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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. Bioconjugation of Antibodies to Horseradish Peroxidase (HRP).

    PubMed

    Hnasko, Robert M

    2015-01-01

    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 convenient means to maintain antibody affinity while imparting a functional reporter used for antigen detection. In this chapter, we describe a process by which Sulfo-SMCC is used to generate a stable maleimide-activated HRP that is reactive with sulfhydryl groups generated in antibodies by SATA-mediated thiolation.

  3. Horseradish-Peroxidase-Catalyzed Tyrosine Click Reaction.

    PubMed

    Sato, Shinichi; Nakamura, Kosuke; Nakamura, Hiroyuki

    2017-03-02

    The efficiency of protein chemical modification on tyrosine residues with N-methylluminol derivatives was drastically improved by using horseradish peroxidase (HRP). In the previous method, based on the use of hemin and H2 O2 , oxidative side reactions such as cysteine oxidation were problematic for functionalization of proteins selectively on tyrosine residues. Oxidative activation of N-methylluminol derivatives with a minimum amount of H2 O2 prevented the occurrence of oxidative side reactions under HRP-catalyzed conditions. As probes for HRP-catalyzed protein modification, N-methylluminol derivatives showed much higher efficiency than tyramide without inducing oligomerization of probe molecules. Tyrosine modification also proceeded in the presence of β-nicotinamide adenine dinucleotide (NADH, H2 O2 -free conditions).

  4. Immobilization of horseradish peroxidase onto kaolin.

    PubMed

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

    2016-03-01

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

  5. 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.

  6. Secretion-related uptake of horseradish peroxidase in neurohypophysial axons

    PubMed Central

    1976-01-01

    During secretion of the neurohypophysial hormones, oxytocin and vasopressin, secretory granule membrane is added to the plasma membrane of the axon terminals. It is generally assumed that subsequent internalization of this additional membrane occurs by endocytosis. In order to study this process, we have traced the uptake of intravenously injected horseradish peroxidase by neurohypophysial axons in rats and golden hamsters. Peroxidase reaction product within the secretory axons was found mainly in vacuolar and C-shaped structures of a size comparable with or larger than the neurosecretory granules. Our observations suggest that these large horseradish peroxidase (HRP)- impregnated vacuoles arise directly by a form of macropinocytosis. Morphometric analysis indicated that this form of membrane retrieval increased significantly after the two types of stimuli used, reversible hemorrhage and electrical stimulation of the pituitary stalk. Microvesicular uptake of HRP was found to be comparatively less. PMID:181385

  7. The Reaction of Coumarins with Horseradish Peroxidase 1

    PubMed Central

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

    1975-01-01

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

  8. Detoxification of pesticides aqueous solution using horseradish peroxidase.

    PubMed

    El-Said, Saad Mohamed

    2013-03-15

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

  9. An analysis of horseradish peroxidase enzyme for effluent treatment

    PubMed Central

    Nunavath, Hanumalal; Banoth, Chandrasekhar; Talluri, Venkateswar Rao; Bhukya, Bhima

    2016-01-01

    The present study explains computational methods to design thermostable horseradish peroxidase enzyme using the crystal structure available from Protein Data Bank (PDB ID: 6ATJ). Multiple mutations were introduced to the original enzyme and developed a model by using Modeler9.14. After designing the model functional effect was confirmed in terms of protein ligand binding by molecular docking using Autodock 4.2. The implementation of modeling steps is demonstrated in the context of performing mutations for particular amino acid residue on the ligand pocket of the horseradish peroxidase, to derive the desired ligand binding properties. The docking investigation of modelled HRP with Quercetindihydroxide using Autodock 4.2 software that six amino acid residues, P139, H42, A31, L174, A38, and G169 are involved in hydrogen bonding. More importantly, it provides insight into understanding and properly interpreting the data produced by these methods. The 3D model was docked with Quercetindihydroxide (a known horseradish modulator) to understand molecular interactions at the active site region. PMID:28293074

  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. Relative binding affinities of monolignols to horseradish peroxidase

    SciTech Connect

    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 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. 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.

  12. Relative binding affinities of monolignols to horseradish peroxidase

    DOE PAGES

    Sangha, Amandeep K.; Petridis, Loukas; Cheng, Xiaolin; ...

    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

  13. Feruloylated arabinoxylans are oxidatively cross-linked by extracellular maize peroxidase but not by horseradish peroxidase.

    PubMed

    Burr, Sally J; Fry, Stephen C

    2009-09-01

    Covalent cross-linking of soluble extracellular arabinoxylans in living maize cultures, which models the cross-linking of wall-bound arabinoxylans, is due to oxidation of feruloyl esters to oligoferuloyl esters and ethers. The oxidizing system responsible could be H2O2/peroxidase, O2/laccase, or reactive oxygen species acting non-enzymically. To distinguish these possibilities, we studied arabinoxylan cross-linking in vivo and in vitro. In living cultures, exogenous, soluble, extracellular, feruloylated [pentosyl-3H]arabinoxylans underwent cross-linking, beginning abruptly 8 d after sub-culture. Cross-linking was suppressed by iodide, an H2O2 scavenger, indicating dependence on endogenous H2O2. However, exogenous H2O2 did not cause precocious cross-linking, despite the constant presence of endogenous peroxidases, suggesting that younger cultures contained natural cross-linking inhibitors. Dialysed culture-filtrates cross-linked [3H]arabinoxylans in vitro only if H2O2 was also added, indicating a peroxidase requirement. This cross-linking was highly ionic-strength-dependent. The peroxidases responsible were heat-labile, although relatively heat-stable peroxidases (assayed on o-dianisidine) were also present. Surprisingly, added horseradish peroxidase, even after heat-denaturation, blocked the arabinoxylan-cross-linking action of maize peroxidases, suggesting that the horseradish protein was a competing substrate for [3H]arabinoxylan coupling. In conclusion, we show for the first time that cross-linking of extracellular arabinoxylan in living maize cultures is an action of apoplastic peroxidases, some of whose unusual properties we report.

  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. 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.

  16. Production and purification of the multifunctional enzyme horseradish peroxidase

    PubMed Central

    Spadiut, Oliver; Herwig, Christoph

    2014-01-01

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

  17. Degradation of horseradish peroxidase after microinjection into mammalian cells

    SciTech Connect

    Knowles, S.E.; Hopgood, M.F.; Ballard, F.J.

    1988-01-01

    Horseradish peroxidase (HRP) has been microinjected into mammalian cells in tissue culture by the erythrocyte ghost-mediated technique. This protein was selected because it can be localized and quantified after injection by cytochemical and spectrophotometric methods. HRP labeled by reductive methylation retained full catalytic activity, was efficiently loaded into erythrocyte ghosts, and did not associate to a significant degree with ghost membranes. A combination of cytochemical staining and autoradiography established that HRP injected into rat L6 myoblasts, HE(39)L human diploid fibroblasts, or HeLa cells was intracellular and uniformly distributed throughout the cell, while cell lysis techniques showed that the catalytically active HRP was not membrane bound. Inactivation of labeled HRP after injection paralleled the disappearance of the 40-kDa polypeptide, and was always more rapid than its overall degradation. This difference was associated with a pool of water-insoluble radioactivity in the injected cells. This material was of smaller molecular size than the native protein: many labeled peptides were detected in the range of 10 to 38 kDa. By the use of inhibitors of autophagic proteolysis or lysosomal function it was established that HRP degradation was not subjected quantitatively to the same regulatory processes as the average endogenous protein labeled in the same cultures.

  18. Comparative permeability of different glioma models to horseradish peroxidase.

    PubMed

    Groothuis, D R; Fischer, J M; Vick, N A; Bigner, D D

    1981-01-01

    Seven different experimental glioma models were studied to determine their capillary permeability to horseradish peroxidase. The models were the autochthonous ASV-viral model, intracerebral (ic) and (sc) injections of rat 9L and RG-2 tumor cell lines, the sc rat S-69C15 tumor cell line, and human glioblastoma cell lines in nude mice. The ASV-viral model showed an average HRP permeability of 63% of tumor volume, the ic RG-2 tumors were 100% permeable, and the ic 9L tumors were 41% permeable. The permeability of the marginal zone (brain-tumor interface) showed similar variation from group to group. In contrast, all of the sc tumors were 100% permeable regardless of the cell line used to create the tumors. Our results show the variability between these glioma models, and suggest that RG-2 ic gliomas and all sc gliomas should be optimal to assess the tumoricidal effect of drugs, because access to the tumor compartment from the vascular compartment is complete.

  19. Phenol removal from refinery wastewater by mutant recombinant horseradish peroxidase.

    PubMed

    Asad, Sedigheh; Dabirmanesh, Bahareh; Khajeh, Khosro

    2014-01-01

    Application of mutated recombinant horseradish peroxidase (HRP) for phenol removal from refinery effluents is reported. Recombinant HRP produced in Escherichia coli suffers from the disadvantage of lacking glycosylation, which affects its catalytic efficiency and stability toward inactivating parameters such as increased temperature and enhanced amounts of hydrogen peroxide. In the present study, the previously reported variant (in which Asn268 was substituted with Asp, N268D) with improved stability characteristics and catalytic efficiency was used to remove phenol from a petroleum refinery effluent. The presence and removal of phenol was studied by high-performance liquid chromatography; the precipitated oxidized phenol was also observed and removed from the sample by centrifugation. Results showed that the N268D variant can remove 61%, 67%, and 81% of phenol from effluent in 1, 2, and 16 H, respectively. By exploiting the N268D mutant, removal of 50% phenol could be achieved in 42 Min, which was more than 22 times less than the treatment time required by native recombinant enzyme.

  20. Horseradish peroxidase mediated free radical polymerization of methyl methacrylate.

    PubMed

    Kalra, B; Gross, R A

    2000-01-01

    This paper reports the free radical polymerization of methyl methacrylate (MMA) catalyzed by horseradish peroxidase (HRP). A novel method was developed whereby MMA polymerization can be carried out at ambient temperatures in the presence of low concentrations of hydrogen peroxide and 2,4-pentanedione in a mixture of water and a water-miscible solvent. Polymers of MMA formed were highly stereoregular with predominantly syndiotactic sequences (syn-dyad fractions from 0.82 to 0.87). Analyses of the chloroform-soluble fraction of syndio-PMMA products by GPC showed that they have number-average molecular weights, Mn, that range from 7500 to 75,000. By using 25% v/v of the cosolvents dioxane, tetrahydrofuran, acetone, and dimethylformamide, 85, 45, 7 and 2% product yields, respectively, resulted after 24 h. Increasing the proportion of dioxane to water from 1:3 to 1:1 and 3:1 resulted in a decrease in polymer yield from 45 to 38 and 7%, respectively. Increase in the enzyme concentration from 70 to 80 and 90 mg/mL resulted in increased reaction kinetics. By adjustment of the molar ratio of 2,4-pentanedione to hydrogen peroxide between 1.30:1.0 and 1.45:1.0, the product yields and Mn values were increased. On the basis of the catalytic properties of HRP and studies herein, we believe that the keto-enoxy radicals from 2,4-pentanedione are the first radical species generated. Then, initiation may take place through this radical or by the radical transfer to another molecule.

  1. Descending projections to the mammillary nuclei in the rat, as studied by retrograde and anterograde transport of wheat germ agglutinin-horseradish peroxidase.

    PubMed

    Shibata, H

    1989-07-22

    The cells of origin and projection fields of the descending afferents to the mammillary nuclei were studied in the rat with retrograde and anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase. The subiculum projects bilaterally to the entire medial mammillary nucleus (MM) in a topographic fashion along the two axes: 1) the proximal part of the subiculum along the presubiculo-CA1 axis projects to the caudal and lateral regions of the MM whereas the more distal part of the subiculum projects to the medial region; 2) the septal part of the subiculum projects to the caudodorsal region of the MM whereas the more temporal part projects progressively to the more rostroventral regions. The ventral subiculum also projects ipsilaterally to the ventral and lateral margin of the lateral mammillary nucleus (LM). The presubiculum projects bilaterally to the dorsolateral region of the pars posterior of the MM and ipsilaterally to the LM. The infra-limbic cortex projects bilaterally to the rostrodorsal region of the MM, whereas the retrosplenial cortex (areas 29a and 29b) projects bilaterally to the medial region at the midrostrocaudal and middorsoventral levels of the MM. The nucleus of the diagonal band projects bilaterally to the caudomedial region of the MM, whereas the lateral septal nucleus projects bilaterally to the pars mediana and the mammillary fiber capsule. A part of the anterior hypothalamic area ventromedial to the fornix projects predominantly ipsilaterally to the rostroventral part of the MM, whereas other basal forebrain regions such as the bed nucleus of the stria terminalis, the medial preoptic and anterior hypothalamic areas, and the area of the tuber cinereum send fibers predominantly ipsilaterally to the mammillary fiber capsule. The results reveal a complex organization of the descending projections to the mammillary nuclei, which may reflect the complex functions of these nuclei within the limbic circuitry.

  2. 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.

  3. Comparison of lignin peroxidase and horseradish peroxidase for catalyzing the removal of nonylphenol from water.

    PubMed

    Dong, Shipeng; Mao, Liang; Luo, Siqiang; Zhou, Lei; Feng, Yiping; Gao, Shixiang

    2014-02-01

    Concentrations of aqueous-phase nonylphenol (NP), a well-known endocrine-disrupting chemical, are shown to be reduced effectively via reaction with lignin peroxidase (LiP) or horseradish peroxidase (HRP) and hydrogen peroxide. We systematically assessed their reaction efficiencies at varying conditions, and the results have confirmed that the catalytic performance of LiP toward NP was more efficient than that of HRP under experimental conditions. Mass spectrum analysis demonstrated that polymerization through radical-radical coupling mechanism was the pathway leading to NP transformation. Our molecular modeling with the assistance of ab initio suggested the coupling of NP likely proceeded via covalent bonding between two NP radicals at their unsubstituted carbons in phenolic rings. Data from acute immobilization tests with Daphnia confirm that NP toxicity is effectively eliminated by LiP/HRP-catalyzed NP removal. The findings in this study provide useful information for understanding LiP/HRP-mediated NP reactions, and comparison of enzymatic performance can present their advantages for up-scale applications in water/wastewater treatment.

  4. DNA-directed immobilization of horseradish peroxidase onto porous SiO2 optical transducers

    NASA Astrophysics Data System (ADS)

    Shtenberg, Giorgi; Massad-Ivanir, Naama; Engin, Sinem; Sharon, Michal; Fruk, Ljiljana; Segal, Ester

    2012-08-01

    Multifunctional porous Si nanostructure is designed to optically monitor enzymatic activity of horseradish peroxidase. First, an oxidized PSi optical nanostructure, a Fabry-Pérot thin film, is synthesized and is used as the optical transducer element. Immobilization of the enzyme onto the nanostructure is performed through DNA-directed immobilization. Preliminary studies demonstrate high enzymatic activity levels of the immobilized horseradish peroxidase, while maintaining its specificity. The catalytic activity of the enzymes immobilized within the porous nanostructure is monitored in real time by reflective interferometric Fourier transform spectroscopy. We show that we can easily regenerate the surface for consecutive biosensing analysis by mild dehybridization conditions.

  5. Oxidation of Indole-3-Acetic Acid-Amino Acid Conjugates by Horseradish Peroxidase

    PubMed Central

    Park, Ro Dong; Park, Chang Kyu

    1987-01-01

    The stability of 21 amino acid conjugates of indole-3-acetic acid (IAA) toward horseradish peroxidase (HRP) was studied. The IAA conjugates of Arg, Ile, Leu, Tyr, and Val were oxidized readily by peroxidase. Those of Ala, β-Ala, Asp, Cys, Gln, Glu, Gly, and Lys were not degraded and their recovery was above 92% after 1 hour incubation with HRP. A correlation between the stability of IAA conjugates toward peroxidase-catalyzed oxidation and the hydrophobicity of the amino acid moiety conjugated to IAA was demonstrated. Polar amino acid conjugates of IAA are more resistant to HRP-catalyzed oxidation. PMID:16665529

  6. Mechanism of NADPH oxidation catalyzed by horse-radish peroxidase and 2,4-diacetyl-[2H]heme-substituted horse-radish peroxidase.

    PubMed

    De Sandro, V; Dupuy, C; Kaniewski, J; Ohayon, R; Dème, D; Virion, A; Pommier, J

    1991-10-15

    The mechanism of NADPH oxidation catalyzed by horse-radish peroxidase (HRP) and 2,4-diacetyl-[2H]heme-substituted horse-radish peroxidase (DHRP) was studied. The roles of the different H2O2/peroxidase compounds were examined by spectral studies. The oxidized NADPH species were identified using the superoxide dismutase effect and by measuring the stoichiometry between NADPH oxidized and H2O2 used. In the presence of a mediating molecule, like scopoletin, both enzymes acted via a similar mechanism, producing only NADP degrees, which in turn reacted with O2 producing O2-. Consequently H2O2 was completely regenerated in the presence of superoxide dismutase and partially regenerated in its absence. In the absence of a mediating molecule, the H2O2 complex of both enzymes (compound I) catalysed NADPH oxidation by single-electron transfer, producing NADP degrees; compound II of these enzymes catalyzed NADPH oxidation more slowly by a direct two-electron transfer, producing NADPH+. There were difference between HRP and DHRP. HRP compound II was produced by the oxidation of 1 mol NADPH/mole compound I, while DHRP compound II was formed by the spontaneous conversion of compound I to compound II. The NADPH oxidation catalyzed by DHRP compound I did not lead to the formation of compound II. When H2O2 was produced slowly by the glucose/glucose-oxidase system, compound II was never formed and a pure O2- adduct of DHRP (compound III) accumulated.

