseradish peroxidase immobilization on carbon nanodots/CoFe layered double hydroxides: direct electrochemistry and hydrogen peroxide sensing.

    PubMed

    Wang, Yinling; Wang, Zhangcui; Rui, Yeping; Li, Maoguo

    2015-02-15

    Carbon nanodots and CoFe layered double hydroxide composites (C-Dots/LDHs) were prepared via simply mixing C-Dots and CoFe-LDHs. The as-prepared composites were used for the immobilization of horseradish peroxidase (HRP) on the glass carbon (GC) electrode. The electrochemical behavior of the HRP/C-Dots/LDHs/GC electrode and its application as a H2O2 biosensor were investigated. The results indicated that HRP immobilized by C-Dots/LDHs retained the activity of enzyme and displayed quasi-reversible redox behavior and fast electron transfer with an electron transfer rate constant ks of 8.46 s(-1). Under optimum experimental conditions, the HRP/C-Dots/LDHs/GC electrode displayed good electrocatalytic reduction activity and excellent analytic performance toward H2O2. The H2O2 biosensor showed a linear range of 0.1-23.1 μM (R(2) = 0.9942) with a calculated detection limit of 0.04 μM (S/N = 3). In addition, the biosensor exhibited high sensitivity, good selectivity, acceptable reproducibility and stability. The superior properties of this biosensor are attributed to the synergistic effect of HRP, C-Dots and CoFe-LDHs, which has been proved by investigating their electrochemical response to H2O2. Thus the C-Dots and LDHs composites provide a promising platform for the immobilization of redox enzymes and construction of sensitive biosensors.

  7. 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

  8. Dual-signal amplified electrochemiluminescence immunoassay for salbutamol based on quantum dots and gold nanoparticle-labeled horseradish peroxidase.

    PubMed

    Cai, Fudong; Wang, Nan; Dong, Tiantian; Deng, Anping; Li, Jianguo

    2015-09-01

    This study describes a novel electrochemical immunosensor to amplify the electrochemiluminescence (ECL) signal for the ultrasensitive detection of salbutamol (SAL) using quantum dots (QDs) and gold nanoparticle (AuNP) conjugated horseradish peroxidase (HRP). The electrochemical detection was based on the HRP catalyzed consumption of self-produced H2O2, which has been extensively used as a co-reactant of QDs, by o-phenylenediamine (OPD). The enzymatic reaction rate is proportional to the amount of HRP bound to the electrode. In the presence of a SAL standard solution, the immobilized SAL coating antigens competed with the SAL solution for the Ab-AuNPs-HRP complexes. With an increase in the SAL concentration, the amount of immobilized HRP decreases, which leads to an increase in the ECL intensity. Under optimized conditions, the ECL intensity changes linearly with the logarithm of the SAL concentration in the range of 0.05-500 ng mL(-1) with a detection limit of 0.017 ng mL(-1) (S/N = 3). The ECL immunosensor possesses high sensitivity, satisfactory reproducibility and selectivity, and may provide a feasible route for practical application.

  9. Covalent attachment of cholesterol oxidase and horseradish peroxidase on perlite through silanization: activity, stability and co-immobilization.

    PubMed

    Torabi, Seyed-Fakhreddin; Khajeh, Khosro; Ghasempur, Salehe; Ghaemi, Nasser; Siadat, Seyed-Omid Ranaei

    2007-08-31

    In the present work, co-immobilization of cholesterol oxidase (COD) and horseradish peroxidase (POD) on perlite surface was attempted. The surface of perlite were activated by 3-aminopropyltriethoxysilane and covalently bonded with COD and POD via glutaraldehyde. Enzymes activities have been assayed by spectrophotometric technique. The stabilities of immobilized COD and POD to pH were higher than those of soluble enzymes and immobilization shifted optimum pH of enzymes to the lower pH. Heat inactivation studies showed improved thermostability of the immobilized COD for more than two times, but immobilized POD was less thermostable than soluble POD. Also activity recovery of immobilized COD was about 50% since for immobilized POD was 11%. The K(m) of immobilized enzymes was found slightly lower than that of soluble enzymes. Immobilized COD showed inhibition in its activity at high cholesterol concentration which was not reported for soluble COD before. Co-immobilized enzymes retained 65% of its initial activity after 20 consecutive reactor batch cycles.

  10. Polymerase chain reaction-mediated gene synthesis: synthesis of a gene coding for isozyme c of horseradish peroxidase.

    PubMed Central

    Jayaraman, K; Fingar, S A; Shah, J; Fyles, J

    1991-01-01

    The synthesis of a gene coding for horseradish peroxidase (HRP, isozyme c; EC 1.11.1.7) is described using a polymerase chain reaction (PCR)-mediated gene synthesis approach developed in our laboratory. In this approach, all the oligonucleotides making up the gene are ligated in a single step by using the two outer oligonucleotides as PCR primers and the crude ligation mixture as the target. The PCR facilitates synthesis and purification of the gene simultaneously. The gene for HRP was synthesized by ligating all 40 oligonucleotides in a single step followed by PCR amplification. The gene was also synthesized from its fragments by using an overlap extension method similar to the procedure as described [Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K. & Pease, L. R. (1989) Gene 77, 61-68]. A method for combining different DNA fragments, in-frame, by using the PCR was also developed and used to synthesize the HRP gene from its gene fragments. This method is applicable to the synthesis of even larger genes and to combine any DNA fragments in-frame. After the synthesis, preliminary characterization of the HRP gene was also carried out by the PCR to confirm the arrangement of oligonucleotides in the gene. This was done by carrying out the PCR with several sets of primers along the gene and comparing the product sizes with the expected sizes. The gene and the fragments generated by PCR were cloned in Escherichia coli and the sequence was confirmed by manual and automated DNA sequencing. Images PMID:1851991

  11. 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.

  12. Horseradish peroxidase-catalyzed oxidation of chlorophyll a with hydrogen peroxide: characterization of the products and mechanism of the reaction.

    PubMed

    Hynninen, Paavo H; Kaartinen, Vesa; Kolehmainen, Erkki

    2010-05-01

    Horseradish peroxidase was verified to catalyze, without any phenol, the hydrogen peroxide oxidation of chlorophyll a (Chl a), solubilized with Triton X-100. The 13(2)(S) and 13(2)(R) diastereomers of 13(2)-hydroxyChl a were characterized as major oxidation products (ca. 60%) by TLC on sucrose, UV-vis, (1)H, and (13)C NMR spectra, as well as fast-atom bombardment MS. A minor amount of the 15(2)-methyl, 17(3)-phytyl ester of Mg-unstable chlorin was identified on the basis of its UV-vis spectrum and reactivity with diazomethane, which converted it to the 13(1),15(2)-dimethyl, 17(3)-phytyl ester of Mg-purpurin 7. The side products (ca. 10%) were suggested to include the 17(3)-phytyl ester of Mg-purpurin 18, which is known to form easily from the Mg-unstable chlorin. The side products also included two red components with UV-vis spectral features resembling those of pure Chl a enolate anion. Hence, the two red components were assigned to the enolate anions of Chl a and pheophytin a or, alternatively, two different complexes of the Chl a enolate ion with Triton X-100. All the above products characterized by us are included in our published free-radical allomerization mechanism of Chl a, i.e. oxidation by ground-state dioxygen. The HRP clearly accelerated the allomerization process, but it did not produce bilins, that is, open-chain tetrapyrroles, the formation of which would require oxygenolysis of the chlorin macrocycle. In this regard, our results are in discrepancy with the claim by several researchers that 'bilirubin-like compounds' are formed in the HRP-catalyzed oxidation of Chl a. Inspection of the likely reactions that occurred on the distal side of the heme in the active centre of HRP provided a reasonable explanation for the observed catalytic effect of the HRP on the allomerization of Chl. In the active centre of HRP, the imidazole nitrogen of His-42 was considered to play a crucial role in the C-13(2) deprotonation of Chl a, which resulted in the Chl a

  13. Afferent projections to the ventral tegmental area of Tsai and interfascicular nucleus: a horseradish peroxidase study in the rat.

    PubMed

    Phillipson, O T

    1979-09-01

    Using the retrograde transport of horseradish peroxidase (HRP), a study has been made of projections to the ventral tegmental area of Tsai (VTA) and related dopaminergic cell groups (A 10). In order to minimise the possibility of damage to fibres of passage, a technique was evolved for the microiontophoresis of HRP such that minimal current strengths and durations were applied. In addition to a sham injection, control injections were also made to the medial lemnisuc, red nucleus, deep tegmental decussations, mesencephalic reticular formation and brachium conjunctivum. Following HRP injections confined to the areas of the VTA containing the dopamine cell groups, labelled neurons appeared in prefrontal cortex, dorsal bank of rhinal sulcus, nucleus accumbens, bed nucleus of stria terminalis, amygdala, diagonal band of Broca, substantis innominata, magnocellular preoptic area, medial and lateral preoptic areas, anterior, lateral and postero-dorsal hypothalamus, lateral habenular, nucleus parafascicular nucleus of thalamus, superior colliculus, nucleus raphe dorsalis, nucleus raphe nagnus and pontis, dorsal and ventral parabrachial nuclei, locus coeruleus and deep cerebellar nuclei. Regions containing catecholamine groups A 1, A 5, A 6, A 7, A 9, A 13 and the serotonin group B 7 corresponded to the topography of labeled cell groups. Injections of HRP to the interfascicular nucleus resulted in labeling predominantly confined to the medial habenular and median raphe nuclei. The results are discussed in relation to the known connections of these regions. Other regions of the brain labelled by VTA injections are assessed in relation to control injections and the limitations of the HRP technique. A review of the organisation of some of these afferents in relation to the known cortical-subcortical-mesencephalic projection systems, suggests that the VTA is in a position to recieve information from a massively convergent system derived ultimately from the entire archi-, paleo

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. Fabrication of an electrochemical platform based on the self-assembly of graphene oxide-multiwall carbon nanotube nanocomposite and horseradish peroxidase: direct electrochemistry and electrocatalysis

    NASA Astrophysics Data System (ADS)

    Zhang, Qian; Yang, Shaojun; Zhang, Jing; Zhang, Ling; Kang, Pingli; Li, Jinghong; Xu, Jingwei; Zhou, Hua; Song, Xi-Ming

    2011-12-01

    A novel hybrid nanomaterial (GO-MWNTs) was explored based on the self-assembly of multiwall carbon nanotubes (MWNTs) and graphene oxide (GO). Compared with pristine MWNTs, such a nanocomposite could be well dispersed in aqueous solution and exhibit a negative charge. Driven by the electrostatic interaction, positively charged horseradish peroxidase (HRP) could then be immobilized onto GO-MWNTs at the surface of a glassy carbon (GC) electrode to form a HRP/GO-MWNT/GC electrode under mild conditions. TEM was used to characterize the morphology of the GO-MWNT nanocomposite. UV-vis and FTIR spectra suggested that HRP was immobilized onto the hybrid matrix without denaturation. Furthermore, the immobilized HRP showed enhanced direct electron transfer for the HRP-Fe(III)/Fe(II) redox center. Based on the direct electron transfer of the immobilized HRP, the HRP/GO-MWNT/GC electrode exhibited excellent electrocatalytic behavior to the reduction of H2O2 and NaNO2, respectively. Therefore, GO-MWNTs could provide a novel and efficient platform for the immobilization and biosensing of redox enzymes, and thus may find wide potential applications in the fabrication of biosensors, biomedical devices, and bioelectronics.

  19. Naked-eye sensitive detection of alkaline phosphatase (ALP) and pyrophosphate (PPi) based on a horseradish peroxidase catalytic colorimetric system with Cu(ii).

    PubMed

    Shi, Dongmin; Sun, Yue; Lin, Lin; Shi, Chunjun; Wang, Guangfeng; Zhang, Xiaojun

    2016-10-01

    In this paper, a novel colorimetric method for the detection of alkaline phosphatase (ALP) and pyrophosphate (PPi) was designed based on a Cu(2+)-horseradish peroxidase (HRP)-3,3',5,5'-tetra-methylbenzidine (TMB)-H2O2 system. In the presence of ALP, l-ascorbic acid-2-phosphate (AAP) could be hydrolyzed to ascorbic acid which could reduce Cu(2+) to Cu(+) to inhibit the enzymatic activity of HRP in the colorimetric system. The change in absorbance was found to be proportional to the ALP concentration with a linear detection range and a limit of detection of 5.4 mU mL(-1). In the presence of PPi, because Cu(2+) was chelated by PPi, the conversion of Cu(ii) by AA was effectively inhibited. The color of the HRP-TMB-H2O2 system with Cu(2+) showed blue. The HRP-TMB-H2O2 system with the Cu(2+) colorimetric system could also detect PPi with a satisfying result. In summary, this method possesses sensitivity, reproducibility, and cost-effectiveness without labelling and separation and the use of a colorimetric method is more in line with the requirements of on-site detection and green chemistry. PMID:27412643

  20. Spin state and axial ligand bonding in the hydroxide complexes of metmyoglobin, methemoglobin, and horseradish peroxidase at room and low temperatures.

    PubMed

    Feis, A; Marzocchi, M P; Paoli, M; Smulevich, G

    1994-04-19

    Absorption and resonance Raman spectra using Soret excitation of alkaline metmyoglobin (metMb), methemoglobin (metHb), and horseradish peroxidase (HRP) were obtained at room and low temperature. At 298 K both metMb and metHb exhibit two isotope-sensitive bands assigned to high- and low-spin nu(Fe-OH) stretching modes, respectively, which are correlated with the spin-state population. The low-spin stretch occurs 60 cm-1 to higher energy than the corresponding high-spin vibration. When the temperature is lowered, only the low-spin species is observed. HRP exhibits at both 298 and 20 K only the low-spin nu(Fe-OH) stretching mode, which occurs 50 cm-1 to lower energy than the corresponding modes observed in the globins. This is explained in the context of a strong hydrogen bond between the hydroxyl ligand and the distal His42 and/or Arg38. Lowering temperature causes in all of the examined proteins a strengthening of the Fe-OH bond and a contraction of the core of about 0.01 A, as determined by the upshifting of the low-spin nu(Fe-OH) stretching mode and the core size marker bands. Both effects are ascribed to an increase of the packing forces.

  1. 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.

  2. Morphophysiology of synaptic transmission between type I hair cells and vestibular primary afferents. An intracellular study employing horseradish peroxidase in the lizard, Calotes versicolor.

    PubMed

    Schessel, D A; Ginzberg, R; Highstein, S M

    1991-03-22

    Intracellular records with glass microelectrodes filled with horseradish peroxidase (HRP) were taken from primary afferents of the horizontal semicircular canal in the lizard, Calotes versicolor. A coefficient of variation (CV) of the interspike intervals of spontaneous action potentials (APs) was calculated and correlated with the terminal morphologies of afferents within the canal crista. Irregular fibers with CV greater than 0.4 always correlated with a nerve chalice or calyx afferent terminal expansion surrounding one or more type I hair cells; more regular fibers with CV less than 0.4 always correlated with a dimorphic or bouton only terminal expansion of afferents. Afferents with a CV greater than 0.4 demonstrated miniature excitatory postsynaptic potentials (mEPSPs) that summated to initiate APs. APs were blocked by tetrodotoxin and mEPSP frequency was modulated by caloric stimulation. Cobalt application reversibly blocked mEPSPs. Electron microscopic examination of physiologically studied afferents with CV greater than 0.4 revealed synaptic profiles consisting of typical synaptic bodies and synaptic vesicles in the type I hair cell presynaptic to the nerve chalice. Examples of the interspike baseline in regular and irregular afferents suggest differential modes of impulse initiation in these two fiber types.

  3. Projections from the medial cortex in the brain of lizards: correlation of anterograde and retrograde transport of horseradish peroxidase with Timm staining.

    PubMed

    Olucha, F; Martinez-Garcia, F; Poch, L; Schwerdtfeger, W K; Lopez-Garcia, C

    1988-10-22

    Efferent projections of the medial cortex of the lizards Podarcis hispanica and Gallotia stehlinii were studied by examining the transport of horseradish peroxidase; results were correlated with those from Timm-stained sections. Two efferent systems were found. The first reaches the distal part of the outer plexiform layer in the medial, dorsomedial, and dorsal cortices, i.e., zones that are negative to Timm staining, and possibly originates from horizontal fusiform neurons. The second reaches the Timm-positive zones in the cortex and septum and is topographically arranged: the vertical portion of the intermediate and caudal medial cortex and the entire rostral medial cortex project to the inner two-thirds of the outer plexiform layer of the dorsomedial cortex and of the medial subfield of the dorsal cortex; to the paraventricular zone of the inner plexiform layer of the medial cortex; and bilaterally to the dorsal part of the dorsal precommissural septum. The dorsal part of the intermediate and caudal medial cortex and the ventralmost folded part of its caudal edge project rostrally to the juxtasomatic zone of the outer plexiform layer and the entire inner plexiform layer of the intermediate and lateral subfields of the dorsal cortex and to the ventral part of the dorsal septum. In its intense Timm reaction and its ultrastructural properties, as reported in earlier studies, the Timm-positive fiber system of the lizard brain shows a close resemblance to the mossy fiber system of the mammalian hippocampus.

  4. 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.

  5. Realization of an ultra-sensitive hydrogen peroxide sensor with conductance change of horseradish peroxidase-immobilized polyaniline and investigation of the sensing mechanism.

    PubMed

    Fang, Kuan-Chung; Hsu, Chen-Pin; Kang, Yen-Wen; Fang, Jung-Ying; Huang, Chih-Cheng; Hsu, Chia-Hsien; Huang, Yu-Fen; Chen, Chih-Chen; Li, Sheng-Shian; Andrew Yeh, J; Yao, Da-Jeng; Wang, Yu-Lin

    2014-05-15

    In this study, we fabricate an ultra-sensitive hydrogen peroxide sensor by using horseradish peroxidase (HRP)-immobilized conducting polymer, polyaniline (PANI). With the proposed detection mechanism, hydrogen peroxide first oxidizes HRP, which then oxidizes polyaniline, thus resulting in decreased conductivity of the polyaniline thin film. The reduced HRP can be further oxidized by hydrogen peroxide and the cycle of the oxidation/reduction would continue until all hydrogen peroxide are reacted, leading to the high sensitivity of the sensor due to the signal contributed from all hydrogen peroxide molecule. The detection limit of this sensor is only 0.7 nM. The detectable concentration of H2O2 is from 0.7 nM to 1 μM. Beyond 1 μM, the sensor gradually saturates and some H2O2 remains, indicating the inhibition of HRP activity at high concentration of H2O2. There is no response to hydrogen peroxide once the PANI is standalone without HRP immobilized, showing the enzymatic reaction is required in the process of hydrogen peroxide detection. The simple process for the sensor fabrication allows the sensor to be cost-effective and disposable. This electronic hydrogen peroxide sensor is promising in applications for low concentration hydrogen peroxide detections, such as the reactive oxygen species (ROS) in oxidative stress studies.

  6. 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.

  7. Colorimetric assay for T4 polynucleotide kinase activity based on the horseradish peroxidase-mimicking DNAzyme combined with λ exonuclease cleavage.

    PubMed

    Jiang, Cheng; Yan, Chunyan; Jiang, Jianhui; Yu, Ruqin

    2013-03-01

    T4 polynucleotide kinase (PNK) plays a critical role in various cellular events. Here, we describe a novel colorimetric strategy for estimating the activity of PNK and screening its inhibitors taking advantage of the efficient cleavage of λ exonuclease and the horseradish peroxidase-mimicking DNAzyme (HRPzyme) signal amplification. A label-free hairpin DNA with the sequence of HRPzyme was utilized in the assay. The 5'-hydroxyl terminal of the hairpin DNA was firstly phosphorylated in the presence of PNK and then digested by λ exonuclease. As a result, the blocked 'HRPzyme' sequence of the hairpin DNA was released due to the removal of its completely complementary sequence. Using this strategy, the assay for PNK activity was successfully translated into the detection of HRPzyme. Because of the completely blocking and efficiently releasing of HRPzyme, the colorimetric method exhibited an excellent performance in PNK analysis with a low detection limit of 0.06 U mL(-1) and a wide detection range from 0.06 to 100 U mL(-1). Additionally, the effects of different inhibitors on PNK activity were also evaluated. The proposed strategy holds great potential in the development of high-throughput phosphorylation investigation as well as in the screening of the related drugs.

  8. Low Concentration of Silver Nanoparticles Not Only Enhances the Activity of Horseradish Peroxidase but Alter the Structure Also

    PubMed Central

    Karim, Zoheb; Adnan, Rohana; Ansari, Mohd Saquib

    2012-01-01

    Chemical synthesis of Ag-NPs was carried out using reduction method. The reduction mechanistic approach of silver ions was found to be a basic clue for the formation of the Ag-NPs. The nanoparticles were characterized by UV-vis, FT-IR and TEM analysis. We had designed some experiments in support of our hypothesis, “low concentrations of novel nanoparticles (silver and gold) increases the activity of plant peroxidases and alter their structure also”, we had used Ag-NPs and HRP as models. The immobilization/interaction experiment had demonstrated the specific concentration range of the Ag-NPs and within this range, an increase in HRP activity was reported. At 0.08 mM concentration of Ag-NPs, 50% increase in the activity yield was found. The U.V-vis spectra had demonstrated the increase in the absorbance of HRP within the reported concentration range (0.06–0.12 mM). Above and below this concentration range there was a decrease in the activity of HRP. The results that we had found from the fluorescence spectra were also in favor of our hypothesis. There was a maximum increase in ellipticity and α-helix contents in the presence of 0.08 mM concentration of Ag-NPs, demonstrated by circular dichroism (CD) spectra. Finally, incubation of a plant peroxidase, HRP with Ag-NPs, within the reported concentration range not only enhances the activity but also alter the structure. PMID:22848490

  9. 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

  10. 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

  11. 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

  12. Immobilization of horseradish peroxidase to a nano-Au monolayer modified chitosan-entrapped carbon paste electrode for the detection of hydrogen peroxide.

    PubMed

    Lei, Cun-Xi; Hu, Shun-Qin; Shen, Guo-Li; Yu, Ru-Qin

    2003-04-10

    A procedure for fabricating an enzyme electrode has been described based on the effective immobilization of horseradish peroxidase (HRP) to a nano-scaled particulate gold (nano-Au) monolayer modified chitosan-entrapped carbon paste electrode (CCPE). The high affinity of chitosan entrapped in CCPE for nano-Au associated with its amino groups has been utilized to realize the use of nano-Au as an intermediator to retain high bioactivity of the enzyme. Hydrogen peroxide (H(2)O(2)) was determined in the presence of hydroquinone as a mediator to transfer electrons between the electrode and HRP. The HRP immobilized on nano-Au displayed excellent electrocatalytical activity to the reduction of H(2)O(2). The effects of experimental variables such as the operating potential of the working electrode, mediator concentration and pH of measuring solution were investigated for optimum analytical performance by using an amperometric method. The enzyme electrode provided a linear response to hydrogen peroxide over a concentration range of 1.22 x 10(-5)-2.43 x 10(-3) mol l(-1) with a sensitivity of 0.013 A l mol(-1) cm(-2) and a detection limit of 6.3 micromol l(-1) based on signal per noise =3. The apparent Michaelis-Menten constant (K(m)(app)) for the sensor was found to be 0.36 mmol l(-1). The lifetime, fabrication reproducibility and measurement repeatability were evaluated with satisfactory results. The analysis results of real sample by this sensor were in satisfactory agreement with those of the potassium permanganate titration method. PMID:18968988

  13. 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.

  14. Inactivation of Escherichia coli glutamine synthetase by xanthine oxidase, nicotinate hydroxylase, horseradish peroxidase, or glucose oxidase: effects of ferredoxin, putidaredoxin, and menadione.

    PubMed

    Stadtman, E R; Wittenberger, M E

    1985-06-01

    Previous studies have shown that several mixed-function oxidation (MFO) systems are capable of catalyzing the inactivation of glutamine synthetase (GS) [R.L. Levine, C. N. Oliver, R. M. Fulks, and E. R. Stadtman (1978) Proc. Natl. Acad. Sci. USA 78, 2120-2124] and a number of the other enzymes [L. Fucci, C. N. Oliver, M. J. Coon, and E. R. Stadtman (1983) Proc. Natl. Acad. Sci. USA 80, 1521-1525]. It has now been found that in the presence of Fe(III), O2, and an appropriate electron donor (hypoxanthine or NADPH, respectively) glutamine synthetase is also inactivated by either milk xanthine oxidase or Clostridial nicotinate hydroxylase. Inactivation of glutamine synthetase by either of these flavoproteins is greatly stimulated by the presence of electron carrier proteins possessing nonheme-iron-sulfur (NHIS) clusters (i.e., ferredoxin or putidaredoxin) or by the presence of menadione. The inactivation reactions are partially inhibited by free radical scavengers, superoxide dismutase, (SOD), histidine, mannitol, dimethyl sulfoxide, and dimethylthiourea, and are inhibited completely by either Mn(II), EDTA, or catalase. The sensitivity to SOD inhibition is greatly suppressed when the xanthine oxidase system is supplemented with either ferredoxin or redoxin. In the presence of the latter NHIS-proteins (and only when they are present), MFO systems, comprised of either horseradish peroxidase and H2O2 or glucose oxidase, O2, and glucose, can also catalyze the inactivation of GS. The ability of ferredoxin and putidaredoxin to promote oxidation modification of GS by any one of these MFO systems suggests that proteins with NHIS centers may mediate the generation (or stabilization) of highly reactive radical intermediates.

  15. 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.

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

    PubMed

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

    2002-05-01

    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

  17. 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

  18. 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

  19. 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

  20. 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

  1. Origin of cerebellar projections to the region of the oculomotor complex, medial pontine reticular formation, and superior colliculus in New World monkeys: a retrograde horseradish peroxidase study.

    PubMed

    Gonzalo-Ruiz, A; Leichnetz, G R; Smith, D J

    1988-02-22

    Cerebellar projections to oculomotor-related brainstem regions were studied in four groups of New World (capuchin, squirrel) monkeys by using the retrograde transport of horseradish peroxidase (HRP) to determine the origin of the principal cerebellar influence on eye movement. Group A monkeys had HRP injections or transcannular HRP gel implants into the oculomotor complex (OMC), the largest of which involved adjacent paraoculomotor nuclei (e.g., ventral periaqueductal gray, PAG; nucleus of Darkschewitsch, ND; medial accessory nucleus of Bechterew, MAB; dorsomedial parvicellular red nucleus, dmPRN). All of these cases contained large numbers of retrogradely labeled cells in cell group Y. Whereas the smallest OMC injection only labeled a few cells in the dentate nucleus (DN), injections involving paraoculomotor nuclei produced labeling in all of the cerebellar nuclei except the basal interstitial nucleus (BIN). Injections extending into the ND and MAB produced particularly heavy labeling within the interposed nuclei. Group B monkeys had injections/implants into the medial pontine tegmentum and dorsomedial basilar pons. The pontine tegmental cases contained labeled cells in all cerebellar nuclei, but the DN was the most heavily labeled when the implant involved the nucleus reticularis tegmenti pontis (NRTP). Cases with injections into the caudal medial pontine tegmentum (nucleus reticularis pontis caudalis, NRPC), including the physiological paramedian pontine reticular formation (PPRF), but not NRTP, contained the largest number of labeled cells in the fastigial nucleus (FN) and lacked retrograde labeling in the DN. Dorsomedial basilar pontine cases contained almost no labeled cells in the FN, anterior interpositus nucleus (AIN), and posterior interpositus nucleus (PIN) but did contain DN labeling when the injection involved the NRTP. Two dorsomedial pontine tegmental cases and one dorsomedial basilar pontine case had more labeled cells in the BIN than in other cases

  2. Peripheral injury and anterograde transport of wheat germ agglutinin-horse radish peroxidase to the spinal cord.

    PubMed

    Valtschanoff, J G; Weinberg, R J; Rustioni, A

    1992-10-01

    Previous observations have revealed labeling in the extracellular space surrounding boutons and unmyelinated fibers in superficial laminae of the spinal cord after injection of the tracer wheat germ agglutinin conjugated to horseradish peroxidase in dorsal root ganglia. The degree of extracellular labeling appeared related to the extent of the damage to the ganglia at the time of the injection. To determine whether injury might produce extracellular labeling, we investigated the effects of unilateral nerve crush or transection on spinal labeling after bilateral injections of the tracer into sciatic nerves. Confirming previous reports, labeling was confined to small dorsal root ganglion cells and to spinal laminae I and II, suggesting a selective affinity of this tracer for unmyelinated fibers. Labeling of both ganglion neurons and superficial spinal laminae was increased on the injured side, probably as a result of increased efficiency of receptor-mediated endocytosis. Electron microscopical observations revealed that the tracer was largely confined to unmyelinated dorsal root fibers bilaterally; a higher percentage of these fibers were labeled on the injured side. In the dorsal horn, the tracer was predominantly within unmyelinated axons and their terminals on the control side, whereas most of the labeling was extracellular and transneuronal on the injured side. The extracellular labeling surrounded unmyelinated fibers and their terminals in the spinal cord, but was excluded from the synaptic cleft. The demonstration that injury is accompanied by significantly increased release of this tracer from the terminals of unmyelinated fibers into the extracellular space suggests that endogenous substances may be released after peripheral lesions as a central signal of injury.

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

    PubMed

    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 Trp(117) 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 (His(170) and Asp(247)) might trigger miss-coordination of the heme iron atom by His(170) residue and further enzyme activity loss.

  4. 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.

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

    PubMed

    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 Trp(117) 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 (His(170) and Asp(247)) might trigger miss-coordination of the heme iron atom by His(170) residue and further enzyme activity loss. PMID:25827765

  6. Production of endothelial cell-enclosing alginate-based hydrogel fibers with a cell adhesive surface through simultaneous cross-linking by horseradish peroxidase-catalyzed reaction in a hydrodynamic spinning process.

    PubMed

    Liu, Yang; Sakai, Shinji; Taya, Masahito

    2012-09-01

    We developed an alginate-based hydrogel fiber enabling to enclose endothelial cells, degradable on-demand by alginate lyase, and having a cell adhesive surface. The hydrogel fiber was obtained by extruding an aqueous solution of 4% (w/v) alginate derivative possessing phenolic hydroxyl moieties (Alg-Ph) and horseradish peroxidase (HRP) into a flow of aqueous solution containing 0.3 mM H(2)O(2) and gelatin derivative possessing Ph moieties (Gelatin-Ph). In the process, cross-linking of Alg-Ph resulting in a hydrogel fiber and immobilization of Gelatin-Ph on the surface of the hydrogel fiber were simultaneously accomplished by an HRP-catalyzed cross-linking reaction between Ph moieties. The diameter of the hydrogel fiber and the quantity of immobilized Gelatin-Ph on the fiber were controllable by changing the flow rates of the solutions and the concentration of HRP in the Alg-Ph-containing solution, respectively. The viability of the human endothelial cells enclosed in the hydrogel fibers obtained by 10 s of flowing in the H(2)O(2)-containing solution was 87.1%. In addition, the cells harvested from the hydrogel fibers through degradation using alginate lyase grew on tissue culture dishes in the same fashion as the cells seeded by a conventional subculture protocol. Human smooth muscle cells adhered, grew and achieved confluence on the surface of the hydrogel fibers. By degrading the hydrogel fibers using alginate lyase, a tubular cell construct was successfully obtained.

  7. Relative number of cells projecting from contralateral and ipsilateral nucleus isthmi to loci in the optic tectum is dependent on visuotopic location: horseradish peroxidase study in the leopard frog.

    PubMed

    Dudkin, E A; Gruberg, E R

    1999-11-15

    The leopard frog optic tectum is the principal target of the contralateral retina. The retinal terminals form a topographic map of the visual field. The tectum also receives bilateral topographic input from a midbrain structure called nucleus isthmi. In this study we determined the relative strength of n. isthmi projections to different loci in the tectum. Horseradish peroxidase (HRP) was applied at single superficial tectal locations in a series of leopard frogs. The application sites were distributed across the tectum. Retrogradely filled cells were counted in ipsilateral and contralateral nucleus isthmi. Although all regions of the tectum receive input from both n. isthmi, the relative number of labeled cells in the two n. isthmi is dependent on visuotopic location. Input to the rostromedial tectum representing the visual field ipsilateral to the labeled tectum comes primarily from the contralateral n. isthmi. Input to the caudolateral tectum representing the visual field contralateral to the labeled tectum originates mostly from the ipsilateral n. isthmi. Tectal application sites representing the visual midline had approximately equal numbers of labeled cells in the two n. isthmi. The results are similar at postapplication survival times ranging from 2 to 14 days. Using application of HRP to rostral tectum and application of nuclear yellow to caudal tectum, we show that the anisotropy in isthmi labeling is not due to take up of these labels by isthmotectal fibers passing through the application sites that terminate elsewhere.

  8. 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.

  9. 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

  10. 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

  11. Poria weirii as a Possible Commercial Source of Peroxidase

    PubMed Central

    Koenigs, J. W.

    1972-01-01

    Poria weirii produced peroxidase in yields amounting to 35% of those obtained from the same weight of horseradish roots. The three isozymes detected were distinct from those of horseradish. PMID:5018622

  12. Quantification of Horseradish Peroxidase Delivery into the Arterial Wall In Vivo as a Model of Local Drug Treatment: Comparison Between a Porous and a Gel-Coated Balloon Catheter

    SciTech Connect

    Dick, Armin; Kromen, Wolfgang; Juengling, Eberhard; Grosskortenhaus, Stephanie; Kammermeier, Helmut; Vorwerk, Dierk; Guenther, Rolf W.