  7. Detection of zeptomolar concentrations of alkaline phosphatase based on a tyrosinase and horse-radish peroxidase bienzyme biosensor.

    PubMed

    Ruan, C; Li, Y

    2001-07-06

    A bienzyme biosensor based on tyrosinase and horse-radish peroxidase is described in a flow injection analysis and cyclic voltammetry for measurement of phenol. Tyrosinase and horse-radish peroxidase were immobilized on the surface of a glassy carbon electrode by bovine serum albumin and glutaric dialdehyde. Phenol was oxidized by tyrosinase and horse-radish peroxidase via catechol to o-quinone in the presence of oxygen and hydrogen peroxide. The o-quinone was reduced to produce catechol (the substrate recycling) on the electrode surface. The enhanced sensitivity of the bienzyme electrode to phenol was observed in the flow injection system comparing with tyrosinase and horse-radish peroxidase monoenzyme electrodes. The mechanisms for enhanced amperometric response to phenol of bienzyme electrode were discussed. The biosensor was used to detect alkaline phosphatase (ALP). A detection limit of 1.4x10(-15) M ALP (140 zmol/100 mul) was obtained after 1 h incubation with phenyl phosphate.

  8. Nonidet P40 and the retrograde transport of horseradish peroxidase in undamaged visceral nerves.

    PubMed

    Rogers, R C; Liholtz, L A; Ebly, E M

    1982-06-01

    The cell bodies of origin of peripheral nerves, in particular visceral nerves, are often difficult to identify using standard horseradish peroxidase (HRP) methods. The non-ionic surfactant Nonidet-P40, when applied to intact peripheral nerve along with HRP, allows the investigator to examine the neurons of origin of the nerve without cutting the fibers or injecting label into its peripheral terminal field.

  9. 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).

  10. Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins

    SciTech Connect

    Storrie, B.; Sachdeva, M.; Viers, V.S.

    1984-02-01

    We used Chinese hamster ovary cells, a cell line of fibroblastic origin, to investigate whether lysosomes are an exocytic compartment. To label lysosomal contents, Chinese hamster ovary cells were incubated with the solute marker horseradish peroxidase. After an 18-h uptake period, horseradish peroxidase was found in lysosomes by cell fractionation in Percoll gradients and by electron microscope cytochemistry. Over a 24-h period, lysosomal horseradish peroxidase was quantitatively retained by Chinese hamster ovary cells and inactivated with a t 1/2 of 6 to 8 h. Lysosomes were radioiodinated in situ by soluble lactoperoxidase internalized over an 18-h uptake period. About 70% of the radioiodine incorporation was pelleted at 100,000 X g under conditions in which greater than 80% of the lysosomal marker enzyme beta-hexosaminidase was released into the supernatant. By one-dimensional electrophoresis, about 18 protein species were present in the lysosomal membrane fraction, with radioiodine incorporation being most pronounced into species of 70,000 to 75,000 daltons. After a 30-min or 2-h chase at 37 degrees C, radioiodine that was incorporated into lysosomal membranes and contents was retained in lysosomes. These observations indicate that lysosomes labeled by fluid-phase pinocytosis are a terminal component of endocytic pathways in fibroblasts.

  11. Fluorescent derivatization of aromatic carboxylic acids with horseradish peroxidase in the presence of excess hydrogen peroxide.

    PubMed

    Odo, Junichi; Inoguchi, Masahiko; Aoki, Hiroyuki; Sogawa, Yuto; Nishimura, Masahiro

    2015-01-01

    The fluorescent derivatization of aromatic carboxylic acids by the catalytic activity of horseradish peroxidase (HRP) in the presence of excess H2O2 was investigated. Four monocarboxylic acids, nine dicarboxylic acids, and two tricarboxylic acids, all of which are non- or weakly fluorescent, were effectively converted into fluorescent compounds using this new method. This technique was further developed for the fluorometric determination of trace amounts of terephthalic acid (3c) and lutidinic acid (2b), and linear calibration curves for concentrations between 2.5 and 20.0 nmol of terephthalic acid (3c) and 1.0 and 10.0 nmol of lutidinic acid (2b) were demonstrated. Compound III, an intermediate of HRP, played an essential role in this process. Additionally, lactoperoxidase and manganese peroxidase, peroxidases similar to HRP, showed successful fluorescent derivatization of nicotinic acid (1b), lutidinic acid (2b), and hemimellitic acid (4a) in the presence of excess H2O2.

  12. Gamma irradiation effect on the enzymatic activities of horseradish and apple peroxidases

    NASA Astrophysics Data System (ADS)

    Constantinovici, M.; Oancea, D.; Zaharescu, T.

    2009-01-01

    The behavior at low-dose exposure (0.033-0.4 kGy) of horseradish peroxidase (HRP) and of two different purified fractions of apple (Jonathan cultivar) peroxidases (named APR 1S and APR 2S) was studied. The HRP solutions were added with either 0.32 M fructose or glucose in order to study their effect on enzymes activity response under γ ( 137Cs, dose rate 0.4 kGy/h) irradiation. The obtained results showed similar behavior between HRP-sugar-added solution and apple fraction with higher oligosaccharides content (APR 2S) undergoing low-dose treatment. The same pattern was observed between unglycosylated HRP and APR 1S with lower oligosaccharides content. These similarities gave us the possibility to conclude that the presence of oligosaccharides, in more or less quantities, influences in the same way the peroxidases activity, from different plant species, exposed to γ irradiation.

  13. Fluorescence decrease of conjugated polymers by the catalytic activity of horseradish peroxidase and its application in phenolic compounds detection.

    PubMed

    González-Sánchez, M I; Laurenti, M; Rubio-Retama, J; Valero, E; Lopez-Cabarcos, E

    2011-04-11

    We report the fluorescence decrease of the water-soluble π-π-conjugated polymer poly(2-methoxy-5-propyloxy sulfonate phenylene vinylene, MPS-PPV) by the catalytic activity of horseradish peroxidase in the presence of H(2)O(2). MPS-PPV acts as a donor substrate in the catalytic cycle of horseradish peroxidase where the electron-deficient enzymatic intermediates compounds I and II can subtract electrons from the polymer leading to its fluorescence decrease. The addition of phenolic drug acetaminophen to the former solution favors the decrease of the polymer fluorescence, which indicates the peroxidase-catalyzed co-oxidation of MPS-PPV and acetaminophen. The encapsulation of horseradish peroxidase within polyacrylamide microgels allows the isolation of intermediates compound I and compound II from the polymer, leading to a fluorescence decrease that is only due to the product of biocatalytic acetaminophen oxidation. This system could be used to develop a new device for phenolic compounds detection.

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

    PubMed

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

    2007-08-25

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

  15. Transformation of dissolved organic matter by oxidative polymerization with horseradish peroxidase.

    PubMed

    Jee, S H; Kim, Y J; Ko, S O

    2010-01-01

    Dissolved organic matter (DOM) has significant influence on the transport and fate of contaminants in multiple phases and it has potential hazard by acting as a precursor of disinfection by-products in water supply. The changes in DOM characteristics, especially by oxidative polymerization might result in different behaviour in the interaction with many contaminants. The aim of this work was to verify the catalytic effects of peroxidase on oxidative polymerization of humic and fulvic substances by examination of the structural characteristics. Transformation of humic acid (HA) and fulvic acid (FA) by oxidative polymerization catalyzed by horseradish peroxidase and hydrogen peroxide were investigated. Size exclusion chromatography, excitation-emission matrices spectra (EEMs), synchronous fluorescence spectra, and infrared spectroscopy was used to evaluate the structural transformation of HA and FA. Molecular weight of HA and FA was continuously changed and their weight-average molecular weight (MWw) reached maximum after 8 h. The MWw of HA and FA were proportionally increased with a dosage of horseradish peroxidase and hydrogen peroxide, indicating that HA and FA was transformed into larger and complex molecules. Spectroscopic results indicated that HA and FA structure contains strong polycyclic aromatic structures with more aromatic rings and a higher degree of conjugation.

  16. 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.

  17. Oxidation of phenols by horseradish peroxidase and lactoperoxidase compound II--kinetic considerations.

    PubMed

    Zahida, M S; Deva, W; Peerzada, G M; Behere, D V

    1998-12-01

    Oxidation of para substituted phenols by horseradish peroxidase compound II (HRP-II) and lactoperoxidase compound II (LPO-II) were studied using stopped flow technique. Apparent second order rate constants (kapp) of the reactions were determined. The kinetics of oxidation of phenols by HRP-II and LPO-II have been compared with the oxidation potentials of the substrates. Reorganization energies of electron-transfer of phenols to the enzymes were estimated from the variation of second order rate constants with the thermodynamic driving force.

  18. Absorption of horse-radish peroxidase by the conjunctival epithelium of monkeys and rabbits.

    PubMed

    Steuhl, P; Rohen, J W

    1983-01-01

    Horse-radish peroxidase was instilled into the conjunctival sac of rabbits and Cynomolgus monkeys. After an interval of 5, 30 or 60 min the conjunctival epithelium was studied by electron microscopy. The tracer was found to be absorbed predominantly by type-V cells, which are rich in mitochondria; this process was found to occur more rapidly in the rabbit than in the monkey. The particles were primarily incorporated into pinocytotic vesicles and phagosomes and were then either digested by phagolysosomes or transported through the basal portion of the surface epithelial cells into the expanding intercellular spaces distal to the junctional complexes.

  19. Hydrodynamics of horseradish peroxidase revealed by global analysis of multiple fluorescence probes.

    PubMed Central

    Brunet, J E; Vargas, V; Gratton, E; Jameson, D M

    1994-01-01

    Previous fluorescence studies of horseradish peroxidase conjugated with protoporphyrin IX suggested that the protein behaved hydrodynamically as a prolate ellipsoid of axial ratio 3 to 1. The present study, designed to further investigate the hydrodynamics of this protein, exploits a series of probes, noncovalently bound to the heme binding site of apo-horseradish peroxidase, having different orientations of the excitation and emission transition dipoles with respect to the protein's rotational axes. The probes utilized included protoporphyrin IX and the naphthalene probes 1-anilino-8-naphthalene sulfonate, 2-p-toluidinyl-6-naphthalene sulfonate, and 4,4'-bis(1-anilino-8-naphthalene sulfonate). Time-resolved data were obtained using multifrequency phase fluorometry. The global analysis approach to the determination of molecular shape using multiple probes was evaluated by utilizing all data sets while maintaining a constant molecular shape for the protein. The results indicated that, in such analyses, probes exhibiting a single exponential decay and limited local motion have the major weight in the evaluation of the axial ratio. Probes that show complex decay patterns and local motions, such as the naphthalene derivatives, give rise to significant uncertainties in such global treatments. By explicitly accounting for the effect of such local motion, however, the shape of the protein can be reliably recovered. PMID:8161698

  20. 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.

  1. Advantages of soybean peroxidase over horseradish peroxidase as the enzyme label in chemiluminescent enzyme-linked immunosorbent assay of sulfamethoxypyridazine.

    PubMed

    Sakharov, Ivan Yu; Berlina, Anna N; Zherdev, Anatoly V; Dzantiev, Boris B

    2010-03-24

    An indirect competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) of sulfamethoxypyridazine (SMP) was developed. The conjugates of streptavidin with cationic horseradish peroxidase (HRP) and anionic soybean peroxidase (SbP) were used in CL-ELISA for the detection of biotinylated anti-SMP antibodies. For streptavidin-HRP conjugate-catalyzed chemiluminescence measured 20 s after the initiation of the enhanced chemiluminescence reaction (ECR), the limit of detection (IC(10)), the IC(50) value, and the working range in CL-ELISA of SMP are 0.3, 12.4, and 1.2-85.0 ng/mL, respectively. An increase in the time interval between the ECR initiation and the luminescence measurement results in the loss in the quality of analytical measurements because of the time-dependent quenching of chemiluminescence typical of the HRP-catalyzed ECR. In the case of SbP-based CL-ELISA of SMP, the limit of detection, the IC(50) value, and the working range (0.025, 0.17, and 0.045-0.63 ng/mL, respectively) are better than those for HRP-based CL-ELISA. Furthermore, the analytical parameters of SbP-based CL-ELISA remain unchanged during a long period of time (for at least 30 min). The recovery values from four spiked milk samples with different concentrations of SMP in SbP-based CL-ELISA vary from 70 to 130%.

  2. The mormyrid brainstem--II. The medullary electromotor relay nucleus: an ultrastructural horseradish peroxidase study.

    PubMed

    Elekes, K; Ravaille, M; Bell, C C; Libouban, S; Szabo, T

    1985-06-01

    The medullary relay nucleus of the mormyrid weakly electric fish Gnathonemus petersii is a stage in the command pathway for the electric organ discharge. It receives input from the presumed command or pacemaker nucleus and projects to the electromotoneurons in the spinal cord. Its fine structure and synaptology were investigated by electron microscopy. The origin of the terminals contacting the cell membrane of the neurons of this nucleus was determined by horseradish peroxidase (HRP) injections into different brain structures, namely into the bulbar command- and mesencephalic command-associated nuclei. Twenty-five to thirty large cells of about 45 micron in diameter constitute the medullary electromotor relay. Each cell has a kidney-shaped, lobulated nucleus, a large myelinated axon with a short initial segment and several long, richly arborizing primary dendrites. Many, if not all, cells are interconnected with large somatosomatic or dendrosomatic, dendrodendritic and dendroaxonic gap junctions. These junctions often occur in serial or triadic arrangements. The relay cells receive large club endings as well as small boutons. The club endings are found mainly on the soma and primary dendrites and are morphologically mixed synapses. The boutons are characterized by synapses which are only chemical and are distributed all over the cell membrane, but with a definitely higher frequency on secondary dendrites and more distal parts of dendritic processes. Horseradish peroxidase injections into the mesencephalic command-associated nucleus reveal a large number of labelled boutons on the secondary dendrites of the relay cells. Injections into the bulbar command-associated nucleus label the same type of boutons as mesencephalic injections, but also label club endings on relay cell soma and primary dendrites. The results support the conclusion made on the basis of previous light microscopical observations that boutons originate from the bulbar command-associated nucleus

  3. Lipid peroxidation induced by indomethacin with horseradish peroxidase and hydrogen peroxide: involvement of indomethacin radicals.

    PubMed

    Miura, Toshiaki; Muraoka, Sanae; Fujimoto, Yukio

    2002-06-01

    Some of the side-effects of using indomethacin (IM) involve damage to the gastric mucosa and liver mitochondria. On the other hand, neutrophils infiltrate inflammatory sites to damage the tissues through the generation of reactive oxygen species by myeloperoxidase. The stomach and intestine have large amounts of peroxidase. These findings suggest that peroxidases are involved in tissue damage induced by IM. To clarify the basis for the tissue damage induced by IM in the presence of horseradish peroxidase (HRP) and H2O2 (HRP-H2O2), lipid peroxidation was investigated. When IM was incubated with liver microsomes in the presence of HRP-H2O2 and ADP-Fe3+, lipid peroxidation was time-dependent. Catalase and desferrioxamine almost completely inhibited lipid peroxidation, indicating that H2O2 and iron are necessary for lipid peroxidation. Of interest, superoxide dismutase strongly inhibited lipid peroxidation, and it also inhibited the formation of bathophenanthroline-Fe2+, indicating that reduction of the ferric ion was due to superoxide (O2-). ESR signals of IM radicals were detected during the interaction of IM with HRP-H2O2. However, the IM radical by itself did not reduce the ferric ion. These results suggest that O2- may be generated during the interaction of IM radicals with H2O2. Ferryl species, which are formed during the reduction of iron by O2-, probably are involved in lipid peroxidation.

  4. X-ray absorption studies of metalloprotein structure: cytochrome P-450, horseradish peroxidase, plastocyanin and laccase

    SciTech Connect

    Penner-Hahn, J.E.

    1984-03-01

    Extended x-ray absorption fine structure (EXAFS) has been developed to determine the structure of metalloproteins. EXAFS data have been collected and analysed for four states in the catalytic cycle of bacterial cytochrome P-450/sub CAM/. This data demonstrates that sulfur is retained as an axial ligand in the reduced forms of the enzyme. EXAFS and edge data have been analysed for the high-valent states of horseradish peroxidase (HRP), and for high-valent iron-porphyrin model compounds. These data provide the first direct confirmation of the presence of a ferryl Fe=O coordination in HRP and in some of the model compounds. The polarized single-crystal EXAFS spectra of plastocyanin have been measured as a function of both orientation and temperature. These data demonstrate that at room temperature the relative motions of the Cu and the S(Met) are essentially uncorrelated.

  5. 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

  6. The electromotor system of the electric eel investigated with horseradish peroxidase as a retrograde tracer.

    PubMed

    Bennett, M V; Sandri, C

    1989-05-29

    The electromotor system of the electric eel, Electrophorus electricus, was studied by injection of horseradish peroxidase as a retrograde tracer. The electromotor neurons, which innervate the electrocytes, comprise a midline nucleus, largely dorsal to the spinal canal. Spinal motoneurons lie ventrolaterally. The electromotor and skeletal motor neuron populations correspond to the acetylcholinesterase-negative and -positive cells previously described. The medullary relay neurons were labeled following HRP injection into the spinal cord at a level where electromotor neurons occurred, but not after injection into the cord in the abdominal region rostral to these cells. Other medullary neurons, presumably bulbospinal motor fibers, were labeled after both levels of spinal cord injection. The results suggest that these axosomatic synapses, which are electrically transmitting but morphologically mixed, take up retrograde tracers in a manner similar to chemical synapses and that tracer uptake is at least largely at terminal regions.