    1999-09-15

    Purpose: To quantify horseradish peroxidase (HRP) delivery into the arterial wall, as a model of local drug delivery, and to compare two different percutaneous delivery balloons. Methods: Perforated and hydrophilic hydrogel-coated balloon catheters were used to deliver HRP in aqueous solution into the wall of porcine iliac arteries in vivo. HRP solutions of 1 mg/ml were used together with both perforated and hydrophilic hydrogel-coated balloon catheters and 40 mg/ml HRP solutions were used with the hydrogel-coated balloon only. The amount of HRP deposited in the arterial wall was then determined photospectrometrically. Results: Using the 1 mg/ml HRP solution, the hydrogel-coated balloon absorbed 0.047 mg HRP into the coating. Treatment with this balloon resulted in a mean vessel wall concentration of 7.4 {mu}g HRP/g tissue {+-} 93% (standard deviation) (n 7). Treatment with the hydrogel-coated balloon that had absorbed 1.88 mg HRP into the coating (using the 40 mg/ml HRP solution) led to a mean vessel wall concentration of 69.5 {mu}g HRP/g tissue {+-} 74% (n = 7). Treatment with the perforated balloon using 1 mg/ml aqueous HRP solution led to a mean vessel wall concentration of 174 {mu}g/g {+-} 81% (n = 7). Differences between the hydrogel-coated and perforated balloons (1 mg/g solutions of HRP) and between hydrogel-coated balloons (0.047 mg vs 1.88 mg absorbed into the balloon coating) were significant (p < 0.05; two-sided Wilcoxon test). Conclusions: The use of a perforated balloon catheter allowed the delivery of a higher total amount of HRP compared with the hydrogel-coated balloon, but at the cost of a higher systemic HRP application. To deliver 174 {mu}g HRP per gram of vessel wall with the perforated balloon, 6.5 {+-} 1.5 mg HRP were lost into the arterial blood (delivery efficiency range = 0.2%-0.3%). With 0.047 mg HRP loaded into the coating of the hydrogel balloon, 7.4 {mu}g HRP could be applied to 1 g of vessel wall (delivery efficiency 1.7%), and with 1

  13. Homology of Plant Peroxidases: AN IMMUNOCHEMICAL APPROACH.

    PubMed

    Conroy, J M; Borzelleca, D C; McDonell, L A

    1982-01-01

    Antisera specific for the basic peroxidase from horseradish (Amoracea rusticana) were used to examine homology among horseradish peroxidase isoenzymes and among basic peroxidases from root plants. The antisera cross-reacted with all tested isoperoxidases when measured by both agar diffusion and quantitative precipitin reactions. Precipitin analyses provided quantitative measurements of homology among these plant peroxidases. The basic radish (Raphanus sativus L. cv. Cherry Belle) peroxidase had a high degree of homology (73 to 81%) with the basic peroxidase from horseradish. Turnip (Brassica rapa L. cv. Purple White Top Globe) and carrot (Daucus carota L. cv. Danvers) basic peroxidases showed less cross-reaction (49 to 54% and 41 to 46%, respectively). However, the cross-reactions of antisera with basic peroxidases from different plants were greater than were those observed with acidic horseradish isoenzymes (30 to 35%). These experiments suggest that basic peroxidase isoenzymes are strongly conserved during evolution and may indicate that the basic peroxidases catalyze reactions involved in specialized cellular functions. Anticatalytic assays were poor indicators of homology. Even though homology among isoperoxidases was detected by other immunological methods, antibodies inhibited only the catalytic activity of the basic peroxidase from radish.

  14. Oscillations in the peroxidase-oxidase reaction: a comparison of different peroxidases.

    PubMed

    Kummer, U; Valeur, K R; Baier, G; Wegmann, K; Olsen, L F

    1996-04-17

    The nonlinear behavior of the peroxidase-oxidase reaction was studied using structurally different peroxidases. For the first time sustained oscillations with peroxidases other than horseradish peroxidase in a single-enzyme system were observed. All peroxidases that showed significant oxidase activity were able to generate sustained oscillations. When adjusting the overall reaction rate, either of the two modifiers 2,4-dichlorophenol or Methylene blue could be omitted from the reaction. Due to the observation of different enzyme intermediates when using different peroxidases, we conclude that the mechanisms responsible for oscillatory kinetics may vary from one peroxidase to the other.

  15. 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

  16. 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

  17. Evidence for peroxidase activity in Caralluma umbellata.

    PubMed

    Achar, Raghu Ram; Venkatesh, B K; Sharanappa, P; Priya, B S; Swamy, S Nanjunda

    2014-08-01

    Vast applications of peroxidases create an increasing demand to characterize peroxidases from new sources with more applicability potential. The aim of the present study was to check the presence of peroxidase activity from Caralluma umbellata. This is the first report on the C. umbellata peroxidase (CUP). The presence of peroxidase was revealed by the histochemical analysis of the stem sections, zymographic studies, and in vitro peroxidase activity assay using various reducing substrates viz., 2, 2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), guaiacol, o-dianisidine, and ferulic acid. The band pattern in zymogram confirms that CUP has a molecular weight less than that of horseradish peroxidase (44 kDa). Comparative evaluation of peroxidase activity of CUP with respect to horseradish peroxidase (HRP) indicates that CUP catalyzes ABTS and ferulic acid in a similar pattern as HRP but with guaiacol, the extent of catalysis shown by CUP over HRP is high. The standard inhibitors sodium azide and sodium meta bisulphite inhibited CUP activity in a dose dependent manner.

  18. 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.

  19. 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.

  20. 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

  1. Role of. pi. -cation radicals in the enzymatic cycles of peroxidases, catalases, and nitrite and sulfite reductases

    SciTech Connect

    Hanson, L K; Chang, C K; Davis, M S; Fajer, J

    1980-01-01

    Charge iterative extended Hueckel calculations, and magnetic and optical results on porphyrins, chlorins, and isobacteriochlorins (1) suggest that the catalytic cycles of the enzymes horseradish peroxidase, catalase, Neurospora crassa catalase, and nitrite and sulfite reductases proceed via ..pi..-cation radicals of their prosthetic groups; (2) offer distinguishing features for the optical and magnetic spectra of these radicals, pertinent to their detection as enzymatic intermediates; (3) reconcile the seemingly contradictory optical and NMR data on Compounds I of horseradish peroxidase; and (4) predict that the axial ligation of the heme differs for horseradish peroxidase and catalase.

  2. 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.

  3. 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

  4. Antigenic relationships between petunia peroxidase a and specific peroxidase isoenzymes in other Solanaceae.

    PubMed

    Hendriks, T; de Jong, A; Wijsman, H J; van Loon, L C

    1990-07-01

    A highly specific rabbit antiserum raised against peroxidase (PRXa) from petunia (Petunia hybrida) was used to investigate the antigenic relatedness of peroxidases in the Solanaceae. After SDS-PAGE of crude leaf extracts from a large number of species of this family, immunoblotting revealed that cross-reacting protein bands were present in all species tested. In order to determine whether these protein bands represent peroxidases, the peroxidase isoenzymes in thorn apple (Datura stramonium L.), tobacco (Nicotiana tabacum L.), sweet pepper (Capsicum annuum L.), potato (Solanum tuberosum L.), and tomato (Lycopersicon esculentum Mill.) were further analyzed. Immunoblots obtained after native PAGE revealed that the antiserum only recognized fast-moving peroxidase isoenzymes that are localized in the apoplast. Despite their serological relatedness, these peroxidases differed with respect to heat stability and apparent molecular weight. Differences in avidity for the petunia PRXa antiserum were suggested by immunoprecipitation with antibodies bound to protein A-Sepharose. The antiserum did not react with peroxidases from horseradish (Armoracea rusticana Gaertn., Mey and Scherb), turnip (Brassica napus L.), African marigold (Tagetes cresta L.), maize (Zea mays L.), and oats (Avena sativa L.). Apparently, the Solanaceae contain orthologous genes encoding the fast-moving anionic peroxidases homologous to petunia PRXa. PMID:24220819

  5. Antigenic relationships between petunia peroxidase a and specific peroxidase isoenzymes in other Solanaceae.

    PubMed

    Hendriks, T; de Jong, A; Wijsman, H J; van Loon, L C

    1990-07-01

    A highly specific rabbit antiserum raised against peroxidase (PRXa) from petunia (Petunia hybrida) was used to investigate the antigenic relatedness of peroxidases in the Solanaceae. After SDS-PAGE of crude leaf extracts from a large number of species of this family, immunoblotting revealed that cross-reacting protein bands were present in all species tested. In order to determine whether these protein bands represent peroxidases, the peroxidase isoenzymes in thorn apple (Datura stramonium L.), tobacco (Nicotiana tabacum L.), sweet pepper (Capsicum annuum L.), potato (Solanum tuberosum L.), and tomato (Lycopersicon esculentum Mill.) were further analyzed. Immunoblots obtained after native PAGE revealed that the antiserum only recognized fast-moving peroxidase isoenzymes that are localized in the apoplast. Despite their serological relatedness, these peroxidases differed with respect to heat stability and apparent molecular weight. Differences in avidity for the petunia PRXa antiserum were suggested by immunoprecipitation with antibodies bound to protein A-Sepharose. The antiserum did not react with peroxidases from horseradish (Armoracea rusticana Gaertn., Mey and Scherb), turnip (Brassica napus L.), African marigold (Tagetes cresta L.), maize (Zea mays L.), and oats (Avena sativa L.). Apparently, the Solanaceae contain orthologous genes encoding the fast-moving anionic peroxidases homologous to petunia PRXa.

  6. 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.

  7. Improved avidin-biotin-peroxidase complex (ABC) staining.

    PubMed

    Cattoretti, G; Berti, E; Schiró, R; D'Amato, L; Valeggio, C; Rilke, F

    1988-02-01

    A considerable intensification of the avidin-biotin-peroxidase complex staining system (ABC) was obtained by sequentially overlaying the sections to be immunostained with an avidin-rich and a biotin-rich complex. Each sequential addition contributed to the deposition of horseradish peroxidase on the immunostained site and allowed the subsequent binding of a complementary complex. With this technique a higher dilution of the antisera could be used and minute amounts of antigen masked by the fixative could be demonstrated on paraffin sections.

  8. Effect of reaction conditions on phenol removal by polymerization and precipitation using Coprinus cinereus peroxidase.

    PubMed

    Masuda, M; Sakurai, A; Sakakibara, M

    2001-03-01

    The quantitative relationships between removal efficiency of phenol and reaction conditions were investigated using Coprinus cinereus peroxidase. The most effective ratio of hydrogen peroxide to phenol was nearly 1/1 (mol/mol) at an adequate enzyme dose. 12.2 U of the enzyme was needed to remove 1 mg of phenol when our peroxidase preparation was used. At an insufficient peroxidase dose, the optimum pH value was 9.0, and lowering the reaction temperature led to the improvement of removal efficiency. At an excess peroxidase dose, almost 100% removal of phenol was obtained over a wide range of pH (5-9) and temperature (0-60 degrees C). Despite the presence of culture medium components, it was shown that Coprinus cinereus peroxidase had the same phenol polymerization performance as horseradish peroxidase or Arthromyces ramosus peroxidase.

  9. Vanadate as an inhibitor of plant and mammalian peroxidases.

    PubMed

    Serra, M A; Sabbioni, E; Marchesini, A; Pintar, A; Valoti, M

    Vanadate ions are shown to inhibit horseradish, squash, and rat intestinal peroxidases by following the reaction spectrophotometrically in a wide range of vanadate concentrations. I50 in phosphate buffer were 43, 9.4, and 535 microM, respectively. No inhibitory effect was found on cow milk lactoperoxidase and beef liver catalase. Gel filtration of peroxidases in the presence of vanadate, as carried out by radioactive 48V for horseradish peroxidases (either in aerobic or anoxic conditions) and neutron activation analysis (NAA) for squash peroxidase, demonstrated a binding of vanadium to these enzymes in stoichiometric amounts. Electron paramagnetic resonance spectra of the eluted peaks for the former peroxidase indicated that vanadium is in the +5 oxidation state, but an equilibrium between V (V) and V (IV) in the assay conditions cannot be discarded. Although the inhibitory mechanism remains obscure, some hypotheses are considered. The potential implications that the inhibitory effect of vanadium might have on plant and animal metabolism are also discussed. PMID:2484422

  10. Amino acid sequence of Coprinus macrorhizus peroxidase and cDNA sequence encoding Coprinus cinereus peroxidase. A new family of fungal peroxidases.

    PubMed

    Baunsgaard, L; Dalbøge, H; Houen, G; Rasmussen, E M; Welinder, K G

    1993-04-01

    Sequence analysis and cDNA cloning of Coprinus peroxidase (CIP) were undertaken to expand the understanding of the relationships of structure, function and molecular genetics of the secretory heme peroxidases from fungi and plants. Amino acid sequencing of Coprinus macrorhizus peroxidase, and cDNA sequencing of Coprinus cinereus peroxidase showed that the mature proteins are identical in amino acid sequence, 343 residues in size and preceded by a 20-residue signal peptide. Their likely identity to peroxidase from Arthromyces ramosus is discussed. CIP has an 8-residue, glycine-rich N-terminal extension blocked with a pyroglutamate residue which is absent in other fungal peroxidases. The presence of pyroglutamate, formed by cyclization of glutamine, and the finding of a minor fraction of a variant form lacking the N-terminal residue, indicate that signal peptidase cleavage is followed by further enzymic processing. CIP is 40-45% identical in amino-acid sequence to 11 lignin peroxidases from four fungal species, and 42-43% identical to the two known Mn-peroxidases. Like these white-rot fungal peroxidases, CIP has an additional segment of approximately 40 residues at the C-terminus which is absent in plant peroxidases. Although CIP is much more similar to horseradish peroxidase (HRP C) in substrate specificity, specific activity and pH optimum than to white-rot fungal peroxidases, the sequences of CIP and HRP C showed only 18% identity. Hence, CIP qualifies as the first member of a new family of fungal peroxidases. The nine invariant residues present in all plant, fungal and bacterial heme peroxidases are also found in CIP. The present data support the hypothesis that only one chromosomal CIP gene exists. In contrast, a large number of secretory plant and fungal peroxidases are expressed from several peroxidase gene clusters. Analyses of three batches of CIP protein and of 49 CIP clones revealed the existence of only two highly similar alleles indicating less

  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.

  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. Stability properties of an ancient plant peroxidase.

    PubMed

    Loughran, N B; O'Connell, M J; O'Connor, B; O'Fágáin, C

    2014-09-01

    Plant (Class III) peroxidases have numerous applications throughout biotechnology but their thermal and oxidative stabilities may limit their usefulness. Horseradish peroxidase isoenzyme C (HRPC) has good catalytic turnover and is moderately resistant to heat and to excess (oxidizing) concentrations of hydrogen peroxide. In contrast, HRP isoenzyme A2 (HRP A2) has better oxidative but poorer thermal stability, while soybean peroxidase (SBP) displays enhanced thermal stability. Intrigued by these variations amongst closely related enzymes, we previously used maximum likelihood methods (with application of Bayesian statistics) to infer an amino acid sequence consistent with their most recent common ancestor, the 'Grandparent' (GP). Here, we report the cloning and expression of active recombinant GP protein in Escherichia coli. GP's half-inactivation temperature was 45 °C, notably less than HRP C's, but its resistance to excess H2O2 was 2-fold greater. This resurrected GP protein enables a greater understanding of plant peroxidase evolution and serves as a test-bed to explore their ancestral properties.

  14. Stability properties of an ancient plant peroxidase.

    PubMed

    Loughran, N B; O'Connell, M J; O'Connor, B; O'Fágáin, C

    2014-09-01

    Plant (Class III) peroxidases have numerous applications throughout biotechnology but their thermal and oxidative stabilities may limit their usefulness. Horseradish peroxidase isoenzyme C (HRPC) has good catalytic turnover and is moderately resistant to heat and to excess (oxidizing) concentrations of hydrogen peroxide. In contrast, HRP isoenzyme A2 (HRP A2) has better oxidative but poorer thermal stability, while soybean peroxidase (SBP) displays enhanced thermal stability. Intrigued by these variations amongst closely related enzymes, we previously used maximum likelihood methods (with application of Bayesian statistics) to infer an amino acid sequence consistent with their most recent common ancestor, the 'Grandparent' (GP). Here, we report the cloning and expression of active recombinant GP protein in Escherichia coli. GP's half-inactivation temperature was 45 °C, notably less than HRP C's, but its resistance to excess H2O2 was 2-fold greater. This resurrected GP protein enables a greater understanding of plant peroxidase evolution and serves as a test-bed to explore their ancestral properties. PMID:24919139

  15. 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.

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

    PubMed

    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, we characterize the H2O2-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 Coprinus 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.

  17. 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

  18. 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

  19. Mouse Splenic Peroxidase and Its Role in Bactericidal Activity 1

    PubMed Central

    Strauss, R. R.; Paul, B. B.; Jacobs, A. A.; Sbarra, A. J.

    1972-01-01

    Spleen cell suspensions from AKR and CD-1 mice contain peroxidase activity as determined by guaiacol oxidation. This activity is found predominately in the 20,000 × g pellet fraction of spleen cell homogenates. In the presence of H2O2 and chloride ion at acidic pH, splenic peroxidase mediates the oxidation of d- or l-alanine to CO2, NH3, and acetaldehyde. The same reaction mixture without added amino acid can kill both gram-positive and gram-negative bacteria. The conditions for both reactions are similar. Both have an absolute requirement for H2O2 and chloride ion, neither is active at neutral or alkaline pH, and both are inhibited by the sulfonic amino acid taurine. In these aspects, splenic peroxidase is qualitatively similar in its activity to myeloperoxidase (MPO) from polymorphonuclear leukocytes. It is quantitatively different from MPO in that the latter is more potent on a per guaiacol unit basis with respect to both amino acid oxidation and bactericidal activity. Still another quantitative difference is that splenic peroxidase requires 0.1 m NaCl for activity, whereas MPO functions with as little as 0.005 m NaCl. Splenic peroxidase and MPO both appear to differ qualitatively from horseradish peroxidase in that the latter enzyme does not mediate amino acid oxidation. PMID:4632463

  20. Peroxidase-catalyzed color removal from bleach plant effluent.

    PubMed

    Paice, M G; Jurasek, L

    1984-05-01

    Effluent from the caustic extraction stage of a bleach plant is highly colored due to the presence of dissolved products from lignin chlorination and oxidation. Color removal from the effluent by hydrogen peroxide at neutral pH was catalyzed by addition of horseradish peroxidase. The catalysis with peroxidase (20 mg/L) was observed over a wide range of peroxide concentrations (0.1mM-500mM), but the largest effect was between 1mM and 100mM. The pH optimum for catalysis was around 5.0, while the basal rate of noncatalyzed peroxide color removal simply increased with pH within the range tested (3-10). Peroxidase catalysis at pH 7.6 reached a maximum at 40 degrees C in 4 h assays with 10mM peroxide, and disappeared above 60 degrees C. Compared with mycelial color removal by Coriolus versicolor, the rate of color removal by peroxide plus peroxidase was initially faster (first 4 h), but the extent of color removal after 48 h was higher with the fungal treatment. Further addition of peroxidase to the enzyme-treated effluent did not produce additional catalysis. Thus, the peroxide/peroxidase system did not fully represent the metabolic route used by the fungus.

  1. 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.

  2. NMR studies of recombinant Coprinus peroxidase and three site-directed mutants. Implications for peroxidase substrate binding.

    PubMed

    Veitch, N C; Tams, J W; Vind, J; Dalbøge, H; Welinder, K G

    1994-06-15

    Proton nuclear magnetic resonance spectroscopy has been used to characterise and compare wild-type fungal and recombinant Coprinus cinereus peroxidase (CIP) and three mutants in which Gly156 and/or Asn157 was replaced by Phe. Analysis of one- and two-dimensional NMR spectra of recombinant CIP was undertaken for comparison with the fungal enzyme and in order to establish a meaningful basis for solution studies of CIP mutants. Proton resonance assignments of haem and haem-linked residues obtained for the cyanide-ligated form of recombinant CIP revealed a high degree of spectral similarity with those of lignin and manganese-dependent peroxidases and extend previously reported NMR data for fungal CIP. The three mutants examined by NMR spectroscopy comprised site-specific substitutions made to a region of the structure believed to form part of the peroxidase haem group access channel for substrate and ligand molecules. Proton resonances of the aromatic side-chains of Phe156 and Phe157 were found to have similar spectral characteristics to those of two phenylalanine residues known to be involved in the binding of aromatic donor molecules to the plant peroxidase, horseradish peroxidase isoenzyme C. The results are discussed in the context of complementary reactivity studies on the mutants in order to develop a more detailed understanding of aromatic donor molecule binding to fungal and plant peroxidases.

  3. Sequence and tissue-specific expression of a putative peroxidase gene from wheat (Triticum aestivum L.).

    PubMed

    Hertig, C; Rebmann, G; Bull, J; Mauch, F; Dudler, R

    1991-01-01

    We have used a cDNA clone encoding a pathogen-induced putative wheat peroxidase to screen a genomic library of wheat (Triticum aestivum L. cv. Cheyenne) and isolated one positive clone, lambda POX1. Sequence analysis revealed that this clone contains a gene encoding a putative peroxidase with a calculated pI of 8.1 which exhibits 58% and 83% sequence identity to the amino acid sequence of the turnip (Brassica rapa) peroxidase and a pathogen-induced putative wheat peroxidase, respectively. The two introns in the wheat gene are at the same positions as introns in the peroxidase genes of tomato and horseradish. Results of S1-mapping experiments suggest that this gene is neither pathogen- nor wound-induced in leaves but is constitutively expressed in roots.

  4. Sequence and tissue-specific expression of a putative peroxidase gene from wheat (Triticum aestivum L.).

    PubMed

    Hertig, C; Rebmann, G; Bull, J; Mauch, F; Dudler, R

    1991-01-01

    We have used a cDNA clone encoding a pathogen-induced putative wheat peroxidase to screen a genomic library of wheat (Triticum aestivum L. cv. Cheyenne) and isolated one positive clone, lambda POX1. Sequence analysis revealed that this clone contains a gene encoding a putative peroxidase with a calculated pI of 8.1 which exhibits 58% and 83% sequence identity to the amino acid sequence of the turnip (Brassica rapa) peroxidase and a pathogen-induced putative wheat peroxidase, respectively. The two introns in the wheat gene are at the same positions as introns in the peroxidase genes of tomato and horseradish. Results of S1-mapping experiments suggest that this gene is neither pathogen- nor wound-induced in leaves but is constitutively expressed in roots. PMID:1653627

  5. 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

  6. Barley peroxidase isozymes

    NASA Astrophysics Data System (ADS)

    Laugesen, Sabrina; Bak-Jensen, Kristian Sass; Hägglund, Per; Henriksen, Anette; Finnie, Christine; Svensson, Birte; Roepstorff, Peter

    2007-12-01

    Thirteen peroxidase spots on two-dimensional gels were identified by comprehensive proteome analysis of the barley seed. Mass spectrometry tracked multiple forms of three different peroxidase isozymes: barley seed peroxidase 1, barley seed-specific peroxidase BP1 and a not previously identified putative barley peroxidase. The presence of multiple spots for each of the isozymes reflected variations in post-translational glycosylation and protein truncation. Complete sequence coverage was achieved by using a series of proteases and chromatographic resins for sample preparation prior to mass spectrometric analysis. Distinct peroxidase spot patterns divided the 16 cultivars tested into two groups. The distribution of the three isozymes in different seed tissues (endosperm, embryo, and aleurone layer) suggested the peroxidases to play individual albeit partially overlapping roles during germination. In summary, a subset of three peroxidase isozymes was found to occur in the seed, whereas products of four other barley peroxidase genes were not detected. The present analysis documents the selective expression profiles and post-translational modifications of isozymes from a large plant gene family.

  7. 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

  8. 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

  9. Purification, crystallization, and characterization of peroxidase from Coprinus cinereus.

    PubMed

    Morita, Y; Yamashita, H; Mikami, B; Iwamoto, H; Aibara, S; Terada, M; Minami, J

    1988-04-01

    Peroxidase (donor: H2O2 oxidoreductase [EC 1.11.1.7]) was purified from a culture broth of an inkcap Basidiomycete, Coprinus cinereus S.F. Gray. A single component containing a low amount of carbohydrate was isolated by affinity chromatography on concanavalin A-Sepharose and crystallized from ammonium sulfate solution. The enzyme is an acidic protein (pI 3.5) and consists of a single polypeptide chain having the molecular weight of 41,600 daltons. The enzyme contains one protohemin per molecule and exhibits the characteristic absorption, circular dichroism, and magnetic circular dichroism spectra of a heme-protein. The Coprinus peroxidase forms two characteristic intermediate compounds, I and II, and the rate constants for hydrogen peroxide and guaiacol had similar values to those for higher plant peroxidases. The ferric enzyme formed a cyanide compound with a dissociation constant similar to those for higher plant enzyme, but the dissociation constant of the ferrous enzyme-cyanide was large. The chemical composition of Coprinus peroxidase showed 381 amino acid residues, 1 glucosamine, 3 true sugars, 3 calcium, and 1 non-heme iron other than 1 protohemin. The secondary structure of the fungal enzyme was very similar to that of horseradish peroxidase.

  10. 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.

  11. Structure of soybean seed coat peroxidase: A plant peroxidase with unusual stability and haem-apoprotein interactions

    PubMed Central

    Henriksen, Anette; Mirza, Osman; Indiani, Chiara; Teilum, Kaare; Smulevich, Giulietta; Welinder, Karen G.; Gajhede, Michael

    2001-01-01

    Soybean seed coat peroxidase (SBP) is a peroxidase with extraordinary stability and catalytic properties. It belongs to the family of class III plant peroxidases that can oxidize a wide variety of organic and inorganic substrates using hydrogen peroxide. Because the plant enzyme is a heterogeneous glycoprotein, SBP was produced recombinant in Escherichia coli for the present crystallographic study. The three-dimensional structure of SBP shows a bound tris(hydroxymethyl)aminomethane molecule (TRIS). This TRIS molecule has hydrogen bonds to active site residues corresponding to the residues that interact with the small phenolic substrate ferulic acid in the horseradish peroxidase C (HRPC):ferulic acid complex. TRIS is positioned in what has been described as a secondary substrate-binding site in HRPC, and the structure of the SBP:TRIS complex indicates that this secondary substrate-binding site could be of functional importance. SBP has one of the most solvent accessible δ-meso haem edge (the site of electron transfer from reducing substrates to the enzymatic intermediates compound I and II) so far described for a plant peroxidase and structural alignment suggests that the volume of Ile74 is a factor that influences the solvent accessibility of this important site. A contact between haem C8 vinyl and the sulphur atom of Met37 is observed in the SBP structure. This interaction might affect the stability of the haem group by stabilisation/delocalisation of the porphyrin π-cation of compound I. PMID:11266599

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

    PubMed

    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 Fe(2+) 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 Fe(2+) under alkaline condition.

  13. Purification, characterization and evaluation of extracellular peroxidase from two Coprinus species for aqueous phenol treatment.

    PubMed

    Ikehata, Keisuke; Buchanan, Ian D; Pickard, Michael A; Smith, Daniel W

    2005-11-01

    Non-ligninolytic fungal peroxidases produced by Coprinus cinereus UAMH 4103 and Coprinus sp. UAMH 10067 were purified, characterized and evaluated as cost-effective alternatives to horseradish peroxidase for aqueous phenol treatment. Purified Coprinus peroxidases exhibited a molecular weight of 36 kDa on matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Although the catalytic properties of the two Coprinus peroxidases were nearly identical in both crude and purified forms, the stabilities were substantially different. The peroxidase from Coprinus sp. UAMH 10067 was more stable at 50 degrees C and under basic conditions (up to pH 10) than the enzyme from C. cinereus UAMH 4103. The former enzyme also performed better at pH 9 than the latter one in aqueous phenol treatment. The phenol removal efficiency of the Coprinus peroxidase was comparable to those of previously studied plant peroxidases. The broader working pH and higher thermal and alkaline stability of the peroxidase from Coprinus sp. UAMH 10067 may be advantageous for its application to industrial wastewater treatment.

  14. Mechanism of reaction of chlorite with mammalian heme peroxidases.

    PubMed

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

    2014-06-01

    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.

  15. 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.

  16. Specific Binding of Peroxidase-Labeled Myelin Basic Protein in Allergic Encephalomyelitis

    PubMed Central

    Johnson, Anne B.; Wiśniewski, Henryk M.; Raine, Cedric S.; Eylar, E. H.; Terry, Robert D.

    1971-01-01

    A conjugate of horseradish peroxidase and the encephalitogenic basic protein from myelin has been used to study the antigen reactivity of tissue in the autoimmune disease, experimental allergic encephalomyelitis. Control conjugates were also prepared of peroxidase and bovine serum albumin and of peroxidase and lysozyme, another basic protein. The basic protein from myelin conjugate was specifically bound by lymph node cells from rabbits immunized against the basic protein. Some of these cells appeared to be plasma cells. The conjugate was also specifically bound by occasional cells in the spinal-cord infiltrates of animals with early signs of allergic encephalomyelitis. These cells resembled large lymphocytes and plasma cells. There was no difference between the binding of basic protein of bovine and rabbit origin. The findings suggest the possibility that a local release of antibody within the target organ may play a role in the pathogenesis of allergic encephalomyelitis. Images PMID:5288245

  17. Specific binding of peroxidase-labeled myelin basic protein in allergic encephalomyelitis.

    PubMed

    Johnson, A B; Wiśniewski, H M; Raine, C S; Eylar, E H; Terry, R D

    1971-11-01

    A conjugate of horseradish peroxidase and the encephalitogenic basic protein from myelin has been used to study the antigen reactivity of tissue in the autoimmune disease, experimental allergic encephalomyelitis. Control conjugates were also prepared of peroxidase and bovine serum albumin and of peroxidase and lysozyme, another basic protein. The basic protein from myelin conjugate was specifically bound by lymph node cells from rabbits immunized against the basic protein. Some of these cells appeared to be plasma cells. The conjugate was also specifically bound by occasional cells in the spinal-cord infiltrates of animals with early signs of allergic encephalomyelitis. These cells resembled large lymphocytes and plasma cells. There was no difference between the binding of basic protein of bovine and rabbit origin. The findings suggest the possibility that a local release of antibody within the target organ may play a role in the pathogenesis of allergic encephalomyelitis.

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. Role of peroxidase inhibition by insulin in the bovine thyroid cell proliferation mechanism.

    PubMed

    Krawiec, León; Pizarro, Ramón A; Aphalo, Paula; de Cavanagh, Elena M V; Pisarev, Mario A; Juvenal, Guillermo J; Policastro, Lucía; Bocanera, Laura V

    2004-07-01

    Monolayer primary cultures of thyroid cells produce, in the presence of insulin, a cytosolic inhibitor of thyroid peroxidase (TPO), lacto peroxidase (LPO), horseradish peroxidase (HRPO) and glutathione peroxidase (GPX). The inhibitor, localized in the cytosol, is thermostable and hydrophylic. Its molecular mass is less than 2 kDa. The inhibitory activity, resistant to proteolytic and nucleolytic enzymes, disappears with sodium metaperiodate treatment, as an oxidant of carbohydrates, supporting its oligosaccharide structure. The presence of inositol, mannose, glucose, the specific inhibition of cyclic AMP-dependent protein kinase and the disappearance of peroxidase inhibition by alkaline phosphatase and alpha-mannosidase in purified samples confirms its chemical structure as inositol phosphoglycan-like. Purification by anionic interchange shows that the peroxidase inhibitor elutes like the two subtypes of inositol phosphoglycans (IPG)P and A, characterized as signal transducers of insulin action. Insulin significantly increases the concentration of the peroxidase inhibitor in a thyroid cell culture at 48 h. The addition of both isolated substances to a primary thyroid culture produces, after 30 min, a significant increase in hydrogen peroxide (H2O2) concentration in the medium, concomitantly with the disappearance of the GPX activity in the same conditions. The presence of insulin or anyone of both products, during 48 h, induces cell proliferation of the thyroid cell culture. In conclusion, insulin stimulates thyroid cell division through the effect of a peroxidase inhibitor, as its second messenger. The inhibition of GPX by its action positively modulates the H2O2 level, which would produce, as was demonstrated by other authors, the signal for cell proliferation.

  4. 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.

  5. 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

  6. Ca2+ activation of wheat peroxidase: a possible physiological mechanism of control.

    PubMed

    Converso, D A; Fernández, M E

    1996-09-01

    Peroxidation of substrates such as ascorbic acid, pyrogallol, or ferulic acid, as well as indole acetic acid oxidation catalyzed by wheat germ peroxidase (WGP)2 C2, were found to be activated by Ca2+. This activation is independent of the stabilizing effect of structural Ca2+ reported for peroxidases. Steady state kinetics of ferulic acid oxidation catalyzed by WGP C2 showed an increase in the rate of compound I formation and of compound II decomposition in the presence of the ion, evidenced as an increase in rate constants k1, from 8.9 x 10(5) to 4.5 x 10(5) M-1 cm-1, and k3, from 4.4 x 10(5) to 1.1 x 10(6) M-1 cm-1. The dissociation constant Kd, for the cyanide derivative of the enzyme showed a marked decrease from 220 to 34 microM in the presence of Ca2+, thus implying an effect of the ion in the H2O2 binding step. In the presence of Ca2+, a conformational change in the protein was revealed by tryptophan fluorescence, providing a basis for the activation mechanism. Other peroxidases such as horseradish peroxidase and WGP C3 were not activated by Ca2+. The results suggest the existence of a physiological mechanism of control of peroxidase isozymes activity mediated by Ca2+.