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

    PubMed

    Didenko, Vladimir V; Baskin, David S

    2006-03-01

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

  8. Proximity does not contribute to activity enhancement in the glucose oxidase-horseradish peroxidase cascade

    NASA Astrophysics Data System (ADS)

    Zhang, Yifei; Tsitkov, Stanislav; Hess, Henry

    2016-12-01

    A proximity effect has been invoked to explain the enhanced activity of enzyme cascades on DNA scaffolds. Using the cascade reaction carried out by glucose oxidase and horseradish peroxidase as a model system, here we study the kinetics of the cascade reaction when the enzymes are free in solution, when they are conjugated to each other and when a competing enzyme is present. No proximity effect is found, which is in agreement with models predicting that the rapidly diffusing hydrogen peroxide intermediate is well mixed. We suggest that the reason for the activity enhancement of enzymes localized by DNA scaffolds is that the pH near the surface of the negatively charged DNA nanostructures is lower than that in the bulk solution, creating a more optimal pH environment for the anchored enzymes. Our findings challenge the notion of a proximity effect and provide new insights into the role of DNA scaffolds.

  9. Gelatin-loaded p(HEMA-GMA) cryogel for high-capacity immobilization of horseradish peroxidase.

    PubMed

    Soomro, Rabel; Perçin, Işık; Memon, Najma; Iqbal Bhanger, Muhammad; Denizli, Adil

    2016-11-01

    Poly(2-hydroxyethyl methacrylate-glycidyl methacrylate) [p(HEMA-GMA)] cryogel discs were prepared under sub-zero temperatures. Gelatin was attached covalently on the p(HEMA-GMA) cryogel discs and reversible immobilization of horseradish peroxidase (HRP) was performed. The p(HEMA-GMA) cryogel discs were characterized by swelling tests, scanning electron microscopy, and surface area measurements. HRP immobilization capacity of p(HEMA-GMA)/gelatin cryogel discs was 24.8 mg/g. Removal of phenol from aqueous solutions was performed using HRP immobilized p(HEMA-GMA)/gelatin cryogel. It was observed that within 2 h of contact time, the percentage of phenol removal reaches up to 91% in the presence of H2O2.

  10. Chemical kinetics and interactions involved in horseradish peroxidase-mediated oxidative polymerization of phenolic compounds.

    PubMed

    Cheng, Wenjing; Harper, Willie F

    2012-03-10

    The primary objective of this research was to evaluate various factors that affect the reaction rate of oxidative coupling (OXC) reaction of phenolic estrogens catalyzed by horseradish peroxidase (HRP). Kinetic parameters were obtained for the conversion of phenol as well as natural and synthetic estrogens estrone (E(1)), 17β-estradiol (E(2)), estriol (E(3)), and 17α-ethinylestradiol (EE(2)). Molecular orbital theory and Autodock software were employed to analyze chemical properties and substrate binding characteristics. Reactions were first order with respect to phenolic concentration and reaction rate constants (k(r)) were determined for phenol, E(3), E(1), E(2) and EE(2) (in increasing order). Oxidative coupling was controlled by enzyme-substrate interactions, not collision frequency. Docking simulations show that higher binding energy and a shorter binding distance both promote more favorable kinetics. This research is the first to show that the OXC of phenolics is an entropy-driven and enthalpy-retarded process.

  11. FETAL RAT INTESTINAL ABSORPTION OF HORSERADISH PEROXIDASE FROM SWALLOWED AMNIOTIC FLUID

    PubMed Central

    Orlic, Donald; Lev, Robert

    1973-01-01

    Horseradish peroxidase (HRP) injected into amniotic fluid is swallowed by rat fetuses and within 3–6 h reaches the gut lumen. This macromolecular protein is then absorbed by the columnar lining cells via a system of apical cytoplasmic tubules formed by invaginations of the plasma membrane. From cytoplasm subjacent to the brush border HRP is transported, within vacuoles, to the supranuclear region, where some is retained for at least 18 h, and to interepithelial spaces. Extracellular enzyme is then found throughout the epithelial basement membrane and between connective tissue cells of the mucosal and submucosal layers Finally, HRP can be detected within lumina of blood and lymphatic capillaries, strongly suggesting that it is transported from the intestine to the circulation. PMID:4118449

  12. 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.

  13. EFFECT OF PHYSICAL FACTORS ON REACTIONS OF HORSE-RADISH PEROXIDASE COMPLEXES WITH REDUCED CYTOCHROME C.

    PubMed

    FARWELL, R W; ACKERMAN, E

    1963-11-01

    This investigation concerns the effect of certain physical factors-viscosity, dielectric constant, ionic strength, and temperature of the medium-on the reaction of hydrogen peroxide and ferrocytochrome c in the presence of the enzyme horse-radish peroxidase. From study of the effects of viscosity and dielectric constant, it was concluded that the reaction between the secondary complex of hydrogen peroxide and enzyme on the one hand and ferrocytochrome c on the other is controlled by diffusion in media of high viscosity and by electrostatic effects at low viscosities. With respect to ionic strength, the data at pH 4.7 indicated a dipole-dipole interreaction. The temperature dependence of the over-all reaction had a Q(10) of 1.25.

  14. Proximity does not contribute to activity enhancement in the glucose oxidase–horseradish peroxidase cascade

    PubMed Central

    Zhang, Yifei; Tsitkov, Stanislav; Hess, Henry

    2016-01-01

    A proximity effect has been invoked to explain the enhanced activity of enzyme cascades on DNA scaffolds. Using the cascade reaction carried out by glucose oxidase and horseradish peroxidase as a model system, here we study the kinetics of the cascade reaction when the enzymes are free in solution, when they are conjugated to each other and when a competing enzyme is present. No proximity effect is found, which is in agreement with models predicting that the rapidly diffusing hydrogen peroxide intermediate is well mixed. We suggest that the reason for the activity enhancement of enzymes localized by DNA scaffolds is that the pH near the surface of the negatively charged DNA nanostructures is lower than that in the bulk solution, creating a more optimal pH environment for the anchored enzymes. Our findings challenge the notion of a proximity effect and provide new insights into the role of DNA scaffolds. PMID:28004753

  15. Carbon based electrodes modified with horseradish peroxidase immobilized in conducting polymers for acetaminophen analysis.

    PubMed

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

    2013-04-11

    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.

  16. 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

  17. Caralluma umbellata Peroxidase: Biochemical Characterization and Its Detoxification Potentials in Comparison with Horseradish Peroxidase.

    PubMed

    Achar, Raghu Ram; Venkatesh, B K; Vivek, H K; Priya, B S; Swamy, S Nanjunda

    2017-02-01

    Caralluma umbellata peroxidase (CUP) is an acidic heme-containing protein having a molecular weight of ~42 kDa and is specific to guaiacol. It is not a glycoprotein. It was purified to 12.5-fold purity with 6.16 % yield. Its activity is dependent on hydrogen peroxide and has an optimum pH and temperature of 6.2 and 45 °C respectively. It can decolorize dyes, viz., Aniline Blue, Reactive Black 5, and Reactive Blue 19 but not Congo Red, while HRP can decolorize Congo Red also. It has lignin-degrading potentiality as it can decompose veratryl alcohol. Detoxification of phenol was more by CUP compared to HRP while with p-nitrophenol HRP has a greater detoxification rate. Based on our results, CUP was identified to be capable of oxidizing a variety of hazardous substances and also a lignin-degrading plant biocatalyst.

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

    SciTech Connect

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

    1989-04-05

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

  19. 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

  20. 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.

  1. 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

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

    PubMed

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

    2015-09-02

    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.

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

    PubMed

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

    2016-05-18

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

  4. A novel horseradish peroxidase biosensor towards the detection of dopamine: a voltammetric study.

    PubMed

    Raghu, P; Reddy, T Madhusudana; Gopal, P; Reddaiah, K; Sreedhar, N Y

    2014-04-10

    A polymerized film of glycine (Gly) was prepared on the surface of carbon paste electrode (CPE) through the cyclic voltammetry (CV) technique. A novel biosensor for the determination of dopamine (DA) has been constructed based on horseradish peroxidase (HRP) and multiwalled carbon nanotubes (MWCNTs) immobilizing on Poly (Gly)/CPE through silica sol-gel (SiSG) entrapment. CV measurements were employed in order to understand the feasibility of poly (Gly) as an electron carrier between the immobilized peroxidase and the surface of CPE. By using differential pulse voltammetry (DPV) the calibration curves of DA was obtained in the range of 15-865 μM. The limit of detection (LOD) and limit of quantification (LOQ) of DA was found to be 6×10⁻⁷ M and 2×10⁻⁶ M respectively. The apparent Michaelis-Menten constant (Km(app)) was found to be 0.5 mM and illustrated that the good biological activity of the fixed enzyme. Electrochemical impedance spectroscopy (EIS) results confirmed the rapid electron transfer and also the immobilization of enzyme on the electrode surface. The biosensor showed high sensitivity, selectivity and reproducibility. This method has been used to determine DA in the presence of various interferences and in clinical preparations.

  5. In situ simultaneous protein-polysaccharide bioconjugation and hydrogelation using horseradish peroxidase.

    PubMed

    Sakai, Shinji; Matsuyama, Tomohiro; Hirose, Keisuke; Kawakami, Koei

    2010-05-10

    We propose the peroxidase-catalyzed simultaneous conjugation and hydrogelation of polysaccharide and protein derivatives, each possessing phenolic hydroxyl (Ph) moieties, as a novel route for obtaining protein-polysaccharide conjugate hydrogels. We used alginate, gelatin, and albumin derivatives bearing Ph moieties (Alg-Ph, Gela-Ph, and Alb-Ph) to demonstrate the feasibility. The gelation time of conjugate gels decreased with decreasing H(2)O(2) concentration and with increasing horseradish peroxidase concentration. Gelation time was controllable from a few seconds to 6 min. The repulsion force detected at 40% compression of a conjugate gel obtained from a mixture of Alg-Ph and Gela-Ph at 1.0% (w/v), respectively, was more than 2.8 times larger than that detected for gels produced from 3.0% (w/v) Gela-Ph or 2.0% (w/v) Alg-Ph alone. Cell adhesiveness of gels was tunable by changing the type of protein derivative. A gel from Gela-Ph and Alg-Ph showed higher cell adhesiveness than Alg-Ph gel, but a gel produced from Alb-Ph and Alg-Ph showed a lower cell adhesiveness than Alg-Ph gel. The conjugate gel was degradable by degrading alginate molecules using the nonproteolytic enzyme alginate lyase. The tunable gelation, mechanical properties, and cell adhesiveness of polysaccharide-protein conjugate hydrogels obtained through peroxidase-catalyzed gelation indicates great potential for a wide range of applications, such as scaffolds for tissue engineering and carriers for drug delivery system.

  6. Kinetics of Horseradish Peroxidase-Catalyzed Nitration of Phenol in a Biphasic System.

    PubMed

    Kong, Mingming; Zhang, Yang; Li, Qida; Dong, Runan; Gao, Haijun

    2017-02-28

    The use of peroxidase in the nitration of phenols is gaining interest as compared with traditional chemical reactions. We investigated the kinetic characteristics of phenol nitration catalyzed by horseradish peroxidase (HRP) in an aqueous-organic biphasic system using n-butanol as the organic solvent and NO2(-) and H2O2 as substrates. The reaction rate was mainly controlled by the reaction kinetics in the aqueous phase when appropriate agitation was used to enhance mass transfer in the biphasic system. The initial velocity of the reaction increased with increasing HRP concentration. Additionally, an increase in the substrate concentrations of phenol (0-2 mM in organic phase) or H2O2 (0-0.1 mM in aqueous phase) enhanced the nitration efficiency catalyzed by HRP. In contrast, high concentrations of organic solvent decreased the kinetic parameter Vmax/Km. No inhibition of enzyme activity was observed when the concentrations of phenol and H2O2 were at or below 10 mM and 0.1 mM, respectively. On the basis of the peroxidase catalytic mechanism, a double-substrate ping-pong kinetic model was established. The kinetic parameters were Km(H2O2)= 1.09 mM, Km(PhOH) = 9.45 mM, and Vmax = 0.196 mM/min. The proposed model was well fit to the data obtained from additional independent experiments under the suggested optimal synthesis conditions. The kinetic model developed in this paper lays a foundation for further comprehensive study of enzymatic nitration kinetics.

  7. 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

  8. Time-resolved fluorescence observation of di-tyrosine formation in horseradish peroxidase upon ultrasound treatment leading to enzyme inactivation

    NASA Astrophysics Data System (ADS)

    Tsikrika, Konstantina; Lemos, M. Adília; Chu, Boon-Seang; Bremner, David H.; Hungerford, Graham

    2017-02-01

    The application of ultrasound to a solution can induce cavitional phenomena and generate high localised temperatures and pressures. These are dependent of the frequency used and have enabled ultrasound application in areas such as synthetic, green and food chemistry. High frequency (100 kHz to 1 MHz) in particular is promising in food chemistry as a means to inactivate enzymes, replacing the need to use periods of high temperature. A plant enzyme, horseradish peroxidase, was studied using time-resolved fluorescence techniques as a means to assess the effect of high frequency (378 kHz and 583 kHz) ultrasound treatment at equivalent acoustic powers. This uncovered the fluorescence emission from a newly formed species, attributed to the formation of di-tyrosine within the horseradish peroxidase structure caused by auto-oxidation, and linked to enzyme inactivation.

  9. Differences between the endocytosis of horseradish peroxidase and its conjugate with wheat germ agglutinin by cultured fibroblasts.

    PubMed

    Stieber, A; Gonatas, J O; Gonatas, N K

    1984-04-01

    A covalent conjugate of wheat germ agglutinin (WGA) with horseradish peroxidase (HRP) was used for a morphologic study of its adsorptive endocytosis by cultured human fibroblasts. Initial binding at 4 degrees C of the conjugate was observed over the entire plasma membrane, including "coated" and smooth pits. Endocytosis of HRP and the WGA-HRP conjugate was observed in lysosomes, but only the conjugate was seen in a cisterna of the Golgi apparatus (GERL), and in adjacent coated vesicles.

  10. [Neuroanatomical study of experimental tremor produced by VMT lesion in monkeys--a horseradish peroxidase study].

    PubMed

    Takahashi, T

    1988-01-01

    Destruction of the ventromedial tegmentum (VMT) of the midbrain in monkeys is known to produce tremors similar to those seen in Parkinson's disease. To elucidate such tremorgenic mechanisms, 50% horseradish peroxidase (HRP) was injected into the VMT target region in three monkeys (macaca fuscata fuscata) and eleven adult cats. The volume injected varied between 0.05 and 0.1 microliter. The results suggest that afferent fibers to the thalamus, which passed through the VMT region, contains tractus cerebellothalamicus and nigrothalamic fibers. A large number of labelled cells were found in the ipsilateral nucleus dorsalis raphae, indicating that serotonergic neurons are related to the experimental tremors. Many labelled terminals were observed in the ipsilateral nucleus subthalamicus in the monkey, but in cats no terminals were found. This suggests that VMT region in the monkeys contains nigrosubthalamic fibers. The experimental tremors produced by destruction of the VMT region in the monkeys appears to be due to combined destruction of the tractus cerebellothalamicus, nigrothalamic fibers, tractus nigrostriatus, ascending serotonergic neurons from the nucleus dorsalis raphae and nigrosubthalamic fibers.

  11. 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.

  12. Improved activity of immobilized horseradish peroxidase on gold nanoparticles in the presence of bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Ni, Yuyang; Li, Jun; Huang, Zhenzhen; He, Ke; Zhuang, Jiaqi; Yang, Wensheng

    2013-11-01

    The using of macromolecular additives is known to be a simple and effective way to improve the activity of immobilized enzymes on solid support, yet the mechanism has not been well understood. Taking horseradish peroxidase (HRP) as an example, only 30 % of its catalytic activity was kept after being immobilized on the surface of 25-nm Au nanoparticles, mainly attributed to the conformational change of the heme-containing active site. The catalytic activity of HRP was significantly improved to 80 % when a certain amount of bovine serum albumin (BSA) was added at the initial stage of the immobilization. Systematic spectral investigation indicated that the addition of BSA inhibited the tertiary structure change around the active site, which was a prerequisite for improved activity of the immobilized HRP. Steady-state kinetic analyses revealed that the introduction of BSA could effectively improve the turnover rate of substrate to product in spite of slight reduced affinity to substrates, which also contributed to the improved catalytic activity.

  13. 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.

  14. 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.

  15. Stability and structural changes of horseradish peroxidase: microwave versus conventional heating treatment.

    PubMed

    Lopes, Lucas C; Barreto, Maria T M; Gonçalves, Karen M; Alvarez, Heiddy M; Heredia, Montserrat Fortuny; de Souza, Rodrigo Octavio M A; Cordeiro, Yraima; Dariva, Cláudio; Fricks, Alini T

    2015-02-01

    Effects of conventional heating (CH) and microwave (MW) on the structure and activity of horseradish peroxidase (HRP) in buffer solution were studied. CH incubation between 30 and 45 °C increased activity of HRP, reaching 170% of residual activity (RA) after 4-6 h at 45 °C. CH treatment at 50 and 60 °C caused HRP inactivation: RA was 5.7 and 16.7% after 12 h, respectively. Secondary and tertiary HRP structural changes were analyzed by circular dichroism (CD) and intrinsic fluorescence emission, respectively. Under CH, activation of the enzyme was attributed to conformational changes in secondary and tertiary structures. MW treatment had significant effects on the residual activity of HRP. MW treatment at 45 °C/30W followed by CH treatment 45 °C regenerated the enzyme activity. The greatest loss in activity occurred at 60 °C/60 W/30 min (RA 16.9%); without recovery of the original activity. The inactivation of MW-treated HRP was related to the loss of tertiary structure, indicating changes around the tryptophan environment.

  16. 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

  17. Horseradish peroxidase dye tracing and embryonic statoacoustic ganglion cell transplantation in the rat auditory nerve trunk.

    PubMed

    Palmgren, Björn; Jin, Zhe; Jiao, Yu; Kostyszyn, Beata; Olivius, Petri

    2011-03-04

    At present severe damage to hair cells and sensory neurons in the inner ear results in non-treatable auditory disorders. Cell implantation is a potential treatment for various neurological disorders and has already been used in clinical practice. In the inner ear, delivery of therapeutic substances including neurotrophic factors and stem cells provide strategies that in the future may ameliorate or restore hearing impairment. In order to describe a surgical auditory nerve trunk approach, in the present paper we injected the neuronal tracer horseradish peroxidase (HRP) into the central part of the nerve by an intra cranial approach. We further evaluated the applicability of the present approach by implanting statoacoustic ganglion (SAG) cells into the same location of the auditory nerve in normal hearing rats or animals deafened by application of β-bungarotoxin to the round window niche. The HRP results illustrate labeling in the cochlear nucleus in the brain stem as well as peripherally in the spiral ganglion neurons in the cochlea. The transplanted SAGs were observed within the auditory nerve trunk but no more peripheral than the CNS-PNS transitional zone. Interestingly, the auditory nerve injection did not impair auditory function, as evidenced by the auditory brainstem response. The present findings illustrate that an auditory nerve trunk approach may well access the entire auditory nerve and does not compromise auditory function. We suggest that such an approach might compose a suitable route for cell transplantation into this sensory cranial nerve.

  18. The effect on structural and solvent water molecules of substrate binding to ferric horseradish peroxidase.

    PubMed

    Simpson, Niall; Adamczyk, Katrin; Hithell, Gordon; Shaw, Daniel J; Greetham, Gregory M; Towrie, Michael; Parker, Anthony W; Hunt, Neil T

    2015-01-01

    Ultrafast, multi-dimensional infrared spectroscopy, in the form of 2D-IR and pump-probe measurements, has been employed to investigate the effect of substrate binding on the structural dynamics of the horseradish peroxidase (HRP) enzyme. Using nitric oxide bound to the ferric haem of HRP as a sensitive probe of local dynamics, we report measurements of the frequency fluctuations (spectral diffusion) and vibrational lifetime of the NO stretching mode with benzohydroxamic acid (BHA) located in the substrate-binding position at the periphery of the haem pocket, in both D2O and H2O solvents. The results reveal that, with BHA bound to the enzyme, the local structural dynamics are insensitive to H/D exchange. These results are in stark contrast to those found in studies of the substrate-free enzyme, which demonstrated that the local chemical and dynamic environment of the haem ligand is influenced by water molecules. In light of the large changes in solvent accessibility caused by substrate binding, we discuss the potential for varying roles for the solvent in the haem pocket of HRP at different stages along the reaction coordinate of the enzymatic mechanism.

  19. 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.

  20. 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.

  1. Horseradish peroxidase immobilized on copper surfaces and applications in selective electrocatalysis of p-dihydroxybenzene

    NASA Astrophysics Data System (ADS)

    Wang, Chuntao; Luo, Xiaoxiao; Jia, Zehui; Shi, Qinghua; Zhu, Ritao

    2017-06-01

    Horseradish Peroxidase (HRP) was immobilized on copper surfaces with the linker of L-Cysteine (L-Cys) self-assembled films to form Cu/L-Cys/HRP electrodes. The activity of HRP can be preserved by the Cu/L-Cys self-assembled films. The Cu/L-Cys/HRP electrodes can be used for the selective electrocatalytic oxidase of p-dihydroxybenzen in absent of H2O2. The optimum pH for electrocatalyzing p-dihydroxybenzen was 5.5 or 7.0, which corresponds to the isoelectric points of L-Cys and HRP, respectively. X-ray photoelectron spectroscopy (XPS) provided the evidence that L-Cys linked with Cu surface by the Cusbnd S bond. Fourier transform infrared spectroscopy (FTIR) analyses indicated that aromatic plane of p-dihydroxybenzen was connected parallel to porphyrin ring of heme in HRP. Quantum chemical calculation of density functional theory (DFT) revealed that symmetry of molecular structure and minimum space steric hindrance for p-dihydroxybenzen were benefit to combination with HRP. Moreover, the lowest energy of LUMO and most negative charges of oxygen atom on hydroxyl group of p-dihydroxybenzen were advantage to lose the hydrogen atom of hydroxyl group to be oxided.

  2. Vagal innervation of intestines: afferent pathways mapped with new en bloc horseradish peroxidase adaptation.

    PubMed

    Wang, Feng-Bin; Powley, Terry L

    2007-08-01

    Neural tracers have not typically been employed to determine the pathways followed by axons between their perikarya and target tissues. We have adapted the tetramethylbenzidine method for horseradish peroxidase (HRP) to stain fibers en bloc in organs and thus to delineate axonal trajectories. We have also applied this protocol to characterize the pathways that vagal afferents follow to the intestines. The protocol confirms that the proximal segment of the duodenum receives afferents carried in the vagal hepatic branch and demonstrates that vagal afferents innervating the remainder of the small and large intestines course through multiple fascicles derived from the celiac branches of the abdominal vagus. These fascicles divide, intermingle, and reorganize along the abdominal aorta and superior mesenteric artery (SMA), but not along the inferior mesenteric artery, and then project to the intestines with secondary arteries that branch from the SMA. The inferior pancreaticoduodenal, jejunal, middle colic, right colic, and ileocecocolic arteries all carry vagal afferents to segments of the intestines. As the arteries derived from the SMA divide repeatedly into successively finer branches and course to the intestines, the vagal afferent fascicles (typically a pair) running with each arterial branch also divide. These divisions generate sets/pairs of finer fascicles coursing with even the highest order arterial radicles. The vagal fascicles enter the intestinal wall with the vessels and appear to innervate the organ near the point of entry. The results verify the practicality and sensitivity of the en bloc HRP technique and suggest that the protocol could delineate other peripheral pathways.

  3. 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.

  4. 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.

  5. Adsorption and inactivation behavior of horseradish peroxidase on cellulosic fiber surfaces.

    PubMed

    Di Risio, Sabina; Yan, Ning

    2009-10-15

    The physical immobilization behavior of horseradish peroxidase (HRP) on cellulosic fiber surfaces was characterized using adsorption and inactivation isotherms measured by the depletion method followed by fitting of Langmuir's and Freundlich's models to the experimental data. The adsorption and inactivation behavior of simpler and relatively non-porous high and low crystalline cellulosic substrates (microcrystalline cellulose and regenerated cellulose) as well as more complex and porous cellulosic pulp fibers (bleached kraft softwood fibers) were investigated. The effect of the sorbent surface energy on HRP adsorption was demonstrated by increasing the hydrophobicity of the cellulosic fibers using an internal sizing agent. The influence of the fiber surface charge density on HRP adsorption was studied via modification of the cellulosic fibers using TEMPO (2,2,6,6-tetramethyl-1-piperidiniloxy radical)-mediated oxidation methods. Results showed that hydrophobic interactions had a much larger effect on HRP adsorption than electrostatic interactions. More hydrophobic fiber surfaces (lower polar surface energy) result in larger enzyme-fiber binding affinity constants and higher binding heterogeneity. It was also found that oxidation of the cellulosic fiber substrate reduces enzyme adsorption affinity but significantly increases the loading capacity per unit weight of the surface.

  6. Structural Stabilization and Functional Improvement of Horseradish Peroxidase upon Modification of Accessible Lysines: Experiments and Simulation

    PubMed Central

    Mogharrab, Navid; Ghourchian, Hedayatollah; Amininasab, Mehriar

    2007-01-01

    Horseradish peroxidase (HRP) is an important heme enzyme with enormous medical diagnostic, biosensing, and biotechnological applications. Thus, any improvement in the applicability and stability of the enzyme is potentially interesting. We previously reported that covalent attachment of an electron relay (anthraquinone 2-carboxylic acid) to the surface-exposed Lys residues successfully improves electron transfer properties of HRP. Here we investigated structural and functional consequences of this modification, which alters three accessible charged lysines (Lys-174, Lys-232, and Lys-241) to the hydrophobic anthraquinolysine residues. Thermal denaturation and thermoinactivation studies demonstrated that this kind of modification enhances the conformational and operational stability of HRP. The melting temperature increased 3°C and the catalytic efficiency enhanced by 80%. Fluorescence and circular dichroism investigations suggest that the modified HRP benefits from enhanced aromatic packing and more buried hydrophobic patches as compared to the native one. Molecular dynamics simulations showed that modification improves the accessibility of His-42 and the heme prosthetic group to the peroxide and aromatic substrates, respectively. Additionally, the hydrophobic patch, which functions as a binding site or trap for reducing aromatic substrates, is more extended in the modified enzyme. In summary, this modification produces a new derivative of HRP with enhanced electron transfer properties, catalytic efficiency, and stability for biotechnological applications. PMID:17114227

  7. Promoting immobilization and catalytic activity of horseradish peroxidase on mesoporous silica through template micelles.

    PubMed

    Wan, Mi Mi; Lin, Wei Gang; Gao, Ling; Gu, Hui Cheng; Zhu, Jian Hua

    2012-07-01

    New concept on the promotion of immobilization and catalytic activity of enzyme on mesoporous silica through template micelles is proposed and realized in this paper. Proper P123 templates are controllable retained in the as-synthesized SBA-15, not only to anchor the horseradish peroxidase (HRP) guest, but also to establish the crowding-like microenvironment around the enzyme. The influence of retaining templates on the pore structure of SBA-15, immobilization, and catalytic activity of HRP is studied, and the possible process of template removal is proposed. Ethanol refluxing of 6 h is conformable to prepare the optimal mesoporous support characterized with the retained templates of about 8%. With the assistance of retained templates in SBA-15, up to 49 mg g(-1) of HRP can be immobilized, 100% more than that on calcined SBA-15. Furthermore, the thermal stability, the resistance of pH variation and denaturing agent urea, and the recycle usage of HRP immobilized are obviously elevated, paving a novel and low-cost route to develop enzyme catalysts.

  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. 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.

  10. 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

    2011-05-09

    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.

  11. Structural insights into the effects of charge-reversal substitutions at the surface of horseradish peroxidase

    PubMed Central

    Navapour, Leila; Mogharrab, Navid

    2016-01-01

    Horseradish peroxidase (HRP), has gained significant interests in biotechnology, especially in biosensor field and diagnostic test kits. Hence, its solvent-exposed lysine residues 174, 232, and 241 have been frequently modified with the aim of improving its stability and catalytic efficiency. In this computational study, we investigated the effects of Lys-to-Glu substitutions on HRP structure to model charge-reversal manipulations at the enzyme surface. Simulation results implied that upon these substitutions, the number of stable hydrogen bonds and α-helical content of HRP are increased and the proximal Ca2+ binding pocket becomes more integrated. The results revealed that although Glu174-heme hydrogen bond is lost after mutation, formation of a new hydrogen bonding network contributes to the stability of heme-protein linkage. Together, it may be concluded that these substitutions enhance the stability of the protein moiety as well as the heme-protein non-covalent interactions. In the enzyme active site, we observed increased accessibility of peroxide binding site and heme prosthetic group to the peroxide and aromatic substrates, respectively. Results also demonstrated that the bottleneck entry of the peroxide-binding site has become wider and more flexible upon substitutions. Moreover, the hydrophobic patch functioning as a binding site or trap for reducing aromatic substrates is more extended in mutated enzyme. These observations suggest that the reactivity of the enzyme to its substrates has increased. Together, the results of this simulation study could provide possible structural clues to explain those experimental observations in which the protein stability achieved upon manipulation of charge distribution on protein surface. PMID:28097171

  12. Mode of bindings of zinc oxide nanoparticles to myoglobin and horseradish peroxidase: A spectroscopic investigations

    NASA Astrophysics Data System (ADS)

    Mandal, Gopa; Bhattacharya, Sudeshna; Ganguly, Tapan

    2011-07-01

    The interactions between two heme proteins myoglobin (HMb) and horseradish peroxidase (HRP) with zinc oxide (ZnO) nanoparticles are investigated by using UV-vis absorption, steady state fluorescence, synchronous fluorescence, time-resolved fluorescence, FT-IR, atomic force microscopy (AFM) and circular dichroism (CD) techniques under physiological condition of pH˜7.4. The presence of mainly static mode in fluorescence quenching mechanism of HMb and HRP by ZnO nanoparticle indicates the possibility of formation of ground state complex. The processes of bindings of ZnO nanoparticles with the two proteins are spontaneous molecular interaction procedures. In both cases hydrogen bonding plays a major role. The circular dichroism (CD) spectra reveal that a helicity of the proteins is reduced by increasing ZnO nanoparticle concentration although the α-helical structures of HMb and HRP retain their identity. On binding to the ZnO nanoparticles the secondary structure of HRP molecules (or HMb molecules) remains unchanged while there is a substantial change in the environment of the tyrosin active site in case of HRP molecules and tryptophan active site in case of HMb molecules. Tapping mode atomic force microscopy (AFM) was applied for the investigation the structure of HRP adsorbed in the environment of nanoparticles on the silicon and on the bare silicon. HRP molecules adsorb and aggregate on the mica with ZnO nanoparticle. The aggregation indicates an attractive interaction among the adsorbed molecules. The molecules are randomly distributed on the bare silicon wafer. The adsorption of HRP in the environment of ZnO nanoparticle changes drastically the domains due to a strong interaction between HRP and ZnO nanoparticles. Similar situation is observed in case of HMb molecules. These findings demonstrate the efficacy of biomedical applications of ZnO nanoparticles as well as in elucidating their mechanisms of action as drugs in both human and plant systems.

  13. Immobilization of horseradish peroxidase on nanoporous copper and its potential applications.

    PubMed

    Qiu, Huajun; Lu, Lu; Huang, Xirong; Zhang, Zhonghua; Qu, Yinbo

    2010-12-01

    Nanoporous copper (NPC) with a pore size of 100-200 nm was prepared by simply dealloying Al(60)Cu(40) alloy in a 5 wt.% HCl solution. The NPC was characterized by scanning electron microscopy and nitrogen adsorption techniques. Horseradish peroxidase (HRP) was immobilized on NPC by adsorption. Compared with free enzyme, the thermal stability of the immobilized enzyme was greatly improved due to the multiple attachments between the enzyme molecule and the NPC surface. After 2h incubation at 50 degrees C, the immobilized HRP retained ca. 90% of the initial activity while only ca. 10% initial activity remained for the free enzyme. The interaction between HRP and the porous surface also made the K(m) and K(cat) values of the immobilized enzyme increase (from 0.43 to 0.80 mM) and decrease (from 8.1 x 10(3) to 2.2 x 10(3)min(-1)), respectively. Based on the good electric conductivity and electrocatalytic activity of the NPC electrode, an electrochemical biosensor for O-phenylenediamine (OPD) was made. The calibration curve of the biosensor was linear from 0.5 microM to 14.5 microM OPD with a sensitivity of 0.37 microA microM(-1). The stability and reproducibility of the biosensor were also demonstrated to be good. When positioned at -0.45 V for 200 s, its current response toward 10 microM OPD remained ca. 80% of its initial value. For five HRP-loaded NPC electrodes, the relative standard deviation (RSD) of the current response toward 10 microM OPD was ca. 4.5%. All these results indicated that NPC was a good support for the HRP immobilization and its low price would facilitate its large-scale application.

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

    PubMed

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

    2016-04-07

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

  15. 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.

  16. Morphology of motoneurons in different subdivisions of the rat facial nucleus stained intracellularly with horseradish peroxidase.

    PubMed

    Friauf, E

    1986-11-08

    Horseradish peroxidase was injected into single facial motoneurons of the rat. Neurons were identified by antidromic stimulation of either the buccal or the marginal mandibular or the posterior auricular nerve branches. Motoneuronal cell bodies supplying the buccal branch were located in the lateral subdivision of the facial nucleus, those supplying the marginal mandibular branch were in the intermediate subdivision, and those supplying the posterior auricular branch were in the medial subdivision. Eleven motoneurons were reconstructed with a computer-assisted technique. Their soma diameters averaged 20 microns; the average number of primary dendrites was 7.9 and the combined lengths of the dendritic trees averaged 17,650 microns. There was no distinction between the three motoneuron groups in terms of these and other quantitative data. However, on the basis of reconstructed dendritic tree orientation (i.e., dendritic distribution), major differences were observed between motoneurons of the three groups. Dendrites from all groups extended beyond the boundaries of the facial nucleus into the reticular formation. The border between the intermediate and the lateral subdivision was crossed by some dendrites but the overlap was small. In contrast, no dendrite of a motoneuron in the medial subdivision entered the intermediate subdivision and vice versa. The dendritic extent was totally restricted by the borders between these two subdivisions. Outside the Nissl-defined nuclear border, however, dendrites from cells in adjacent subdivisions overlapped. It is concluded that the medial subdivision of the facial nucleus can be distinguished from the intermediate and lateral subdivisions not only by its sharp Nissl-defined border but also by the discrete organization of its dendritic field.