  7. Evidence for an unusual electronic structure of wheat germ peroxidase compound I.

    PubMed

    Converso, D A; Fernández, M E

    1998-09-01

    Oxidized states of wheat germ peroxidase isozyme C2 (WGP C2) were investigated by means of electronic absorption spectroscopy. Addition of one molar equivalent of H2O2 to ferric WGP C2 led to the formation of an oxidized species with an absorption spectrum very similar to that of peroxidase compound II, with a Soret maximum at 411 nm and visible maxima at 523 and 553 nm. The transformation took place with an isosbestic point at 409 nm. Stopped flow spectroscopy showed no inflection points for the formation of this species when it was registered at 420 nm, and we could verify the persistence of the isosbestic point from 20 ms to 10 s. The oxidized species decays spontaneously to ferric enzyme in a double-exponential manner. By adding excess H2O2 to the system we obtained an inactive derivative identical to horseradish peroxidase P-670. In the presence of one equivalent of reducing substrate and excess H2O2 compound III was formed. The results so indicate that the species obtained in the reaction of WGP C2 with equimolecular amounts of H2O2 is compound I. The resulting compound I spectrum was identical to that of cytochrome c peroxidase, suggesting the formation of a protein radical rather than the typical pi cation radical, a feature which had not been described before for a plant peroxidase.

  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.

  9. 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

  10. Hierarchical hybrid peroxidase catalysts for remediation of phenol wastewater.

    PubMed

    Duan, Xiaonan; Corgié, Stéphane C; Aneshansley, Daniel J; Wang, Peng; Walker, Larry P; Giannelis, Emmanuel P

    2014-04-01

    We report a new family of hierarchical hybrid catalysts comprised of horseradish peroxidase (HRP)-magnetic nanoparticles for advanced oxidation processes and demonstrate their utility in the removal of phenol from water. The immobilized HRP catalyzes the oxidation of phenols in the presence of H2 O2 , producing free radicals. The phenoxy radicals react with each other in a non-enzymatic process to form polymers, which can be removed by precipitation with salts or condensation. The hybrid peroxidase catalysts exhibit three times higher activity than free HRP and are able to remove three times more phenol from water compared to free HRP under similar conditions. In addition, the hybrid catalysts reduce substrate inhibition and limit inactivation from reaction products, which are common problems with free or conventionally immobilized enzymes. Reusability is improved when the HRP-magnetic nanoparticle hybrids are supported on micron-scale magnetic particles, and can be retained with a specially designed magnetically driven reactor. The performance of the hybrid catalysts makes them attractive for several industrial and environmental applications and their development might pave the way for practical applications by eliminating most of the limitations that have prevented the use of free or conventionally immobilized enzymes.

  11. Effects of terbium (III) on signaling molecules in horseradish.

    PubMed

    Wang, Lihong; Zhang, Xuanbo; Zhou, Qing; Huang, Xiaohua

    2015-03-01

    Rare earth elements, especially terbium (Tb), are high-valence heavy metal elements that accumulate in the environment, and they show toxic effects on plants. Signaling molecules regulate many physiological and biochemical processes in plants. How rare earth elements affect signaling molecules remains largely unknown. In the present study, the effects of Tb(3+) on some extracellular and intracellular signaling molecules (gibberellic acid, abscisic acid, auxin, H2O2, and Ca(2+)) in horseradish leaves were investigated by using high-performance liquid chromatography, X-ray energy spectrometry, and transmission electron microscopy, and Tb(3+) was sprayed on the surface of leaves. Tb(3+) treatment decreased the auxin and gibberellic acid contents and increased the abscisic acid content. These changes in the contents of phytohormones (gibberellic acid, abscisic acid, and auxin) triggered excessive production of intracellular H2O2. Consequently, the increase in H2O2 content stimulated the influx of extracellular Ca(2+) and the release of Ca(2+) from Ca(2+) stores, leading to Ca(2+) overload and the resulting inhibition of physiological and biochemical processes. The effects outlined above were more evident with increasing the concentration of Tb(3+) sprayed on horseradish leaves. Our data provide a possible underlying mechanism of Tb(3+) action on plants.

  12. Oligosynaptic pathways possibly relaying visceral and/or gustatory information to the olfactory bulb in the hedgehog tenrec.

    PubMed

    Künzle, H; Radtke-Schuller, S

    2001-04-27

    Using anterograde and retrograde transport of wheat germ agglutinin we showed that the parabrachial nucleus, known to receive second order visceral and gustatory afferents, might project directly to the anterior olfactory nucleus which is connected with the olfactory bulb (OfB). Only a small bulbar region is targeted directly by parabrachial fibers. This region is located immediately adjacent to the accessory OfB and may be closely related to, if not identical with the modified glomerular complex. To further substantiate the presence of true parabrachio-bulbar projections thyrosine hydroxylase immunohistochemistry was employed. The absence of immunoreactive neurons in the parabrachial nucleus and the different distribution patterns of immunoreactive fibers and axons labeled with wheat germ agglutinin conjugated to horseradish peroxidase in the target areas make it unlikely that catecholaminergic fibers were involved in the projections shown.

  13. Analysis of an Experimental Cortical Network: ii) Connections of Visual Areas 17 and 18 After Neonatal Injections of Ibotenic Acid

    PubMed Central

    Innocenti, G. M.; Berbel, P.

    1991-01-01

    Lesions of cortical areas 17 and 18 were produced in newborn kittens by local injections of the excitotoxin ibotenic acid. In the adult this results in a microcortex which consists of superficial layers I, II and III, in the absence of granular and infragranular layers. Horseradish peroxidase, alone or wheat germ agglutinin conjugated, was injected in the microcortex or in the contralateral, intact areas 17 and 18. The microcortex maintains several connections characteristic of normal areas 17 and 18 of the cat. It receives afferents from the dLGN, and several visual areas of the ipsilateral and contralateral hemisphere. However, it has lost its projections to dLGN, superior colliculus, and, at least in part, those to contralateral visual areas. Thus some parts of the microcortex receive from, but do not project into, the corpus callosum. In addition, the microcortex maintains afferents from ipsilateral and contralateral auditory areas AI and AII which are normally eliminated in development. PMID:1714302

  14. Dihydrotetramethylrosamine: a long wavelength, fluorogenic peroxidase substrate evaluated in vitro and in a model phagocyte.

    PubMed

    Whitaker, J E; Moore, P L; Haugland, R P; Haugland, R P

    1991-03-15

    Dihydrotetramethylrosamine, a fluorogenic substrate for peroxidase, and its fluorescent oxidation product, tetramethylrosamine chloride, were evaluated. The substrate is colorless and nonfluorescent while the oxidized dye absorbs at 550 nm and emits at 574 nm in both methanol and water. In vitro assays demonstrated that the substrate was oxidized to the fluorophore by horseradish peroxidase in the presence of hydrogen peroxide. In vivo uptake and oxidation of the substrate by Amoeba proteus was characterized by the initial appearance of fluorescent phagocytic vacuoles with subsequent localization in vesicular organelles the size and shape of protozoan mitochondria. Similar staining patterns occurred in cells incubated with substrate, oxidized rosamine or rhodamine 123, a known mitochondrial stain. PMID:2018489

  15. 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

  16. Effects of rare earth elements on the distribution of mineral elements and heavy metals in horseradish.

    PubMed

    Wang, Lihong; Huang, Xiaohua; Zhou, Qing

    2008-09-01

    In order to investigate the effects of rare earth elements (REEs) on horseradish, the distribution of the mineral elements and heavy metals in different organs of horseradish have been studied by using inductively coupled plasma-atomic emission spectrometry (ICP-AES). Meanwhile, three variable major parameters, namely the concentration of REEs, the type of REEs, and the growth stage of plant were chosen. The results indicated that the test REEs, Ce(III) and Tb(III), could be accumulated in leaves, stems and roots of horseradish. In addition, we found that the content of mineral elements was increased in horseradish treated with 20mgl(-1) of Ce(III), but not those with the 20mgl(-1) of Tb(III). Moreover, the content of mineral elements in horseradish was decreased with the increasing concentration of REEs (100, 300mgl(-1)). Furthermore, we found that there were the opposite effects on the content of the heavy metals in horseradish treated with REEs. Finally, we found that the effect of REEs on the accumulation of REEs, and the content of mineral elements or heavy metals of horseradish during vigorous growth stage, no matter positive or negative, was more obvious than that of the other growth stages. These results demonstrated that the distribution behaviors of mineral elements and heavy metals in horseradish can be affected by the type and concentration of REEs, and the growth period of plant.

  17. IgG abzymes with peroxidase and oxidoreductase activities from the sera of healthy humans.

    PubMed

    Tolmacheva, Anna S; Blinova, Elena A; Ermakov, Evgeny A; Buneva, Valentina N; Vasilenko, Nataliya L; Nevinsky, Georgy A

    2015-09-01

    We present the evidence showing that small fractions of electrophoretically homogeneous immunoglobulin G (IgGs) from the sera of healthy humans and their Fab and F(ab)2 fragments oxidize 3,3'-diaminobenzidine through a peroxidase activity in the presence of H2 O2 and through an oxidoreductase activity in the absence of H2 O2 . During purification on protein G-Sepharose and gel filtration, the polyclonal IgGs partially lose the Me(2+) ions. After extensive dialysis of purified Abs against agents chelating metal ions, the relative peroxidase activity decreased dependently of IgG analyzed from 100 to ~10-85%, while oxidoreductase activity from 100 to 14-83%. Addition of external metal ions to dialyzed and non-dialyzed IgGs leads to a significant increase in their activity. Chromatography of the IgGs on Chelex non-charged with Cu(2+) ions results in the adsorption of a small IgG fraction bound with metal ions (~5%), while Chelex charged with Cu(2+) ions bind additionally ~38% of the total IgGs. Separation of Abs on both sorbents results in IgG separation to many different subfractions demonstrating various affinities to the chelating resin and different levels of the specific oxidoreductase and peroxidase activities. In the presence of external Cu(2+) ions, the specific peroxidase activity of several IgG subfractions achieves 20-27 % as compared with horseradish peroxidase (HRP, taken for 100%). The oxidoreductase activity of these fractions is ~4-6-fold higher than that for HRP. Antioxidant enzymes such as superoxide dismutases, catalases, and glutathione peroxidases are known to represent critical defence mechanisms for preventing oxidative modifications of DNA, proteins, and lipids. Peroxidase and oxidoreductase activities of human IgGs could also play an important role in the protection of organisms from oxidative stress and toxic compounds.

  18. 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

  19. Catalytic Profile of Arabidopsis Peroxidases, AtPrx-2, 25 and 71, Contributing to Stem Lignification

    PubMed Central

    Shigeto, Jun; Nagano, Mariko; Fujita, Koki; Tsutsumi, Yuji

    2014-01-01

    Lignins are aromatic heteropolymers that arise from oxidative coupling of lignin precursors, including lignin monomers (p-coumaryl, coniferyl, and sinapyl alcohols), oligomers, and polymers. Whereas plant peroxidases have been shown to catalyze oxidative coupling of monolignols, the oxidation activity of well-studied plant peroxidases, such as horseradish peroxidase C (HRP-C) and AtPrx53, are quite low for sinapyl alcohol. This characteristic difference has led to controversy regarding the oxidation mechanism of sinapyl alcohol and lignin oligomers and polymers by plant peroxidases. The present study explored the oxidation activities of three plant peroxidases, AtPrx2, AtPrx25, and AtPrx71, which have been already shown to be involved in lignification in the Arabidopsis stem. Recombinant proteins of these peroxidases (rAtPrxs) were produced in Escherichia coli as inclusion bodies and successfully refolded to yield their active forms. rAtPrx2, rAtPrx25, and rAtPrx71 were found to oxidize two syringyl compounds (2,6-dimethoxyphenol and syringaldazine), which were employed here as model monolignol compounds, with higher specific activities than HRP-C and rAtPrx53. Interestingly, rAtPrx2 and rAtPrx71 oxidized syringyl compounds more efficiently than guaiacol. Moreover, assays with ferrocytochrome c as a substrate showed that AtPrx2, AtPrx25, and AtPrx71 possessed the ability to oxidize large molecules. This characteristic may originate in a protein radical. These results suggest that the plant peroxidases responsible for lignin polymerization are able to directly oxidize all lignin precursors. PMID:25137070

  20. Catalytic profile of Arabidopsis peroxidases, AtPrx-2, 25 and 71, contributing to stem lignification.

    PubMed

    Shigeto, Jun; Nagano, Mariko; Fujita, Koki; Tsutsumi, Yuji

    2014-01-01

    Lignins are aromatic heteropolymers that arise from oxidative coupling of lignin precursors, including lignin monomers (p-coumaryl, coniferyl, and sinapyl alcohols), oligomers, and polymers. Whereas plant peroxidases have been shown to catalyze oxidative coupling of monolignols, the oxidation activity of well-studied plant peroxidases, such as horseradish peroxidase C (HRP-C) and AtPrx53, are quite low for sinapyl alcohol. This characteristic difference has led to controversy regarding the oxidation mechanism of sinapyl alcohol and lignin oligomers and polymers by plant peroxidases. The present study explored the oxidation activities of three plant peroxidases, AtPrx2, AtPrx25, and AtPrx71, which have been already shown to be involved in lignification in the Arabidopsis stem. Recombinant proteins of these peroxidases (rAtPrxs) were produced in Escherichia coli as inclusion bodies and successfully refolded to yield their active forms. rAtPrx2, rAtPrx25, and rAtPrx71 were found to oxidize two syringyl compounds (2,6-dimethoxyphenol and syringaldazine), which were employed here as model monolignol compounds, with higher specific activities than HRP-C and rAtPrx53. Interestingly, rAtPrx2 and rAtPrx71 oxidized syringyl compounds more efficiently than guaiacol. Moreover, assays with ferrocytochrome c as a substrate showed that AtPrx2, AtPrx25, and AtPrx71 possessed the ability to oxidize large molecules. This characteristic may originate in a protein radical. These results suggest that the plant peroxidases responsible for lignin polymerization are able to directly oxidize all lignin precursors.

  1. A putative peroxidase cDNA from turnip and analysis of the encoded protein sequence.

    PubMed

    Romero-Gómez, S; Duarte-Vázquez, M A; García-Almendárez, B E; Mayorga-Martínez, L; Cervantes-Avilés, O; Regalado, C

    2008-12-01

    A putative peroxidase cDNA was isolated from turnip roots (Brassica napus L. var. purple top white globe) by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Total RNA extracted from mature turnip roots was used as a template for RT-PCR, using a degenerated primer designed to amplify the highly conserved distal motif of plant peroxidases. The resulting partial sequence was used to design the rest of the specific primers for 5' and 3' RACE. Two cDNA fragments were purified, sequenced, and aligned with the partial sequence from RT-PCR, and a complete overlapping sequence was obtained and labeled as BbPA (Genbank Accession No. AY423440, named as podC). The full length cDNA is 1167bp long and contains a 1077bp open reading frame (ORF) encoding a 358 deduced amino acid peroxidase polypeptide. The putative peroxidase (BnPA) showed a calculated Mr of 34kDa, and isoelectric point (pI) of 4.5, with no significant identity with other reported turnip peroxidases. Sequence alignment showed that only three peroxidases have a significant identity with BnPA namely AtP29a (84%), and AtPA2 (81%) from Arabidopsis thaliana, and HRPA2 (82%) from horseradish (Armoracia rusticana). Work is in progress to clone this gene into an adequate host to study the specific role and possible biotechnological applications of this alternative peroxidase source. PMID:18686036

  2. Haem iron-containing peroxidases.

    PubMed

    Isaac, I S; Dawson, J H

    1999-01-01

    Peroxidases are enzymes that utilize hydrogen peroxide to oxidize substrates. A histidine residue on the proximal side of the haem iron ligates most peroxidases. The various oxidation states and ligand complexes have been spectroscopically characterized. HRP-I is two oxidation states above ferric HRP. It contains an oxoferryl (= oxyferryl) iron with a pi-radical cation that resides on the haem. HRP-II is one oxidation state above ferric HRP and contains an oxoferryl iron. HRP-III is equivalent to the oxyferrous state. Only compounds I and II are part of the peroxidase reaction cycle. CCP-ES contains an oxoferryl iron but the radical cation resides on the Trp-191 residue and not on the haem. CPO is the only known peroxidase that is ligated by a cysteine residue rather than a histidine residue, on the proximal side of the haem iron. CPO is a more versatile enzyme, catalysing numerous types of reaction: peroxidase, catalase and halogenation reactions. The various CPO species are less stable than other peroxidase species and more elusive, thus needing further characterization. The roles of the amino acid residues on the proximal and distal sides of the haem need more investigation to further decipher their specific roles. Haem proteins, especially peroxidases, are structure-function-specific. PMID:10730188

  3. Relationships of the visual cortex in the marsupial brush-tailed possum, Trichosurus vulpecula, a horseradish peroxidase and autoradiographic study.

    PubMed Central

    Haight, J R; Sanderson, K J; Neylon, L; Patten, G S

    1980-01-01

    The ascending and descending relationships of visual cortex in Trichosurus were determined using HRP and autoradiographic methods. The visual thalamus, LGNd and LP, was found to project to three cytoarchitecturally distinct areas of cortex with each of these regions displaying differing patterns of connectivity with other centres. The striate and peristriate cortices received homotypically organized projections from LGNd as well as projections from both divisions of LP. The posterior parietal cortex received projections from both LGNd and LP as well as from the latero-intermediate, posterior, ventroanterior and intralaminar thalamic nuclei. The cortical projection fields of LGNd and LP were co-extensive. Descending projections to thalamic centres were reciprocal with the exception of a descending only projection to the thalamic reticular nucleus. The striatogeniculate projection was homotypically organized as was the striatocollicular projection which extended only to the superficial layers of that nucleus. The posterior parietal projection to the superior colliculus involved the deeper layers of that nucleus. The entire visual cortex projected to the pretectum and to the pontine nuclei. The organization of visual cortex in Trichosurus differs from that reported in the American marsupial, Didelphis, in that the LGNd and LP complexes possess a very broad and co-extensive cortical projection, a finding substantially different to what has been reported in placental mammals as well. The organization of thalamic relationships with the posterior parietal cortex would also appear to be unusual with this region receiving convergent projections from at least seven separate thalamic nuclei. Images Fig. 1 Fig. 6 Fig. 11 PMID:7216909

  4. 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. --------------------------------------------------------------------------------

  5. 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

  6. 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

  7. 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.

  8. 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

  9. Highly sensitive and robust peroxidase-like activity of porous nanorods of ceria and their application for breast cancer detection.

    PubMed

    Tian, Zhimin; Li, Jing; Zhang, Zhiyun; Gao, Wei; Zhou, Xuemei; Qu, Yongquan

    2015-08-01

    Porous nanorods of ceria (PN-Ceria), a novel ceria nanostructure with a large surface area and a high surface Ce(3+) fraction, exhibited strong intrinsic peroxidase activity toward a classical peroxidase substrate in the presence of H2O2. Peroxidase-like activity of ceria originated from surface Ce(3+) species as the catalytic center, thereby explaining the high performance of PN-Ceria as an artificial enzyme mimicking peroxidase. Compared with the natural enzyme horseradish peroxidase (HRP), PN-Ceria showed several advantages such as low cost, easy storage, high sensitivity, and, prominently, chemical and catalytic stability under harsh conditions. Importantly, the enzymatic activity of PN-Ceria remained nearly constant and stable over a wide range of temperature and pH values, ensuring the accuracy and reliability of measurements of its peroxidase-like activity. A PN-Ceria based novel diagnostic system was developed for breast cancer detection with a higher sensitivity than the standard HRP detection system. Our work has laid a solid foundation for the development of PN-Ceria as a novel diagnostic tool for clinical use.

  10. Magnetosomes extracted from Magnetospirillum magneticum strain AMB-1 showed enhanced peroxidase-like activity under visible-light irradiation.

    PubMed

    Li, Kefeng; Chen, Chuanfang; Chen, Changyou; Wang, Yuzhan; Wei, Zhao; Pan, Weidong; Song, Tao

    2015-05-01

    Magnetosomes are intracellular structures produced by magnetotactic bacteria and are magnetic nanoparticles surrounded by a lipid bilayer membrane. Magnetosomes reportedly possess intrinsic enzyme mimetic activity similar to that found in horseradish peroxidase (HRP) and can scavenge reactive oxygen species depending on peroxidase activity. Our previous study has demonstrated the phototaxis characteristics of Magnetospirillum magneticum strain AMB-1 cells, but the mechanism is not well understood. Therefore, we studied the relationship between visible-light irradiation and peroxidase-like activity of magnetosomes extracted from M. magneticum strain AMB-1. We then compared this characteristic with that of HRP, iron ions, and naked magnetosomes using 3,3',5,5'-tetramethylbenzidine as a peroxidase substrate in the presence of H2O2. Results showed that HRP and iron ions had different activities from those of magnetosomes and naked magnetosomes when exposed to visible-light irradiation. Magnetosomes and naked magnetosomes had enhanced peroxidase-like activities under visible-light irradiation, but magnetosomes showed less affinity toward substrates than naked magnetosomes under visible-light irradiation. These results suggested that the peroxidase-like activity of magnetosomes may follow an ordered ternary mechanism rather than a ping-pong mechanism. This finding may provide new insight into the function of magnetosomes in the phototaxis in magnetotactic bacteria.

  11. Isolation and sequencing of cDNA clones encoding ethylene-induced putative peroxidases from cucumber cotyledons.

    PubMed

    Morgens, P H; Callahan, A M; Dunn, L J; Abeles, F B

    1990-05-01

    A cDNA library from ethephon-treated cucumber cotyledons (Cucumis sativus L. cv. Poinsett 76) was constructed. Two cDNA clones encoding putative peroxidases were isolated by means of a synthetic probe based on a partial amino acid sequence of a 33 kDa cationic peroxidase that had been previously shown to be induced by ethylene. DNA sequencing indicates that the two clones were derived from two closely related RNA species that are related to published plant peroxidase sequences. Southern analysis indicates that there are 1-5 copies in a haploid genome of a gene homologous to the cDNA clones. The deduced amino acid sequences are homologous with a tobacco (55% sequence identity), a horseradish (53%), a turnip (45%), and a potato (41%) peroxidase. The cloned sequences do not encode the 33 kDa peroxidase from which the original synthetic probe was been derived, but rather other putative peroxidases. An increase in the level of mRNA is evident by 3 hours after ethephon or ethylene treatment and plateaus by 15 hours. PMID:2102850

  12. Targeted systemic mesenchymal stem cell delivery using hyaluronate - wheat germ agglutinin conjugate.

    PubMed

    Kim, Yun Seop; Kong, Won Ho; Kim, Hyemin; Hahn, Sei Kwang

    2016-11-01

    A variety of receptors for hyaluronate (HA), a natural linear polysaccharide, were found in the body, which have been exploited as target sites for HA-based drug delivery systems. In this work, mesenchymal stem cells (MSCs) were surface-modified with HA - wheat germ agglutinin (WGA) conjugate for targeted systemic delivery of MSCs to the liver. WGA was conjugated to HA by coupling reaction between aldehyde-modified HA and amine group of WGA. The conjugation of WGA to HA was corroborated by gel permeation chromatography (GPC) and the successful surface modification of MSCs with HA-WGA conjugate was confirmed by confocal microscopy. The synthesized HA-WGA conjugate could be incorporated onto the cellular membrane by agglutinating the cell-associated carbohydrates. Fluorescent imaging for in vivo biodistribution visualized the targeted delivery of the HA-WGA/MSC complex to the liver after intravenous injection. This new strategy for targeted delivery of MSCs using HA-WGA conjugate might be successfully exploited for various regenerative medicines including cell therapy. PMID:27569867

  13. Soybean agglutinin-conjugated silver nanoparticles nanocarriers in the treatment of breast cancer cells.

    PubMed

    Casañas Pimentel, Rocio Guadalupe; Robles Botero, Viviana; San Martín Martínez, Eduardo; Gómez García, Consuelo; Hinestroza, Juan Paulo

    2016-01-01

    Silver nanoparticles (AgNPs) induce diverse cell-death mechanisms, similar to those promoted by anticancer chemotherapeutics; however, they have not been tested in vivo because their action is not limited to cancer cells. Therefore, in vivo evaluations of their effectiveness should be developed with targeting systems. Breast cancer shows changes in the sugar expression patterns on cell surfaces, related to cancer progression and metastases; those changes have been identified previously by the specific binding of soybean agglutinin (SBA). Here is proposed the use of SBA to target the AgNP activity in breast cancer. For that, the present work reports the synthesis of AgNPs (3.89 ± 0.90 nm) through the polyol method, the generation of AgNP nanocarriers, and the bioconjugation protocol of the nanocarrier with SBA. The free AgNPs, the AgNP nanocarriers, and the SBA-bioconjugated AgNP nanocarriers were tested for cytotoxicity in breast cancerous (MDA-MB-231and MCF7) and non cancerous (MCF 10A) cells, using the MTT assay. AgNPs demonstrated cytotoxic activity in vitro, the non cancerous cells (MCF 10A) being more sensible than the cancerous cells (MDA-MB-231 and MCF7) showing LD(50) values of 128, 205, and 319 μM Ag, respectively; the nanoencapsulation decreased the cytotoxic effect of AgNPs in non cancerous cells, maintaining or increasing the effect on the cancer-derived cells, whereas the SBA-bioconjugation allowed AgNP cytotoxic activity with a similar behavior to the nanocarriers. Future experiments need to be developed to evaluate the targeting effect of the SBA-bioconjugated AgNP nanocarriers to study their functionality in vivo.

  14. Pd-Ir Core-Shell Nanocubes: A Type of Highly Efficient and Versatile Peroxidase Mimic.

    PubMed

    Xia, Xiaohu; Zhang, Jingtuo; Lu, Ning; Kim, Moon J; Ghale, Kushal; Xu, Ye; McKenzie, Erin; Liu, Jiabin; Ye, Haihang

    2015-10-27

    Peroxidase mimics with dimensions on the nanoscale have received great interest as emerging artificial enzymes for biomedicine and environmental protection. While a variety of peroxidase mimics have been actively developed recently, limited progress has been made toward improving their catalytic efficiency. In this study, we report a type of highly efficient peroxidase mimic that was engineered by depositing Ir atoms as ultrathin skins (a few atomic layers) on Pd nanocubes (i.e., Pd-Ir cubes). The Pd-Ir cubes exhibited significantly enhanced efficiency, with catalytic constants more than 20- and 400-fold higher than those of the initial Pd cubes and horseradish peroxidase (HRP), respectively. As a proof-of-concept demonstration, the Pd-Ir cubes were applied to the colorimetric enzyme-linked immunosorbent assay (ELISA) of human prostate surface antigen (PSA) with a detection limit of 0.67 pg/mL, which is ∼110-fold lower than that of the conventional HRP-based ELISA using the same set of antibodies and the same procedure.

  15. Purification and partial characterization of three turnip (Brassicanapus L. var. esculenta D.C.) peroxidases.

    PubMed

    Duarte-Vázquez, M A; García-Almendárez, B; Regalado, C; Whitaker, J R

    2000-05-01

    Three turnip peroxidases (fractions C1, C2, and C3) were partially purified and characterized, to permit study of their feasibility for use in clinical and enzyme immunoassays. These fractions represented 20% of the initial activity, and fractions C1 and C2 were purified to homogeneity. The optimum pH was between 5.0 and 5.5, while optimum temperature ranged from 40 to 55 degrees C. The ABTS K(m) values for the two acidic fractions (C2 and C3) were 0.70 and 0.42 mM, respectively; about 5 times lower than that reported for the acidic commercial horseradish peroxidase (HRP). Fraction C3 had 4 times higher K(m) value than commercial cationic HRP. The molecular weights determined by SDS-PAGE ranged from 39.2 to 42.5 kDa. Activation energies for inactivation were 113 (C1), 130 (C2), and 172 kJ/mol (C3) which are higher or comparable to other peroxidase isoenzymes reported. Fractions C1 and C3 represent an alternative source of peroxidase because of their higher purification yield and specific activity, when compared to fraction C2. PMID:10820061

  16. 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

  17. 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

  18. Purification and characterization of a new cationic peroxidase from fresh flowers of Cynara scolymus L.

    PubMed

    López-Molina, Dorotea; Heering, Hendrik A; Smulevich, Giulietta; Tudela, José; Thorneley, Roger N F; García-Cánovas, Francisco; Rodríguez-López, José Neptuno

    2003-03-01

    A basic heme peroxidase isoenzyme (AKPC) has been purified to homogeneity from artichoke flowers (Cynara scolymus L.). The enzyme was shown to be a monomeric glycoprotein, M(r)=42300+/-1000, (mean+/-S.D.) with an isoelectric point >9. The native enzyme exhibits a typical peroxidase ultraviolet-visible spectrum with a Soret peak at 404 nm (epsilon=137,000+/-3000 M(-1) cm(-1)) and a Reinheitzahl (Rz) value (A(404nm)/A(280nm)) of 3.8+/-0.2. The ultraviolet-visible absorption spectra of compounds I, II and III were typical of class III plant peroxidases but unlike horseradish peroxidase isoenzyme C, compound I was unstable. Resonance Raman and UV-Vis spectra of the ferric form show that between pH 5.0 and 7.0 the protein is mainly 6 coordinate high spin with a water molecule as the sixth ligand. The substrate-specificity of AKPC is characteristic of class III (guaiacol-type) peroxidases with chlorogenic and caffeic acids, that are abundant in artichoke flowers, as particularly good substrates at pH 4.5. Ferric AKPC reacts with hydrogen peroxide to yield compound I with a second-order rate constant (k(+1)) of 7.4 x 10(5) M(-1) s(-1) which is significantly slower than that reported for most other class III peroxidases. The reaction of ferric and ferrous AKPC with nitric oxide showed a potential use of this enzyme for quantitative spectrophotometric determination of NO and as a component of novel NO sensitive electrodes.

  19. Tissue printing for myrosinase activity in roots of turnip and Japanese radish and horseradish: a technique for localizing myrosinases.

    PubMed

    Hara, M; Eto, H; Kuboi, T

    2001-02-01

    A method for analyzing the tissue distribution of myrosinase activity in Brassicaceous plants was developed. This technique is based on 'tissue printing' to visualize enzyme activity. The freshly-cut surface (transverse direction) of the root of three species, Japanese radish (Raphanus sativus), turnip (Brassica campestris) and Japanese horseradish (wasabi, Wasabia japonica), was pressed onto a polyvinylidene difluoride (PVDF) filter to immobilize the proteins onto the membrane. The sites of myrosinase activity on the membranes were visualized by the sinigrin-glucose oxidase-peroxidase system. Signals for myrosinase activity were observed in both the epidermis and vascular cambium of the root of the Japanese radish, turnip and wasabi. Measurement of myrosinase activity in protein extracts indicated that the level of myrosinase activity in the peeling, which consisted of the epidermis, cortex and vascular cambium, was much higher than that in the peeled root of the three species. These results support the image that myrosinase activity, obtained in tissue printing, corresponded well with the tissue distribution of myrosinase activity. PMID:11166428

  20. 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.

  1. In vitro and in vivo stimulation of neutrophil migration and lymphocyte transformation by thiamine related to inhibition of the peroxidase/H2O2/halide system.

    PubMed Central

    Theron, A; Anderson, R; Grabow, G; Meiring, J L

    1981-01-01

    The effects of thiamine on neutrophil functions and mitogen-induced lymphocyte transformation were investigated in vitro and in vivo in adult volunteers following the injection of 50 mg thiamine intramuscularly. Thiamine caused stimulation of neutrophil motility in vitro and in vivo and increased lymphocyte transformation in vivo. Enhancement of these functions was related to inhibition of neutrophil post-phagocytic iodination of Candida albicans by the MPO/H2O2/halide system. The horseradish peroxidase/-H2O2/125 I-mediated iodination of bovine serum albumin was also inhibited by thiamine concentrations which caused increased neutrophil motility. It was found that preincubation of neutrophils and lymphocytes with the horseradish peroxidase/H2O2/halide system caused considerable inhibition of the migratory and proliferative responses respectively. Inclusion of thiamine at concentrations which were found to inhibit the peroxidase/-H2O2/halide system protected the neutrophil migratory and lymphocyte proliferative responses from inactivation by this system. It is suggested that thiamine may cause increased neutrophil migration and lymphocyte transformation by protecting these cells from toxic oxidative products generated by the peroxidase/H2O2/halide system. PMID:6273033

  2. Interaction between La(III) and proteins on the plasma membrane of horseradish

    NASA Astrophysics Data System (ADS)

    Yang, Guang-Mei; Chu, Yun-Xia; Lv, Xiao-Fen; Zhou, Qing; Huang, Xiao-Hua

    2012-06-01

    Lanthanum (La) is an important rare earth element in the ecological environment of plant. The proteins on the plasma membrane control the transport of molecules into and out of cell. It is very important to investigate the effect of La(III) on the proteins on the plasma membrane in the plant cell. In the present work, the interaction between La(III) and proteins on the plasma membrane of horseradish was investigated using optimization of the fluorescence microscopy and fluorescence spectroscopy. It is found that the fluorescence of the complex system of protoplasts and 1-aniline Kenai-8-sulfonic acid in horseradish treated with the low concentration of La(III) is increased compared with that of the control horseradish. The opposite effect is observed in horseradish treated with the high concentration of La(III). These results indicated that the low concentration of La(III) can interact with the proteins on the plasma membrane of horseradish, causing the improvement in the structure of proteins on the plasma membrane. The high concentration of La(III) can also interact with the proteins on the plasma membrane of horseradish, leading to the destruction of the structure of proteins on the plasma membrane. We demonstrate that the proteins on the plasma membrane are the targets of La(III) action on plant cell.

  3. Naturally-occurring tetrahydro-β-carboline alkaloids derived from tryptophan are oxidized to bioactive β-carboline alkaloids by heme peroxidases.