  17. Reagentless amperometric immunosensors based on direct electrochemistry of horseradish peroxidase for determination of carcinoma antigen-125.

    PubMed

    Dai, Zong; Yan, Feng; Chen, Jin; Ju, Huangxian

    2003-10-15

    A novel strategy for immunoassay and the preparation of reagentless immunosensors was proposed. This strategy was based on the immobilization of antigen and the direct electrochemistry of horseradish peroxidase (HRP) that was labeled to an antibody. A reagentless immunosensor for carcinoma antigen-125 (CA 125) determination was developed. The immunosensor was prepared by immobilizing CA 125 with titania sol-gel on a glassy carbon electrode by the vapor deposition method. The incubation of the immunosensor in phosphate buffer solution (PBS) including HRP-labeled CA 125 antibody led to the formation of a HRP-modified surface. The immobilized HRP displayed its direct electrochemistry with a rate constant of 3.04 +/- 1.21 s(-1). With a competition mechanism, a differential pulse voltammetric determination method for CA 125 was established by the peak current decrease of the immobilized HRP. The current decrease resulted from the competitive binding of the CA 125 in sample solution and the immobilized CA 125 to the limited amount of HRP-labeled CA 125 antibody. Under optimal conditions, the current decrease was proportional to CA 125 concentration ranging from 2 to 14 units mL(-1) with a detection limit of 1.29 units mL(-1) at a current decrease by 10%. The CA 125 immunosensor showed good accuracy and acceptable precision and fabrication reproducibility with intraassay CVs of 8.7 and 5.5% at 8 and 14 units mL(-1) CA 125 concentrations, respectively, and interassay CV of 19.8% at 8 units mL(-1). The storage stability was acceptable in a pH 7.0 PBS at 4 degrees C for 15 days. The proposed method provided a new promising platform for clinical immunoassay.

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

    PubMed Central

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

    2013-01-01

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

  19. 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

    2016-03-07

    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.

  20. A comparative study of free and immobilized soybean and horseradish peroxidases for 4-chlorophenol removal: protective effects of immobilization.

    PubMed

    Bódalo, Antonio; Bastida, Josefa; Máximo, M Fuensanta; Montiel, M Claudia; Gómez, María; Murcia, M Dolores

    2008-10-01

    Horseradish peroxidase (HRP) and soybean peroxidase (SBP) were covalently immobilized onto aldehyde glass through their amine groups. The activity yield and the protein content for the immobilized SBP were higher than for the immobilized HRP. When free and immobilized peroxidases were tested for their ability to remove 4-chlorophenol from aqueous solutions, the removal percentages were higher with immobilized HRP than with free HRP, whereas immobilized SBP needs more enzyme to reach the same conversion than free enzyme. In the present paper the two immobilized derivatives are compared. It was found that at an immobilized enzyme concentration in the reactor of 15 mg l(-1), SBP removed 5% more of 4-chlorophenol than HRP, and that a shorter treatment was necessary. Since immobilized SBP was less susceptible to inactivation than HRP and provided higher 4-chlorophenol elimination, this derivative was chosen for further inactivation studies. The protective effect of the immobilization against the enzyme inactivation by hydrogen peroxide was demonstrated.

  1. Pelvic nerve innervation of the external sphincter of urethra as suggested by urodynamic and horse-radish peroxidase studies.

    PubMed

    Morita, T; Nishizawa, O; Noto, H; Tsuchida, S

    1984-03-01

    In view of the fact that the detrusor vesicae and external urethral sphincter perform closely synergic functions in micturition, experiments were conducted to explore the action of the pelvic efferent neurons on the external urethral sphincter. The pelvic efferent neurons are generally recognized, by urodynamic assessments and histochemical study with the technique of retrograde axonal transport of horse-radish peroxidase, to innervate the vesical detrusor. In 7 of 15 adult dogs studied, the external urethral sphincter continued to show a normal synergic electromyogram pattern with enhanced electrical activity on vesical distention and disappearance of discharges on vesical contraction even after bilateral transection of the pudendal nerves. The electrical discharges ceased in the sphincter only after subsequent bilateral pelvic neurotomy. Horse-radish peroxidase-positive cells were demonstrated in the intermediolateral and intermediomedial nuclei and in the Onuf nucleus of the sacral cord in approximately half the dogs whose pelvic nerve was injected with the plant peroxidase. The results suggest that the pelvic nerve may contain somatic fibers innervating the external urethral sphincter.

  2. 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

  3. 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.

  4. Theoretical study of model compound I complexes of horseradish peroxidase and catalase.

    PubMed Central

    Du, P; Loew, G H

    1995-01-01

    Theoretical studies of the electronic structure and spectra of models for the ferric resting state and Compound I intermediates of horseradish peroxidase (HRP-I) and catalase (CAT-I) have been performed using the INDO-RHF/CI method. The goals of these studies were twofold: i) to determine whether the axial ligand of HRP is best described as imidazole or imidazolate, and ii) to address the long-standing question of whether HRP-I and CAT-I are a1u and a2u tau cation radicals. Only the imidazolate HRP-I model led to a calculated electronic spectra consistent with the experimentally observed significant reduction in the intensity of the Soret band compared with the ferric resting state. These results provide compelling evidence for significant proton transfer to the conserved Asp residue by the proximal histidine. The origin of the observed reduction of the Soret band intensity in HRP-I and CAT-I spectra has been examined and found to be caused by the mixing of charge transfer transitions into the predominantly porphyrin tau-tau transitions. For both HRP-I and CAT-I, the a1u porphyrin tau cation state is the lowest energy, and it is further stabilized by both the anionic form of the ligand and the porphyrin ring substituents of protoporphyrin-IX. The calculated values of quadrupole-splitting observed in the Mossbauer resonance of HRP-I and CAT-I are similar for the a1u and a2u tau cation radicals. Electronic spectrum of the a1u tau cation radical of HRP-I are more similar to the observed spectra, whereas the spectra of both a1u tau and a2u tau cation radicals of CAT-I resemble the observed spectra. These results also indicate the limitations of using any one observable property to try to distinguish between these states. Taken together, comparison of calculated and observed properties indicate that there is no compelling reason to invoke the higher energy a2u tau cation radical as the favored state in HRP-I and CAT-I. Both ground-state properties and electronic spectra

  5. Preparation of goat and rabbit anti-camel immunoglobulin G whole molecule labeled with horseradish peroxidase

    PubMed Central

    Abdel-Rahman, Eman Hussein; El-Jakee, Jakeen Kamal; Hatem, Mahmoud Essam; Ata, Nagwa Sayed; Fouad, Ehab Ali

    2017-01-01

    Aim: As the labeled anti-camel immunoglobulins (Igs) with enzymes for enzyme-linked immunosorbent assay (ELISA) are unavailable in the Egyptian market, the present investigation was directed for developing local labeled anti-camel IgG with horseradish peroxidase (HRP) to save hard curacy. Materials and Methods: For purification of camel IgG whole molecule, camel sera was preliminary precipitated with 50% saturated ammonium sulfate and dialyzed against 15 mM phosphate-buffered saline pH 7.2 then concentrated. This preparation was further purified by protein A sepharose affinity column chromatography. The purity of the eluted camel IgG was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresi. Anti-camel IgG was prepared by immunization of goats and rabbits separately, with purified camel IgG. The anti-camel IgG was purified by protein A sepharose affinity column chromatography. Whole molecule anti-camel IgG was conjugated with HRP using glutraldehyde based assay. Sensitivity and specificity of prepared conjugated secondary antibodies were detected using positive and negative camel serum samples reacted with different antigens in ELISA, respectively. The potency of prepared conjugated antibodies was evaluated compared with protein A HRP. The stability of the conjugate at −20°C during 1 year was assessed by ELISA. Results: The electrophoretic profile of camel IgG showed four bands of molecular weight 63, 52, 40 and 33 kDa. The recorded sensitivity and specificity of the product are 100%. Its potency is also 100% compared to 58-75% of commercial protein A HRP. The conjugates are stable for 1 year at −20°C as proved by ELISA. Conclusion: Collectively, this study introduces goat and rabbit anti-camel IgG whole molecules with simple, inexpensive method, with 100% sensitivity, 100% specificity and stability up to 1 year at −20°C. The important facet of the current study is saving hard curacy. Future investigations are necessary for preparation of Ig

  6. 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.

  7. Assay of H2O2 production by macrophages and neutrophils with homovanillic acid and horse-radish peroxidase.

    PubMed

    Ruch, W; Cooper, P H; Baggiolini, M

    1983-10-28

    A simple and sensitive method for measurement of the release of H2O2 from phagocytic cells is described. The assay is based on the H2O2-dependent oxidation of homovanillic acid (3-methoxy-4-hydroxyphenylacetic acid, HVA) to a highly fluorescent dimer (2,2'-dihydroxy-3,3'-dimethoxydiphenyl-5,5'-diacetic acid) which is mediated by horse-radish peroxidase. A linear relationship between fluorescence (lambda ex = 312 nm and lambda em = 420 nm) and amount of H2O2 was found in the range of 0.1-10 nmoles per 2.25 ml assay. The method was reliable for monitoring H2O2 production in large numbers of cell samples, as suspensions or monolayers, over periods of time extending between minutes and several hours. At concentrations optimal for detection of cellular release of H2O2, HVA and horse-radish peroxidase were devoid of cytotoxic effects. The time course of H2O2 release by mouse peritoneal and bone marrow-derived macrophages and by human neutrophils was determined following stimulation with zymosan particles or phorbol myristate acetate, and the dependence of H2O2 release on cell number and stimulus dosage was studied.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  9. Mechanism of action of collagenase on the blood-brain barrier permeability. Increase of endothelial cell pinocytotic activity as shown with horse-radish peroxidase as a tracer.

    PubMed

    Godeau, G; Robert, A M

    1979-12-01

    The ultrastructural mechanism of the protease induced blood-brain barrier permeability-increase was studied with horse-radish peroxidase as a tracer. After intravenous injection of collagenase or pronase, a significantly increased number of pinocytotic vesicles was found in brain capillary endothelial cells. alpha-Chymotrypsine did not exert such an action.

  10. 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.

  11. Application of 4-iodophenol-enhanced luminol chemiluminescence to direct detection of horseradish peroxidase encapsulated in liposomes.

    PubMed

    Kamidate, Tamio; Maruya, Masumi; Tani, Hirofumi; Ishida, Akihiko

    2009-09-01

    4-Iodophenol was applied to an enhancer in the direct detection of horseradish peroxidase (HRP) encapsulated in liposomes by using luminol chemiluminescence (CL). Luminol, 4-iodophenol and hydrogen peroxide permeate into the inner phase of liposomes containing HRP, resulting in the progress of 4-iodophenol-enhanced luminol CL catalyzed by HRP in liposomes. The CL intensity observed in liposomes was a factor of 150 greater than that observed in a lipid-free bulk solution. The detection limit in the direct detection of HRP encapsulated in liposomes was sensitive by a factor of 30 compared with that in a lipid-free bulk solution. 4-Iodophenol effectively functioned as an enhancer in HRP-catalyzed luminol CL in liposomes.

  12. The effect of chemical modification with pyromellitic anhydride on structure, function, and thermal stability of horseradish peroxidase.

    PubMed

    Hassani, Leila

    2012-06-01

    The stability of enzymes remains a critical issue in biotechnology. Compared with the strategies for obtaining stable enzymes, chemical modification is a simple and effective technique. In the present study, chemical modification of horseradish peroxidase (HRP) was carried out with pyromellitic anhydride. HRP has achieved a prominent position in the pharmaceutical, chemical, and biotechnological industries. In this study, the effect of chemical modification on thermal stability, structure, and function of the enzyme was studied by fluorescence, circular dichroism, and absorbance measurements. The results indicated a decrease in compactness of the structure and a considerable enhancement in thermal stability of HRP below 60 °C. It seems the charge replacement and introduction of the bulky group bring about the observed structural and the functional changes.

  13. Central projection of rat sciatic nerve fibres as revealed by Ricinus communis agglutinin and horseradish peroxidase tracers.

    PubMed

    Leong, S K; Tan, C K

    1987-10-01

    The central projection of afferent fibres in the rat sciatic nerve has been studied by means of the suicide transport of a lectin, Ricinus communis agglutinin 60 (RCA 60), and the transganglionic transport of horseradish peroxidase (HRP). The results obtained from these two methods are similar; however, the RCA method gave a more consistent and better localisation of the primary afferent terminals than the HRP method. The present study has shown that primary afferents from the sciatic nerve project predominantly to the ipsilateral gracile nucleus. In addition, they also project to several other brainstem nuclei; these include the contralateral nucleus gracilis, the ipsilateral main cuneate nucleus, the external cuneate nucleus and the presumptive nucleus z.

  14. Horseradish Peroxidase (HRP) Immobilized Poly(aniline-co-m-aminophenol) Film Electrodes–fabrication and Evaluation as Hydrogen Peroxide Sensor

    PubMed Central

    Seo, Kang-Deuk; Lee, Kwang-Pill; Gopalan, Anantha Iyengar; Chung, Sang J.; Lim, Yong Taik; Choi, Seong-Ho

    2007-01-01

    Enzyme modified electrodes were fabricated with poly (aniline-co-m-aminophenol). Electrochemical polymerization of aniline and m-aminophenol was performed to get the film of copolymer on the surface of gold electrode. Modified electrodes were fabricated by two methods, physical entrapment and covalent cross-linking. In one of the method, gold nanoparticles were loaded into the copolymer film and horseradish peroxidase (HRP) was immobilized into the Au nanoparticle loaded copolymer film through physical entrapment. In the other method, the amino and -OH groups in the copolymer are utilized to form covalent functionalization with HRP via glutaric dialdehyde as cross-linker/mediator. The conducting copolymer/enzyme modified electrodes prepared by physical entrapment/covalent functionalization of enzyme were tested for electrocatalytic activities towards sensing of H2O2. Amperometric results indicate that enzyme modified electrode via physical entrapment possesses better electrocatalytic performance over covalent functionalized enzyme electrode.

  15. [Oxidative destruction of estradiol after treatment with hydrogen peroxide catalyzed by horseradish peroxidase and methemoglobin].

    PubMed

    Petrenko, Iu M; Matiushin, A I; Titov, V Iu

    1999-01-01

    It is shown that estradiol in the presence of horse radish peroxidase interacts with hydrogen peroxide, which is evidenced by an increase in its optical density at 280 nm. The photometering of samples containing estradiol and horse radish peroxidase upon their titration with hydrogen peroxide indicated that the increase in optical density stops after introducing hydrogen peroxide equimolar in concentration to estradiol. The stoichiometric ratio of estradiol consumed during oxidative destruction to hydrogen peroxide was 1:1. In the presence of ascorbate, the oxidative destruction of estradiol by the action of hydrogen peroxide, catalyzed by horse radish peroxidase, was observed only after a latent period and showed the same regularities as in the absence of ascorbate. It was found by calorimetry that, during the latent period, estradiol catalyzes the degradation of hydrogen peroxide and ascorbate without undergoing oxidative destruction. The substrates of the peroxidase reaction benzidine, 1-naphthol, and phenol interact with hydrogen peroxide in the presence of ascorbate and horse radish peroxidase in a similar way. Presumably, upon interaction with hydrogen peroxide in the presence of horse radish peroxidase, estradiol, like other substrates of this reaction, undergoes oxidative destruction by the mechanism of peroxidase reaction. It is shown that oxidative destruction of estradiol by the action of hydrogen peroxide can also be catalyzed by methemoglobin by the same mechanism. These data are important for understanding the role of estradiol in the organism and the pathways of its metabolic conversions.

  16. 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

  17. Spectrometric assay for horseradish peroxidase activity based on the linkage of conjugated system formed by oxidative decarboxylation

    NASA Astrophysics Data System (ADS)

    Yamaguchi, Masaki; Sato, Shingo

    2017-03-01

    Horseradish peroxidase (HRP)-catalyzed oxidation of 2,2-bis[3-acethylfilicinic acid-5-yl]acetic acid (BAFA, 4) produces Dehydro-3,3'-diacetyl-5,5'-methylenedifilicinic acid (DDMF, 3). A new photometric hydrogen donor (4) for peroxidase (POD)-catalyzed oxidation was demonstrated to be potentially useful for spectrocolorimetric and spectrofluorometric determination of HRP. Our developed colorimetric (absorption at 483 nm) and fluorometric (emission at 529 nm) systems both gave a linear calibration curve for HRP (y = 0.0025 × + 0.0237; R2 = 0.9997, and y = 0.241 × + 3.194; R2 = 0.9914, respectively) in the same concentration range of 9.1 × 10- 8-1.1 × 10- 6 nmol/L. The calculated Km and Vmax values of 5.10 × 10- 4 M and 5.13 × 10- 6 M/min, respectively. The results indicate that the quantification of HRP using BAFA 4 as hydrogen donor is possible.

  18. Sensitive mRNA detection by fluorescence in situ hybridization using horseradish peroxidase-labeled oligodeoxynucleotides and tyramide signal amplification.

    PubMed

    van de Corput, M P; Dirks, R W; van Gijlswijk, R P; van Binnendijk, E; Hattinger, C M; de Paus, R A; Landegent, J E; Raap, A K

    1998-11-01

    With the ongoing progress in human genome projects, many genes are discovered whose function and/or expression pattern are not known. Most of these genes are expressed in relatively low abundance compared to housekeeping genes such as elongation factor-1alpha and beta-actin. Gene expression is studied by Northern blot assays or by semiquantitative PCR methods. Another method is the visualization of transcripts in tissue or cell cultures by fluorescence in situ hybridization (FISH). However, for low-abundance RNA detection, this method is hampered by its limited detection sensitivity and by the interference of background signals with specific hybridization signals. Background signals are introduced by nonspecific hybridization of probe sequences or nonspecific binding of antibodies used for visualization. To eliminate background signals derived from both sources and to benefit from the peroxidase-driven tyramide signal amplification (TSA), we directly conjugated horseradish peroxidase (HRP) to oligodeoxynucleotides (ODNs) and used these probes to study in the bladder cancer cell line 5637 the expression of various cytokine genes which, according to Northern hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) assays, are expressed at levels up to 10,000-fold less than abundantly expressed housekeeping genes. The results show that reduction of probe complexity and the limited use of immunocytochemical detection layers strongly reduces noise signals derived from nonspecific binding of nucleic acid probe and antibodies. The use of the HRP-ODNs in combination with TSA allowed detection of low-abundance cytokine mRNAs by FISH.

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

    PubMed Central

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

    2003-01-01

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

  20. Retrograde and transganglionic transport of horseradish peroxidase-conjugated cholera toxin B subunit, wheatgerm agglutinin and isolectin B4 from Griffonia simplicifolia I in primary afferent neurons innervating the rat urinary bladder.

    PubMed

    Wang, H F; Shortland, P; Park, M J; Grant, G

    1998-11-01

    In the present study, we investigated and compared the ability of the cholera toxin B subunit, wheat germ agglutinin and isolectin B4 from Griffonia simplicifolia I conjugated to horseradish peroxidase, to retrogradely and transganglionically label visceral primary afferents after unilateral injections into the rat urinary bladder wall. Horseradish peroxidase histochemical or lectin-immunofluorescence histochemical labelling of bladder afferents was seen in the L6-S1 spinal cord segments and in the T13-L2 and L6-S1 dorsal root ganglia. In the lumbosacral spinal cord, the most intense and extensive labelling of bladder afferents was seen when cholera toxin B subunit-horseradish peroxidase was injected. Cholera toxin B subunit-horseradish peroxidase-labelled fibres were found in Lissauer's tract, its lateral and medial collateral projections, and laminae I and IV-VI of the spinal gray matter. Labelled fibres were numerous in the lateral collateral projection and extended into the spinal parasympathetic nucleus. Labelling from both the lateral and medial projections extended into the dorsal grey commissural region. Wheat germ agglutinin-horseradish peroxidase labelling produced a similar pattern but was not as dense and extensive as that of cholera toxin B subunit-horseradish peroxidase. The isolectin B4 from Griffonia simplicifolia I-horseradish peroxidase-labelled fibres, on the other hand, were fewer and only observed in the lateral collateral projection and occasionally in lamina I. Cell profile counts showed that a larger number of dorsal root ganglion cells were labelled with cholera toxin B subunit-horseradish peroxidase than with wheat germ agglutinin- or isolectin B4-horseradish peroxidase. In the L6-S1 dorsal root ganglia, the majority (81%) of the cholera toxin B subunit-, and almost all of the wheat germ agglutinin- and isolectin B4-immunoreactive cells were RT97-negative (an anti-neurofilament antibody that labels dorsal root ganglion neurons with

  1. Transient-state and steady-state kinetics of the oxidation of aliphatic and aromatic thiols by horseradish peroxidase.

    PubMed

    Burner, U; Obinger, C

    1997-07-14

    In the course of oxidation of thiols by peroxidases thiyl radicals are formed which are known to undergo several free-radical conjugative reactions, among others leading to hydrogen peroxide formation. The present paper for the first time presents a comparative transient-state and steady-state investigation of the reaction of 15 aliphatic and aromatic mono- and dithiols with horseradish peroxidase (HRP). Both sequential-stopped-flow spectrophotometric investigations of the reaction of HRP intermediates Compound I (k2) and Compound II (k3) with thiols and measurements of the overall thiol oxidation and the simultaneous oxygen consumption in the presence and absence of exogenously added hydrogen peroxide (10 microM) have been performed. With HRP as thiyl radical generator it was shown that three groups of thiols have to be distinguished: (i) Aromatic thiols (e.g. thiophenol, 2-mercaptopurine) were excellent electron donors of both Compounds (k2: 10(4)-10(7) M(-1) s(-1) and k3: 10(3)-10(6) M(-1) s(-1)); however, the overall reaction was shown to depend on addition of hydrogen peroxide, indicating insufficient peroxide regeneration by arylthiyl radicals. (ii) Aliphatic thiols which were extremely bad substrates (k3 < 10 M(-1) s(-1)) for HRP (e.g. homocysteine, glutathione) and/or have a pK(a,SH) > 9.5 (e.g. N-acetylcysteine, alpha-lipoic acid) were also shown to depend on exogenously added H2O2 to maintain the peroxidasic reaction, whereas (iii) with those thiols with rates of k3 between 11 and 1600 M(-1) s(-1) (e.g. cysteine, cysteamine, cysteine methyl ester, cysteine ethyl ester) and/or with a pK(a,SH) < 8 (penicillamine) thiol oxidation was independent of exogenously added hydrogen peroxide, indicating sufficient hydrogen peroxide regeneration.

  2. 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

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

    PubMed

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

    2003-04-20

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

  4. How Modification of Accessible Lysines to Phenylalanine Modulates the Structural and Functional Properties of Horseradish Peroxidase: A Simulation Study

    PubMed Central

    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

  5. 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.

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

    NASA Astrophysics Data System (ADS)

    Zhang, Junli; Li, Baoxin

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

  7. 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.

  8. Direct electrochemistry of horseradish peroxidase immobilized on the layered calcium carbonate-gold nanoparticles inorganic hybrid composite.

    PubMed

    Li, Feng; Feng, Yan; Wang, Zhen; Yang, Limin; Zhuo, Linhai; Tang, Bo

    2010-06-15

    A mediator-free hydrogen peroxide (H(2)O(2)) biosensor was fabricated based on immobilization of horseradish peroxidase (HRP) on layered calcium carbonate-gold nanoparticles (CaCO(3)-AuNPs) inorganic hybrid composite. The proposed biosensor showed a strong electrocatalytic activity toward the reduction of H(2)O(2), which could be attributed to the favored orientation of HRP in the well-confined surface as well as the high electrical conductivity of the resulting CaCO(3)-AuNPs inorganic hybrid composite. The hybrid composite was obtained by the adsorption of AuNPs onto the surfaces of layered CaCO(3) through electrostatic interaction. The key analytical parameters relative to the biosensor performance such as pH and applied potential were optimized. The developed biosensor also exhibited a fast amperometric response (3s), a good linear response toward H(2)O(2) over a wide range of concentration from 5.0x10(-7) to 5.2x10(-3)M, and a low detection limit of 1.0x10(-7)M. The facile, inexpensive and reliable sensing platform based on layered CaCO(3)-AuNPs inorganic hybrid composite should hold a huge potential for the fabrication of more other biosensors.

  9. Effect of maternal obstructive cholestasis during pregnancy on the biliary transport of horseradish peroxidase in the rat offspring.

    PubMed

    Monte, Maria J; Villanueva, Gloria R; Macias, Rocio I R; Vazquez, David J; Toledo, Marta; Dominguez, Mercedes; Marin, Jose J G

    2003-09-01

    MOCP (maternal obstructive cholestasis during pregnancy) induces a reversible impairment in bile formation in young rats born to these mothers. The aim of the present study was to gain information on the effects of MOCP on the maturation of pathways involved in protein secretion into bile in young (4-week-old) rats. The amount of hepatic alpha-tubulin and the structure of the microtubular network were apparently not affected by MOCP. HRP (horseradish peroxidase) was used as a model protein, and its secretion into bile after administration through the jugular vein was measured. In adult (8-week-old) rats, two peaks of HRP output into bile were observed following administration: an early peak presumably due to paracellular transfer, and a late peak presumably due to transcytosis. In young rats (4 weeks old), the early peak was similar to that of adult animals, and was not affected by MOCP. However, the late peak was markedly smaller in young control rats, and was further reduced by MOCP. Brefeldin A decreased, whereas taurocholate did not change, the early peak, whereas both affected the transcytotic transport of HRP. Brefeldin A delayed HRP secretion (similarly in control and MOCP groups), without affecting cumulative output, whereas taurocholate accelerated the transcytotic transport of HRP in the control group, but not in the MOCP group. These results suggest that MOCP affects the maturation of hepatocyte mechanisms involved in the transcytotic secretion of HRP into bile.

  10. Horseradish peroxidase functionalized gold nanorods as a label for sensitive electrochemical detection of alpha-fetoprotein antigen.

    PubMed

    Guo, Jinjin; Han, Xiaowei; Wang, Junchun; Zhao, Junqing; Guo, Zilin; Zhang, Yuzhong

    2015-12-15

    In this study, a novel tracer, horseradish peroxidase (HRP) functionalized gold nanorods (Au NRs) nanocomposites (HRP-Au NRs), was designed to label the signal antibodies for sensitive electrochemical measurement of alpha-fetoprotein (AFP). The preparation of HRP-Au NRs nanocomposites and the labeling of secondary antibody (Ab2) were performed by one-pot assembly of HRP and Ab2 on the surface of Au NRs. The immunosensor was fabricated by assembling carbon nanotubes (CNTs), Au NRs, and capture antibodies (Ab1) on the glassy carbon electrode. In the presence of AFP antigen, the labels were captured on the surface of the Au NRs/CNTs via specific recognition of antigen-antibody, resulting in the signal intensity being clearly increased. Differential pulse voltammetry (DPV) was employed to record the response signal of the immunosensor in phosphate-buffered saline (PBS) containing hydrogen peroxide (H2O2) and 3,3',5,5'-tetramethylbenzidine (TMB). Under optimal conditions, the signal intensity was linearly related to the concentration of AFP in the range of 0.1-100 ng ml(-1), and the limit of detection was 30 pg ml(-1) (at signal/noise [S/N] = 3). Furthermore, the immunoassay method was evaluated using human serum samples, and the recovery obtained was within 99.0 and 102.7%, indicating that the immunosensor has potential clinical applications.

  11. Highly Chemiluminescent Graphene Oxide Hybrids Bifunctionalized by N-(Aminobutyl)-N-(Ethylisoluminol)/Horseradish Peroxidase and Sensitive Sensing of Hydrogen Peroxide.

    PubMed

    Liu, Xiaoying; Han, Zhili; Li, Fang; Gao, Lingfeng; Liang, Gaolin; Cui, Hua

    2015-08-26

    N-aminobutyl-N-ethylisoluminol and horseradish peroxidase bifunctionalized graphene oxide hybrids (ABEI-GO@HRP) were prepared through a facile and green strategy for the first time. The hybrids exhibited excellent chemiluminescence (CL) activity over a wide range of pH from 6.1 to 13.0 when reacted with H2O2, whereas ABEI functionalized GO had no CL emission at neutral pH and showed more than 2 orders of magnitude lower CL intensity than ABEI-GO@HRP at pH 13.0. Such strong CL emission from ABEI-GO@HRP was probably due to that HRP and GO facilitated the formation of O2(•-), - CO4(•2-), HO(•), and π-C═C(•) in the CL reaction, and GO as a reaction interface promoted the electron transfer of the radical-involved reaction. By virtue of ABEI-GO@HRP as a platform, an ultrasensitive, selective, and reagentless CL sensor was developed for H2O2 detection. The CL sensor exhibited a detection limit of 47 fM at physiological pH, which was more than 2 orders of magnitude lower than previously reported methods. This work reveals that bifunctionalization of GO by ABEI and HRP leads to excellent CL feature and enzyme selectivity, which can be used as an ideal platform for developing novel analytical methods.

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

    PubMed

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

    2014-10-13

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

  13. Comparative cytotoxicity and ROS generation by curcumin and tetrahydrocurcumin following visible-light irradiation or treatment with horseradish peroxidase.

    PubMed

    Atsumi, Toshiko; Tonosaki, Keiichi; Fujisawa, Seiichiro

    2007-01-01

    In order to clarify the cytotoxic mechanism of curcumin, a well-known chemopreventive agent, the cytotoxicity (by MTT method), intracellular glutathione (using GSH detection kit) and intracellular reactive oxygen species (ROS) levels (with a flow cytometer), were measured in curcumin- and tetrahydrocurcumin (TH-curcumin)-treated cancer (HSG) and normal (HGF) cells under two different oxidation conditions: irradiation with visible light (VL) and enzymatic oxidation with horseradish peroxidase (HRP)/H2O2. The cytotoxicity of curcumin was highly enhanced by VL-irradiation, whereas that of TH-curcumin was enhanced by HRP/H2O2 treatment. The cytotoxicity of curcumin against HGF cells was greater than that against HSG cells. Curcumin significantly reduced the intracellular GSH level significantly under VL-irradiation, and increased it under HRP/H2O2, whereas TH-curcumin had no effect with either oxidation treatment. HRP/H2O2 treatment of TH-curcumin enhanced generation of ROS; in contrast, VL-irradiation of curcumin was considered to produce ROS preferably. In conclusion, curcumin was highly photo-toxic, caused a decrease in GSH and mediated ROS generation. In contrast, the cytotoxicity of TH-curcumin was enhanced by enzymatic oxidation. A low-level pro-oxidant intracellular milieu induced by TH-curcumin could be effectively useful for cancer prevention.

  14. 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.

  15. Combined cross-linked enzyme aggregates of horseradish peroxidase and glucose oxidase for catalyzing cascade chemical reactions.

    PubMed

    Nguyen, Le Truc; Yang, Kun-Lin

    2017-05-01

    Cascade reactions involved unstable intermediates are often encountered in biological systems. In this study, we developed combined cross-linked enzyme aggregates (combi-CLEA) to catalyze a cascade reaction which involves unstable hydrogen peroxide as an intermediate. The combi-CLEA contains two enzymes̶ glucose oxidase (GOx) and horseradish peroxidase (HRP) which are cross-linked together as solid aggregates. The first enzyme GOx catalyzes the oxidation of glucose and produces hydrogen peroxide, which is used by the second enzyme HRP to oxidize 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). The apparent reaction rate of the cascade reaction reaches 10.5±0.5μM/min when the enzyme ratio is 150:1 (GOx:HRP). Interestingly, even in the presence of catalase, an enzyme that quickly decomposes hydrogen peroxide, the reaction rate only decreases by 18.7% to 8.3±0.3μM/min. This result suggests that the intermediate hydrogen peroxide is not decomposed by catalase due to a short diffusion distance between GOx and HRP in the combi-CLEA. Scanning electron microscopy images suggest that combi-CLEA particles are hollow spheres and have an average diameter around 250nm. Because of their size, combi-CLEA particles can be entrapped inside a nylon membrane for detecting glucose by using the cascade reaction.

  16. 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.

  17. Comparative studies with penicillinase, horseradish peroxidase, and alkaline phosphatase as enzyme labels in developing enzyme immunoassay of cortisol.

    PubMed

    Kumari, G Lakshmi; Dhir, Ravindra N

    2003-01-01

    Relative merit of different enzyme labels for measuring cortisol directly in serum by competitive enzyme immunoassay (ELISA) was examined. Cortisol-21-hemisuccinate was labeled separately with penicillinase, horseradish peroxidase (HRP), and alkaline phosphatase (ALP) under identical reaction conditions. Antibody developed in rabbits against cortisol-3-0-(carboxymethyl)-oxime-bovine serum albumin was used to coat polystyrene tubes that were precoated with anti-rabbit gamma globulin (ARGG). Cortisol standards were prepared in steroid-free human serum in buffer (1:4) contaning 8-anilino-1-naphthalene sulfonic acid (8-ANS). Assay buffer also consisted 8-ANS. The assay involved adding standard cortisol or serum sample to antibody-coated tubes, followed by addition of enzyme label and buffer, and incubation for 2 h at 37 degrees C. The whole procedure took 3 h for completion. All three labels proved to be sensitive, with a slope around -2.0. Although penicillinase as an enzyme label was highly sensitive and stable compared with others, the assays were not always accurate and precise, especially at low concentrations of cortisol. This was mainly due to the color reagent used for measuring penicillinase activity. Serum samples that underwent 2-3 freeze-thaw cycles gave high values with HRP label compared with ALP. Therefore, utilizing ALP as an enzyme label, an ELISA was developed and its performance was comparable with some of the commercial kits already in the market.

  18. 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.

  19. A new phosphothreonine lyase electrochemical immunosensor for detecting Salmonella based on horseradish peroxidase/GNPs-thionine/chitosan.