    PubMed

    Herraiz, Tomás; Galisteo, Juan

    2014-08-15

    β-Carbolines are indole alkaloids that occur in plants, foods, and endogenously in mammals and humans, and which exhibit potent biological, psychopharmacological and toxicological activities. They form from naturally-occurring tetrahydro-β-carboline alkaloids arising from tryptophan by still unknown way and mechanism. Results in this research show that heme peroxidases catalyzed the oxidation of tetrahydro-β-carbolines (i.e. 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid and 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid) into aromatic β-carbolines (i.e. norharman and harman, respectively). This oxidation followed a typical catalytic cycle of peroxidases through redox intermediates I, II, and ferric enzyme. Both, plant peroxidases (horseradish peroxidase, HRP) and mammalian peroxidases (myeloperoxidase, MPO and lactoperoxidase, LPO) catalyzed the oxidation in an efficient manner as determined by kinetic parameters (VMAX and KM). Oxidation of tetrahydro-β-carbolines was inhibited by peroxidase inhibitors such as sodium azide, ascorbic acid, hydroxylamine and excess of H2O2. The formation of aromatic β-carbolines by heme peroxidases can help to explain the presence and activity of these compounds in biological systems.

  4. Screening of Coprinus species for the production of extracellular peroxidase and evaluation of the enzyme for the treatment of aqueous phenol.

    PubMed

    Ikehata, K; Buchanan, I D

    2002-12-01

    The production of extracellular peroxidase by twenty-five strains of Coprinus species was investigated for the purpose of its application to the removal of phenolic and other aromatic compounds from industrial waste streams. After initial screening experiments, the production of peroxidase by three superior strains of C cinererus UAMH 4103, UAMH 7907 and IFO 30116 was monitored over a 15-day period. Peroxidase activity was detected after 3 days of growth and had reached itspeak another 6 days later. The peroxidase activity appeared to increase with a corresponding depletion of glucose concentration and rapidly declined immediately after the exhaustion of glucose. The effectiveness of the cultivated C. cinereus peroxidase (CIP) for the removal of aqueous phenol was evaluated in the presence and in the absence of additives including polyethylene glycol (PEG) and chitosan, and compared with those of purified horseradish peroxidase (HRP) and Arthromyces ramosus peroxidase (ARP). The addition of PEG and chitosan enhanced the efficiency of phenol transformation catalyzed by CIP by the factor of 1.5 and 1.3, respectively. Although the efficiency of phenol transformation was higher with CIP than those with purified HRP and ARP in the absence of addtives, its superiority diminished in the presence of PEG. This suggests that the by-products of fungal culture in the crude CIP solution, presumably polycarbohydrates and proteins, have protective effects on the enzyme against inactivation during catalytic transformation of phenol, and the addition of PEG provides small effects on further protection.

  5. A sensitive fluorescent assay for thiamine based on metal-organic frameworks with intrinsic peroxidase-like activity.

    PubMed

    Tan, Hongliang; Li, Qian; Zhou, Zhengchen; Ma, Chanjiao; Song, Yonghai; Xu, Fugang; Wang, Li

    2015-01-26

    Metal-organic frameworks (MOFs) with tunable structures and properties have recently been emerged as very interesting functional materials. However, the catalytic properties of MOFs as enzymatic mimics remain to be further investigated. In this work, we for the first time demonstrated the peroxidase-like activity of copper-based MOFs (HKUST-1) by employing thiamine (TH) as a peroxidase substrate. In the presence of H2O2, HKUST-1 can catalyze efficiently the conversion of non-fluorescent TH to strong fluorescent thiochrome. The catalytic activity of HKUST-1 is highly dependent on the temperature, pH and H2O2 concentrations. As a peroxidase mimic, HKUST-1 not only has the features of low cost, high stability and easy preparation, but also follows Michaelis-Menten behaviors and shows stronger affinity to TH than horseradish peroxidase (HRP). Based on the peroxidase-like activity of HKUST-1, a simple and sensitive fluorescent method for TH detection has been developed. As low as 1 μM TH can be detected with a linear range from 4 to 700 μM. The detection limit for TH is about 50 fold lower than that of HRP-based fluorescent assay. The proposed method was successfully applied to detect TH in tablets and urine samples and showed a satisfactory result. We believed that the present work could improve the understanding of catalytic behaviors of MOFs as enzymatic mimics and find out a wider application in bioanalysis.

  6. Projections of brainstem core cholinergic and non-cholinergic neurons of cat to intralaminar and reticular thalamic nuclei.

    PubMed

    Paré, D; Smith, Y; Parent, A; Steriade, M

    1988-04-01

    We combined the retrograde transport of wheat germ agglutinin conjugated with horseradish peroxidase with choline acetyltransferase immunohistochemistry to study the projections of cholinergic and non-cholinergic neurons of the upper brainstem core to rostral and caudal intralaminar thalamic nuclei, reticular thalamic complex and zona incerta in the cat. After wheat germ agglutinin-horseradish peroxidase injections in the rostral pole of the reticular thalamic nucleus, the distribution and amount of retrogradely labeled brainstem neurons were similar to those found after tracer injection in thalamic relay nuclei (see preceding paper). After wheat germ agglutinin-horseradish peroxidase injections in the caudal intralaminar centrum medianum-parafascicular complex, rostral intralaminar central lateral-paracentral wing, and zona incerta, the numbers of retrogradely labeled brainstem neurons were more than three times higher than those found after injections in thalamic relay nuclei. The larger numbers of horseradish peroxidase-positive brainstem reticular neurons after tracer injections in intralaminar or zona incerta injections results from a more substantial proportion of labeled neurons in the central tegmental field at rostral midbrain (perirubral) levels and in the ventromedial part of the pontine reticular formation, ipsi- and contralaterally to the injection site. Of all retrogradely labeled neurons in the caudal midbrain core at the level of the cholinergic peribrachial area and laterodorsal tegmental nucleus, 45-50% were also choline acetyltransferase-positive after the injections into central lateral-paracentral and reticular nuclei, while only 25% were also choline acetyltransferase-positive after the injection into the centrum medianum-parafascicular complex. These findings are discussed in the light of physiological evidence of brainstem cholinergic mechanisms involved in the blockade of synchronized oscillations and in activation processes of

  7. 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.

  8. Peroxidase-catalyzed removal of phenols from a petroleum refinery wastewater.

    PubMed

    Wagner, M; Nicell, J A

    2001-01-01

    The phenol content of a petroleum refinery wastewater was reduced below the discharge limit following treatment with horseradish peroxidase and H2O2. Approximately 58% of COD, 78% of BOD5, and 95% of toxicity were removed along with the phenols. As a result of treatment, phenols were transformed into less biodegradable compounds which could be removed by subsequent coagulation and precipitation. Optimization of the peroxide concentration led to 20% enzyme savings. The use of PEG and chitosan as protective additives resulted in 4 and 25-fold reductions in enzyme requirements, respectively. Phenol removal did not appear to be adversely affected by the presence of other hydrocarbons that are frequently present in refinery wastewaters.

  9. 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.

  10. Evolutionary Divergence of Arabidopsis thaliana Classical Peroxidases.

    PubMed

    Kupriyanova, E V; Mamoshina, P O; Ezhova, T A

    2015-10-01

    Polymorphisms of 62 peroxidase genes derived from Arabidopsis thaliana were investigated to evaluate evolutionary dynamics and divergence of peroxidase proteins. By comparing divergence of duplicated genes AtPrx53-AtPrx54 and AtPrx36-AtPrx72 and their products, nucleotide and amino acid substitutions were identified that were apparently targets of positive selection. These substitutions were detected among paralogs of 461 ecotypes from Arabidopsis thaliana. Some of these substitutions are conservative and matched paralogous peroxidases in other Brassicaceae species. These results suggest that after duplication, peroxidase genes evolved under the pressure of positive selection, and amino acid substitutions identified during our study provided divergence of properties and physiological functions in peroxidases. Our predictions regarding functional significance for amino acid residues identified in variable sites of peroxidases may allow further experimental assessment of evolution of peroxidases after gene duplication.

  11. Toxic effects of heavy metal terbium ion on the composition and functions of cell membrane in horseradish roots.

    PubMed

    Yang, Qing; Wang, Lihong; Zhou, Qing; Huang, Xiaohua

    2015-01-01

    The environmental safety of rare earth elements (REEs), especially the toxic effect of REEs on plants, has attracted increasing attention. However, the cellular mechanism of this toxic effect remains largely unknown. Here, the toxic effects of heavy REE terbium ion [Tb(III)] on the cell membrane of horseradish roots were investigated by using electron microscope autoradiography (EMARG) and histochemical methods. The results indicated that Tb(III) was distributed in the extracellular and intracellular spaces of the roots after horseradish was treated with Tb(III). Moreover, the percentage contents of the unsaturated fatty acids in the membrane lipids, the current of the outward K(+) channel and the average diameter of membrane proteins in the roots of horseradish treated with Tb(III) were decreased; on the contrary, the percentage contents of the saturated fatty acids and malondialdehyde in the roots of horseradish treated with Tb(III) were increased. Furthermore, the contents of intracellular N, P, Mg and Fe in the roots of horseradish treated with Tb(III) were decreased, while the contents of intracellular K and Ca in the roots of horseradish treated with Tb(III) were increased. Finally, the effects of Tb(III) on horseradish roots were increased with increasing concentration or duration of Tb(III) treatment. In conclusion, after horseradish was treated with Tb(III), Tb(III) could enter the cells of horseradish roots and lead to the toxic effects on horseradish, which caused the oxidation of the unsaturated fatty acids in the membrane lipids, the changes in the membrane proteins (including the outward K(+) channel), the decrease in the membrane fluidity, and then the inhibition of the intracellular/extracellular-ion exchange in horseradish roots.

  12. 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

  13. Artificial Peroxidase/Oxidase Multiple Enzyme System Based on Supramolecular Hydrogel and Its Application as a Biocatalyst for Cascade Reactions.

    PubMed

    Qu, Rui; Shen, Liangliang; Qu, Aoting; Wang, Ruolin; An, Yingli; Shi, Linqi

    2015-08-01

    Inspired by delicate structures and multiple functions of natural multiple enzyme architectures such as peroxisomes, we constructed an artificial multiple enzyme system by coencapsulation of glucose oxidases (GOx) and artificial peroxidases in a supramolecular hydrogel. The artificial peroxidase was a functional complex micelle, which was prepared by the self-assembly of diblock copolymer and hemin. Compared with catalase or horseradish peroxidase (HRP), the functional micelle exhibited comparable activity and better stability, which provided more advantages in constructing a multienzyme with a proper oxidase. The hydrogel containing the two catalytic centers was further used as a catalyst for green oxidation of glucose, which was a typical cascade reaction. Glucose was oxidized by oxygen (O2) via the GOx-mediated reaction, producing toxic intermediate hydrogen peroxide (H2O2). The produced H2O2 further oxidized peroxidase substrates catalyzed by hemin-micelles. By regulating the diffusion modes of the enzymes and substrates, the artificial multienzyme based on hydrogel could successfully activate the cascade reaction, which the soluble enzyme mixture could not achieve. The hydrogel, just like a protective covering, protected oxidases and micelles from inactivation via toxic intermediates and environmental changes. The artificial multienzyme could efficiently achieve the oxidation task along with effectively eliminating the toxic intermediates. In this way, this system possesses great potentials for glucose detection and green oxidation of a series of substrates related to biological processes. PMID:26173996

  14. 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

  15. 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

  16. 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.

  17. Artificial Peroxidase/Oxidase Multiple Enzyme System Based on Supramolecular Hydrogel and Its Application as a Biocatalyst for Cascade Reactions.

    PubMed

    Qu, Rui; Shen, Liangliang; Qu, Aoting; Wang, Ruolin; An, Yingli; Shi, Linqi

    2015-08-01

    Inspired by delicate structures and multiple functions of natural multiple enzyme architectures such as peroxisomes, we constructed an artificial multiple enzyme system by coencapsulation of glucose oxidases (GOx) and artificial peroxidases in a supramolecular hydrogel. The artificial peroxidase was a functional complex micelle, which was prepared by the self-assembly of diblock copolymer and hemin. Compared with catalase or horseradish peroxidase (HRP), the functional micelle exhibited comparable activity and better stability, which provided more advantages in constructing a multienzyme with a proper oxidase. The hydrogel containing the two catalytic centers was further used as a catalyst for green oxidation of glucose, which was a typical cascade reaction. Glucose was oxidized by oxygen (O2) via the GOx-mediated reaction, producing toxic intermediate hydrogen peroxide (H2O2). The produced H2O2 further oxidized peroxidase substrates catalyzed by hemin-micelles. By regulating the diffusion modes of the enzymes and substrates, the artificial multienzyme based on hydrogel could successfully activate the cascade reaction, which the soluble enzyme mixture could not achieve. The hydrogel, just like a protective covering, protected oxidases and micelles from inactivation via toxic intermediates and environmental changes. The artificial multienzyme could efficiently achieve the oxidation task along with effectively eliminating the toxic intermediates. In this way, this system possesses great potentials for glucose detection and green oxidation of a series of substrates related to biological processes.

  18. Projections of physiologically defined subdivisions of the inferior colliculus in the mustached bat: targets in the medial geniculate body and extrathalamic nuclei.

    PubMed

    Wenstrup, J J; Larue, D T; Winer, J A

    1994-08-01

    This study examined the output of the central nucleus of the inferior colliculus to the medial geniculate body and other parts of the nervous system in the mustached bat (Pteronotus parnellii). Small deposits of anterograde tracers (horseradish peroxidase, [3H]leucine, Phaseolus vulgaris leucoagglutinin, wheat germ agglutinin conjugated to horseradish peroxidase, or biocytin) were made at physiologically defined sites in the central nucleus representing major components of the bat's echolocation signal. The topography, frequency specificity, and axonal morphology of these outputs were studied. The medial geniculate body was a major target of inferior collicular neurons, with three distinct input patterns. The projection to the ventral division was tonotopically organized, but had a relatively sparse contribution from neurons representing frequency modulated components of the biosonar pulse. The second input was to the rostral medial geniculate body, in which projections from inferior collicular neurons representing constant frequency sonar components were separated from those representing frequency modulated components. A third input was to the suprageniculate nucleus, which received strong, topographically arranged projections. Inputs to the dorsal nucleus and medial division were also observed. Extrathalamic regions receiving input included the pontine gray, external nucleus of the inferior colliculus, pericollicular tegmentum, nucleus of the brachium of the inferior colliculus, and pretectum. These central nucleus projections differed in organization and the structure of axon terminals, suggesting different physiological influences on their target nuclei. These results demonstrate that the central nucleus has divergent projections to various sensory and premotor nuclei, besides its well-established projection to the medial geniculate body.

  19. 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

  20. PeroxiBase: the peroxidase database.

    PubMed

    Passardi, Filippo; Theiler, Grégory; Zamocky, Marcel; Cosio, Claudia; Rouhier, Nicolas; Teixera, Felipe; Margis-Pinheiro, Marcia; Ioannidis, Vassilios; Penel, Claude; Falquet, Laurent; Dunand, Christophe

    2007-06-01

    Peroxidases (EC 1.11.1.x), which are encoded by small or large multigenic families, are involved in several important physiological and developmental processes. Analyzing their evolution and their distribution among various phyla could certainly help to elucidate the mystery of their extremely widespread and diversified presence in almost all living organisms. PeroxiBase was originally created for the exhaustive collection of class III peroxidase sequences from plants (Bakalovic, N., Passardi, F., et al., 2006. PeroxiBase: a class III plant peroxidase database. Phytochemistry 67, 534-539). The extension of the class III peroxidase database to all proteins capable to reduce peroxide molecules appears as a necessity. Our database contains haem and non-haem peroxidase sequences originated from annotated or not correctly annotated sequences deposited in the main repositories such as GenBank or UniProt KnowledgeBase. This new database will allow obtaining a global overview of the evolution the protein families and superfamilies capable of peroxidase reaction. In this rapidly growing field, there is a need for continual updates and corrections of the peroxidase protein sequences. Following the lack of unified nomenclature, we also introduced a unique abbreviation for each different family of peroxidases. This paper thus aims to report the evolution of the PeroxiBase database, which is freely accessible through a web server (http://peroxibase.isb-sib.ch). In addition to new categories of peroxidases, new specific tools have been created to facilitate query, classification and submission of peroxidase sequences.

  1. 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.

  2. 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.

  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.

  4. 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

  5. Turning points in the evolution of peroxidase-catalase superfamily: molecular phylogeny of hybrid heme peroxidases.

    PubMed

    Zámocký, Marcel; Gasselhuber, Bernhard; Furtmüller, Paul G; Obinger, Christian

    2014-12-01

    Heme peroxidases and catalases are key enzymes of hydrogen peroxide metabolism and signaling. Here, the reconstruction of the molecular evolution of the peroxidase-catalase superfamily (annotated in pfam as PF00141) based on experimentally verified as well as numerous newly available genomic sequences is presented. The robust phylogenetic tree of this large enzyme superfamily was obtained from 490 full-length protein sequences. Besides already well-known families of heme b peroxidases arranged in three main structural classes, completely new (hybrid type) peroxidase families are described being located at the border of these classes as well as forming (so far missing) links between them. Hybrid-type A peroxidases represent a minor eukaryotic subfamily from Excavates, Stramenopiles and Rhizaria sharing enzymatic and structural features of ascorbate and cytochrome c peroxidases. Hybrid-type B peroxidases are shown to be spread exclusively among various fungi and evolved in parallel with peroxidases in land plants. In some ascomycetous hybrid-type B peroxidases, the peroxidase domain is fused to a carbohydrate binding (WSC) domain. Both here described hybrid-type peroxidase families represent important turning points in the complex evolution of the whole peroxidase-catalase superfamily. We present and discuss their phylogeny, sequence signatures and putative biological function.

  6. 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

  7. BSA-templated MnO2 nanoparticles as both peroxidase and oxidase mimics.

    PubMed

    Liu, Xing; Wang, Qi; Zhao, Huihui; Zhang, Lichun; Su, Yingying; Lv, Yi

    2012-10-01

    Inorganic nanomaterials that mimic enzymes are fascinating as they potentially have improved properties relative to native enzymes, such as greater resistance to extremes of pH and temperature and lower sensitivity to proteases. Although many artificial enzymes have been investigated, searching for highly-efficient and stable catalysts is still of great interest. In this paper, we first demonstrated that bovine serum albumin (BSA)-stabilized MnO(2) nanoparticles (NPs) exhibited highly peroxidase-, oxidase-, and catalase-like activities. The activities of the BSA-MnO(2) NPs were evaluated using the typical horseradish peroxidase (HRP) substrates o-phenylenediamine (OPD) and 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of either hydrogen peroxide or dissolved oxygen. These small-sized BSA-MnO(2) NPs with good dispersion, solubility and biocompatibility exhibited typical Michaelis-Menten kinetics and high affinity for H(2)O(2), OPD and TMB, indicating that BSA-MnO(2) NPs can be used as satisfactory enzyme mimics. Based on these findings, BSA-MnO(2) NPs were used as colorimetric immunoassay tags for the detection of goat anti-human IgG in place of HRP. The colorimetric immunoassay using BSA-MnO(2) NPs has the advantages of being fast, robust, inexpensive, easily prepared and with no HRP and H(2)O(2) being needed. These water-soluble BSA-MnO(2) NPs may have promising potential applications in biotechnology, bioassays, and biomedicine.

  8. Nanogold-actuated biomimetic peroxidase for sensitized electrochemical immunoassay of carcinoembryonic antigen in human serum.

    PubMed

    Chen, Huafeng; Tang, Juan; Su, Biling; Chen, Guonan; Huang, Jianxin; Tang, Dianping

    2010-09-30

    A new electrochemical immunoassay protocol for the determination of carcinoembryonic antigen (CEA) in human serum was developed by means of immobilizing horseradish peroxidase-labeled anti-CEA antibodies (HRP-anti-CEA) on the nanogold/nano-Fe(3)O(4) particles-functionalized polyion complex (PIC) membrane. With a one-step immunoassay format, the formed immunocomplex inhibited partly the bioactive center of the immobilized HRP, and decreased the HRP toward the reduction of H(2)O(2). Nanogold-actuated nano-Fe(3)O(4) mimetic peroxidase was used for the sensitivity improvement of the electrochemical immunosensor with Prussian blue (PB) as mediators. Under optimal conditions, the electrochemical immunosensor displayed a wide dynamic range of 0.1-220 ng mL(-1) with a relative low detection limit (LOD) of 10 pg mL(-1) CEA (at 3σ). Intra- and inter-assay show good precisions with a coefficient of variation (CV) lower than 7.8% and 7.3%, respectively. No significant difference at the 95% confidence level was encountered in the analysis of 17 human serum specimens between the electrochemical immunosensor and automatic electrochemiluminescent (ECL) analyzer for the determination of CEA.

  9. Directing the oligomer size distribution of peroxidase-mediated cross-linked bovine alpha-lactalbumin.

    PubMed

    Heijnis, Walter H; Wierenga, Peter A; van Berkel, Willem J H; Gruppen, Harry

    2010-05-12

    Enzymatic protein cross-linking is a powerful tool to change protein functionality. For optimal functionality in gel formation, the size of the cross-linked proteins needs to be controlled, prior to heating. In the current study, we addressed the optimization of the horseradish peroxidase-mediated cross-linking of calcium-depleted bovine alpha-lactalbumin. To characterize the formed products, the molecular weight distribution of the cross-linked protein was determined by size exclusion chromatography. At low ionic strength, more dimers of alpha-lactalbumin are formed than at high ionic strength, while the same conversion of monomers is observed. Similarly, at pH 5.9 more higher oligomers are formed than at pH 6.8. This is proposed to be caused by local changes in apo alpha-lactalbumin conformation as indicated by circular dichroism spectroscopy. A gradual supply of hydrogen peroxide improves the yield of cross-linked products and increases the proportion of higher oligomers. In conclusion, this study shows that the size distribution of peroxidase-mediated cross-linked alpha-lactalbumin can be directed toward the protein oligomers desired.

  10. 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.

  11. (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.

  12. Peroxidase enzymes regulate collagen extracellular matrix biosynthesis.

    PubMed

    DeNichilo, Mark O; Panagopoulos, Vasilios; Rayner, Timothy E; Borowicz, Romana A; Greenwood, John E; Evdokiou, Andreas

    2015-05-01

    Myeloperoxidase and eosinophil peroxidase are heme-containing enzymes often physically associated with fibrotic tissue and cancer in various organs, without any direct involvement in promoting fibroblast recruitment and extracellular matrix (ECM) biosynthesis at these sites. We report herein novel findings that show peroxidase enzymes possess a well-conserved profibrogenic capacity to stimulate the migration of fibroblastic cells and promote their ability to secrete collagenous proteins to generate a functional ECM both in vitro and in vivo. Mechanistic studies conducted using cultured fibroblasts show that these cells are capable of rapidly binding and internalizing both myeloperoxidase and eosinophil peroxidase. Peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl 4-hydroxylase-dependent manner that does not require ascorbic acid. This response was blocked by the irreversible myeloperoxidase inhibitor 4-amino-benzoic acid hydrazide, indicating peroxidase catalytic activity is essential for collagen biosynthesis. These results suggest that peroxidase enzymes, such as myeloperoxidase and eosinophil peroxidase, may play a fundamental role in regulating the recruitment of fibroblast and the biosynthesis of collagen ECM at sites of normal tissue repair and fibrosis, with enormous implications for many disease states where infiltrating inflammatory cells deposit peroxidases.

  13. Screening of postharvest agricultural wastes as alternative sources of peroxidases: characterization and kinetics of a novel peroxidase from lentil ( Lens culinaris L.) stubble.

    PubMed

    Hidalgo-Cuadrado, Nazaret; Pérez-Galende, Patricia; Manzano, Teresa; De Maria, Cándido Garcia; Shnyrov, Valery L; Roig, Manuel G

    2012-05-16

    Aqueous crude extracts of a series of plant wastes (agricultural, wild plants, residues from sports activities (grass), ornamental residues (gardens)) from 17 different plant species representative of the typical biodiversity of the Iberian peninsula were investigated as new sources of peroxidases (EC 1.11.1.7). Of these, lentil (Lens culinaris L.) stubble crude extract was seen to provide one of the highest specific peroxidase activities, catalyzing the oxidation of guaiacol in the presence of hydrogen peroxide to tetraguaiacol, and was used for further studies. For the optimum extraction conditions found, the peroxidase activity in this crude extract (110 U mL(-1)) did not vary for at least 15 months when stored at 4 °C (k(inact) = 0.146 year(-1), t(1/2 inact) = 4.75 year), whereas, for comparative purposes, the peroxidase activity (60 U mL(-1)) of horseradish (Armoracia rusticana L.) root crude extract, obtained and stored under the same conditions, showed much faster inactivation kinetics (k(inact) = 2.2 × 10(-3) day(-1), t(1/2 inact) = 315 days). Using guaiacol as an H donor and a universal buffer (see above), all crude extract samples exhibited the highest peroxidase activity in the pH range between 4 and 7. Once semipurified by passing the crude extract through hydrophobic chromatography on phenyl-Sepharose CL-4B, the novel peroxidase (LSP) was characterized as having a purity number (RZ) of 2.5 and three SDS-PAGE electrophoretic bands corresponding to molecular masses of 52, 35, and 18 kDa. The steady-state kinetic study carried out on the H(2)O(2)-mediated oxidation of guaiacol by the catalytic action of this partially purified peroxidase pointed to apparent Michaelian kinetic behavior (K(m)(appH(2)O(2)) = 1.87 mM; V(max)(appH(2)O(2)) = 6.4 mM min(-1); K(m)(app guaicol) = 32 mM; V(max)(app guaicol) = 9.1 mM min(-1)), compatible with the two-substrate ping-pong mechanism generally accepted for peroxidases. Finally, after the effectiveness of the crude

  14. 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

  15. 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.

  16. 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

  17. Purification and properties of a neutral peroxidase isozyme from turnip (Brassica napus L. Var. Purple Top White Globe) roots.

    PubMed

    Duarte-Vázquez, M A; García-Almendárez, B E; Regalado, C; Whitaker, J R

    2001-09-01

    A neutral peroxidase isozyme (pI 7.2) from turnip roots (TNP) was purified to homogeneity and partially characterized. TNP is a monomeric glycoprotein with 9.1% carbohydrate content and a molecular weight of 36 kDa. Optimum pH values for activity using 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and guaiacol as H donors were 4.5 and 5.5, whereas the K(m) values were 0.7 and 3.7 mM, respectively. The ABTS K(m) was approximately 7 times higher than that reported for basic commercial horseradish peroxidase (HRP-C). TNP retained approximately 70% activity after 11 min of heating at 65 degrees C, whereas the activation energy for inactivation (132 kJ/mol) was higher than or comparable to that of other peroxidases. The low ABTS K(m) and high specific activity (1930 units/mg) gave a high catalytic efficiency (500 M(-1) s(-1)). These properties make TNP an enzyme with a high potential as an alternative to HRP in various applications. PMID:11559153

  18. Adapting protein solubility by glycosylation. N-glycosylation mutants of Coprinus cinereus peroxidase in salt and organic solutions.

    PubMed

    Tams, J W; Vind, J; Welinder, K G

    1999-07-13

    Protein solubility is a fundamental parameter in biology and biotechnology. In the present study we have constructed and analyzed five mutants of Coprinus cinereus peroxidase (CIP) with 0, 1, 2, 4 and 6 N-glycosylation sites. All mutants contain Man(x)(GlcNAc)(2) glycans. The peroxidase activity was the same for wild-type CIP and all the glycosylation mutants when measured with the large substrate 2,2'-azino-bis(-3-ethylbenzthiazoline-6-sulfonic acid). The solubility of the five CIP mutants showed a linear dependence on the number of carbohydrate residues attached to the protein in buffered solution of both ammonium sulfate (AMS) and acetone, increasing in AMS and decreasing in acetone. Moreover, the change in free energy of solvation appears to be a constant, though with opposite signs in these solvents, giving DeltaDeltaG degrees (sol)=-0.32+/-0.05 kJ/mol per carbohydrate residue in 2.0 M AMS, a value previously obtained comparing ordinary and deglycosylated horseradish peroxidase, and 0. 37+/-0.10 kJ/mol in 60 v/v% acetone.

  19. 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.

  20. 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.

  1. Resonance Raman study of the active site of Coprinus cinereus peroxidase.

    PubMed

    Smulevich, G; Feis, A; Focardi, C; Tams, J; Welinder, K G

    1994-12-27

    Resonance Raman (RR) spectra for the resting state ferric and the reduced ferrous forms of recombinant Coprinus cinereus peroxidase (CIP), obtained with different excitation wavelengths and in polarized light, are reported. The spectra are compared with those obtained previously for cytochrome c peroxidase expressed in Escherichia coli [(CCP(MI)] and horseradish peroxidase (HRP-C). Although the enzymic properties of CIP and HRP-C are similar, the RR data show that, in terms of the heme cavity structures, CIP and CCP(MI) are much more closely related to each other than to HRP-C. The ferric state of CIP at neutral pH is characteristic mainly of a five-coordinate high spin heme. However, the lower frequency of the v2 mode and a higher frequency of the v(C = C) vinyl stretching modes for CIP as compared to CCP, indicate a higher degree of vibrational coupling between the two modes in CIP. In addition, CIP is rather unstable under low laser power irradiation as an irreversible transition to a six-coordinate high spin heme followed by a second transition to a six-coordinate low spin heme is observed. This instability of CIP as compared to CCP(MI) is proposed to be a consequence of the presence of a distal Phe54 in CIP rather than the homologous Trp51 in CCP, as Trp51 is hydrogen-bonded to a distal water molecule located above the heme Fe thereby preventing its coordination in CCP. In CIP the FeII-His RR band has two components with frequencies at 230 and 211 cm-1.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. 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

  3. 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.

  4. 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.

  5. Thiol-Based Peroxidases and Ascorbate Peroxidases: Why Plants Rely on Multiple Peroxidase Systems in the Photosynthesizing Chloroplast?

    PubMed

    Dietz, Karl-Josef

    2016-01-01

    Photosynthesis is a highly robust process allowing for rapid adjustment to changing environmental conditions. The efficient acclimation depends on balanced redox metabolism and control of reactive oxygen species release which triggers signaling cascades and potentially detrimental oxidation reactions. Thiol peroxidases of the peroxiredoxin and glutathione peroxidase type, and ascorbate peroxidases are the main peroxide detoxifying enzymes of the chloroplast. They use different electron donors and are linked to distinct redox networks. In addition, the peroxiredoxins serve functions in redox regulation and retrograde signaling. The complexity of plastid peroxidases is discussed in context of suborganellar localization, substrate preference, metabolic coupling, protein abundance, activity regulation, interactions, signaling functions, and the conditional requirement for high antioxidant capacity. Thus the review provides an opinion on the advantage of linking detoxification of peroxides to different enzymatic systems and implementing mechanisms for their inactivation to enforce signal propagation within and from the chloroplast.

  6. Redesign of cytochrome c peroxidase into a manganese peroxidase: role of tryptophans in peroxidase activity.

    PubMed

    Gengenbach, A; Syn, S; Wang, X; Lu, Y

    1999-08-31

    Trp191Phe and Trp51Phe mutations have been introduced into an engineered cytochrome c peroxidase (CcP) containing a Mn(II)-binding site reported previously (MnCcP; see Yeung, B. K.-S., et al. (1997) Chem. Biol. 5, 215-221). The goal of the present study is to elucidate the role of tryptophans in peroxidase activity since CcP contains both Trp51 and Trp191 while manganese peroxidase (MnP) contains phenylalanine residues at the corresponding positions. The presence of Trp191 in CcP allows formation of a unique high-valent intermediate containing a ferryl oxo and tryptophan radical called compound I'. The absence of a tryptophan residue at this position in MnP is the main reason for the formation of an intermediate called compound I which contains a ferryl oxo and porphyrin pi-cation radical. In this study, we showed that introduction of the Trp191Phe mutation to MnCcP did not improve MnP activity (specific activity: MnCcP, 0.750 micromol min-1 mg-1; MnCcP(W191F), 0.560 micromol min-1 mg-1. k(cat)/K(m): MnCcP, 0.0517 s-1 mM-1; MnCcP(W191F), 0.0568 s-1 mM-1) despite the fact that introduction of the same mutation to WTCcP caused the formation of a transient compound I (decay rate, 60 s-1). However, introducing both the Trp191Phe and Trp51Phe mutations not only resulted in a longer lived compound I in WTCcP (decay rate, 18 s-1), but also significantly improved MnP activity in MnCcP (MnCcP(W51F, W191F): specific activity, 8.0 micromol min-1 mg-1; k(cat)/K(m), 0. 599 s-1 mM-1). The increase in activity can be attributed to the Trp51Phe mutation since MnCcP(W51F) showed significantly increased MnP activity relative to MnCcP (specific activity, 3.2 micromol min-1 mg-1; k(cat)/K(m), 0.325 s-1 mM-1). As with MnP, the activity of MnCcP(W51F, W191F) was found to increase with decreasing pH. Our results demonstrate that, while the Trp191Phe and Trp51Phe mutations both play important roles in stabilizing compound I, only the Trp51Phe mutation contributes significantly to

  7. Determination of hydrogen peroxide concentrations by flow injection analysis based on the enhanced chemiluminescent reaction using peroxidase

    SciTech Connect

    Eremin, S.A.; Vlasenko, S.B.; Osipov, A.P.; Eremina, I.D.; Egerov, A.M. )

    1989-01-01

    The technique of flow injection analysis was employed in the determination of hydrogen peroxide. The method was based on the chemiluminescence reaction of luminol with H{sub 2}O{sub 2} which is catalyzed by horseradish peroxidase and enhanced by p-iodophenol. Hydrogen peroxide was linearly detected in the range 10{sup {minus}6}M-10{sup {minus}4}M by measuring the maximum intensity of light emitted. The detection limit is about 1 10{sup {minus}6}M hydrogen peroxide. Transition metal cations at millimolar concentrations do not have any interference on the determination of hydrogen peroxide by FIA based on the enhanced chemiluminescent reaction. This technique is relatively rapid and simple, and permits measurement of up to 80 samples/hr using generally available equipment.

  8. 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

  9. 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.

  10. 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.

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

    PubMed

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

    2016-04-21

    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.

  12. Roles of distal arginine in activity and stability of Coprinus cinereus peroxidase elucidated by kinetic and NMR analysis of the Arg51Gln, -Asn, -Leu, and -Lys mutants.

    PubMed

    Schiødt, Christine B; Veitch, Nigel C; Welinder, Karen G

    2007-02-01

    In heme peroxidases, a distal His residue plays an essential role in the initial two electron oxidation of resting state enzyme to compound I by hydrogen peroxide. A distal Arg residue assists in this process. The contributions of the charge, H-bonding capacity, size, and mobility of this Arg residue to Coprinus cinereus peroxidase (CIP) reactivity and stability have been examined by substituting Arg51 with Gln (retains H-bond donor at N epsilon position), Asn (small size, H-bond donor and acceptor), Leu (similar to Asn, but hydrophobic), and Lys (charge and H-bond donor, but at N zeta position). UV-visible spectroscopy was used to monitor pH-linked heme changes, compound I formation and reduction, fluoride binding, and thermostability. (1)H NMR spectroscopy enabled heme pocket differences in both resting and cyanide-ligated states of the enzymes to be evaluated and compared with wild-type CIP. We found that the H-bonding capacity of distal Arg is key to fast compound I formation and ligand binding to heme, whereas charge is important for lowering the pK(a) of distal His and for the binding and stabilisation of anionic ligands at heme iron. The properties of the distal Arg residue in CIP, cytochrome c peroxidase (CCP) and horseradish peroxidase (HRP) differ significantly in their pH induced transitions and dynamics.

  13. Wheat germ agglutinin-conjugated PLGA nanoparticles for enhanced intracellular delivery of paclitaxel to colon cancer cells.

    PubMed

    Wang, Chunxia; Ho, Paul C; Lim, Lee Yong

    2010-11-15

    The purpose of this study was to investigate the potentiation of the anticancer activity and enhanced cellular retention of paclitaxel-loaded PLGA nanoparticles after surface conjugation with wheat germ agglutinin (WGA) against colon cancer cells. Glycosylation patterns of representative colon cancer cells confirmed the higher expression levels of WGA-binding glycoproteins in the Caco-2 and HT-29 cells, than in the CCD-18Co cells. Cellular uptake and in vitro cytotoxicity of WNP (final formulation) against colon cell lines was evaluated alongside control formulations. Confocal microscopy and quantitative analysis of intracellular paclitaxel were used to monitor the endocytosis and retention of nanoparticles inside the cells. WNP showed enhanced anti-proliferative activity against Caco-2 and HT-29 cells compared to corresponding nanoparticles without WGA conjugation (PNP). The greater efficacy of WNP was associated with higher cellular uptake and sustained intracellular retention of paclitaxel, which in turn was attributed to the over-expression of N-acetyl-D-glucosamine-containing glycoprotein on the colon cell membrane. WNP also demonstrated increased intracellular retention in the Caco-2 (30% of uptake) and HT-29 (40% of uptake) cells, following post-uptake incubation with fresh medium, compared to the unconjugated PNP nanoparticles (18% in Caco-2) and (27% in HT-29), respectively. Cellular trafficking study of WNP showed endocytosed WNP could successful escape from the endo-lysosome compartment and release into the cytosol with increasing incubation time. It may be concluded that WNP has the potential to be applied as a targeted delivery platform for paclitaxel in the treatment of colon cancer. PMID:20804835

  14. (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.

  15. 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.

  16. Guaiacol peroxidase zymography for the undergraduate laboratory.

    PubMed

    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 detect peroxidase activity and furthermore, to analyze the total protein profile. After the assay, students may estimate the apparent molecular mass of the enzyme and discuss its structure. After the 4-h experiment, students gain knowledge concerning biological sample preparation, gel preparation, electrophoresis, and the importance of specific staining procedures for the detection of enzymatic activity.

  17. Irregular-shaped platinum nanoparticles as peroxidase mimics for highly efficient colorimetric immunoassay.

    PubMed

    Gao, Zhuangqiang; Xu, Mingdi; Hou, Li; Chen, Guonan; Tang, Dianping

    2013-05-01

    Enzyme-linked immunosorbent assay (ELISA) methods based on natural enzyme-labeled probes have been applied in the immunoassays, but most have some inevitable limitations (e.g. harsh preparation, purification and storage) and are unsuitable for routine use. Herein we synthesized a new class of irregular-shaped platinum nanoparticles (ISPtNP) with a mean length of 7.0 nm and a narrowing width from 2.0 to 5.0 nm along the longitudinal axes, which were utilized as peroxidase-like mimics for the development of colorimetric immunoassays. Compared with bioactive horseradish peroxidase (HRP), the synthesized ISPtNP exhibited a low Km value (~0.12 mM) and a high Kcat value (~2.27×10(4)s(-1)) for 3,3',5,5'-tetramethylbenzidine (TMB) with strong thermal stability and pH tolerance. The catalytic mechanism of the ISPtNP toward TMB/H2O2 was for the first time discussed and deliberated in this work. Based on a sandwich-type assay format, two types of colorimetric immunoassay protocols were designed and developed for the detection of rabbit IgG (RIgG, as a model) by using the synthesized ISPtNP and conventional HRP as the labeling of detection antibodies, respectively. Similar detection limits (LODs) of 2.5 ng mL(-1) vs. 1.0 ng mL(-1) were obtained toward RIgG with the ISPtNP labeling compared to HRP format. Intra- and inter-assay coefficients of variation were less than 13%. Importantly, the ISPtNP-based assay system could be suitable for use in a mass production of miniaturized lab-on-a-chip devices and open new opportunities for protein diagnostics and biosecurity.

  18. 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

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

    PubMed

    Lalwani, Gaurav; Xing, Weiliang; Sitharaman, Balaji

    2014-10-01

    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.

  20. High-yield reactivation of anionic tobacco peroxidase overexpressed in Escherichia coli.

    PubMed

    Zakharova, G S; Poloznikov, A A; Chubar, T A; Gazaryan, I G; Tishkov, V I

    2015-09-01

    Anionic tobacco peroxidase (TOP) is extremely active in chemiluminescence reaction of luminol oxidation without addition of enhancers and more stable than horseradish peroxidase under antibody conjugation conditions. In addition, recombinant TOP (rTOP) produced in Escherichia coli is known to be a perfect direct electron transfer catalyst on electrodes of various origin. These features make the task of development of a high-yield reactivation protocol for rTOP practically important. Previous attempts to reactivate the enzyme from E. coli inclusion bodies were successful, but the reported reactivation yield was only 14%. In this work, we thoroughly screened the refolding conditions for dilution protocol and compared it with gel-filtration chromatography. The impressive reactivation yield in the dilution protocol (85%) was achieved for 8 μg/mL solubilized rTOP protein and the refolding medium containing 0.3 mM oxidized glutathione, 0.05 mM dithiothreitol, 5 mM CaCl2, 5% glycerol in 50 mM Tris-HCl buffer, pH 9.6, with 1 μM hemin added at the 24th hour of incubation. A practically important discovery was a 30-40% increase in the reactivation yield upon delayed addition of hemin. The reactivation yield achieved is one of the highest reported in the literature on protein refolding by dilution. The final yield of purified active non-glycosylated rTOP was ca. 60 mg per L of E. coli culture, close to the yield reported before for tomato and tobacco plants overexpressing glycosylated TOP (60 mg/kg biomass) and much higher than for the previously reported refolding protocol (2.6 mg per L of E. coli culture).

  1. Heme-linked ionizations of myeloperoxidase detected by Raman difference spectroscopy. A comparison with plant and yeast peroxidases.

    PubMed Central

    Stump, R F; Deanin, G G; Oliver, J M; Shelnutt, J A

    1987-01-01

    The pH-dependence of the oxidation state marker line v4 of human leucocyte myeloperoxidase is determined in the absence of chloride using Raman difference spectroscopy (RDS). A transition in the frequency of v4 with pK of 4.2 +/- 0.3 is found. The pK compares favorably with that previously determined by spectrophotometric titration and kinetic studies. The shift in v4 across the transition is -1.3 cm-1. The shift in v4 and other Raman marker lines indicates enhanced pi charge in the chlorin ring below the transition. The low frequencies of the oxidation state marker lines indicate that a structural change occurs near the chromophore, which results in the formation of a more pi-charge donating protein environment for the chlorin ring at low pH. The Raman results are discussed in terms of a proposed catalytic control mechanism based on charge stabilization of the energy of ring charge-depleted ferryl intermediates of the reaction with peroxide. The myeloperoxidase findings are compared with similar RDS results for ferrous horseradish peroxidase and ferric cytochrome c peroxidase. PMID:3034344

  2. Peroxidase-catalyzed metabolism of etoposide (VP-16-213) and covalent binding of reactive intermediates to cellular macromolecules

    SciTech Connect

    Haim, N.; Nemec, J.; Roman, J.; Sinha, B.K.

    1987-11-15

    The horseradish peroxidase- and prostaglandin synthetase-catalyzed oxidative metabolism of the highly active anticancer drug, etoposide (VP-16-213), has been studied in vitro. This oxidation of VP-16 resulted in the formation of VP-16 quinone, an aromatic VP-16 derivative and the corresponding aromatic VP-16 quinone. This oxidative metabolism of VP-16 also resulted in the formation of reactive species that covalently bound to exogenously added DNA and heat-inactivated microsomal proteins. The peroxidase-catalyzed binding was time dependent and required the presence of cofactors (hydrogen peroxide or arachidonic acid). The prostaglandin synthetase/arachidonic acid-catalyzed metabolism and binding of VP-16 were inhibited by indomethacin, an inhibitor of the cyclooxygenase, and were shown to involve the peroxidative arm of prostaglandin synthetase. Our studies show that the protein covalent binding species were formed as a result of O-demethylation of the drug as shown by the loss of specifically labeled (O-/sup 14/CH/sub 3/) radioactivity from O-methoxy group and by incubating proteins with VP-16 quinones. In contrast, the covalent binding intermediates for DNA appeared to be different and VP-16-derived quinone methides are suggested as DNA binding species. Co-oxidation of VP-16 and the related drug, VM-26, during prostaglandin biosynthesis may be an important pathway for the metabolism of these agents and may play a role in their cytotoxic properties.

  3. TiO2@CeOx core-shell nanoparticles as artificial enzymes with peroxidase-like activity.

    PubMed

    Artiglia, Luca; Agnoli, Stefano; Paganini, Maria Cristina; Cattelan, Mattia; Granozzi, Gaetano

    2014-11-26

    The Ce4+↔Ce3+ redox switch is at the basis of an all-inorganic catalytic cycle that is capable of mimicking the activity of several natural redox enzymes. The efficiency of these artificial enzymes (nanozymes) strongly depends on the Ce4+/Ce3+ ratio. By capitalizing on the results obtained on oxide/oxide model systems, we implemented a simple and effective procedure to obtain conformal TiO2@CeOx core-shell nanoparticles whose thickness is controlled with single-layer precision. Since the Ce3+ species are stabilized only at the interface by the electronic hybridization with the TiO2 states, the modulation of the shell thickness offers a simple method to tailor the Ce4+/Ce3+ ratio and therefore the catalytic properties. The activity of these nanoparticles as artificial peroxidase-like enzymes was tested, showing exceptional performances, even better than natural horseradish peroxidase enzyme. The main advantage with respect to other oxide/oxide nanozymes is that our nanoparticles, having a tunable Ce4+/Ce3+ ratio, are efficient already at low H2O2 concentrations.

  4. Filling polymersomes with polymers by peroxidase-catalyzed atom transfer radical polymerization.

    PubMed

    Dinu, Maria Valentina; Spulber, Mariana; Renggli, Kasper; Wu, Dalin; Monnier, Christophe A; Petri-Fink, Alke; Bruns, Nico

    2015-03-01

    Polymersomes that encapsulate a hydrophilic polymer are prepared by conducting biocatalytic atom transfer radical polymerization (ATRP) in these hollow nanostructures. To this end, ATRPase horseradish peroxidase (HRP) is encapsulated into vesicles self-assembled from poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PDMS-b-PMOXA) diblock copolymers. The vesicles are turned into nanoreactors by UV-induced permeabilization with a hydroxyalkyl phenone and used to polymerize poly(ethylene glycol) methyl ether acrylate (PEGA) by enzyme-catalyzed ATRP. As the membrane of the polymersomes is only permeable for the reagents of ATRP but not for macromolecules, the polymerization occurs inside of the vesicles and fills the polymersomes with poly(PEGA), as evidenced by (1) H NMR. Dynamic and static light scattering show that the vesicles transform from hollow spheres to filled spheres during polymerization. Transmission electron microscopy (TEM) and cryo-TEM imaging reveal that the polymersomes are stable under the reaction conditions. The polymer-filled nanoreactors mimic the membrane and cytosol of cells and can be useful tools to study enzymatic behavior in crowded macromolecular environments.

  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. Recognition of glycoprotein peroxidase via Con A-carrying self-assembly layer on gold.

    PubMed

    Liu, Songqin; Wang, Kewei; Du, Dan; Sun, Yueming; He, Lin

    2007-07-01

    We have successfully fabricated a self-assembled layer of concanavalin A (Con A) on a gold surface for recognition of glycoproteins. The type IV Con A is covalently bound to 11-mercaptoundecanoic acid (MUA) on gold with a 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU) linkage. The binding interaction between glycoproteins and self-assembled Con A is studied using horseradish peroxidase (HRP) as a model glycoprotein. Voltammetric, electrochemical impedance studies, and photometric activity measurements show the presence of both specific and nonspecific bindings of HRP to the Con A interface. The specific binding is attributed to the Con A-sugar interaction where Con A selectively recognizes the glycosylation sites of HRP. The catalytic current of the HRP-loaded electrode, because of catalytic oxidation of thionine in the presence of hydrogen peroxide (H2O2), is found to be proportional to the HRP concentrations in the incubation solution. A linear correlation coefficient of 0.993 was obtained over a wide HRP concentration range of 12.5 microg/mL to 1 mg/mL. The approach described in this study provides a simple yet selective means to immobilize glycoproteins on a solid support. The specific binding achieved is desirable in biosensor fabrication, glycoprotein separation, recognition, and purification as well as in drug-releasing systems.

  7. 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.

  8. 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

  9. 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…

  10. 3-(10'-Phenothiazinyl)propionic acid is a potent primary enhancer of peroxidase-induced chemiluminescence and its application in sensitive ELISA of methylglyoxal-modified low density lipoprotein.

    PubMed

    Sakharov, Ivan Yu; Demiyanova, Alexandra S; Gribas, Anastasia V; Uskova, Natalia A; Efremov, Evgeny E; Vdovenko, Marina M

    2013-10-15

    Using a full factorial design the optimization of experimental conditions of enhanced chemiluminescence reaction (ECR) catalyzed by horseradish peroxidase (HRP) in the presence of 3-(10'-phenothiazinyl)propionic acid (PPA) as a primary enhancer was performed. The effect of concentrations of PPA, hydrogen peroxide, MORPH, luminol, and Tris on a ratio of peroxidase-catalyzed CL to background was studied. The detection limit value of HRP in ECR with PPA was 0.09 pM. Using PPA the ultra-sensitive chemiluminescent ELISA for determination of methylglyoxal-modified low density lipoprotein was developed. The detection limit value for the developed method was 0.5 ng mL(-1). The obtained results open up very promising perspectives for using PPA to improve the sensitivity of enzyme immunoassay kits.

  11. Peroxidase-benzhydroxamic acid complexes: spectroscopic evidence that a Fe-H2O distance of 2.6 A can correspond to hexa-coordinate high-spin heme.

    PubMed

    Smulevich, G; Feis, A; Indiani, C; Becucci, M; Marzocchi, M P

    1999-02-01

    Resonance Raman (RR) spectra have been obtained for single-crystal horseradish peroxidase isozyme C complexed with benzhydroxamic acid (BHA). The data are compared with those obtained in solution by both RR and electronic absorption spectroscopies at room and low (12-80 K) temperatures. Moreover, the analysis has been extended to Coprinus cinereus peroxidase complexed with BHA. The results obtained for the two complexes are very similar and are consistent with the presence of an aqua six-coordinate high-spin heme. Therefore it can be concluded that despite the rather long Fe-H2O distance of 2.6-2.7 A found by X-ray crystallography in both complexes, the distal water molecule can still coordinate to the heme iron.

  12. 3-(10'-Phenothiazinyl)propionic acid is a potent primary enhancer of peroxidase-induced chemiluminescence and its application in sensitive ELISA of methylglyoxal-modified low density lipoprotein.

    PubMed

    Sakharov, Ivan Yu; Demiyanova, Alexandra S; Gribas, Anastasia V; Uskova, Natalia A; Efremov, Evgeny E; Vdovenko, Marina M

    2013-10-15

    Using a full factorial design the optimization of experimental conditions of enhanced chemiluminescence reaction (ECR) catalyzed by horseradish peroxidase (HRP) in the presence of 3-(10'-phenothiazinyl)propionic acid (PPA) as a primary enhancer was performed. The effect of concentrations of PPA, hydrogen peroxide, MORPH, luminol, and Tris on a ratio of peroxidase-catalyzed CL to background was studied. The detection limit value of HRP in ECR with PPA was 0.09 pM. Using PPA the ultra-sensitive chemiluminescent ELISA for determination of methylglyoxal-modified low density lipoprotein was developed. The detection limit value for the developed method was 0.5 ng mL(-1). The obtained results open up very promising perspectives for using PPA to improve the sensitivity of enzyme immunoassay kits. PMID:24054611

  13. 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

  14. Purification and some properties of peroxidase isozymes from pineapple stem.

    PubMed

    Sung, H Y; Yu, R H; Chang, C T

    1993-01-01

    The enzyme peroxidase is widely distributed among the higher plants. Isozymes of peroxidase are known to occur in a variety of tissues in a large number of plant species. In this study, peroxidase isozymes were purified from the extract of pineapple stem through successive steps of ammonium sulfate fractionation, CM-Sepharose CL-6B chromatographies and DEAE-Sepharose CL-6B chromatographies. By these steps, twelve isozymes of peroxidase were obtained. Some properties of the isozymes were studied and compared.

  15. 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.

  16. A peroxidase isoenzyme secreted by turnip (Brassica napus) hairy-root cultures: inactivation by hydrogen peroxide and application in diagnostic kits.

    PubMed

    Agostini, Elizabeth; Hernández-Ruiz, Josefa; Arnao, Marino B; Milrad, Silvia R; Tigier, Horacio A; Acosta, Manuel

    2002-02-01

    We have purified various peroxidase isoenzymes from roots and hairy-root cultures of turnip (Brassica napus) which could potentially be used for commercial applications such as an enzyme immunoassays, diagnostic test kits, wastewater treatment and soil remediation. One of them, a basic peroxidase called HR2, was secreted into the medium of turnip hairy-root cultures. HR2 had a pI of 9.6, a molecular mass of 39.3 kDa and showed great thermostability. The inactivation of HR2 by H2O2 in the absence of reductant substrates was studied. Under these conditions H2O2 acted as a suicide substrate. The kinetic constants calculated have been compared with those of a basic isoperoxidase from horseradish (Armoracia sp.) roots (HRP-C), which is commonly used in commercial kits. The results for HR2 indicated that it was more resistant to inactivation because it presented a lower inactivation efficiency and a higher value for the partition ratio (r=1250) than those described for HRP-C. These results make turnip peroxidase HR2 suitable for use in systems in which high H2O2 concentrations are found. Such an application is demonstrated, namely an enzymic diagnostic kit for determination of uric acid in which HR2 was found to be as efficient as the enzyme originally included in standard kits. PMID:11834124

  17. [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.

  18. Isolation and characterization of the first putative peroxidase gene from oilseed rape (Brassica napus) which is highly homologous to HRPC.

    PubMed

    Wu, Weisheng; Lu, Jie; Wei, Yamin; Wang, Jin; Lin, Juan; Cao, Shuwen; Sun, Xiaofen; Tang, Kexuan

    2006-06-01

    A new gene, designated as BnPrx (GenBank Accession No. DQ078754), was isolated from oilseed rape (Brassica napus) by SMART Rapid Amplification of cDNA Ends (RACE). The full-length cDNA is 1307 bp long and contains a 1062 bp open reading frame (ORF), which encodes a 354 amino acid peroxidase precursor, with a 31 aa N-terminal signal peptide and a 15 aa C-terminal propeptide. The putative protein has a molecular weight of 38.86 kDa and a calculated pI of 5.85. BnPrx shares high identity with HRPC (89%). BnPrx possesses all active residues and two Ca(2+) sites present in Horseradish peroxidase isoenzymes C (HRPC) as well as six N-glycosylation sites. The predicted 3-D structure of BnPrx is very similar to that of HRPC. Assisted by genomic walking technology, the genomic DNA of BnPrx was also cloned, consisting of 3 introns and 4 exons. Thirty-two TATA boxes, 18 CAAT boxes and many cis-elements, such as WUN, MeJR, were found in its promoter region. Southern blot analysis indicated that BnPrx belonged to a small gene family. Northern blot analysis revealed that BnPrx was constitutively expressed in all tested tissues, including roots, stems and leaves, with the high expression in leaves and stems. The expression of BnPrx could be induced by methyl jasmonate (MeJA), salicylic acid (SA), cold and H(2)O(2). The cloning and characterizing of BnPrx might not only help us understand the physiological function and molecular evolution of the large peroxidase gene family more comprehensively, but also provide an alternative way of seeking a more effective and economical substitute for HRPC. PMID:16855866

  19. Fe-TAML encapsulated inside mesoporous silica nanoparticles as peroxidase mimic: femtomolar protein detection.

    PubMed

    Kumari, Sushma; Dhar, Basab B; Panda, Chakadola; Meena, Abhishek; Sen Gupta, Sayam

    2014-08-27

    Peroxidase, such as horseradish peroxidase (HRP), conjugated to antibodies are routinely used for the detection of proteins via an ELISA type assay in which a critical step is the catalytic signal amplification by the enzyme to generate a detectable signal. Synthesis of functional mimics of peroxidase enzyme that display catalytic activity which far exceeds the native enzyme is extremely important for the precise and accurate determination of very low quantities of proteins (fM and lower) that is necessary for early clinical diagnosis. Despite great advancements, analyzing proteins of very low abundance colorimetrically, a method that is most sought after since it requires no equipment for the analysis, still faces great challenges. Most reported HRP mimics that show catalytic activity greater than native enzyme (∼10-fold) are based on metal/metal-oxide nanoparticles such as Fe3O4. In this paper, we describe a second generation hybrid material developed by us in which approximately 25,000 alkyne tagged biuret modified Fe-tetraamido macrocyclic ligand (Fe-TAML), a very powerful small molecule synthetic HRP mimic, was covalently attached inside a 40 nm mesoporous silica nanoparticle (MSN). Biuret-modified Fe-TAMLs represent one of the best small molecule functional mimics of the enzyme HRP with reaction rates in water close to the native enzyme and operational stability (pH, ionic strength) far exceeding the natural enzyme. The catalytic activity of this hybrid material is around 1000-fold higher than that of natural HRP and 100-fold higher than that of most metal/metal oxide nanoparticle based HRP mimics reported to date. We also show that using antibody conjugates of this hybrid material it is possible to detect and, most importantly, quantify femtomolar quantities of proteins colorimetrically in an ELISA type assay. This represents at least 10-fold higher sensitivity than other colorimetric protein assays that have been reported using metal/metal oxide

  20. 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

  1. Revisiting the Non-Animal Peroxidase Superfamily.

    PubMed

    Lazzarotto, Fernanda; Turchetto-Zolet, Andreia Carina; Margis-Pinheiro, Márcia

    2015-12-01

    Peroxidases reduce peroxide through substrate oxidation in order to alleviate oxidative stress in aerobic organisms. Since the initial description of the non-animal peroxidase superfamily, great effort has been made to characterize this large and heterogeneous group of proteins. Next generation sequencing data have permitted an in-depth study of the molecular evolution of this superfamily and allowed us to perform a phylogenetic reconstruction. Through this analysis, we identified two additional class I members and, here, we discuss the similarities and differences among members of this class. Our results provide new insights into the organization of these antioxidant enzymes, allowing us to propose a new model for the emergence and evolution of this superfamily.

  2. Conversion of aminonitrotoluenes by fungal manganese peroxidase.

    PubMed

    Scheibner, K; Hofrichter, M

    1998-01-01

    Preparations of extracellular manganese peroxidase from the white-rot fungus Nematoloma frowardii and the litter decaying fungus Stropharia rugosoannulata converted rapidly the main intermediates of the explosive 2,4,-trinitrotoluene--the aminonitrotoluenes. In a cell-free system, 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene and 2,6-diamino-4-nitrotoluene were degraded without formation of identifiable metabolites. Radioactive experiments using a complex mixture of uniform ring-labeled 14C-TNT reduction products demonstrated the partial direct mineralization of these compounds by manganese peroxidase. The extent of aminonitrotoluene conversion as well as the release of 14CO2 from TNT reduction products were considerably enhanced in the presence of thiols like reduced glutathione or the amino acid L-cystein, which probably act as secondary mediators.

  3. Insecticidal activity and fungitoxicity of plant extracts and components of horseradish (Armoracia rusticana) and garlic (Allium sativum).

    PubMed

    Tedeschi, Paola; Leis, Marilena; Pezzi, Marco; Civolani, Stefano; Maietti, Annalisa; Brandolini, Vincenzo

    2011-01-01

    To avoid environmental pollution and health problems caused by the use of traditional synthetic pesticides, there is a trend to search for naturally occurring toxicants from plants. Among the compounds discussed for anti-fungal and insecticidal activity, the natural extracts from garlic and horseradish have attracted considerable attention. The objective of this study is to determine the insecticidal and anti-fungal activity of Armoracia rusticana and Allium sativum L. extracts against larvae of Aedes albopictus (Skuse) and some pathogenic fungi. For the insecticidal test, horseradish and garlic extracts were prepared from fresh plants (cultivated in Emilia Romagna region) in a solution of ethanol 80 % and the two different solutions were used at different concentrations (for the determination of the lethal dose) against the fourth instar mosquito's larvae. The fungicidal test was carried out by the agar plates technique using garlic and horseradish extracts in a 10 % ethanol solution against the following organisms: Sclerotium rolfsii Sacc., Trichoderma longibrachiatum, Botrytis cinerea Pers., Fusarium oxysporum Schlecht. and Fusarium culmorum (Wm. G. Sm.) Sacc. The first results demonstrated that the horseradish ethanol extracts present only a fungistatic activity against Sclerotium rolfsii Sacc., Fusarium oxysporum Schlecht. and F. culmorum (Wm.G. Sm) Sacc. while garlic extracts at the same concentration provided a good fungicidal activity above all against Botrytis cinerea Pers. and S. rolfsii. A. rusticana and A. sativum preparations showed also an interesting and significant insecticidal activity against larvae of A. albopictus, even if horseradish presented a higher efficacy (LC₅₀ value of 2.34 g/L), approximately two times higher than garlic one (LC₅₀ value of 4.48 g/L).

  4. 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

  5. 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.

  6. Directed evolution of a fungal peroxidase.

    PubMed

    Cherry, J R; Lamsa, M H; Schneider, P; Vind, J; Svendsen, A; Jones, A; Pedersen, A H

    1999-04-01

    The Coprinus cinereus (CiP) heme peroxidase was subjected to multiple rounds of directed evolution in an effort to produce a mutant suitable for use as a dye-transfer inhibitor in laundry detergent. The wild-type peroxidase is rapidly inactivated under laundry conditions due to the high pH (10.5), high temperature (50 degrees C), and high peroxide concentration (5-10 mM). Peroxidase mutants were initially generated using two parallel approaches: site-directed mutagenesis based on structure-function considerations, and error-prone PCR to create random mutations. Mutants were expressed in Saccharomyces cerevisiae and screened for improved stability by measuring residual activity after incubation under conditions mimicking those in a washing machine. Manually combining mutations from the site-directed and random approaches led to a mutant with 110 times the thermal stability and 2.8 times the oxidative stability of wild-type CiP. In the final two rounds, mutants were randomly recombined by using the efficient yeast homologous recombination system to shuffle point mutations among a large number of parents. This in vivo shuffling led to the most dramatic improvements in oxidative stability, yielding a mutant with 174 times the thermal stability and 100 times the oxidative stability of wild-type CiP.

  7. Enzyme immunoassay of mumps virus in cell culture with peroxidase-labelled virus specific monoclonal antibodies and its application for determination of antibodies.

    PubMed

    van Tiel, F H; Kraaijeveld, C A; Baller, J; Harmsen, T; Oosterlaken, T A; Snippe, H

    1988-10-01

    Mumps neutralizing monoclonal antibodies (MAs) were purified and labelled with horseradish peroxidase and used to detect virus-infected Vero cells, which were seeded as monolayers in wells of 96-well plates. This direct enzyme immunoassay (EIA) in cell culture proved to be a sensitive method for detection and titration of mumps virus and it may be useful for diagnostic purposes. The EIA is also suitable for the rapid determination of neutralizing antibodies. Neutralization of mumps virus by preincubation with either monoclonal or polyclonal antibodies was indicated by inhibition of the absorbance at 450 nm as measured with a multichannelled photometer. The EIA (duration 2 days) for determination of mumps neutralizing antibodies is an attractive alternative for the plaque reduction test (duration 6 days).

  8. Biochemical and pathological studies on peroxidases -an updated review.

    PubMed

    Khan, Amjad A; Rahmani, Arshad H; Aldebasi, Yousef H; Aly, Salah M

    2014-09-01

    Peroxidases represent a family of isoenzymes actively involved in oxidizing reactive oxygen species, innate immunity, hormone biosynthesis and pathogenesis of several diseases. Different types of peroxidases have organ, tissues, cellular and sub-cellular level of specificities in their function. Different diseases lead to varied expressions of peroxidases based on several mechanisms proposed. Several researches are going on to understand its deficiency, over-expression and malfunction in relation with different diseases. Some common diseases of mankind like cancer, cardiovascular diseases and diabetes directly or indirectly involve the role of peroxidases. So the status of peroxidase levels may also function as a marker of different diseases. Although many types of diseases in human beings have a strong correlation with tissue specific peroxidases, the clear role of these oxido-reductases is not yet fully understood. Here we are focusing on the role of peroxidases in relations with different diseases occurring due to oxidative stress.

  9. Enzyme Technology of Peroxidases: Immobilization, Chemical and Genetic Modification

    NASA Astrophysics Data System (ADS)

    Longoria, Adriana; Tinoco, Raunel; Torres, Eduardo

    An overview of enzyme technology applied to peroxidases is made. Immobilization on organic, inorganic, and hybrid supports; chemical modification of amino acids and heme group; and genetic modification by site-directed and random mutagenesis are included. Different strategies that were carried out to improve peroxidase performance in terms of stability, selectivity, and catalytic activity are analyzed. Immobilization of peroxidases on inorganic and organic materials enhances the tolerance of peroxidases toward the conditions normally found in many industrial processes, such as the presence of an organic solvent and high temperature. In addition, it is shown that immobilization helps to increase the Total Turnover Number at levels high enough to justify the use of a peroxidase-based biocatalyst in a synthesis process. Chemical modification of peroxidases produces modified enzymes with higher thermostability and wider substrate variability. Finally, through mutagenesis approaches, it is possible to produce modified peroxidases capable of oxidizing nonnatural substrates with high catalytic activity and affinity.

  10. 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.

  11. Innervation of propatagial musculature in a flying squirrel, Glaucomys volans (Rodentia, Sciuridae).

    PubMed

    Chickering, J G; Sokoloff, A J

    1996-01-01

    The propatagium of gliding and flying mammals is of both functional and phylogenetic interest. The innervation of the propatagial muscle, platysma II, was studied with the axonal tracer wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) in a flying squirrel, Glaucomys volans. Injections of WGA-HRP into the proximal third of platysma II labeled motoneurons in the lateral part of the medial subdivision of the ipsilateral facial nucleus and in the ipsilateral ventral horn of the brachial enlargement. Injections into distal regions of platysma II labeled motoneurons in the ipsilateral ventral horn of spinal segments C5-C8 but not in the facial nucleus. Injections along the whole length of the muscle labeled afferent axons in the ipsilateral dorsal horn of spinal segments C4-T1. These results demonstrate a mixed facial and spinal motor innervation of propatagial musculature in the flying squirrel and indicate that this pattern of mixed innervation is more widespread among flying and gliding mammals than previously reported. Mixed facial and cervical propatagial innervation, independently derived in different flying and gliding mammals, may represent a common solution in the design of the propatagium. These findings complicate the use of propatagial muscle innervation patterns for the establishment of phylogenetic relationships among flying and gliding mammals. PMID:8834780

  12. Distribution of cortical neurons projecting to dorsal column nuclear complex and spinal cord in the hedgehog tenrec, Echinops telfairi.

    PubMed

    Künzle, H; Rehkämper, G

    1992-01-01

    Using retrograde axonal flow and wheatgerm agglutinin conjugated to horseradish peroxidase, we studied the distribution of cortical neurons giving rise to spinal and dorsal column nuclear projections, and correlated the regions involved in the projections with the cytoarchitectonic areas recently identified in the lesser hedgehog tenrec, Echinops telfairi (Insectivora). Labeled cortical neurons were most numerous following injections of tracer into higher cervical segments, whereas almost none were found following thoracic injections. The cortical labeling appeared more prominent ipsilaterally than contralaterally after spinal injections, although it was more prominent on the contralateral side after injection into the dorsal column nuclear complex. The majority of labeled neurons found in lamina V occupied the neocortex adjacent to the interhemispheric fissure along the rostrocaudal extent of the small corpus callosum. This location corresponded to an intermediate rostrocaudal portion of the hemisphere, and particularly to area 2 of Rehkämper. In some cases, adjacent portions of areas 1 and 3 were also involved, as well as neocortical regions of the lateral hemisphere. The present data did not suggest a somatotopic organization of the projections; likewise, evidence for the presence of more than one somatosensorimotor representation was sparse.