    PubMed

    Lu, Dingqiang; Pang, Guangchang; Xie, Junbo

    2017-03-01

    In the current study, a novel double-layer gold nanoparticles- electrochemical immunosensor electrode (DGN-EIE) immobilized with Salmonella plasmid virulence C (SpvC) antibody 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, the SpvC monoclonal antibodies (derived from Balb/c mice) were prepared and screened with a high affinity to SpvC. To evaluate the quality of DGN-EIE, the amperometric I-t curve method was applied to determine Salmonella in PBS. The results showed that the response current had a good linear correlation with the bacterial quantity ranged from 1.0 × 10(1)-5.0 × 10(4) cfu/mL. The lowest detection limit was found at 5 cfu/mL. Furthermore, the proposed immunosensor has been demonstrated with high sensitivity, good selectivity and reproducibility. Apparently, DGN-EIE may be a very useful tool for monitoring the bacteria.

  20. 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.

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

    PubMed Central

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

    2016-01-01

    Intercellular protein-protein interactions (PPIs) enable communication between cells in diverse biological processes, including cell proliferation, immune responses, infection and synaptic transmission, but they are challenging to visualize because existing techniques1,2,3 have insufficient sensitivity and/or specificity. Here we report split horseradish peroxidase (sHRP) as a sensitive and specific tool for detection of intercellular PPIs. The two sHRP fragments, engineered through screening of 17 cut sites in HRP followed by directed evolution, reconstitute into an active form when driven together by an intercellular PPI, producing bright fluorescence or contrast for electron microscopy. Fusing the sHRP fragments to the proteins neurexin (NRX) and neuroligin (NLG), which bind each other across the synaptic cleft4, enabled sensitive visualization of synapses between specific sets of neurons, including two classes of synapses in the mouse visual system. sHRP should be widely applicable for studying mechanisms of communication between a variety of cell types. PMID:27240195

  2. Haem propionates control oxidative and reductive activities of horseradish peroxidase by maintaining the correct orientation of the haem.

    PubMed Central

    Adak, S; Banerjee, R K

    1998-01-01

    The role of haem propionates in oxidative and reductive reactions catalysed by horseradish peroxidase (HRP) was studied after successful reconstitution of ferric protoporphyrin IX dimethyl ester (PPDME) into the apoperoxidase. The reconstituted enzyme oxidizes neither guaiacol (aromatic electron donor) nor iodide or thiocyanate (inorganic donor). Although the reconstituted enzyme binds guaiacol with a similar Kd (13 mM) to that of the native enzyme (10 mM), the Kd for SCN- binding (5 mM) is decreased 20-fold compared with that of the native enzyme (100 mM). This indicates that haem propionates hinder the entry or binding of inorganic anion to the active site of the native HRP. However, the reconstituted enzyme is catalytically inactive as it does not form spectroscopically detectable compound II with H2O2. CD measurements indicate a significant loss of haem CD spectrum of the reconstituted enzyme at 409 nm, suggesting a loss of asymmetry of the haem-protein interaction. Thus the inability of the reconstituted enzyme to form catalytic intermediates results from the change in orientation of the haem due to loss of interactions via the haem propionates. HRP also catalyses reductive reactions such as reduction of iodine (I+) in the presence of EDTA and H2O2. The reconstituted enzyme cannot catalyse I+ reduction because of the loss of I+ binding to the haem propionate. Since I+ reduction requires formation of the catalytically active enzyme-I+-EDTA ternary complex, the loss of reductive activity is primarily due to the loss of active enzyme formation. Haem propionates thus play a vital role in the oxidative and reductive reactions of HRP by favouring the formation of catalytic intermediates with H2O2 by maintaining the correct orientation of the haem with respect to the surrounding residues. PMID:9693101

  3. 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

  4. Development of horseradish peroxidase-based cross-linked enzyme aggregates and their environmental exploitation for bioremediation purposes.

    PubMed

    Bilal, Muhammad; Iqbal, Hafiz M N; Hu, Hongbo; Wang, Wei; Zhang, Xuehong

    2017-03-01

    In the present study, horseradish peroxidase (HRP), in-house isolated crude cocktail enzyme, from Armoracia rusticana was cross-linked using a new type of cross-linking agent, i.e., ethylene glycol-bis [succinic acid N-hydroxysuccinimide, (EG-NHS)], which is mild in nature as compared to the glutaraldehyde (GA). The HRP-immobilized cross-linked enzyme aggregates (HRP-CLEAs) were developed using a wider range of EG-NHS and notably no adverse effect was observed. In a comparative evaluation, in the case of EG-NHS, a high-level stability in the residual activity was recorded, whereas a sharp decrease was observed in the case of glutaraldehyde. Following initial cross-linker evaluation, the HRP-CLEAs were tested to investigate their bio-catalytic efficacy for bioremediation purposes using a newly developed packed bed reactor system (PBRS). A maximal of 94.26% degradation of textile-based methyl orange dye was recorded within the shortest time frame, following 91.73% degradation of basic red 9, 84.35% degradation of indigo, 81.47% degradation of Rhodamin B, and 73.6% degradation of Rhodamine 6G, respectively, under the same working environment. Notably, the HRP-CLEAs retained almost 60% of its original activity after methyl orange dye degradation in seven consecutive cycles using PBRS. Furthermore, after HRP-CLEAs-mediated treatment in the PBRS, a significant toxicity reduction in the dye samples was recorded as compared to their pristine counterparts. In conclusion, the results suggest that the newly developed HRP-CLEAs have a great potential for industrial exploitation, to tackle numerous industrial dye-based emergent pollutants.

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

    PubMed

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

    1991-05-15

    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.

  6. 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.

  7. 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.

  8. 3,3',5,5' - Tetramethylbenzidine as an Ames test negative chromogen for horse-radish peroxidase in enzyme-immunoassay.

    PubMed

    Bos, E S; van der Doelen, A A; van Rooy, N; Schuurs, A H

    1981-01-01

    The use of 3,3',5,5' - tetramethylbenzidine as non-mutagenic chromogen for the end point determination in enzyme-immunoassay (EIA) is described. In sandwich EIAs for HCG and HBsAg and in a competitive EIA for testosterone, the colour yield with TMB was superior to that obtained with o-phenylene diamine (OPD), which was by far the best chromogen for horse-radish peroxidase until now. This led to an improvement of sensitivity and precision of the assays and makes EIA even more competitive with other types of immunoassays.

  9. 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.

  10. The permeability alteration of brain and spinal cord vasculature to horseradish peroxidase during experimental decompression sickness as compared to the alteration in permeability induced by hyperosmolar solution.

    PubMed

    Lehtosalo, J; Panula, P; Laitinen, L A

    1982-01-01

    The permeability of microvasculature in the cerebral cortex, neostriatum, and spinal cord to i.v. injected horseradish peroxidase (HRP) has been investigated in rats following experimental compression to 6.1 bars (abs.) air for 90 min, and subsequent decompression to the ambient pressure in 1 min. For comparison, 1 ml of 2.0 M urea was injected into the right common carotid artery of rats during 15 s. After exposure to compression-decompression, under the light microscope focal leaky areas were found in all the regions examined. The leakage was most prominent in the grey matter of the spinal cord, and the cerebral cortex. In decompressed rats, arterioles were most often the site of peroxidase extravasation, whereas extravasation of HRP was less frequently displayed by capillaries and venules. In urea-treated rats, capillaries and venules frequently displayed extravasation of HRP as well. Parenchymal cells accumulated the trace adjacent to the leaky areas. Under the electron microscope, the extravasation of HRP was associated with peroxidase-containing pleomorphic vesicular structures in the endothelium, both in decompressed and urea-injected rats. Moreover, in contrast to decompressed rats, the junctions between endothelial cells were penetrated by the trace in urea-treated rats. Accordingly, the results indicate that during decompression sickness the pathway for the extravasation of proteins is through vesicular transfer, whereas the injection of hyperosmolar urea induces extravasation, both through vesicular transfer and junctions between the endothelial cells.

  11. An amperometric biosensor based on the coimmobilization of horseradish peroxidase and methylene blue on a beta-type zeolite modified electrode.

    PubMed

    Liu, B; Liu, Z; Chen, D; Kong, J; Deng, J

    2000-07-01

    A new biosensor for the amperometric detection of hydrogen peroxide was developed based on the coimmobilization of horseradish peroxidase (HRP) and methylene blue on a beta-type zeolite modified glassy carbon electrode without the commonly used bovine serum albumin-glutaraldehyde. The intermolecular interaction between enzyme and zeolite matrix was investigated using FT-IR. The cyclic voltammetry and amperometric measurement demonstrated that methylene blue co-immobilized with HRP in this way displayed good stability and could efficiently transfer electrons between immobilized HRP and the electrode. The sensor responded rapidly to H2O2 in the linear range from 2.5 x 10(-6) to 4.0 x 10(-3) M with a detection limit of 0.3 microM. The sensor was stable in continuous operation.

  12. Hofmeister effects in biology: effect of choline addition on the salt-induced super activity of horseradish peroxidase and its implication for salt resistance of plants.

    PubMed

    Pinna, M C; Bauduin, P; Touraud, D; Monduzzi, M; Ninham, B W; Kunz, W

    2005-09-01

    The effect of choline addition on the salt-induced super activity of horseradish peroxidase (HRP) is investigated. HRP is presented in the literature as an efficient H(2)O(2) scavenger, and choline is the precursor of glycine betaine, a strong osmoprotectant molecule. Both the regulations of H(2)O(2) and of osmoprotectant concentrations are implicated in plants in order to counteract salt-induced cell damage. For the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), sulfate anions were found to play a crucial role in the increase of HRP activity. This induced super activity can be strongly reduced by adding choline chloride. The phenomena provide an example of physicochemical Hofmeister effects playing a central regulatory role in an important biological system.

  13. Electron paramagnetic resonance and ultraviolet/visible study of compounds I and II in the horseradish peroxidase-H 2O 2-silk fiber reaction system

    NASA Astrophysics Data System (ADS)

    Oliva, C.; Freddi, G.; Repetto, S.; D'Ambrosio, A.

    2003-06-01

    The enzymatic oxidation of silk with H2O2 in the presence of horseradish peroxidase (HRP) has been investigated. Two intermediate complexes have been observed during this reaction. Both can be attributed to Fe4+ ions axially bonded to an oxygen atom and to a porphyrin radical (Prad ). In the most unstable of them, indicated as compound II, the chemical bond between [FeIVO]2+ and Prad was weaker than in the other, indicated as compound I. The former compound disappeared within 1 h of the reaction, at difference with the latter, traces of which were observed even after 3 weeks with dried samples. However, the chemical bond between [FeIVO]2+ and Prad in compound I weakened during the sample ageing. All these phenomena have been enlightened by electron paramagnetic resonance (EPR) and spectrophotometric ultraviolet/visible (UV/Vis) measurements.

  14. 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.

  15. 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.

  16. Selective decrease of small sensory neurons in lumbar dorsal root ganglia labeled with horseradish peroxidase after ND:YAG laser irradiation of the tibial nerve in the rat

    SciTech Connect

    Wesselmann, U.; Lin, S.F.; Rymer, W.Z. )

    1991-02-01

    Recent electrophysiological evidence indicates that Q-switched Nd:YAG laser irradiation might have selective effects on neural impulse transmission in small slow conducting sensory nerve fibers as compared to large diameter afferents. In an attempt to clarify the ultimate fate of sensory neurons after laser application to their peripheral axons, we have used horseradish peroxidase (HRP) as a cell marker to retrogradely label sensory neurons innervating the distal hindlimb in the rat. Pulsed Nd:YAG laser light was applied to the tibial nerve at pulse energies of 70 or 80 mJ/pulse for 5 min in experimental rats. Seven days later HRP was applied to the left (laser-treated) and to the contralateral (untreated) tibial nerve proximal to the site of laser irradiation. In control animals the numbers of HRP-labeled dorsal root ganglion cells were not significantly different between the right and the left side. In contrast, after previous laser irradiation labeling was always less on the laser-treated side (2183 +/- 513 cells, mean +/- SEM) as compared to the untreated side (3937 +/- 225). Analysis of the dimensions of labeled cells suggested that the reduction of labeled cells on the laser-treated side was mainly due to a deficit in small sensory neurons. Since the conduction velocity of nerve fibers is related to the size of their somata, our histological data imply that laser light selectively affects retrograde transport mechanisms for HRP in slow conducting sensory nerve fibers.

  17. Spinal projections from the lower brain stem in the cat as demonstrated by the horseradish peroxidase technique. II. Projections from the dorsolateral pontine tegmentum and raphe nuclei.

    PubMed

    Tohyama, M; Sakai, K; Touret, M; Salvert, D; Jouvet, M

    1979-11-02

    The descending projections to the spinal cord arising from the dorsolateral pontine tegmentum and brain stem raphe nuclei have been investigated by means of the horseradish peroxidase (HRP) technique. Particular attention was taken to clarify the cells of origin and the funicular trajectory of these spinal projections. After injections of HRP into the spinal cord, a significant of HRP labeled neurons were observed in the following dorsolateral pontine tegmental structures: (1) an area ventral to the nucleus cuneiformis; (2) principal locus coeruleus; (3) locus coeruleus a; (4) locuse subcoeruleus; (5) Kölliker-Fuse nucleus; and (6) nucleus parabrachialis lateralis. As a rule, the projections are ipsilateral and descendaphe-spinal projections, we have demonstrated that the nucleus raphe dorsalis also sends axons to the cervical segment of the spinal cord. Furthermore, in accord with previous reports, HRP labeled cells were also identified in the nucleus raphe magnus, pallidus and obscurus, but not in the nucleus raphe centralis superior and pontis. On the whole the present study further clarified the organization of spinal projections from the dorsolateral pons and raphe nuclei and provided some additional anatomical data for the physiology of the tegmentospinal and raphe-spinal projections.

  18. Horseradish peroxidase-labeled silver/reduced graphene oxide thin film-modified screen-printed electrode for detection of carcinoembryonic antigen.

    PubMed

    Lee, S X; Lim, H N; Ibrahim, I; Jamil, A; Pandikumar, A; Huang, N M

    2017-03-15

    In this study, a disposable and simple electrochemical immunosensor was fabricated for the detection of carcinoembryonic antigen. In this method, silver nanoparticles (AgNPs) were mixed with reduced graphene oxide (rGO) to modify the surface of screen-printed carbon electrode (SPE). Initially, AgNPs-rGO modified-SPEs were fabricated by using simple electrochemical deposition method. Then the carcinoembryonic antigen (CEA) was immobilized between the primary antibody and horseradish peroxidase (HRP)-conjugated secondary antibody onto AgNPs-rGO modified-SPEs to fabricate a sandwich-type electrochemical immunosensor. The proposed method could detect the CEA with a linear range of 0.05-0.50µgmL(-1) and a detection limit down to 0.035µgmL(-1) as compared to its non-sandwich counterpart, which yielded a linear range of 0.05-0.40µgmL(-1), with a detection limit of 0.042µgmL(-1). The immunosensor showed good performance in the detection of carcinoembryonic antigen, exhibiting a simple, rapid and low-cost. The immunosensor showed a higher sensitivity than an enzymeless sensor.

  19. A morphometric study of the endocytosis of wheat germ agglutinin-horseradish peroxidase conjugates by retinal ganglion cells in the rat.

    PubMed

    Trojanowski, J Q; Gonatas, N K

    1983-08-08

    In order to elucidate the sequence for the intraneuronal translocation of ligands after internalization in vivo, the adsorptive endocytosis of horseradish peroxidase (HRP) conjugates of the lectin wheat germ agglutinin (WGHRP) by retinal ganglion cells of the rat was studied by ultrastructural morphometry after intravitreal injections of this probe. Retinas were harvested at post-injection survival times of 15 min to 7 days and processed for the electron microscopic visualization of WGHRP in subcellular organelles. The labeled organelles included vesicles, tubules, lysosomes and the cisterns and coated as well as uncoated vesicles of GERL (Golgi Apparatus-Endoplasmic-Reticulum-Lysosomes). For quantitation, labeled organelles were classed as vesicles, lysosomes and GERL. From 15 min to 3 h the number of labeled GERL and vesicles progressively increased to a maximum at 3 h and then declined to zero by 7 days. In contrast, the number of labeled lysosomes continued to increase beyond 3 h to reach a maximum at 24 h before declining to near zero by 7 days. These results are consistent with the hypothesis that the adsorptive endocytosis of WGHRP entails the passage of the ligand through GERL prior to being deposited in lysosomes. They do not exclude the possibility that other endocytic pathways for WGHRP and possible WGHRP-membrane complexes may exist in retinal ganglion cells including a plasma membrane to lysosome route.

  20. 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.

  1. Highly sensitive photoelectrochemical immunoassay with enhanced amplification using horseradish peroxidase induced biocatalytic precipitation on a CdS quantum dots multilayer electrode.

    PubMed

    Zhao, Wei-Wei; Ma, Zheng-Yuan; Yu, Pei-Pei; Dong, Xiao-Ya; Xu, Jing-Juan; Chen, Hong-Yuan

    2012-01-17

    Herein we demonstrate the protocol of a biocatalytic precipitation (BCP)-based sandwich photoelectrochemical (PEC) horseradish peroxidase (HRP)-linked immunoassay on the basis of their synergy effect for the ultrasensitive detection of mouse IgG (antigen, Ag) as a model protein. The hybrid film consisting of oppositely charged polyelectrolytes and CdS quantum dots (QDs) is developed by the classic layer by layer (LbL) method and then employed as the photoactive antibody (Ab) immobilization matrix for the subsequent sandwich-type Ab-Ag affinity interactions. Improved sensitivity is achieved through using the bioconjugates of HRP-secondary antibodies (Ab(2)). In addition to the much enhanced steric hindrance compared with the original one, the presence of HRP would further stimulate the BCP onto the electrode surface for signal amplification, concomitant to a competitive nonproductive absorption that lowers the photocurrent intensity. As a result of the multisignal amplification in this HRP catalyzed BCP-based PEC immunoassay, it possesses excellent analytical performance. The antigen could be detected from 0.5 pg/mL to 5.0 ng/mL with a detection limit of 0.5 pg/mL.