  13. Neuronal network disturbance after focal ischemia in rats

    SciTech Connect

    Kataoka, K.; Hayakawa, T.; Yamada, K.; Mushiroi, T.; Kuroda, R.; Mogami, H. )

    1989-09-01

    We studied functional disturbances following left middle cerebral artery occlusion in rats. Neuronal function was evaluated by (14C)2-deoxyglucose autoradiography 1 day after occlusion. We analyzed the mechanisms of change in glucose utilization outside the infarct using Fink-Heimer silver impregnation, axonal transport of wheat germ agglutinin-conjugated-horseradish peroxidase, and succinate dehydrogenase histochemistry. One day after occlusion, glucose utilization was remarkably reduced in the areas surrounding the infarct. There were many silver grains indicating degeneration of the synaptic terminals in the cortical areas surrounding the infarct and the ipsilateral cingulate cortex. Moreover, in the left thalamus where the left middle cerebral artery supplied no blood, glucose utilization significantly decreased compared with sham-operated rats. In the left thalamus, massive silver staining of degenerated synaptic terminals and decreases in succinate dehydrogenase activity were observed 4 and 5 days after occlusion. The absence of succinate dehydrogenase staining may reflect early changes in retrograde degeneration of thalamic neurons after ischemic injury of the thalamocortical pathway. Terminal degeneration even affected areas remote from the infarct: there were silver grains in the contralateral hemisphere transcallosally connected to the infarct and in the ipsilateral substantia nigra. Axonal transport study showed disruption of the corticospinal tract by subcortical ischemia; the transcallosal pathways in the cortex surrounding the infarct were preserved. The relation between neural function and the neuronal network in the area surrounding the focal cerebral infarct is discussed with regard to ischemic penumbra and diaschisis.

  14. 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

  15. 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.

  16. Development of phosphonate modified Fe 1-x MnxFe2O4 mixed ferrite nanoparticles: novel peroxidase mimetics in enzyme linked immunosorbent assay.

    PubMed

    Bhattacharya, Dipsikha; Baksi, Ananya; Banerjee, Indranil; Ananthakrishnan, Rajakumar; Maiti, Tapas K; Pramanik, Panchanan

    2011-10-30

    A highly facile and feasible strategy on the fabrication of advanced intrinsic peroxidase mimetics based on Mn(2+) doped mixed ferrite (Mn(II)(x)Fe(II)(1-x)Fe(III)(2)O(4)) nanoparticles was demonstrated for the quantitative and sensitive detection of mouse IgG (as a model analyte). Mn(2+) doped Fe(1-x)Mn(x)Fe(2)O(4) nanoparticles were synthesized using varying ratios of Mn(2+):Fe(2+) ions and characterized by the well known complementary techniques. The increase of Mn(2+) proportion had remarkably enhanced the peroxidase activity and magnetism. The catalytic activity of mixed ferrites was found to follow Michaelis-Menten kinetics and was noticeably higher than native Fe(3)O(4). The calculated K(m) and K(cat) exhibited strong affinity with substrates which were remarkably higher than similar sized native magnetite nanoparticles and horseradish peroxidase (HRP). These findings stimulated us to develop carboxyl modified Fe(1-x)Mn(x)Fe(2)O(4) nanoparticles using phosphonomethyl immunodiacetic acid (PMIDA) to engineer PMIDA-Fe(1-x)Mn(x)Fe(2)O(4) fabricated enzyme linked immunosorbent assay (ELISA). Results of both PMIDA-Fe(1-x)Mn(x)Fe(2)O(4) linked ELISA revealed that the enhancements in absorbance during the catalysis of enzyme substrate were linearly proportional to the concentration of mouse IgG within the range between 0.1 μg/ml and 2.5 μg/ml. Further, this detection was ten times lower than previous reports and the detection limit of mouse IgG was 0.1 μg/ml. The advantages of our fabricated artificial peroxidase mimetics are combined of low cost, easy to prepare, better stability and tunable catalytic activity. Moreover, this method provides a new horizon for the development of promising analytical tools in the application of biocatalysis, bioassays, and bioseparation.

  17. In vitro analysis of the monolignol coupling mechanism using dehydrogenative polymerization in the presence of peroxidases and controlled feeding ratios of coniferyl and sinapyl alcohol.

    PubMed

    Moon, Sun-Joo; Kwon, Mi; Choi, Donha; Won, Keehoon; Kim, Yong Hwan; Choi, In-Gyu; Choi, Joon Weon

    2012-10-01

    In this study, dehydrogenative polymers (DHP) were synthesized in vitro through dehydrogenative polymerization using different ratios of coniferyl alcohol (CA) and sinapyl alcohol (SA) (10:0, 8:2, 6:4, 2:8, 0:10), in order to investigate the monolignol coupling mechanism in the presence of horseradish peroxidase (HRP), Coprinus cinereus peroxidase (CiP) or soybean peroxidase (SBP) with H(2)O(2), respectively. The turnover capacities of HRP, CiP and SBP were also measured for coniferyl alcohol (CA) and sinapyl alcohol (SA), and CiP and SBP were found to have the highest turnover capacity for CA and SA, respectively. The yields of HRP-catalyzed DHP (DHP-H) and CiP-catalyzed DHP (DHP-C) were estimated between ca. 7% and 72% based on the original weights of CA/SA in these synthetic conditions. However, a much lower yield of SBP-catalyzed DHP (DHP-S) was produced compared to that of DHP-H and DHP-C. In general, the DHP yields gradually increased as the ratio of CA/SA increased. The average molecular weight of DHP-H also increased with increasing CA/SA ratios, while those of DHP-C and DHP-S were not influenced by the ratios of monolignols. The frequency of β-O-4 linkages in the DHPs decreased with increasing CA/SA ratios, indicating that the formation of β-O-4 linkages during DHP synthesis was influenced by peroxidase type.

  18. The relationship between lignin peroxidase and manganese peroxidase production capacities and cultivation periods of mushrooms.

    PubMed

    Xu, Jian Z; Zhang, Jun L; Hu, Kai H; Zhang, Wei G

    2013-05-01

    Mushrooms are able to secrete lignin peroxidase (LiP) and manganese peroxidase (MnP), and able to use the cellulose as sources of carbon. This article focuses on the relation between peroxidase-secreting capacity and cultivation period of mushrooms with non-laccase activity. Methylene blue and methyl catechol qualitative assay and spectrophotometry quantitative assay show LiP secreting unvaryingly accompanies the MnP secreting in mushroom strains. The growth rates of hyphae are detected by detecting the dry hyphal mass. We link the peroxidase activities to growth rate of mushrooms and then probe into the relationship between them. The results show that there are close relationships between LiP- and/or MnP-secretory capacities and the cultivation periods of mushrooms. The strains with high LiP and MnP activities have short cultivation periods. However, those strains have long cultivation periods because of the low levels of secreted LiP and/or MnP, even no detectable LiP and/or MnP activity. This study provides the first evidence on the imitate relation between the level of secreted LiP and MnP activities and cultivation periods of mushrooms with non-laccase activity. Our study has significantly increased the understanding of the role of LiP and MnP in the growth and development of mushrooms with non-laccase activity.

  19. Roles of apoplastic peroxidases in plant response to wounding.

    PubMed

    Minibayeva, Farida; Beckett, Richard Peter; Kranner, Ilse

    2015-04-01

    Apoplastic class III peroxidases (EC 1.11.1.7) play key roles in the response of plants to pathogen infection and abiotic stresses, including wounding. Wounding is a common stress for plants that can be caused by insect or animal grazing or trampling, or result from agricultural practices. Typically, mechanical damage to a plant immediately induces a rapid release and activation of apoplastic peroxidases, and an oxidative burst of reactive oxygen species (ROS), followed by the upregulation of peroxidase genes. We discuss how plants control the expression of peroxidases genes upon wounding, and also the sparse information on peroxidase-mediated signal transduction pathways. Evidence reviewed here suggests that in many plants production of the ROS that comprise the initial oxidative burst results from a complex interplay of peroxidases with other apoplastic enzymes. Later responses following wounding include various forms of tissue healing, for example through peroxidase-dependent suberinization, or cell death. Limited data suggest that ROS-mediated death signalling during the wound response may involve the peroxidase network, together with other redox molecules. In conclusion, the ability of peroxidases to both generate and scavenge ROS plays a key role in the involvement of these enigmatic enzymes in plant stress tolerance.

  20. 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.

  1. Correlation of quinone reductase activity and allyl isothiocyanate formation among different genotypes and grades of horseradish roots.

    PubMed

    Ku, Kang-Mo; Jeffery, Elizabeth H; Juvik, John A; Kushad, Mosbah M

    2015-03-25

    Horseradish (Armoracia rusticana) is a perennial crop and its ground root tissue is used in condiments because of the pungency of the glucosinolate (GS)-hydrolysis products allyl isothiocyanate (AITC) and phenethyl isothiocyanate (PEITC) derived from sinigrin and gluconasturtiin, respectively. Horseradish roots are sold in three grades: U.S. Fancy, U.S. No. 1, and U.S. No. 2 according to the USDA standards. These grading standards are primarily based on root diameter and length. There is little information on whether root grades vary in their phytochemical content or potential health promoting properties. This study measured GS, GS-hydrolysis products, potential anticancer activity (as quinone reductase inducing activity), total phenolic content, and antioxidant activities from different grades of horseradish accessions. U.S. Fancy showed significantly higher sinigrin and AITC concentrations than U.S. No. 1 ,whereas U.S. No. 1 showed significantly higher concentrations of 1-cyano 2,3-epithiopropane, the epithionitrile hydrolysis product of sinigrin, and significantly higher total phenolic concentrations than U.S. Fancy.

  2. 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.

  3. 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.

  4. The Con-A-peroxidase method for tissue localization of glucosyl and mannosyl groups applied to mouse hepatocytes and chicken erythrocytes.

    PubMed

    Moraes, Alberto S; Mello, Maria Luiza S

    2006-01-01

    A variation of the Concanavalin A (Con-A)-peroxidase labelling method originally described by Kiernan [Localization of alpha-D-glucosyl and alpha-D-mannosyl groups of mucosubstances with Concanavalin A and horseradish peroxidase. Histochemistry 1975;44:39-45] was applied to unsectioned cell preparations, with an emphasis on the nuclear localization of glycoproteins. Mouse liver imprints and chicken blood smears fixed in acetic acid-ethanol solution were studied. Modifications of the method included using increased Con-A concentration, and a range of pH values for the Con-A solutions. The strongest Con-A labelling of both erythrocytes and hepatocytes was obtained after incubation with Con-A at pH 6.5 and with Con-A concentrations at least two-fold greater than those used for tissue sections. These conditions may alter the Con-A conformation, enabling the lectin molecule to enter the cell nucleus and bind to nuclear glycoproteins, thus allowing their localization and quantification.

  5. Purification of peroxidase isoenzymes from turnip roots.

    PubMed

    Hamed, R R; Maharem, T M; Abel Fatah, M M; Ataya, F S

    1998-08-01

    Simple reproducible procedures for purification of the main soluble (S) and ionically bound (IB) cationic peroxidase isoenzymes from turnip roots were established. The procedures included ammonium sulfate precipitation of the isoenzymes, chromatographic separation of the main isoenzymes using cellulose phosphate columns and purification to homogeneity by hydrophobic interaction chromatography on phenyl Sepharose columns. The specific activity of the phenyl Sepharose purified S and IB isoenzymes were 2760 and 896 units/mg protein with 140 and 4.8 fold increase over the crude extract and 38 and 13% recovery. The pH maxima and K(m) for phenol and H2O2 of purified S and IB were determined. PMID:9720311

  6. 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

  7. PeroxiBase: a class III plant peroxidase database.

    PubMed

    Bakalovic, Nenad; Passardi, Filippo; Ioannidis, Vassilios; Cosio, Claudia; Penel, Claude; Falquet, Laurent; Dunand, Christophe

    2006-03-01

    Class III plant peroxidases (EC 1.11.1.7), which are encoded by multigenic families in land plants, are involved in several important physiological and developmental processes. Their varied functions are not yet clearly determined, but their characterization will certainly lead to a better understanding of plant growth, differentiation and interaction with the environment, and hence to many exciting applications. Since there is currently no central database for plant peroxidase sequences and many plant sequences are not deposited in the EMBL/GenBank/DDBJ repository or the UniProt KnowledgeBase, this prevents researchers from easily accessing all peroxidase sequences. Furthermore, gene expression data are poorly covered and annotations are inconsistent. In this rapidly moving field, there is a need for continual updating and correction of the peroxidase superfamily in plants. Moreover, consolidating information about peroxidases will allow for comparison of peroxidases between species and thus significantly help making correlations of function, structure or phylogeny. We report a new database (PeroxiBase) accessible through a web server with specific tools dedicated to facilitate query, classification and submission of peroxidase sequences. Recent developments in the field of plant peroxidase are also mentioned.

  8. Visible-Light-Induced Activity Control of Peroxidase Bound to Fe-Doped Titanate Nanosheets with Nanometric Lateral Dimensions.

    PubMed

    Kamada, Kai; Ito, Daiki; Soh, Nobuaki

    2015-10-21

    Catalytic performance of horseradish peroxidase (HRP) electrostatically adsorbed on nanometric and semiconducting Fe-doped titanate (FT) nanosheets was successfully manipulated by visible light illumination. A colloidal solution of FT with a narrow band gap corresponding to a visible light region was fabricated through a hydrolysis reaction of metals sources. HRP could be easily bound to the FT at pH = 4 through an electrostatic interaction between them, and the formed HRP-FT was utilized for the visible-light-driven enzymatic reaction. Under exposure to visible light with enough energy for band gap excitation of the FT, catalytic activity of HRP-FT was dramatically enhanced as compared with free (unbound) HRP and was simply adjusted by light intensity. In addition, wavelength dependence of an enzymatic reaction rate was analogous to an optical absorption spectrum of the FT. These results substantiated an expected reaction mechanism in which the photoenzymatic reaction was initiated by band gap excitation of FT followed by transferring holes generated in the valence band of irradiated FT to HRP. The excited HRP oxidized substrates (amplex ultrared: AUR) accompanied by two-electron reduction to regenerate the resting state. In addition, the catalytic activity was clearly switched by turning on and off the light source.

  9. Generation of reactive species and fate of thiols during peroxidase-catalyzed metabolic activation of aromatic amines and phenols

    SciTech Connect

    Ross, D.; Moldeus, P.

    1985-12-01

    The horseradish peroxidase (HRP)-catalyzed oxidation of p-phenetidine and acetaminophen was investigated. Studies using the spin probe 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH) suggested these oxidations involve the generation of substrate-derived free radicals. This was confirmed by using glutathione (GSH) in these incubations in the presence of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), DMPO-glutathionyl radical adducts were observed using EPR spectroscopy during HRP-catalyzed oxidation of both p-phenetidine and acetaminophen. Investigations of oxygen uptake and oxidized glutathione (GSSG) formation during HRP-catalyzed oxidations of p-phenetidine and acetaminophen suggested that further reactions of the glutathionyl radical involve glutathione peroxysulfenyl radical and glutathione sulfenyl hydroperoxide production. Quinonoid products of the peroxidatic oxidations of p-phenetidine and acetaminophen, and their interaction with GSH via both conjugation and redox mechanisms are described. The relevance of these reactions of GSH with reactive species as detoxification mechanisms is discussed. 29 references.

  10. 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

  11. Effects of allyl isothiocyanate from horseradish on several experimental gastric lesions in rats.

    PubMed

    Matsuda, Hisashi; Ochi, Momotaro; Nagatomo, Akifumi; Yoshikawa, Masayuki

    2007-04-30

    Allyl isothiocyanate is well known to be a principal pungent constituent of horseradish and an agonist for transient receptor potential (TRP) A1. Ally isothiocyanate markedly inhibited the formation of gastric lesions induced by ethanol (1.5 ml/rat, p.o.), 0.6 M HCl (1.5 ml/rat, p.o.), 1% ammonia (1.5 ml/rat, p.o.), and aspirin (150 mg/kg, p.o.) (ED(50)=1.6, 2.2, 1.7, ca. 6.5 mg/kg, p.o.). It also significantly inhibited the formation of gastric lesions induced by indomethacin (20 mg/kg, p.o.), though the inhibition was ca. 60% at a high dose (40 mg/kg, p.o.). Furthermore, several synthetic isothiocyanate compounds also significantly inhibited ethanol and indomethacin-induced gastric lesions. Whereas, TRPV1 agonists, capsaicin and piperine, inhibited gastric lesions induced by ethanol, 1% ammonia, and aspirin, but had less of an effect on 0.6 M HCl-induced gastric lesions. With regard to mode of action, the protective effects of ally isothiocyanate on ethanol-induced gastric lesions were attenuated by pretreatment with indomethacin, but not with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME), or ruthenium red. Pretreatment with indomethacin reduced the protective effects of piperine, and L-NAME reduced the effects of capsaicin and omeprazole. Furthermore, ruthenium red reduced the effects of capsaicin, piperine, and omeprazole. These findings suggest that endogenous prostaglandins play an important role in the protective effect of allyl isothiocyanate in ethanol-induced gastric lesions different from capsaicin, piperine, and omeprazole.

  12. 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.

  13. The quantum mixed-spin heme state of barley peroxidase: A paradigm for class III peroxidases.

    PubMed Central

    Howes, B D; Schiodt, C B; Welinder, K G; Marzocchi, M P; Ma, J G; Zhang, J; Shelnutt, J A; Smulevich, G

    1999-01-01

    Electronic absorption and resonance Raman (RR) spectra of the ferric form of barley grain peroxidase (BP 1) at various pH values, at both room temperature and 20 K, are reported, together with electron paramagnetic resonance 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 coexists with 6- and 5-cHS hemes; 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 currently 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 may be necessary for the QS state. PMID:10388773

  14. Oxidation of indole-3-acetic acid by peroxidase: involvement of reduced peroxidase and compound III with superoxide as a product.

    PubMed

    Smith, A M; Morrison, W L; Milham, P J

    1982-08-31

    Kinetic and spectral data establish that peroxidase may oxidize indole-3-acetic acid by either of two pathways depending on the enzyme/substrate ratio. When relatively low enzyme/substrate ratios are employed, the oxidation proceeds through a reduced peroxidase in equilibrium compound III shuttle. Conversely, peroxidase operates through the conventionally accepted pathway involving native enzyme and compounds I and II only when high enzyme/substrate ratios are used. Compound III, a specific oxidase, constitutes the dominant steady-state form of peroxidase when the reduced peroxidase in equilibrium compound III shuttle is operational. Activation of this shuttle also produces a flux of superoxide anion radical at the expense of molecular oxygen. Thus, important biological consequences may follow activation of this shuttle under physiological conditions.

  15. One-pot synthesis of porphyrin functionalized γ-Fe2O3 nanocomposites as peroxidase mimics for H2O2 and glucose detection.

    PubMed

    Liu, Qingyun; Zhang, Leyou; Li, Hui; Jia, Qingyan; Jiang, Yanling; Yang, Yanting; Zhu, Renren

    2015-10-01

    Meso-tetrakis(4-carboxyphenyl)-porphyrin-functionalized γ-Fe2O3 nanoparticles (H2TCPP-γ-Fe2O3) were successfully prepared by one-pot method under hydrothermal conditions and were found to possess intrinsic peroxidase-like activity. The H2TCPP-γ-Fe2O3 nanocomposites can catalytically oxidize peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2 to produce a blue color reaction, which can be easily observed by the naked eye. Furthermore, kinetic studies indicate that the H2TCPP-γ-Fe2O3 nanocomposites have an even higher affinity to TMB than that of the natural enzyme, horseradish peroxidase (HRP). On the basis of the high activity, the reaction provides a simple, sensitive and selective method for colorimetric detection of H2O2 over a range of 10-100 μM with a minimum detection limit of 1.73 μM. Moreover, H2TCPP-γ-Fe2O3/glucose oxidase (GOx)/TMB system provides a novel colorimetric sensor for glucose and shows good response toward glucose detection over a range of 5-25 μM with a minimum detection limit of 2.54 μM. The results indicated that it is a simple, cheap, convenient, highly selective, sensitive and easy handling colorimetric assay. Results of a fluorescent probe suggest that the catalase-mimic activity of the H2TCPP-γ-Fe2O3 nanocomposites effectively catalyze the decomposition of H2O2 into H2O and O2.

  16. 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.

  17. 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.

  18. Purification and characterization of peroxidase from papaya (Carica papaya) fruit.

    PubMed

    Pandey, Veda P; Singh, Swati; Singh, Rupinder; Dwivedi, Upendra N

    2012-05-01

    Ripening of papaya fruit was found to be characterized with a decrease in peroxidase activity and its transcript. This peroxidase was purified to homogeneity through successive steps of ammonium sulfate fractionation, ion exchange and molecular exclusion chromatography. The peroxidase was purified 30.22-folds with overall recovery of 44.37% and specific activity of 68.59. Purified peroxidase was found to be a heterotrimer of ~240 kDa, containing two subunits each of 85 and one of 70 kDa. Purified enzyme exhibited pH and temperature optima of 7.0 and 40 °C, respectively. K(m) values for substrates o-dianicidin, guaiacol and ascorbic acid were found to be 0.125, 0.8 and 5.2 mM, respectively. K(m) for H(2)O(2) was found to be 0.25 mM. Salicylic acid was found to activate peroxidase up to 50 μM concentration, beyond which it acted as inhibitor. Ca(2+) and Mg(2+) activated peroxidase while sodium azide, SDS, and Triton X-100 were found to inhibit peroxidase.

  19. Purification and characterization of windmill palm tree (Trachycarpus fortunei) peroxidase.

    PubMed

    Caramyshev, Alexei V; Firsova, Yuliya N; Slastya, Evgen A; Tagaev, Andrei A; Potapenko, Nataly V; Lobakova, Elena S; Pletjushkina, Olga Yu; Sakharov, Ivan Yu

    2006-12-27

    High peroxidase activity was demonstrated to be present in the leaf of several species of cold-resistant palms. Histochemical studies of the leaf of windmill palm tree (Trachycarpus fortunei) showed the peroxidase activity to be localized in hypoderma, epidermis, cell walls, and conducting bundles. However, chlorophyll-containing mesophyll cells had no peroxidase at all. The leaf windmill palm tree peroxidase (WPTP) was purified to homogeneity and had a specific activity of 6230 units/mg, RZ = 3.0, a molecular mass of 50 kDa, and an isoelectric point of pI 3.5. The electronic spectrum of WPTP with a Soret band at 403 nm was typical of plant peroxidases. The N-terminal amino acid sequence of WPTP was determined. The substrate specificity of WPTP was distinct from that of other palm peroxidases, and the best substrate for WPTP was 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid). The palm peroxidase showed an unusually high stability at elevated temperatures and high concentrations of guanidine.

  20. Magnetic circular dichroism studies of myoglobin, hemoglobin and peroxidase at room and low temperatures. Ferrous high spin derivatives.

    PubMed

    Sharonov, Y A; Mineyev, A P; Livshitz, M A; Sharonova, N A; Zhurkin, V B; Lysov, Y P

    1978-04-13

    The magnetic circular dichroism spectra (MCD) recorded for the visible and near-UV regions of high-spin ferrous derivatives of myoglobin, hemoglobin, hemoglobin dimers and isolated chains as well as of horseradish peroxidase at pH 6.8 and 11.4 have been compared at the room and liquid nitrogen temperatures. The MCD of the Q00- and QV-bands have been shown to be sensitive to structural differences in the heme environment of these hemoproteins. The room temperature visible MCD of native hemoglobin differs from that of myoglobin, hemoglobin dimers and isolated chains as well as from that of model pentacoordinated complex. The MCD of hemoglobin is characterized by the greater value of the MCD intensity ratio of derivative shape A-term in the Q00-band to the A-term in the QV-band. The evidneces are presented for the existence of two pH-dependent forms of ferroperoxidase, the neutral peroxidase shows the "hemoglobin-like" MCD, while the alkaline ferroperoxidase is characterized by the "myoglobin-like" MCD spectrum in the visible region. The differences in the MCD of deoxyhemoglobin and neutral ferroperoxidase as compared with other high-spin ferrous hemoproteins are considered to result from the constraints on heme group imposed by quaternary and/or tertiary protein structure. The differences between hemoporteins which are seen at the room temperature become more pronounced at liquid nitrogen temperature. Except the peak at approximately 580 nm in the MCD of deoxymyoglobin and reduced peroxidase at pH 11.4 the visible MCD does not show appreciable temperature dependent C-terms. The nature of the temperature dependent effect at approximately 580 nm is not clear. The Soret MCD of all hemoproteins studied are similar and are predominantly composed of the derivative-shaped C-terms as revealed by the increase of the MCD peaks approximately in accordance with Boltzmann distribution. The interpretation of temperature-dependent MCD observed for the Soret band has been made in

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. Improved operational stability of peroxidases by coimmobilization with glucose oxidase.

    PubMed

    van de Velde, F; Lourenço, N D; Bakker, M; van Rantwijk, F; Sheldon, R A

    2000-08-01

    The operational stability of peroxidases was considerably enhanced by generating hydrogen peroxide in situ from glucose and oxygen. For example, the total turnover number of microperoxidase-11 in the oxidation of thioanisole was increased sevenfold compared with that obtained with continuous addition of H(2)O(2). Coimmobilization of peroxidases with glucose oxidase into polyurethane foams afforded heterogeneous biocatalysts in which the hydrogen peroxide is formed inside the polymeric matrix from glucose and oxygen. The total turnover number of chloroperoxidase in the oxidation of thioanisole and cis-2-heptene was increased to new maxima of 250. 10(3) and 10. 10(3), respectively, upon coimmobilization with glucose oxidase. Soybean peroxidase, which normally shows only classical peroxidase activity, was transformed into an oxygen-transfer catalyst when coimmobilized with glucose oxidase. The combination catalyst mediated the enantioselective oxidation of thioanisole [50% ee (S)] with 210 catalyst turnovers. PMID:10861408

  6. CYTOCHEMICAL LOCALIZATION OF ENDOGENOUS PEROXIDASE IN THYROID FOLLICULAR CELLS

    PubMed Central

    Strum, Judy M.; Karnovsky, Morris J.

    1970-01-01

    Endogenous peroxidase activity in rat thyroid follicular cells is demonstrated cytochemically. Following perfusion fixation of the thyroid gland, small blocks of tissue are incubated in a medium containing substrate for peroxidase, before being postfixed in osmium tetroxide, and processed for electron microscopy. Peroxidase activity is found in thyroid follicular cells in the following sites: (a) the perinuclear cisternae, (b) the cisternae of the endoplasmic reticulum, (c) the inner few lamellae of the Golgi complex, (d) within vesicles, particularly those found apically, and (e) associated with the external surfaces of the microvilli that project apically from the cell into the colloid. In keeping with the radioautographic evidence of others and the postulated role of thyroid peroxidase in iodination, it is suggested that the microvillous apical cell border is the major site where iodination occurs. However, that apical vesicles also play a role in iodination cannot be excluded. The in vitro effect of cyanide, aminotriazole, and thiourea is also discussed. PMID:4190069

  7. 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

  8. 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.

  9. 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.

  10. Iron deficiency differently affects peroxidase isoforms in sunflower.

    PubMed

    Ranieri, A; Castagna, A; Baldan, B; Soldatini, G F

    2001-01-01

    The response of both specific (ascorbate peroxidase, APX) and unspecific (POD) peroxidases and H(2)O(2) content of sunflower plants (Helianthus annuus L. cv. Hor) grown hydroponically with (C) or without (-Fe) iron in the nutrient solution were analysed to verify whether iron deficiency led to cell oxidative status. In -Fe leaves a significant increase of H(2)O(2) content was detected, a result confirmed by electron microscopy analysis. As regards extracellular peroxidases, while APX activity significantly decreased, no change was observed in either soluble guaiacol or syringaldazine-dependent POD activity following iron starvation. Moreover, guaiacol-dependent POD activity was found to decrease in both ionically and covalently-cell-wall bound fractions, while syringaldazine-POD activity decreased only in the covalently-bound fraction. At the intracellular level both guaiacol-POD and APX activities underwent a significant decrease. The overall reduction of peroxidase activity was confirmed by the electrophoretic separation of POD isoforms and, at the extracellular level, by cytochemical localization of peroxidases by diaminobenzidine staining. The electrophoretic separation, besides quantitative differences, also revealed quantitative changes, particularly evident for ionically and covalently-bound fractions. Therefore, in sunflower plants, iron deficiency seems to affect the different peroxidase isoenzymes to different extents and to induce a secondary oxidative stress, as indicated by the increased levels of H(2)O(2). However, owing to the almost completely lack of catalytic iron capable of triggering the Fenton reaction, iron-deficient sunflower plants are probably still sufficiently protected against oxidative stress.

  11. 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.

  12. Glutathione peroxidase and catalase modulate the genotoxicity of arsenite.

    PubMed

    Wang, T S; Shu, Y F; Liu, Y C; Jan, K Y; Huang, H

    1997-09-01

    The X-ray hypersensitive Chinese hamster ovary (CHO) cells, xrs-5, are also more sensitive to sodium arsenite in terms of cell growth and micronucleus induction than CHO-K1 cells. Since reactive oxygen species are suggested to be involved in arsenic toxicity, we have measured antioxidant mechanisms in xrs-5 as well as CHO-K1 cells. There were no apparent differences in the activities of superoxide dismutase, glutathione S-transferase, glutathione reductase, and the levels of glutathione between xrs-5 and CHO-K1 cells. However, the activities of glutathione peroxidase and catalase were 5.4- and 5.8-fold lower, respectively, in xrs-5 cells. The addition of catalase or glutathione peroxidase to cultures reduced the arsenite-induced micronuclei in xrs-5 cells. Whereas, simultaneous treatment with mercaptosuccinate, an inhibitor of glutathione peroxidase, and 3-aminotriazole, an inhibitor of catalase, synergistically increased the arsenite-induced micronuclei. These results suggest that both catalase and glutathione peroxidase are involved in defense against arsenite genotoxicity. The xrs-6 cells, another line of x-ray hypersensitive CHO cells, which had 1.6-fold higher catalase activity and 2.5-fold higher glutathione peroxidase activity than xrs-5 cells, were also more sensitive than CHO-K1 cells but were less sensitive than xrs-5 cells to cell growth inhibition of arsenite. Moreover, a 1.6-fold increase of glutathione peroxidase activity by selenite adaptation effectively removed the arsenite-induced micronuclei in CHO-K1 cells. These results suggest that glutathione peroxidase is more important than catalase in defending against arsenite toxicity. Our results also suggest that increasing the intracellular antioxidant level may have preventive or therapeutic effects in arsenic poisoning.

  13. Looking for Arabidopsis thaliana peroxidases involved in lignin biosynthesis.

    PubMed

    Herrero, Joaquín; Esteban-Carrasco, Alberto; Zapata, José Miguel

    2013-06-01

    Monolignol polymerization into lignin is catalyzed by peroxidases or laccases. Recently, a Zinnia elegans peroxidase (ZePrx) that is considered responsible for monolignol polymerization in this plant has been molecularly and functionally characterized. Nevertheless, Arabidopsis thaliana has become an alternative model plant for studies of lignification, filling the gaps that may occur with Z. elegans. The arabidopsis genome offers the possibility of performing bioinformatic analyses and data mining that are not yet feasible with other plant species, in order to obtain preliminary evidence on the role of genes and proteins. In our search for arabidopsis homologs to the ZePrx, we performed an exhaustive in silico characterization of everything from the protein to the transcript of Arabidopsis thaliana peroxidases (AtPrxs) homologous to ZePrx, with the aim of identifying one or more peroxidases that may be involved in monolignol polymerization. Nine peroxidases (AtPrx 4, 5, 52, 68, 67, 36, 14, 49 and 72) with an E-value greater than 1e-80 with ZePrx were selected for this study. The results demonstrate that a high level of 1D, 2D and 3D homology between these AtPrxs and ZePrx are not always accompanied by the presence of the same electrostatic and mRNA properties that indicate a peroxidase is involved in lignin biosynthesis. In summary, we can confirm that the peroxidases involved in lignification are among AtPrx 4, 52, 49 and 72. Their structural and mRNA features indicate that exert their action in the cell wall similar to ZePrx.

  14. Glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase in tissues of Balb/C mice

    SciTech Connect

    Spallholz, J.E.; Roveri, A.; Yan, L.; Boylan, L.M.; Kang, C.R.; Ursini, F. Univ. of Padua )

    1991-03-11

    The two selenium (Se) enzymes glutathione peroxidase (GPx) and phospholipid hydroperoxide glutathione peroxide (PHGPx) were assayed in tissues and organ homogenates of female Balb/C mice fed torula yeast diets containing 0.008, 0.2 and 1.0 ug/g Se as selenite for nine months. GPx activity was detected in all tissues and organs whereas PHGPx activity was not detectable in lung, large intestine, eye or thymus tissue. GPx activity nearly always exceeded PHGPx activity when tissues or organs contained both enzymes irrespective of dietary Se treatment. GPx activity in tissues was generally more susceptible to the dietary Se deficiency than was PHGPx activity when expressed as a percentage of the activity found in tissues and organs of mice fed supplemented Se diets. This limited study suggests that dietary Se may be more valued in supporting PHGPx activity than GPx activity in the course of a protracted dietary Se deficiency.

  15. In vivo toxicity and immunogenicity of wheat germ agglutinin conjugated poly(ethylene glycol)-poly(lactic acid) nanoparticles for intranasal delivery to the brain

    SciTech Connect

    Liu Qingfeng; Shao Xiayan; Chen Jie; Shen Yehong; Feng Chengcheng; Gao Xiaoling; Zhao Yue; Li Jingwei; Zhang Qizhi Jiang, Xinguo

    2011-02-15

    Biodegradable polymer-based nanoparticles have been widely studied to deliver therapeutic agents to the brain after intranasal administration. However, knowledge as to the side effects of nanoparticle delivery system to the brain is limited. The aim of this study was to investigate the in vivo toxicity and immunogenicity of wheat germ agglutinin (WGA) conjugated poly(ethylene glycol)-poly(lactic acid) nanoparticles (WGA-NP) after intranasal instillation. Sprague-Dawley rats were intranasally given WGA-NP for 7 continuous days. Amino acid neurotransmitters, lactate dehydrogenase (LDH) activity, reduced glutathione (GSH), acetylcholine, acetylcholinesterase activity, tumor necrosis factor {alpha} (TNF-{alpha}) and interleukin-8 (IL-8) in rat olfactory bulb (OB) and brain were measured to estimate the in vivo toxicity of WGA-NP. Balb/C mice were intranasally immunized by WGA-NP and then WGA-specific antibodies in serum and nasal wash were detected by indirect ELISA. WGA-NP showed slight toxicity to brain tissue, as evidenced by increased glutamate level in rat brain and enhanced LDH activity in rat OB. No significant changes in acetylcholine level, acetylcholinesterase activity, GSH level, TNF-{alpha} level and IL-8 level were observed in rat OB and brain for the WGA-NP group. WGA-specific antibodies in mice serum and nasal wash were not increased after two intranasal immunizations of WGA-NP. These results demonstrate that WGA-NP is a safe carrier system for intranasal delivery of therapeutic agents to the brain.

  16. 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.

  17. Cantaloupe melon peroxidase: characterization and effects of additives on activity.

    PubMed

    Lamikanra, O; Watson, M A

    2000-06-01

    Peroxidase in cantaloupe melon (Cucumis melo L. var. reticulatus Naud.), a fruit commonly fresh cut processed, was characterized to determine reaction pathway, optimal conditions for activity and effect of some additives on enzymatic action. Mn2+, CaCl2, NaNO2 and kinetin had partial inhibitory effects on enzyme activity. Activity was effectively inhibited by compounds capable of chelating peroxidase heme iron such as diethyldithiocarbamate and tiron, but unaffected by EDTA. Free radical scavenger, superoxide dismutase, also had no effect on reaction velocity. Enzymatic action was consistent with that of ascorbate peroxidase based on the relatively higher affinity for ascorbate over guaiacol. Optimum activity temperature was 50-55 degrees C. The enzyme was stable at temperatures below 40 degrees C and at 50 degrees C for up to 10 min. Over 90% of total activity was lost at 80 degrees C within 5 min. Broad pH optima, 5.5-7.5 at 50 degrees C and 6-7 at 30 degrees C, were obtained. Peroxidase activity in cantaloupe was higher than those in strawberry (Fragaria ananassa Duch.) and lettuce (Lactuca sativa L.), suggesting a relatively high oxidative stress in fresh cut cantaloupe. The potential use of ascorbate as an additive in fresh cut cantaloupe melon was demonstrated by its ability to preserve color in minimally processed fruits for 25 days at 4 degrees C, possibly as a result of an enhanced antioxidative action of the ascorbate-peroxidase complex and trace metal ion cofactors.

  18. 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.

  19. 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.

  20. Peroxidases from cell suspension cultures of Brassica napus.

    PubMed

    Agostini, E; de Forchetti, S M; Tigier, H A

    2000-08-01

    Cell suspension cultures of Brassica napus were obtained under different hormonal conditions, using 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin as growth regulators. They were analyzed as a culture system for peroxidase production in vitro to avoid many of the problems that affect the production from field-grown roots. Total peroxidase specific activities reached a maximum at the end of exponential growth phase of the cultures. Cultures obtained with 4 mg/l of 2,4-D an without kinetin or with 1 mg/l of 2,4-D and the same amount of kinetin produced twice the total activity of root extracts and, in addition, they released peroxidases to the culture medium, which would be advantageous for the commercial production of the enzyme. Peroxidase patterns, obtained by isoelectric focusing of cell extracts and of culture medium of cell suspension cultures, differed from those of root crude extracts from field-grown plants with additional bands of higher isoelectric points. These cultures showed interesting properties and could be considered an alternative source of peroxidases for commercial production and/or to be applied as a model for physiological research. PMID:10979611

  1. 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.

  2. Electron pathways in catalase and peroxidase enzymic catalysis. Metal and macrocycle oxidations of iron porphyrins and chlorins

    SciTech Connect

    Hanson, L.K.; Chang, C.K.; Davis, M.S.; Fajer, J.

    1981-02-11

    Charge iterative extended Hueckel calculations are presented for compound II, the one-electron oxidation intermediate of horseradish peroxidase (HRP), and for compounds I, the two-electron oxidation transients of HRP and catalase (CAT) observed in the catalytic cycles of the hydroperoxidase enzymes. Compound II is described in terms of a ferryl configuration (O = Fe/sup IV/), and compounds I are described as ferrylporphyrin ..pi..-cation radicals. The validity of the iron ..pi..-cation calculations is supported by favorable comparison of parallel computations for porphyrin ..pi.. cations of diamagnetic metals with new and previously reported ESR results for radicals of zinc tetrabenz-, meso-tetramethyl, (/sup 14/N and /sup 15/N) tetraphenyl-, and magnesium (/sup 1/H and /sup 2/H) octaethylporphyrins. The calculated electronic configurations and unpaired spin density profiles for the ferryl ..pi.. cations satisfactorily account for the physical properties reported for compounds I of HRP (in the native protoporphyrin IX form or reconstituted with deuteroporphyrin), chloroperoxidase, and CAT. The ground states of the ..pi.. cations, a/sub 1u/ or a/sub 2u/, are determined by peripheral substitution and axial ligation, and the axial ligand of CAT I is predicted to differ from that of HRP I. The combination of model studies and calculations suggests that /sup 2/H, /sup 13/C, and /sup 15/N NMR studies of isotopically substituted proto and deutero HRP I would confirm the electronic profiles predicted. /sup 15/N NMR in particular would clearly discriminate between a/sub 1u/ and a/sub 2u/ configurations. As an additional test of the ferryl ..pi..-cation hypothesis, calculations are presented for a proposed ferrylchlorin ..pi.. cation of Neurospora crassa catalase, which contains an iron chlorin prosthetic group. Compound I of this unusual heme is predicted to occupy an a/sub 2/ ground state with the spin distribution and optical spectra reported here for synthetic chlorin

  3. Evidence for the involvement of cell wall peroxidase in the generation of hydroxyl radicals mediating extension growth.

    PubMed

    Liszkay, Anja; Kenk, Barbara; Schopfer, Peter

    2003-08-01

    Hydroxyl radicals (*OH), produced in the cell wall, are capable of cleaving wall polymers and can thus mediate cell wall loosening and extension growth. It has recently been proposed that the biochemical mechanism responsible for *OH generation in the cell walls of growing plant organs represents an enzymatic reaction catalyzed by apoplastic peroxidase (POD). This hypothesis was investigated by supplying cell walls of maize ( Zea mays L.) coleoptiles and sunflower ( Helianthus annuus L.) hypocotyls with external NADH, an artificial substrate known to cause *OH generation by POD in vitro. The effects of NADH on wall loosening, growth, and *OH production in vivo were determined. NADH mediates cell wall extension in vitro and in vivo in an H2O2-dependent reaction that shows the characteristic features of POD. NADH-mediated production of *OH in vivo was demonstrated in maize coleoptiles using electron paramagnetic resonance spectroscopy in combination with a specific spin-trapping reaction. Kinetic properties and inhibitor/activator sensitivities of the *OH-producing reaction in the cell walls of coleoptiles resembled the properties of horseradish POD. Apoplastic consumption of external NADH by living coleoptiles can be traced back to the superimposed action of two enzymatic reactions, a KCN-sensitive reaction mediated by POD operating in the *OH-forming mode, and a KCN-insensitive reaction with the kinetic properties of a superoxide-producing plasma-membrane NADH oxidase the activity of which can be promoted by auxin. Under natural conditions, i.e. in the absence of external NADH, this enzyme may provide superoxide (O2*-) (and H2O2 utilized by POD for) *OH production in the cell wall.

  4. Optimization of extracellular fungal peroxidase production by 2 Coprinus species.

    PubMed

    Ikehata, Keisuke; Pickard, Michael A; Buchanan, Ian D; Smith, Daniel W

    2004-12-01

    Optimum culture conditions for the batch production of extracellular peroxidase by Coprinus cinereus UAMH 4103 and Coprinus sp. UAMH 10067 were explored using 2 statistical experimental designs, including 2-level, 7-factor fractional factorial design and 2-factor central composite design. Of the 7 factors examined in the screening study, the concentrations of carbon (glucose) and nitrogen (peptone or casitone) sources showed significant effects on the peroxidase production by Coprinus sp. UAMH 10067. The optimum glucose and peptone concentrations were determined as 2.7% and 0.8% for Coprinus sp. UAMH 10067, and 2.9% and 1.4% for C. cinereus UAMH 4103, respectively. Under the optimized culture condition the maximum peroxidase activity achieved in this study was 34.5 U x mL(-1) for Coprinus sp. UAMH 10067 and 68.0 U x mL(-1) for C. cinereus UAMH 4103, more than 2-fold higher than the results of previous studies.

  5. 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.

  6. Properties of a cationic peroxidase from Citrus jambhiri cv. Adalia.

    PubMed

    Mohamed, Saleh A; El-Badry, Mohamed O; Drees, Ehab A; Fahmy, Afaf S

    2008-08-01

    The major pool of peroxidase activity is present in the peel of some Egyptian citrus species and cultivars compared to the juice and pulp. Citrus jambhiri cv. Adalia had the highest peroxidase activity among the examined species. Four anionic and one cationic peroxidase isoenzymes from C. jambhiri were detected using the purification procedure including ammonium sulfate precipitation, chromatography on diethylaminoethanol-cellulose, carboxymethyl-cellulose, and Sephacryl S-200 columns. Cationic peroxidase POII is proved to be pure, and its molecular weight was 56 kDa. A study of substrate specificity identified the physiological role of POII, which catalyzed the oxidation of some phenolic substrates in the order of o-phenylenediamine > guaiacol > o-dianisidine > pyrogallol > catechol. The kinetic parameters (K (m), V (max), and V (max)/K (m)) of POII for hydrolysis toward H2O2 and electron donor substrates were studied. The enzyme had pH and temperature optima at 5.5 and 40 degrees C, respectively. POII was stable at 10-40 degrees C and unstable above 50 degrees C. The thermal inactivation profile of POII is biphasic and characterized by a rapid decline in activity on exposure to heat. The most of POII activity (70-80%) was lost at 50, 60, and 70 degrees C after 15, 10, and 5 min of incubation, respectively. Most of the examined metal ions had a very slight effect on POII except of Li+, Zn2+, and Hg2+, which had partial inhibitory effects. In the present study, the instability of peroxidase above 50 degrees C makes the high temperature short time treatment very efficient for the inactivation of peel peroxidase contaminated in orange juice to avoid the formation of off-flavors. PMID:18633734

  7. 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.

  8. [Purification and characterization of peroxidase from Phellinus igniarius (author's transl)].

    PubMed

    Krüger, G; Pfeil, E

    1976-08-01

    A Peroxidase (EC 1.11.1.7) of the basidiomycet Phellinus igniarius was derived from mycel and a medium containing glucose and extract of yeast by using various methods of preparation. The enzyme resists extreme conditions (pH, temperature salt concentration). Its optimum pH for activities is in the acid range. Two isoenzymes were found. The molecular weight, isoelectric point, Michaelis-Menten constant, indolacetic acid oxidase activity and spectral and analytical properties of this peroxidase were determined. It is assumed that the enzyme has an intracellular as well as an extracellular field of activity. PMID:962469

  9. Glutathione peroxidase and iron-thiol dependent lipid peroxidation.

    PubMed

    Punekar, N S; Lardy, H A

    1989-12-01

    Role of glutathione peroxidase in iron-thiol-mediated lipid peroxidation was examined. The enzyme was unable to prevent peroxidation of extracted rat liver microsomal lipids. In contrast, when arachidonic acid was the substrate, glutathione peroxidase did decrease the formation of thiobarbituric acid-reactive material. Superoxide dismutase produced a consistent but partial inhibition of peroxidation and catalase was without effect. Our results suggest that iron-thiol-dependent lipid peroxidation cannot be completely blocked by protective enzymes that are effective in other systems. PMID:2635868

  10. Determination of inhibitory concentrations of antiviral agents in cell culture by use of an enzyme immunoassay with virus-specific, peroxidase-labeled monoclonal antibodies.

    PubMed Central

    van Tiel, F H; Boere, W A; Harmsen, T; Kraaijeveld, C A; Snippe, H

    1985-01-01

    An enzyme immunoassay (EIA) to determine 50% inhibitory concentrations of drugs which suppress Semliki Forest virus replication is described. Inhibition of virus replication was measured in L-cells, seeded as monolayers in 96-well plates by use of horseradish peroxidase-labeled monoclonal antibodies directed against the E1 glycoprotein of Semliki Forest virus. The antiviral agents tested were cycloheximide, tunicamycin, NH4Cl, and disodium cromoglycate. The 50% inhibitory concentration of these antiviral agents was arbitrarily defined as the concentration of drug, in culture medium, associated with 50% reduction of the control absorbance value measured on Semliki Forest virus-infected cells without drug in the culture fluid. Twenty-two hours after infection the 50% inhibitory concentrations of the drugs were 0.2 microgram/ml for cycloheximide, 0.8 microgram/ml for tunicamycin, 0.3 mg/ml for NH4Cl, and 4.9 mg/ml for disodium cromoglycate. These values are similar to those determined by others with conventional methods of virus quantification. This test is sensitive and easy to perform and therefore is suited for large-scale experiments. PMID:3925876

  11. 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.

  12. 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

  13. 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

  14. Effects of different media supplements on the production of an active recombinant plant peroxidase in a Pichia pastoris Δoch1 strain.

    PubMed

    Gmeiner, Christoph; Spadiut, Oliver

    2015-01-01

    Recombinant protein production in microorganisms is one of the most studied areas of research in biotechnology today. In this respect the yeast Pichia pastoris is an important microbial production host due to its capability of secreting the target protein and performing posttranslational modifications. In a recent study, we described the development of a robust bioprocess for a glyco-engineered recombinant P. pastoris strain where the native α-1,6-mannosyltransfrease OCH1 was knocked out (Δoch1 strain). This strain produced the glycosylated enzyme horseradish peroxidase (HRP) with more homogeneous and shorter surface glycans than the respective benchmark strain. However, the recombinant Δoch1 strain was physiologically impaired and thus hard to cultivate. We faced cell cluster formation, cell lysis and consequent intensive foam formation. Thus, 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 in a multivariate manner. However, not only process parameters might influence these characteristics, but also media supplements might have an impact. Here, we describe the effects of different heme-precursors as well as of a protease-inhibitor cocktail on the production of active HRP in therecombinant P. pastoris Δoch1strain.

  15. 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.

  16. 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

  17. 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 864.7675 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7675...

  18. 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 864.7675 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7675...

  19. 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 864.7675 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7675...

  20. 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 864.7675 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7675...

  1. 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 864.7675 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7675...

  2. Interference of peptone and tyrosine with the lignin peroxidase assay.

    PubMed Central

    ten Have, R; Hartmans, S; Field, J A

    1997-01-01

    The N-unregulated white rot fungus Bjerkandera sp. strain BOS55 was cultured in 1 liter of peptone-yeast extract medium to produce lignin peroxidase (LiP). During the LiP assay, the oxidation of veratryl alcohol to veratraldehyde was inhibited due to tyrosine present in the peptone and the yeast extract. PMID:9251220

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. [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.

  9. 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

  10. Pt74Ag26 nanoparticle-decorated ultrathin MoS2 nanosheets as novel peroxidase mimics for highly selective colorimetric detection of H2O2 and glucose.

    PubMed

    Cai, Shuangfei; Han, Qiusen; Qi, Cui; Lian, Zheng; Jia, Xinghang; Yang, Rong; Wang, Chen

    2016-02-14

    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 (˙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.

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. Corticocortical connections of cat primary somatosensory cortex.

    PubMed

    Schwark, H D; Esteky, H; Jones, E G

    1992-01-01

    The organization of corticocortical connections in the representation of the forepaw in cat primary somatosensory cortex (SI) was studied following injections of various tracers into different cortical cytoarchitectonic areas. Small injections of horseradish peroxidase, wheat germ agglutinin-conjugated HRP, Phaseolus vulgaris leukoagglutinin, or fast blue were placed into the representation of the forepaw in areas 3b, 1, or 2. The positions of labeled neurons in SI and the surrounding cortical areas were plotted on flattened surface reconstructions to determine the organization of the corticocortical connections within SI. A strong, reciprocal projection linked the two forepaw representations which have been described in area 3b and the part of area 2 which lies in the anterior bank of the lateral ansate sulcus (see Iwamura and Tanaka 1978a, b). Dense projections also linked these areas with SII, as previously reported (Burton and Kopf 1984a). Additional projections to area 3b arose primarily from areas 3a and 1. Projections to area 2 were more widespread than those to area 3b, and arose from all other areas of SI as well as from areas 4 and 5a. All injections into SI tended to label groups of neurons which lay in mediolateral strips. Corticocortical projection neurons which were most heavily labeled by SI injections were pyramidal cells in layer III. Additional projections from area 2 to 3b, area 5a to 2, and SII to areas 2 and 3b arose from layer VI as well. Although neurons of layers III and VI were always the most densely labeled, large injections into SI labeled neurons in layers II and V as well. PMID:1282890

  16. Origin and distribution of cerebral vascular innervation from superior cervical, trigeminal and spinal ganglia investigated with retrograde and anterograde WGA-HRP tracing in the rat.

    PubMed

    Arbab, M A; Wiklund, L; Svendgaard, N A

    1986-11-01

    Peripheral sources of cerebral vascular innervation have been investigated with retrograde and anterograde neuronal tracing of wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) in the rat. For retrograde identification of sources of innervation, WGA-HRP was applied to the exposed basilar artery through a fine slit in the overlying meninges, and sections of brain and peripheral ganglia were reacted with tetramethylbenzidine for detection of the tracer. A high density of tetramethylbenzidine reaction product was observed around the basilar artery and in the surrounding pial tissue, but the application sites were not completely selective since some tracer always had spread into the ventral brain stem. Retrogradely labelled cell bodies were identified in the superior cervical, stellate, first and second spinal, and trigeminal ganglia, i.e. these ganglia may represent origins of basilar artery innervation. In a second series of experiments, microinjections of WGA-HRP were placed into the indicated ganglia to obtain anterograde labelling of nerve fibres on whole-mounts of the cerebral vessels. Injections into trigeminal ganglia labelled nerve fibres on the ipsilateral half of the circle of Willis, as well as the contralateral anterior cerebral artery and the rostral part of the basilar artery. The first and second spinal ganglia projected to the vertebrobasilar arteries, while the ipsilateral part of the internal carotid (outside the circle of Willis) received fibres from the second spinal ganglion. Nerve fibres originating in trigeminal and spinal ganglia were organised in bundles, and between these a sparse plexus of thin single fibres appeared. Injection of WGA-HRP into superior cervical ganglion labelled a plexus of nerve fibres on the ipsilateral circle of Willis and the (rostral) basilar artery. These experiments demonstrated the origin and distribution of sympathetic and sensory innervation to major cerebral arteries in the rat.

  17. Areal, modular, and connectional organization of visual cortex in a prosimian primate, the slow loris (Nycticebus coucang).

    PubMed

    Preuss, T M; Beck, P D; Kaas, J H

    1993-01-01

    Slow lorises (Nycticebus coucang) are nocturnal prosimian (i.e. strepsirhine) primates, closely related to bushbabies (Galago spp.). We examined the organization of visual cortex in four hemispheres from two slow lorises, using connectional and architectonic techniques. All hemispheres were flattened and sections stained for myelin and cytochrome oxidase (CO). Our results indicate, first, that the primary visual area (V1) in slow lorises has a system of small CO-dense blobs, as has been described in most other anthropoid and prosimian primates examined to date. The second visual area (V2) is characterized by broad, stripe-like zones of dense CO staining separated by zones of lighter staining. Loris V2 stripes are less distinct than those of anthropoid primates, and separate classes of thin and thick dark stripes are not apparent. However, V2 stripes are much better developed than in Galago, where they are virtually absent. Injections of wheat-germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) in area V1 revealed reciprocal connections with area V2, and the middle temporal (MT) and dorsolateral (DL) extrastriate areas. Area MT was also identified by its distinctive, dense myelination. As has been reported in anthropoids, DL can be divided into separate caudal and rostral divisions, which differ in myelin and CO staining, and in the strength of their connections with V1. Taken together, our results suggest that many of the features that characterize visual cortex organization in anthropoid primates are present in prosimians and thus probably evolved early in primate history, prior to the diversification of modern primate groups.

  18. Increased serotonergic innervation of lumbosacral motoneurons of rolling mouse Nagoya in correlation with abnormal hindlimb extension.

    PubMed

    Koyanagi, Y; Sawada, K; Sakata-Haga, H; Jeong, Y-G; Fukui, Y

    2006-12-01

    Rolling Mouse Nagoya (RMN) carries a mutation in a gene encoding for alpha(1A) subunit of P/Q-type Ca(2+) channel (Ca(v)2.1). In addition to ataxia, this mutant mouse exhibits abnormal hindlimb extension, which is characterized by a sustained excessive tone of hindlimb extensor muscles. This study aimed to clarify whether serotonergic (5-HTergic) innervation of the spinal motoneurons was altered in RMN in relation to the abnormal hindlimb extension. The density of 5-HT immunoreactive fibres in the ventral horn of lumbar and sacral regions of spinal cord was significantly greater in RMN than in controls. Retrograde wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) labelling combined with 5-HT immunostaining revealed that the number of 5-HT immunoreactive terminals adjoining femoris quadriceps motoneurons was about 2.5-fold greater in RMN than in controls. Furthermore, 5-HT immunostaining in the lumbar cord ventral horn was examined in three other Ca(v)2.1 mutant mice (tottering, leaner and pogo) as to whether or not they showed the abnormal hindlimb extension. Among these mutants, the increased density of 5-HT immunoreactive fibres was observed in correlation with the presence of the abnormal hindlimb extension. The results suggest an increased 5-HTergic innervation of the lumbosacral motoneurons in correlation with the abnormal hindlimb extension in RMN and other Ca(v)2.1 mutant mice. As 5-HT is known to induce the sustained membrane depolarizations without continuous excitatory synaptic inputs (plateau potentials) in spinal motoneurons, the increased 5-HTergic innervation may cause the sustained excitation of hindlimb extensor motoneurons, resulting in the abnormal hindlimb extension.

  19. The central localization of the vagus nerve in the ferret (Mustela putorius furo) and the mink (Mustela vison).

    PubMed

    Ranson, R N; Butler, P J; Taylor, E W

    1993-05-01

    The location of vagal preganglionic neurones (VPN) has been determined in nine ferrets (Mustela putorius furo) and seven mink (M. vison) using neuronal tract-tracing techniques employing horseradish peroxidase (HRP) and wheat-germ agglutinin conjugated HRP (WGA-HRP) mixtures injected into the nodose ganglion of the vagus nerve. Labelled VPN were located ipsilaterally in the dorsal motor nucleus of the vagus (DmnX), nucleus ambiguus (nA), and reticular formation (rf) of the medulla oblongata. In four of the ferrets, labelled VPN were also identified in the nucleus dorsomedialis (ndm) and the nucleus of the spinal accessory nerve (nspa). In a single mink a few labelled cells were observed in the ndm but no labelled VPN were found in the nspa. Labelling of afferent components of the vagus nerve was seen in two ferrets and two mink with the best labelling obtained following an injection of an HRP/WGA-HRP mixture into the nodose ganglion. Labelled afferents were observed to cross the ipsilateral spinal trigeminal tract (SpV) before entering the tractus solitarius (TS) in regions separate from the motor axons which exit the medulla in separate fasicles. Sensory terminal fields were identified bilaterally in the nucleus of the tractus solitarius (nTS) in both species and bilaterally in the area postrema (ap) of the ferret; however, the contralateral labelling was sparse in comparison to the densely labelled ipsilateral nTS/ap. Maximal terminal labelling was seen in regions just rostral and caudal to obex in both species.

  20. Central pupillary light reflex circuits in the cat: I. The olivary pretectal nucleus.

    PubMed

    Sun, Wensi; May, Paul J

    2014-12-15

    The central pathways subserving the feline pupillary light reflex were examined by defining retinal input to the olivary pretectal nucleus (OPt), the midbrain projections of this nucleus, and the premotor neurons within it. Unilateral intravitreal wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) injections revealed differences in the pattern of retinal OPt termination on the two sides. Injections of WGA-HRP into OPt labeled terminals bilaterally in the anteromedian nucleus, and to a lesser extent in the supraoculomotor area, centrally projecting Edinger-Westphal nucleus, and nucleus of the posterior commissure. Labeled terminals, as well as retrogradely labeled multipolar cells, were present in the contralateral OPt, indicating a commissural pathway. Injections of WGA-HRP into the anteromedian nucleus labeled fusiform premotor neurons within the OPt, as well as multipolar cells in the nucleus of the posterior commissure. Connections between retinal terminals and the pretectal premotor neurons were characterized by combining vitreous chamber and anteromedian nucleus injections of WGA-HRP in the same animal. Fusiform-shaped, retrogradely labeled cells fell within the anterogradely labeled retinal terminal field in the OPt. Ultrastructural analysis revealed labeled retinal terminals containing clear spherical vesicles. They contacted labeled pretectal premotor neurons via asymmetric synaptic densities. These results provide an anatomical substrate for the pupillary light reflex in the cat. Pretectal premotor neurons receive direct retinal input via synapses suggestive of an excitatory drive, and project directly to nuclei containing preganglionic motoneurons. These projections are concentrated in the anteromedian nucleus, indicating its involvement in the pupillary light reflex.

  1. Purification of turnip peroxidase and its kinetic properties.

    PubMed

    Singh, Naresh; Gade, W N; Singh, Jai

    2002-02-01

    Peroxidase from turnip roots was purified using metal affinity chromatography up to a specific activity of 337 units/mg protein with 3.02 RZ and 63.5% recovery. After purification, the enzyme showed 2-3 bands on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme was found to be 37-39 kD with matrix assisted laser desorption ionization mass spectrometer (MALDI-MS). The enzyme showed maximum activity in phosphate buffer, pH 6.0, and lowest activity in borate buffer at the same pH. The Km of the enzyme was found to be 7.07 x 104 mM. Turnip peroxidase also contains an iron moiety which is found to be about 0.28%. The enzyme showed 50% inhibition of its specific activity with ethylene diamine tetraacetic acid (EDTA). PMID:11934076

  2. Molecular Dynamics Simulations of Lignin Peroxidase in Solution

    PubMed Central

    Francesca Gerini, M.; Roccatano, Danilo; Baciocchi, Enrico; Nola, Alfredo Di

    2003-01-01

    The dynamical and structural properties of lignin peroxidase and its Trp171Ala mutant have been investigated in aqueous solution using molecular dynamics (MD) simulations. In both cases, the enzyme retained its overall backbone structure and all its noncovalent interactions in the course of the MD simulations. Very interestingly, the analysis of the MD trajectories showed the presence of large fluctuations in correspondence of the residues forming the heme access channel; these movements enlarge the opening and facilitate the access of substrates to the enzyme active site. Moreover, steered molecular dynamics docking simulations have shown that lignin peroxidase natural substrate (veratryl alcohol) can easily approach the heme edge through the access channel. PMID:12770894

  3. Proteomic identification of a basic peroxidase stabilized within acetylated polymannan polysaccharide of Aloe barbadensis.

    PubMed

    Vittori, Natale; Martín, Mercedes; Sabater, Bartolomé

    2012-01-01

    Acetylated polymannan polysaccharide (ApmP) isolated from Aloe barbadensis Miller contains a stable peroxidase that was solubilized to investigate its biochemical, electrophoretic, immunological, and proteomic properties. In the electrophoretic band corresponding to the solubilized peroxidase, proteomic analysis detected seven tryptic peptides that matched homologous peptides covering one third of the ATP22a peroxidase of Arabidopsis thaliana. All the characteristics tested indicated that the activity stabilized within the ApmP pertains to the basic secretory peroxidase family, which includes members that have several biotechnological uses. Hence ApmP might yield a widely used peroxidase in stabilized form.

  4. Polyphenol oxidase and peroxidase in fruits and vegetables.

    PubMed

    Vámos-Vigyázó, L

    1981-01-01

    Polyphenol oxidases and peroxidases are among the most studied enzymes in fruits and vegetables. Owing to the deleterious effects of discoloration and off-flavor formation induced by their actions, these enzymes have not ceased to be a matter of concern to food technologists, while their versatility as catalyst and their diversity as protein present a challenge to the biochemist. This article gives an account on the present state of knowledge in this field. The occurrence of polyphenol oxidases and peroxidases in food and food raw materials, and their role and importance in food processing are briefly outlined. Results of biochemical research including catalytic properties, substrate specificity, susceptibility towards pH and temperature, action of inhibitors, isolation, purification, and characteristics of the enzymes are given, with special emphasis on recent achievements based on high resolution separation and isoenzyme techniques. Finally, the behavior of polyphenol oxidase and peroxidase in selected major groups of fruits and vegetables is discussed. Some contradictions found in the literature are pointed out and some questions that have not been given the necessary attention by researchers so far are mentioned.

  5. Multifunctional catalytic platform for peroxidase mimicking, enzyme immobilization and biosensing.

    PubMed

    Maroneze, Camila Marchetti; Dos Santos, Glauco P; de Moraes, Vitoria B; da Costa, Luiz P; Kubota, Lauro Tatsuo

    2016-03-15

    A hybrid platform based on ionic liquid-based alkoxysilane functionalized mesoporous silica was applied for the synthesis of supported Pt nanoparticles with peroxidase-like catalytic activity. The positively charged groups (imidazolium) chemically bonded to the surface provide dual-functionality as ion-exchangers to the hybrid material, firstly used for the in situ synthesis of the highly dispersed Pt nanostructures and, secondly, for the immobilization of biological species aiming biosensing purposes. The peroxidase-like catalytic activity of the SiO2/Imi/Pt material was evaluated towards the H2O2-mediated oxidation of a chromogenic peroxidase substrate (TMB), allowing the colorimetric detection of H2O2. Finally, to further explore the practical application of this nanomaterial-based artificial system, glucose oxidase (GOx) was immobilized on the catalytic porous platform and a bioassay for the colorimetric determination of glucose was successfully conducted as a model system. The enzyme-like catalytic properties of the SiO2/Imi/Pt as well as its ability to immobilize and keep active biological entities on the porous structure indicate that this hybrid porous platform is potentially useful for the development of biosensing devices. PMID:26499871

  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. 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

  8. 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.

  9. Production and Characterization of Monoclonal Antibodies to Wall-Localized Peroxidases from Corn Seedlings 1

    PubMed Central

    Kim, Sung-Ha; Terry, Maurice E.; Hoops, Pepper; Dauwalder, Marianne; Roux, Stanley 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. Images Fig. 3 Fig. 4 Fig. 5 Fig. 7 Fig. 8 PMID:11537437

  10. 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.

  11. Biochemical and Pathological Studies on Peroxidases –An Updated Review

    PubMed Central

    Khan, Amjad A.; Rahmani, Arshad H.; Aldebasi, Yousef H.; Aly, Salah M.

    2014-01-01

    Peroxidases represent a family of isoenzymes actively involved in oxidizing reactive oxygen species, innate immunity, hormone biosynthesis and pathogenesis of several diseases. Different types of peroxidases have organ, tissues, cellular and sub-cellular level of specificities in their function. Different diseases lead to varied expressions of peroxidases based on several mechanisms proposed. Several researches are going on to understand its deficiency, over-expression and malfunction in relation with different diseases. Some common diseases of mankind like cancer, cardiovascular diseases and diabetes directly or indirectly involve the role of peroxidases. So the status of peroxidase levels may also function as a marker of different diseases. Although many types of diseases in human beings have a strong correlation with tissue specific peroxidases, the clear role of these oxido-reductases is not yet fully understood. Here we are focusing on the role of peroxidases in relations with different diseases occurring due to oxidative stress. PMID:25168993

  12. Effect of culture temperature on the heterologous expression of Pleurotus eryngii versatile peroxidase in Aspergillus hosts.

    PubMed

    Eibes, G M; Lú-Chau, T A; Ruiz-Dueñas, F J; Feijoo, G; Martínez, M J; Martínez, A T; Lema, J M

    2009-01-01

    Production of recombinant versatile peroxidase in Aspergillus hosts was optimized through the modification of temperature during bioreactor cultivations. To further this purpose, the cDNA encoding a versatile peroxidase of Pleurotus eryngii was expressed under control of the alcohol dehydrogenase (alcA) promoter of Aspergillus nidulans. A dependence of recombinant peroxidase production on cultivation temperature was found. Lowering the culture temperature from 28 to 19 degrees C enhanced the level of active peroxidase 5.8-fold and reduced the effective proteolytic activity twofold. Thus, a maximum peroxidase activity of 466 U L(-1) was reached. The same optimization scheme was applied to a recombinant Aspergillus niger that bore the alcohol dehydrogenase regulator (alcR), enabling transformation with the peroxidase cDNA under the same alcA promoter. However, with this strain, the peroxidase activity was not improved, while the effective proteolytic activity was increased between 3- and 11-fold compared to that obtained with A. nidulans.

  13. 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.

  14. 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.

  15. Mn(II) regulation of lignin peroxidases and manganese-dependent peroxidases from lignin-degrading white rot fungi

    SciTech Connect

    Bonnarme, P.; Jeffries, T.W. )

    1990-01-01

    Two families of peroxidases-lignin peroxidase (LiP) and manganese-dependent lignin peroxidase (MnP)-are formed by the lignin-degrading white rot basidiomycete Phanerochaete chrysosporium and other white rot fungi. Isoenzymes of these enzyme families carry out reactions important to the biodegradation of lignin. This research investigated the regulation of LiP and MnP production by Mn(II). In liquid culture, LiP titers varied as an inverse function of and MnP titers varied as a direct function of the Mn(II) concentration. The extracellular isoenzyme profiles differed radically at low and high Mn(II) levels, whereas other fermentation parameters, including extracellular protein concentrations, the glucose consumption rate, and the accumulation of cell dry weight, did not change significantly with the Mn(II) concentration. In the absence of Mn(II), extracellular LiP isoenzymes predominated, whereas in the presence of Mn(II), MnP isoenzymes were dominant. The release of {sup 14}CO{sub 2} from {sup 14}C-labeled dehydrogenative polymerizate lignin was likewise affected by Mn(II). The rate of {sup 14}CO{sub 2} release increased at low Mn(II) and decreased at high Mn(II) concentrations. This regulatory effect of Mn(II) occurred with five strains of P. chrysosporium, two other species of Phanerochaete, three species of Phlebia, Lentinula edodes, and Phellinus pini.

  16. 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.

  17. 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

  18. 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

  19. Measurement of malondialdehyde, glutathione, and glutathione peroxidase in SLE patients.

    PubMed

    Gheita, Tamer A; Kenawy, Sanaa A

    2014-01-01

    Oxidative stress contributes to chronic inflammation of tissues and plays a central role in immunomodulation, which may lead to autoimmune diseases such as systemic lupus erythematosus (SLE) and antiphospholipid syndrome. Markers of oxidative damage include malondialdehyde (MDA), antioxidant scavengers as glutathione (GSH), and glutathione peroxidase (GSH Px), which all correlate well with SLE disease activity. Amelioration of some clinical manifestations of SLE may be expected by targeting lipid peroxidation with dietary or pharmacological antioxidants. Here, we describe the detection of the key players of oxidant/antioxidant imbalance in SLE.

  20. Acute effects of mustard, horseradish, black pepper and ginger on energy expenditure, appetite, ad libitum energy intake and energy balance in human subjects.

    PubMed

    Gregersen, N T; Belza, A; Jensen, M G; Ritz, C; Bitz, C; Hels, O; Frandsen, E; Mela, D J; Astrup, A

    2013-02-14

    Chilli peppers have been shown to enhance diet-induced thermogenesis (DIT) and reduce energy intake (EI) in some studies, but there are few data on other pungent spices. The primary aim of the present study was to test the acute effects of black pepper (pepper), ginger, horseradish and mustard in a meal on 4 h postprandial DIT. The secondary aim was to examine the effects on subjective appetite measures, ad libitum EI and energy balance. In a five-way placebo-controlled, single-blind, cross-over trial, twenty-two young (age 24·9 (SD 4·6) years), normal-weight (BMI 21·8 (SD 2·1) kg/m²) males were randomly assigned to receive a brunch meal with either pepper (1·3 g), ginger (20 g), horseradish (8·3 g), mustard (21 g) or no spices (placebo). The amounts of spices were chosen from pre-testing to make the meal spicy but palatable. No significant treatment effects were observed on DIT, but mustard produced DIT, which tended to be larger than that of placebo (14 %, 59 (SE 3) v. 52 (SE 2) kJ/h, respectively, P=0·08). No other spice induced thermogenic effects approaching statistical significance. Subjective measures of appetite (P>0·85), ad libitum EI (P=0·63) and energy balance (P=0·67) also did not differ between the treatments. Finally, horseradish decreased heart rate (P=0·048) and increased diastolic blood pressure (P= 0·049) compared with placebo. In conclusion, no reliable treatment effects on appetite, EI or energy balance were observed, although mustard tended to be thermogenic at this dose. Further studies should explore the possible strength and mechanisms of the potential thermogenic effect of mustard actives, and potential enhancement by, for example, combinations with other food components.

  1. 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.

  2. Characterization of Laccases and Peroxidases from Wood-Rotting Fungi (Family Coprinaceae)

    PubMed Central

    Heinzkill, Marion; Bech, Lisbeth; Halkier, Torben; Schneider, Palle; Anke, Timm

    1998-01-01

    Panaeolus sphinctrinus, Panaeolus papilionaceus, and Coprinus friesii are described as producers of ligninolytic enzymes. P. papilionaceus and P. sphinctrinus both produced a laccase. In addition, P. sphinctrinus produced a manganese peroxidase. C. friesii secreted a laccase and two peroxidases similar to the peroxidase of Coprinus cinereus. The purified laccases and peroxidases were characterized by broad substrate specificities, significant enzyme activities at alkaline pH values, and remarkably high pH optima. The two peroxidases of C. friesii remained active at pH 7.0 and 60°C for up to 60 min of incubation. The peroxidases were inhibited by sodium azide and ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), whereas the laccases were inhibited by sodium azide and N,N-diethyldithiocarbamic acid. As determined by native polyacrylamide gel electrophoresis and isoelectric focusing, all three fungi produced laccase isoenzymes. PMID:9572923

  3. Characterization of laccases and peroxidases from wood-rotting fungi (family Coprinaceae).

    PubMed

    Heinzkill, M; Bech, L; Halkier, T; Schneider, P; Anke, T

    1998-05-01

    Panaeolus sphinctrinus, Panaeolus papilionaceus, and Coprinus friesii are described as producers of ligninolytic enzymes. P. papilionaceus and P. sphinctrinus both produced a laccase. In addition, P. sphinctrinus produced a manganese peroxidase. C. friesii secreted a laccase and two peroxidases similar to the peroxidase of Coprinus cinereus. The purified laccases and peroxidases were characterized by broad substrate specificities, significant enzyme activities at alkaline pH values, and remarkably high pH optima. The two peroxidases of C. friesii remained active at pH 7.0 and 60 degrees C for up to 60 min of incubation. The peroxidases were inhibited by sodium azide and ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), whereas the laccases were inhibited by sodium azide and N,N-diethyldithiocarbamic acid. As determined by native polyacrylamide gel electrophoresis and isoelectric focusing, all three fungi produced laccase isoenzymes.

  4. The nop gene from Phanerochaete chrysosporium encodes a peroxidase with novel structural features.

    PubMed

    Larrondo, Luisf; Gonzalez, Angel; Perez Acle, Tomas; Cullen, Dan; Vicuña, Rafael

    2005-07-01

    Inspection of the genome of the ligninolytic basidiomycete Phanerochaete chrysosporium revealed an unusual peroxidase_like sequence. The corresponding full length cDNA was sequenced and an archetypal secretion signal predicted. The deduced mature protein (NoP, novel peroxidase) contains 295 aa residues and is therefore considerably shorter than other Class II (fungal) peroxidases, such as lignin peroxidases and manganese peroxidases. Comparative modeling of NoP was conducted using the crystal structures of Coprinus cinereus and Arthromyces ramosus peroxidases as templates. The model was validated by molecular dynamics and showed several novel structural features. In particular, NoP has only three disulfide bridges and tryptophan replaces the distal phenylalanine within the heme pocket.

  5. [Initiation and inhibition of free-radical processes in biochemical peroxidase systems: a review].

    PubMed

    Metelitsa, D I; Karaseva, E I

    2007-01-01

    The role of complexes containing oxygen or peroxide in monooxygenase systems and models thereof, as well as in peroxidase- and quasi-peroxidase-catalyzed processes, has been reviewed. Pathways of conversion of these intermediate complexes involving single-electron (radical) and two-electron (heterolytic) mechanisms are dealt with. Coupled peroxidase-catalyzed oxidation of aromatic amines and phenols is analyzed; inhibition and activation of peroxidase-catalyzed reactions are characterized quantitatively. Oxidation of chromogenic substrates (ABTS, OPD, and TMB) in the presence of phenolic inhibitors or polydisulfides of substituted phenols is characterized by inhibition constants (Ki, micromol). Activation of peroxidase-catalyzed oxidation of the same substrates is characterized by the degree (coefficient) of activation (alpha, M(-1)), which was determined for 2-aminothiazole, melamine, tetrazole, and its 5-substituted derivatives. Examples of applied use of peroxidase-catalyzed enzyme and model systems are given (oxidation of organic compounds, chemical analysis, enzyme immunoassay, tests for antioxidant activity of biological fluids).

  6. Three differentially expressed basic peroxidases from wound-lignifying Asparagus officinalis.

    PubMed

    Holm, Kirsten B; Andreasen, Per H; Eckloff, Reinhard M G; Kristensen, Brian K; Rasmussen, Søren K

    2003-10-01

    The activity of ionically bound peroxidases from an asparagus spear increased from 5-24 h post-harvest. Isoelectric focusing showed that the post-harvest increase of the total peroxidase activity was due to the increase of several distinct isoperoxidases. Concomitantly, a decrease in the activity of two anionic peroxidases was observed. Peroxidases with pI 5.9, 6.4 and 9.2 were detected only at 24 h post-harvest, whereas four peroxidases, with pI 8.7, 8.1, 7.4, and 6.7, detected throughout the time-course, increased in their activity. Histochemical staining demonstrated that lignin and peroxidase activity were located in the vascular bundles throughout the period of measurement. Lignin was detected in the cell walls of the protoxylem in the vascular bundles of the asparagus stem. A cDNA library of mRNA isolated from asparagus spears 24 h post-harvest was screened for peroxidases using homologous and heterologous probes. Three clones were isolated and the corresponding mature asparagus peroxidases displayed 70%, 76% and 81% amino acid sequence identity to each other. These new asparagus peroxidases are typical class III plant peroxidases in terms of conserved regions with a calculated pI >9.2, which is consistent with most basic peroxidases. One of the genes was shown to be a constitutively expressed single-copy gene, whereas the others showed an increased expression at post-harvest. The highest similarity in the amino acid sequence (71-77%) was found in peroxidases from roots of winter grown turnip TP7, to Arabidopsis AtP49, to an EST sequence from cotton fibres and to TMV-infected tobacco. PMID:12947050

  7. Oxidation of chlorophenols catalyzed by Coprinus cinereus peroxidase with in situ production of hydrogen peroxide.

    PubMed

    Pezzotti, Fabio; Okrasa, Krzysztof; Therisod, Michel

    2004-01-01

    Degradation of 2,6-dichlorophenol (2,6-DCP) was accomplished by oxidation catalyzed by Coprinus cinereus peroxidase. Immobilization of the enzyme in a polyacrylamide matrix enhanced DCP oxidation. Hydrogen peroxide, peroxidase's natural substrate, was produced enzymatically in situ to avoid peroxidase inactivation by its too high concentration. In the case of larger scale utilization, the method would also avoid direct handling of this hazardous reagent.

  8. Accumulation of peroxidase in the cap rays of Acetabularia during the development of gametangia.

    PubMed

    Menzel, D

    1979-06-01

    Accumulation of peroxidase was demonstrated by light and electron microscopy to occur in Acetabularia in certain regions of the cap rays in relation to the development of the gametangia (cysts). Peroxidase was found to be incorporated into special, cell wall-like obstructions that separate the cap rays from the stalk when the secondary nuclei have settled in the cap rays. It is assumed that peroxidase acts as an anti-microbial protectant of the gametangia.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. Polyethylene glycol improves phenol removal by immobilized turnip peroxidase.

    PubMed

    Quintanilla-Guerrero, F; Duarte-Vázquez, M A; García-Almendarez, B E; Tinoco, R; Vazquez-Duhalt, R; Regalado, C

    2008-12-01

    Purified peroxidase from turnip (Brassica napus L. var. esculenta D.C.) was immobilized by entrapment in spheres of calcium alginate and by covalent binding to Affi-Gel 10. Both immobilized Turnip peroxidase (TP) preparations were assayed for the detoxification of a synthetic phenolic solution and a real wastewater effluent from a local paints factory. The effectiveness of phenolic compounds (PC's) removal by oxidative polymerization was evaluated using batch and recycling processes, and in the presence and in the absence of polyethylene glycol (PEG). The presence of PEG enhances the operative TP stability. In addition, reaction times were reduced from 3h to 10 min, and more effective phenol removals were achieved when PEG was added. TP was able to perform 15 reaction cycles with a real industrial effluent showing PC's removals >90% PC's during the first 10 reaction cycles. High PC's removal efficiencies (>95%) were obtained using both immobilized preparations at PC's concentrations <1.2mM. Higher PC's concentrations decreased the removal efficiency to 90% with both preparations after the first reaction cycle, probably due to substrate inhibition. On the other hand, immobilized TP showed increased thermal stability when compared with free TP. A large-scale enzymatic process for industrial effluent treatment is expected to be developed with immobilized TP that could be stable enough to make the process economically feasible. PMID:18502120

  16. Monosaccharide composition and properties of a deglycosylated turnip peroxidase isozyme.

    PubMed

    Duarte-Vázquez, Miguel A; García-Almendárez, Blanca E; Rojo-Domínguez, Arturo; Whitaker, John R; Arroyave-Hernández, C; Regalado, Carlos

    2003-01-01

    A neutral peroxidase isozyme (TP) purified from turnip (Brassica napus L. var. purple top white globe) was partially deglycosylated, using chemical and enzymatic treatment. A 32% carbohydrate removal was achieved by exposing TP to a mixture of PNGase F, O-glycosidase, NANase, GALase III and HEXase I, while m-periodate treatment removed about 88% of TP carbohydrate moiety. The glycoprotein fraction of the TP contained a relatively high mannose and fucose content (37 and 31%, w/w, respectively), 16% (w/w) galactose, and 15% (w/w) GlcNAc. Thus, the carbohydrate moiety was classified as a hybrid type. Partially deglycosylated TP had reduced activity (by 50-85%), was more susceptible to proteolysis, and showed a slight decrease in thermostability compared to the native enzyme. Circular dichroism studies strongly suggested that although the carbohydrate moiety of TP did not influence the conformation of the polypeptide backbone, its presence considerably enhanced protein conformational stability toward heat. Removal of oligosaccharide chains from TP caused a decrease in K(m) and V(max) for hydrogen peroxide. Native and chemically deglycosylated TP were similarly immunodetected by rabbit polyclonal antibodies raised against TP. The results suggest that the carbohydrate moiety of TP is important for peroxidase activity and stability. PMID:12475613

  17. 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

  18. Manganese peroxidases of the white rot fungus Phanerochaete sordida.

    PubMed Central

    Rüttimann-Johnson, C; Cullen, D; Lamar, R T

    1994-01-01

    The ligninolytic enzymes produced by the white rot fungus Phanerochaete sordida in liquid culture were studied. Only manganese peroxidase (MnP) activity could be detected in the supernatant liquid of the cultures. Lignin peroxidase (LiP) and laccase activities were not detected under a variety of different culture conditions. The highest MnP activity levels were obtained in nitrogen-limited cultures grown under an oxygen atmosphere. The enzyme was induced by Mn(II). The initial pH of the culture medium did not significantly affect the MnP production. Three MnP isozymes were identified (MnPI, MnPII, and MnPIII) and purified to homogeneity by anion-exchange chromatography followed by hydrophobic chromatography. The isozymes are glycoproteins with approximately the same molecular mass (around 45 kDa) but have different pIs. The pIs are 5.3, 4.2, and 3.3 for MnPI, MnPII, and MnPIII, respectively. The three isozymes are active in the same range of pHs (pHs 3.0 to 6.0) and have optimal pHs between 4.5 and 5.0. Their amino-terminal sequences, although highly similar, were distinct, suggesting that each is the product of a separate gene. Images PMID:8135519

  19. 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.

  20. 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.

  1. 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.

  2. Comparison of structure and activities of peroxidases from Coprinus cinereus, Coprinus macrorhizus and Arthromyces ramosus.

    PubMed

    Kjalke, M; Andersen, M B; Schneider, P; Christensen, B; Schülein, M; Welinder, K G

    1992-04-17

    Initial structural and kinetic data suggested that peroxidases from Coprinus cinereus, Coprinus macrorhizus and Arthromyces ramosus were similar. Therefore they were characterized more fully. The three peroxidases were purified to RZ 2.5 and showed immunochemical identity as well as an identical M(r) of 38,000, pI about 3.5 and similar amino acid compositions. The N-termini were blocked for amino acid sequencing. The peroxidases had similar retention volumes by anion-exchange and gel-filtration chromatography. All peroxidases showed multiple peaks by Concanavalin A-Sepharose chromatography. The Concanavalin A-Sepharose profiles were different and depended furthermore on a fermentation batch. Tryptic peptide maps were very similar except for one peptide. This peptide contained an N-linked glycan composed of varying ratios of glucosamine and mannose for the three peroxidases. Rate constants and their pH dependence were the same for the three peroxidases using guaiacol or iodide as reducing substrates. We conclude that peroxidases from Coprinus cinereus, Coprinus macrorhizus and Arthromyces ramosus are most likely identical in their amino acid sequences, but deviate in glycosylation which, apparently, has no influence on the reaction rates of the enzyme. We suggest, that the Coprinus fungi express one peroxidase only in contrast to the lignin-degrading white-rot Basidiomycetes, which produce multiple peroxidase isozymes.

  3. Systematic characterization of the peroxidase gene family provides new insights into fungal pathogenicity in Magnaporthe oryzae.

    PubMed

    Mir, Albely Afifa; Park, Sook-Young; Abu Sadat, Md; Kim, Seongbeom; Choi, Jaeyoung; Jeon, Junhyun; Lee, Yong-Hwan

    2015-07-02

    Fungal pathogens have evolved antioxidant defense against reactive oxygen species produced as a part of host innate immunity. Recent studies proposed peroxidases as components of antioxidant defense system. However, the role of fungal peroxidases during interaction with host plants has not been explored at the genomic level. Here, we systematically identified peroxidase genes and analyzed their impact on fungal pathogenesis in a model plant pathogenic fungus, Magnaporthe oryzae. Phylogeny reconstruction placed 27 putative peroxidase genes into 15 clades. Expression profiles showed that majority of them are responsive to in planta condition and in vitro H2O2. Our analysis of individual deletion mutants for seven selected genes including MoPRX1 revealed that these genes contribute to fungal development and/or pathogenesis. We identified significant and positive correlations among sensitivity to H2O2, peroxidase activity and fungal pathogenicity. In-depth analysis of MoPRX1 demonstrated that it is a functional ortholog of thioredoxin peroxidase in Saccharomyces cerevisiae and is required for detoxification of the oxidative burst within host cells. Transcriptional profiling of other peroxidases in ΔMoprx1 suggested interwoven nature of the peroxidase-mediated antioxidant defense system. The results from this study provide insight into the infection strategy built on evolutionarily conserved peroxidases in the rice blast fungus.

  4. 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.

  5. Influence of protein environment on magnetic circular dichroism spectral properties of ferric and ferrous ligand complexes of yeast cytochrome c peroxidase.

    PubMed

    Pond, A E; Sono, M; Elenkova, E A; Goodin, D B; English, A M; Dawson, J H

    1999-01-01

    The addition of exogenous ligands to the ferric and ferrous states of yeast cytochrome c peroxidase (CCP) is investigated with magnetic circular dichroism (MCD) at 4 degrees C to determine the effect the protein environment may exercise on spectral properties. The MCD spectrum of each derivative is directly compared to those of analogous forms of horseradish peroxidase (HRP) and myoglobin (Mb), two well-characterized histidine-ligated heme proteins. The ferric azide adduct of CCP is a hexacoordinate, largely low-spin species with an MCD spectrum very similar to that of ferric azide HRP. This complex displays an MCD spectrum dissimilar from that of the Mb derivative, possibly because of the stabilizing interaction between the azide ligand and the distal arginine of CCP (Arg 48). For the ferric fluoride derivative all three proteins display varied MCD data, indicating that the differences in the distal pocket of each protein influences their respective MCD characteristics. The MCD data for the cyanoferric complexes are similar for all three proteins, demonstrating that a strong field ligand bound in the sixth axial position dominates the MCD characteristics of the derivative. Similarly, the ferric NO complexes of the three proteins show MCD spectra similar in feature position and shape, but vary somewhat in intensity. Reduction of CCP at neutral pH yields a typical pentacoordinate high-spin complex with an MCD spectrum similar to that of deoxyferrous HRP. Formation of the NO and cyanide complexes of ferrous CCP gives derivatives with MCD spectra similar to the analogous forms of HRP and Mb in both feature position and shape. Addition of CO to deoxyferrous CCP results in a ferrous-CO complex with MCD spectral similarity to that of ferrous-CO HRP but not Mb, indicating that interactions between the ligand and the distal residues affects the MCD characteristics. Examination of alkaline (pH 9.7) deoxyferrous CCP indicates that a pH dependent conformational change has

  6. Identification of plant metabolites of environmental contaminants by UPLC-QToF-MS: the in vitro metabolism of triclosan in horseradish.

    PubMed

    Macherius, André; Seiwert, Bettina; Schröder, Peter; Huber, Christian; Lorenz, Wilhelm; Reemtsma, Thorsten

    2014-02-01

    Plants can extensively transform contaminants after uptake through phase I and phase II metabolism to a large diversity of products. UPLC-QToF-MS was used to detect and identify metabolites of the bacteriostatic agent triclosan in a horseradish hairy root culture. Thirty-three metabolites of triclosan were recognized by a stepwise approach of mass defect filtering, multivariate data analysis, and isotope pattern filtering from a data set of several thousands of signals in the exposed culture. Structure proposals were elaborated for 23 triclosan metabolites on the basis of their MS data. The majority were identified as conjugates (phase II metabolites) such as saccharides or sulfosaccharides. Additionally, a disulfosaccharide was identified as a plant metabolite for the first time. Besides that, also conjugates of a phase I metabolite, hydroxytriclosan, were determined in horseradish tissue extracts. Dehalogenation products of triclosan were not observed. The large number of metabolites detected and identified in this study emphasizes the importance of a comprehensive analytical approach in studies on the uptake and fate of organic contaminants in plants.

  7. Apoplastic Peroxidases and Lignification in Needles of Norway Spruce (Picea abies L.).

    PubMed Central

    Polle, A.; Otter, T.; Seifert, F.

    1994-01-01

    The objective of the present study was to investigate the correlation of soluble apoplastic peroxidase activity with lignification in needles of field-grown Norway spruce (Picea abies L.) trees. Apoplastic peroxidases (EC 1.11.1.7) were obtained by vacuum infiltration of needles. The lignin content of isolated cell walls was determined by the acetyl bromide method. Accumulation of lignin and seasonal variations of apoplastic peroxidase activities were studied in the first year of needle development. The major phase of lignification started after bud break and was terminated about 4 weeks later. This phase correlated with a transient increase in apoplastic guaiacol and coniferyl alcohol peroxidase activity. NADH oxidase activity, which is thought to sustain peroxidase activity by production of H2O2, peaked sharply after bud break and decreased during the lignification period. Histochemical localization of peroxidase with guaiacol indicated that high activities were present in lignifying cell walls. In mature needles, lignin was localized in walls of most needle tissues including mesophyll cells, and corresponded to 80 to 130 [mu]mol lignin monomers/g needle dry weight. Isoelectric focusing of apoplastic washing fluids and activity staining with guaiacol showed the presence of strongly alkaline peroxidases (isoelectric point [greater than or equal to] 9) in all developmental stages investigated. New isozymes with isoelectric points of 7.1 and 8.1 appeared during the major phase of lignification. These isozymes disappeared after lignification was terminated. A strong increase in peroxidase activity in autumn was associated with the appearance of acidic peroxidases (isoelectric point [less than or equal to] 3). These results suggest that soluble alkaline apoplastic peroxidases participate in lignin formation. Soluble acidic apoplastic peroxidases were apparently unrelated to developmentally regulated lignification in spruce needles. PMID:12232302

  8. Effect of enzyme impurities on phenol removal by the method of polymerization and precipitation catalyzed by Coprinus cinereus peroxidase.

    PubMed

    Masuda, M; Sakurai, A; Sakakibara, M

    2001-11-01

    The removal of phenol by peroxidase-catalyzed polymerization was examined using the Coprinus cinereus peroxidases at different levels of impurity with respect to contamination. The phenol removal efficiency was improved by lowering the peroxidase purity. Acidic and high molecular weight proteins present as impurities in the peroxidase solution had some positive effect on the phenol-polymerizing reaction. The residual enzyme activity, either only in the solution or both in the solution and on the precipitate during the polymerizing reaction, was measured. The results indicate that the main effect of impurities in the peroxidase solution was the suppression of the adsorption of peroxidase molecules on the polymerized precipitate.

  9. 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.

  10. 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.

  11. Asparagus byproducts as a new source of peroxidases.

    PubMed

    Jaramillo-Carmona, Sara; Lopez, Sergio; Vazquez-Castilla, Sara; Rodriguez-Arcos, Rocio; Jimenez-Araujo, Ana; Guillen-Bejarano, Rafael

    2013-07-01

    Soluble peroxidase (POD) from asparagus byproducts was purified by ion exchange chromatographies, and its kinetic and catalytic properties were studied. The isoelectric point of the purified isoperoxidases was 9.1, and the optimum pH and temperature values were 4.0 and 25 °C, respectively. The cationic asparagus POD (CAP) midpoint inactivation temperature was 57 °C, which favors its use in industrial processes. The Km values of cationic asparagus POD for H₂O₂ and ABTS were 0.318 and 0.634 mM, respectively. The purified CAP is economically obtained from raw materials using a simple protocol and possesses features that make it advantageous for the potential use of this enzyme in a large number of processes with demonstrated requirements of thermostable POD. The results indicate that CAP can be used as a potential candidate for removing phenolic contaminants.

  12. Peroxidase activity and superficial scald development in apple fruit.

    PubMed

    Fernández-Trujillo, J Pablo; Nock, Jacqueline F; Kupferman, Eugene M; Brown, Susan K; Watkins, Christopher B

    2003-11-19

    The relationship between soluble peroxidase (EC 1.11.1.7; POX) activity and the development of a chilling-related disorder, superficial scald, was studied in three apple fruit (Malus x domestica Borkh.) systems: a White Angel x Rome Beauty population with progeny with different scald susceptibilities; Delicious from three harvests with progressively declining scald susceptibility; and the scald-resistant Idared and the scald-susceptible Law Rome. Differences in incidence and severity of scald in progeny from White Angel x Rome Beauty progeny tended to show relationships with POX activity at harvest, but, overall, associations were not consistent. However, greater scald incidence and lower POX activity were found in less mature Delicious fruit than in later harvested fruit. Also, the scald-resistant Iotadared had a much higher POX activity compared with the scald-susceptible Law Rome. A general hypothesis that POX activity is related to scald susceptibility was generally supported, but exceptions were observed.

  13. Insights into the catalytic mechanism of synthetic glutathione peroxidase mimetics.

    PubMed

    Bhowmick, Debasish; Mugesh, Govindasamy

    2015-11-01

    Glutathione Peroxidase (GPx) is a key selenoenzyme that protects biomolecules from oxidative damage. Extensive research has been carried out to design and synthesize small organoselenium compounds as functional mimics of GPx. While the catalytic mechanism of the native enzyme itself is poorly understood, the synthetic mimics follow different catalytic pathways depending upon the structures and reactivities of various intermediates formed in the catalytic cycle. The steric as well as electronic environments around the selenium atom not only modulate the reactivity of these synthetic mimics towards peroxides and thiols, but also the catalytic mechanisms. The catalytic cycle of small GPx mimics is also dependent on the nature of peroxides and thiols used in the study. In this review, we discuss how the catalytic mechanism varies with the substituents attached to the selenium atom.

  14. 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

  15. 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

  16. Involvement of peroxidase activity in developing somatic embryos of Medicago arborea L. Identification of an isozyme peroxidase as biochemical marker of somatic embryogenesis.

    PubMed

    Gallego, Piedad; Martin, Luisa; Blazquez, Antonio; Guerra, Hilario; Villalobos, Nieves

    2014-01-15

    The legume Medicago arborea L. is very interesting as regards the regeneration of marginal arid soils. The problem is that it does not have a good germinative yield. It was therefore decided to regenerate via somatic embryogenesis and find a marker of embryogenic potential. In this study, peroxidase activity was evaluated in non-embryogenic and embryogenic calli from M. arborea L. A decrease in soluble peroxidase activity is observed in its embryonic calli at the time at which the somatic embryos begin to appear. This activity is always lower in embryonic calli than in non-embryonic ones (unlike what happens in the case of wall-bound peroxidases). These results suggest that peroxidases can be considered to be enzymes involved in somatic embryogenesis in M. arborea. In addition, isozyme analyses were carried out on protein extracts using polyacrylamide gel electrophoresis. The band called P5 was detected only in embryogenic cultures at very early stages of development. This band was digested with trypsin and analyzed using linear ion trap (LTQ) mass spectrometer. In P5 isoform a peroxidase-L-ascorbate peroxidase was identified. It can be used as a marker that allows the identification of embryological potential.

  17. Electrostatic control of the tryptophan radical in cytochrome c peroxidase.

    PubMed

    Barrows, Tiffany P; Bhaskar, B; Poulos, Thomas L

    2004-07-13

    Previously a K(+)-binding site, analogous to that found in ascorbate peroxidase (APX), was engineered into cytochrome c peroxidase (CcP) to test the hypothesis that the bound K(+) influences the stability of the Trp191 cation radical formed during the CcP catalytic cycle (Bonagura et al., (1996) Biochemistry 35, 6107 and Bonagura et al., (1999) Biochemistry 38, 5528). Characterization of this mutant, designated CcPK2, showed that the stability of the Trp191 cation radical is dependent on the occupancy of the engineered K(+) site and that the Trp191 radical was much less stable in this mutant than in wild-type CcP. The mutations Met230Leu, Met231Gln, and Met172Ser have now been constructed on the CcPK2 mutant template to test if the Met residues also contribute to the stabilization of the Trp191 cation radical. Crystal structures show that the mutations affect only the local structure near the sites of mutation. Removal of these electronegative residues located less than 8 A from the Trp radical results in a further destabilization of the Trp radical. The characteristic EPR signal associated with the Trp radical is significantly narrowed and is characteristic of a tyrosine radical signal. Double-mixing stopped-flow experiments, where the delay time between the formation of CcP compound I and its mixing with horse heart ferrocytochrome c is varied, show that the stability of the Trp radical decreases as the Met residues are removed from the proximal cavity. When taken together, these results demonstrate a strong correlation between the experimentally determined stability of the Trp191 radical, the enzyme activity, and the calculated electrostatic stabilization of the Trp191 radical. PMID:15236591

  18. The Roles of Glutathione Peroxidases during Embryo Development.

    PubMed

    Ufer, Christoph; Wang, Chi Chiu

    2011-01-01

    Embryo development relies on the complex interplay of the basic cellular processes including proliferation, differentiation, and apoptotic cell death. Precise regulation of these events is the basis for the establishment of embryonic structures and the organ development. Beginning with fertilization of the oocyte until delivery the developing embryo encounters changing environmental conditions such as varying levels of oxygen, which can give rise to reactive oxygen species (ROS). These challenges are met by the embryo with metabolic adaptations and by an array of anti-oxidative mechanisms. ROS can be deleterious by modifying biological molecules including lipids, proteins, and nucleic acids and may induce abnormal development or even embryonic lethality. On the other hand ROS are vital players of various signaling cascades that affect the balance between cell growth, differentiation, and death. An imbalance or dysregulation of these biological processes may generate cells with abnormal growth and is therefore potentially teratogenic and tumorigenic. Thus, a precise balance between processes generating ROS and those decomposing ROS is critical for normal embryo development. One tier of the cellular protective system against ROS constitutes the family of selenium-dependent glutathione peroxidases (GPx). These enzymes reduce hydroperoxides to the corresponding alcohols at the expense of reduced glutathione. Of special interest within this protein family is the moonlighting enzyme glutathione peroxidase 4 (Gpx4). This enzyme is a scavenger of lipophilic hydroperoxides on one hand, but on the other hand can be transformed into an enzymatically inactive cellular structural component. GPx4 deficiency - in contrast to all other GPx family members - leads to abnormal embryo development and finally produces a lethal phenotype in mice. This review is aimed at summarizing the current knowledge on GPx isoforms during embryo development and tumor development with an emphasis on

  19. Characterization of a catalase-peroxidase from the hyperthermophilic archaeon Archaeoglobus fulgidus.

    PubMed

    Kengen, S W; Bikker, F J; Hagen, W R; de Vos, W M; van der Oost, J

    2001-10-01

    A putative perA gene from Archaeoglobus fulgidus was cloned and expressed in Escherichia coli BL21(DE3), and the recombinant catalase-peroxidase was purified to homogeneity. The enzyme is a homodimer with a subunit molecular mass of 85 kDa. UV-visible spectroscopic analysis indicated the presence of protoheme IX as a prosthetic group (ferric heme), in a stoichiometry of 0.25 heme per subunit. Electron paramagnetic resonance analysis confirmed the presence of ferric heme and identified the proximal axial ligand as a histidine. The enzyme showed both catalase and peroxidase activity with pH optima of 6.0 and 4.5, respectively. Optimal temperatures of 70 degrees C and 80 degrees C were found for the catalase and peroxidase activity, respectively. The catalase activity strongly exceeded the peroxidase activity, with Vmax values of 9600 and 36 U mg(-1), respectively. Km values for H2O2 of 8.6 and 0.85 mM were found for catalase and peroxidase, respectively. Common heme inhibitors such as cyanide, azide, and hydroxylamine inhibited peroxidase activity. However, unlike all other catalase-peroxidases, the enzyme was also inhibited by 3-amino-1,2,4-triazole. Although the enzyme exhibited a high thermostability, rapid inactivation occurred in the presence of H2O2, with half-life values of less than 1 min. This is the first catalase-peroxidase characterized from a hyperthermophilic microorganism. PMID:11699646

  20. 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...