  2. 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.

  3. Labelling of neurons in the rat superior cervical ganglion after injection of wheat-germ agglutinin-horseradish peroxidase into the contralateral ganglion: evidence of transneuronal labelling.

    PubMed Central

    Atasever, A; Palaoğlu, S; Erbengi, A; Celik, H H

    1994-01-01

    Recent studies have shown that injection of the tracer wheat-germ agglutinin-horseradish peroxidase (WGA-HRP) into the superior cervical ganglion (SCG) of one side results in labelling of neurons in the contralateral SCG and the stellate ganglion. This study was designed to verify whether or not bilateral projections from the superior cervical ganglion to the midline structures, particularly to the pineal gland, play a role in the transport of WGA-HRP to the contralateral SCG. One group of rats received WGA-HRP injection into the right SCG (group I). Four groups of rats underwent the following operations prior to the injection of WGA-HRP into the right superior cervical ganglion: transection of the external carotid nerve (group II), transection of the internal carotid nerve (group III), transection of the external carotid nerve combined with pinealectomy (group IV), transection of both the internal and the external carotid nerves (group V). The mean number of labelled neurons in the left SCG of each group were found as follows: group I, 1516 +/- 221 (mean +/- S.D.); group II, 861 +/- 122; group III, 543 +/- 99; group IV, 562 +/- 144; group V, 220 +/- 52. The results of this study suggest that the contralateral labelling depends on the transneuronal transport of WGA-HRP through the terminal fields of innervation of the midline structures that receive bilateral projections from both SCGs. Images Fig. 1 Fig. 2 PMID:7512544

  4. Immobilization of horseradish peroxidase on ZnO nanowires/macroporous SiO2 composites for the complete decolorization of anthraquinone dyes.

    PubMed

    Sun, Huaiyan; Jin, Xinyu; Jiang, Feng; Zhang, Ruifeng

    2017-02-21

    A Zinc Oxide (ZnO) nanowires/ macroporous Silicon dioxide (SiO2 ) composite was used as support to immobilize horseradish peroxidase (HRP) simply by in-situ cross-linking method. As cross-linker was adsorbed on the surface of ZnO nanowires, the cross-linked HRP was quite different from the traditional cross-linking enzyme aggregates (CLEAs) on both structure and catalytic performance. Among three epoxy compounds diethylene glycol diglycidyl ether (DDE) was the best cross-linker, with which the loading amount of HRP with pI of 5.3 reached as high as 118.1 mg/g and specific activity was up to 14.9 U/mg-support. The mass-loss of HRP cross-linked with DDE was negligible during 50 h leaching at 4 °C, and the thermal stability of the immobilized HRP was also quite good. The catalytic performance of immobilized HRP to decolorize anthraquinone dye was explored by using Reactive Blue 19 (RB 19) and Acid Violet 109 (AV 109) as model substrates. The results indicated that the immobilized HRP exhibited high decolorization efficiency and good reusability. The decolorization efficiency reached 94.3% and 95.9% for AV 109 and RB 19 within the first 30 min, respectively. A complete decolorization of these two dyes has been realized within 2∼3 hours by using this new biocatalysis system. This article is protected by copyright. All rights reserved.

  5. Carbon Nanotubes Labeled with Aptamer and Horseradish Peroxidase as a Probe for Highly Sensitive Protein Biosensing by Postelectropolymerization of Insoluble Precipitates on Electrodes.

    PubMed

    Li, Jing; Wang, Jingjing; Guo, Xiang; Zheng, Qiong; Peng, Jing; Tang, Hao; Yao, Shouzhuo

    2015-08-04

    Carbon nanotubes (CNTs) labeled with aptamer and horseradish peroxidase (HRP) were used as a probe to amplify the impedimetric sensing of the aptamer-protein (with thrombin as the model) interaction. The HRP-biocatalyzed oxidation of 3,3-diaminobenzidine (DAB) in the presence of H2O2 and the postelectropolymerization of insoluble precipitates produced on the electrode supports were used as a signal amplification route for the sensing process. Thrombin was sensed by aptamer 1 immobilized on a glassy carbon electrode. The multiwalled CNT-aptamer 2-HRP probe was linked to the aptamer 1-thrombin complex through the thrombin-aptamer 2 interaction. The postelectropolymerization of biocatalyzed precipitates of DAB on the electrode greatly increased the electron-transfer resistance at the electrode-solution interface. Cyclic voltammetry and electrochemical impedance spectroscopy were employed to follow the stepwise fabrication of the aptasensor and impedimetric detection of thrombin. Thrombin concentration as low as 0.05 pM could be detected by this method. In addition, the proposed impedimetric aptasensor exhibits good sensitivity (5195 Ω decade(-1)), selectivity, and reproducibility. The aptasensor also has acceptable recovery for thrombin detection in complex protein sample.

  6. 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.

  7. Design and construction of novel molecular conjugates for signal amplification (I): conjugation of multiple horseradish peroxidase molecules to immunoglobulin via primary amines on lysine peptide chains.

    PubMed

    Dhawan, Subhash

    2002-12-01

    Immunoconjugates are widely used for indirect detection of analytes (such as antibodies or antigens) in a variety of immunoassays. However, the availability of functional groups such as primary amines or free sulfhydryls in an immunoglobulin molecule is the limiting factor for optimal conjugation and, therefore, determines the sensitivity of an assay. In the present study, an N-terminal bromoacetylated 20 amino acid peptide containing 20 lysine residues was conjugated to N-succinimidyl-S-acetylthioacetate (SATA)-modified IgG or free sulfhydryl groups on 2-mercaptoethylamine (2-MEA)-reduced IgG molecules via a thioether (S[bond]CH(2)CONH) linkage to introduce multiple reactive primary amines per IgG. These primary amines were then covalently coupled with maleimide-activated horseradish peroxidase (HRP). The poly-HRP-antibody conjugates thus generated demonstrated greater than 15-fold signal amplification upon reaction with orthophenyldiamine substrate. The poly-HRP-antibody conjugates efficiently detected human immunodeficiency virus (HIV)-1 antibodies in plasma specimens with significantly higher sensitivity than conventionally prepared HRP-antibody conjugates in an HIV-1 solid-phase enzyme immunoassay and Western blot analysis. The signal amplification techniques reported here could have the potential for development of highly sensitive immunodiagnostic assay systems.

  8. Removal of estrogenic activity of natural and synthetic hormones from a municipal wastewater: efficiency of horseradish peroxidase and laccase from Trametes versicolor.

    PubMed

    Auriol, Muriel; Filali-Meknassi, Youssef; Adams, Craig D; Tyagi, Rajeshwar D; Noguerol, Tania-Noelia; Piña, Benjamin

    2008-01-01

    Some researches studied the removal of steroid estrogens by enzymatic treatment, however none verified the residual estrogenicity after the enzymatic treatment at environmental conditions. In this study, the residual estrogenic activities of the key natural and synthetic steroid estrogens were investigated following enzymatic treatment with horseradish peroxidase (HRP) and laccase from Trametes versicolor. Synthetic water and municipal wastewater containing environmental concentrations of estrone, 17beta-estradiol, estriol, and 17alpha-ethinylestradiol were treated. Liquid chromatography-mass spectrometry analysis demonstrated that the studied steroid estrogens were completely oxidized in the wastewater reaction mixture after a 1-h treatment with either HRP (8-10 U ml(-1)) or laccase (20 U ml(-1)). Using the recombinant yeast assay, it was also confirmed that both enzymatic treatments were very efficient in removing the estrogenic activity of the studied steroid estrogens. The laccase-catalyzed process seemed to present great advantages over the HRP-catalyzed system for up-scale applications for the treatment of municipal wastewater.

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

    PubMed

    Chang, Qing; Tang, Heqing

    2014-09-29

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

  10. 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

  11. Effect of the structure of imidazolium cations in [BF4](-)-type ionic liquids on direct electrochemistry and electrocatalysis of horseradish peroxidase in Nafion films.

    PubMed

    Lu, Lu; Huang, Xirong; Qu, Yinbo

    2011-10-01

    The direct electrochemistry and bioelectrocatalysis of horseradish peroxidase (HRP) in Nafion films at glassy carbon electrode (GCE) was investigated in three [BF(4)](-)-type room-temperature ionic liquids (ILs) to understand the structural effect of imidazolium cations. The three ILs are 1-ethyl-3-methylimidazolium tetrafluoroborate ([Emim][BF(4)]), 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF(4)]) and 1-hexyl-3-methylimidazolium tetrafluoroborate ([Hmim][BF(4)]). A small amount of water in the three ILs is indispensable for maintaining the electrochemical activity of HRP in Nafion films, and the optimum water contents decrease with the increase of alkyl chain length on imidazole ring. Analysis shows that the optimum water contents are primarily determined by the hydrophilicity of ILs used. In contrast to aqueous medium, ILs media facilitate the direct electron transfer of HRP, and the electrochemical parameters obtained in different ILs are obviously related to the nature of ILs. The direct electron transfer between HRP and GCE is a surface-confined quasi-reversible single electron transfer process. The apparent heterogeneous electron transfer rate constant decreases gradually with the increase of alkyl chain length on imidazole ring, but the changing extent is relatively small. The electrocatalytic reduction current of H(2)O(2) at the present electrode decreases obviously with the increase of alkyl chain length, and the mass transfer of H(2)O(2) via diffusion in ILs should be responsible for the change. In addition, the modified electrode has good stability and reproducibility; the ability to tolerate high levels of F(-) has been greatly enhanced due to the use of Nafion film. When an appropriate mediator is included in the sensing layer, a sensitive nonaqueous biosensor could be fabricated.

  12. New features of site-specific horseradish peroxidase (HRP) glycosylation uncovered by nano-LC-MS with repeated ion-isolation/fragmentation cycles.

    PubMed

    Wuhrer, Manfred; Balog, Crina I A; Koeleman, Carolien A M; Deelder, André M; Hokke, Cornelis H

    2005-05-25

    Horseradish peroxidase (HRP) is widely used in biomedical research as a reporter enzyme in diagnostic assays. In addition, it is of considerable interest as a model glycoprotein with core-xylosylated and -(alpha1-3)-fucosylated N-glycans that form antigenic elements of plant allergens and parasitic helminths. Using a combination of techniques comprising (1) nano-liquid chromatography (LC)-mass spectrometry (MS)/MS with multiple selection/fragmentation cycles of HRP tryptic (glyco-)peptides, (2) nano-electrospray MS of intact HRP, and (3) carbohydrate linkage analysis, it was revealed that most of the HRP N-glycosylation sites can be occupied with an alternative Fuc(1-3)GlcNAc-disaccharide. Two main variants of HRP occur: The major population (approximately 60%) has eight glycosylation sites carrying core(1-3)fucosylated, xylosylated, trimannosyl N-glycans, with the ninth potential N-glycosylation site Asn316 not occupied. Another group of HRP carries seven of the above-mentioned N-glycans, with an eighth N-glycosylation site carrying the alternative Fuc(1-3)GlcNAc-unit (approximately 35%). In addition, minor subsets of HRP were found to contain a xylosylated, trimannosyl N-glycan lacking core-fucosylation as a ninth N-glycan attached to Asn316, which has hitherto been assumed to be unoccupied. The finding of these new features of glycosylation of an already exceptionally well-studied glycoprotein underscores the potential of the nano-LC-MS(n) based analytical approach followed.

  13. Evaluation of lophine derivatives as L-012 (luminol analog)-dependent chemiluminescence enhancers for measuring horseradish peroxidase and H2O2.

    PubMed

    Ichibangase, T; Ohba, Y; Kishikawa, N; Nakashima, K; Kuroda, N

    2014-03-01

    8-Amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione (L-012) was recently synthesized as a new chemiluminescence (CL) probe; the light intensity and the sensitivity of L-012 are higher than those of other CL probes such as luminol. Previously, our group developed four lophine-based CL enhancers of the horseradish peroxidase (HRP)-catalyzed CL oxidation of luminol, namely 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI), 2-(4-hydroxyphenyl)-4,5-di(2-pyridyl)imidazole (HPI), 4-(4,5-diphenyl-1H-imidazol-2-yl)phenylboronic acid (DPA), and 4-[4,5-di(2-pyridyl)-1H-imidazol-2-yl]phenylboronic acid (DPPA), and showed that DPPA was suitable for the photographic detection of HRP. In this study, we replaced luminol with L-012 and evaluated these as L-012-dependent CL enhancers. In addition, to detect HRP and/or H2O2 with higher sensitivity, each detection condition for the L-012-HRP-H2O2 enhanced CL was optimized. All the derivatives enhanced the L-012-dependent CL as well as luminol CL; HPI generated the highest enhanced luminescence. Under optimized conditions for HRP detection, the detection limit of HRP was 0.08 fmol. By contrast, the detection limit of HRP with the enhanced L-012-dependent CL using 4-iodophenol, which is a common enhancer of luminol CL, was 1.1 fmol. With regard to H2O2 detection, the detection limits for enhanced CL with HPI and 4-iodophenol were 0.29 and 1.5 pmol, respectively. Therefore, it is demonstrated that HPI is the most superior L-012-dependent CL enhancer.

  14. 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.

  15. 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.

  16. Interactions of Macromolecular Crowding Agents and Cosolutes with Small-Molecule Substrates: Effect on Horseradish Peroxidase Activity with Two Different Substrates

    PubMed Central

    2015-01-01

    The importance of solution composition on enzymatic reactions is increasingly appreciated, particularly with respect to macromolecular cosolutes. Macromolecular crowding and its effect on enzymatic reactions has been studied for several enzymes and is often understood in terms of changes to enzyme conformation. Comparatively little attention has been paid to the chemical properties of small-molecule substrates for enzyme reactions in crowded solution. In this article, we studied the reaction of horseradish peroxidase (HRP) with two small-molecule substrates that differ in their hydrophobicity. Crowding agents and cosolutes had quite different effects on HRP activity when the substrate used was 3,3′,5,5′-tetramethylbenzidine (TMB, which is hydrophobic) as compared to o-phenylenediamine (OPD, which is more hydrophilic). Reaction rates with TMB were much more sensitive to the presence of crowding agents and cosolutes than OPD, suggesting that the small-molecule substrates may themselves be interacting with crowders and cosolutes. At high polyethylene glycol (PEG) concentrations (25–30 wt/wt %), no reaction was observed for TMB. Even at lower concentrations, Michaelis constants (KM) for HRP with the more hydrophobic substrate increased in the presence of crowding agents and cosolutes, particularly with PEG. Diffusion of TMB and OPD in the PEG and dextran reaction media was evaluated using pulsed field gradient nuclear magnetic resonance (PFG-NMR). The diffusivity of the TMB decreased 3.9× in 10% PEG 8k compared to that in buffer and decreased only 1.7× for OPD. Together, these data suggest that weak attractive interactions between small-molecule substrates and crowders or cosolutes can reduce substrate chemical activity and consequently decrease enzyme activity and that these effects vary with the identity of the molecules involved. Because many enzymes can act on multiple substrates, it is important to consider substrate chemistry in understanding enzymatic

  17. Oxidations of N-(3-indoleethyl) cyclic aliphatic amines by horseradish peroxidase: the indole ring binds to the enzyme and mediates electron-transfer amine oxidation.

    PubMed

    Ling, Ke-Qing; Li, Wen-Shan; Sayre, Lawrence M

    2008-01-23

    Although oxidations of aromatic amines by horseradish peroxidase (HRP) are well-known, typical aliphatic amines are not substrates of HRP. In this study, the reactions of N-benzyl and N-methyl cyclic amines with HRP were found to be slow, but reactions of N-(3-indoleethyl) cyclic amines were 2-3 orders of magnitude faster. Analyses of pH-rate profiles revealed a dominant contribution to reaction by the amine-free base forms, the only species found to bind to the enzyme. A metabolic study on a family of congeneric N-(3-indoleethyl) cyclic amines indicated competition between amine and indole oxidation pathways. Amine oxidation dominated for the seven- and eight-membered azacycles, where ring size supports the change in hybridization from sp3 to sp2 that occurs upon one-electron amine nitrogen oxidation, whereas only indole oxidation was observed for the six-membered ring congener. Optical difference spectroscopic binding data and computational docking simulations suggest that all the arylalkylamine substrates bind to the enzyme through their aromatic termini with similar binding modes and binding affinities. Kinetic saturation was observed for a particularly soluble substrate, consistent with an obligatory role of an enzyme-substrate complexation preceding electron transfer. The significant rate enhancements seen for the indoleethylamine substrates suggest the ability of the bound indole ring to mediate what amounts to medium long-range electron-transfer oxidation of the tertiary amine center by the HRP oxidants. This is the first systematic investigation to document aliphatic amine oxidation by HRP at rates consistent with normal metabolic turnover, and the demonstration that this is facilitated by an auxiliary electron-rich aromatic ring.

  18. Improved transport of horseradish peroxidase after injection with a non-ionic detergent (Nonidet P-40) into mouse cortex and observations on the relationship between spread at the injection site and amount of transported label.

    PubMed

    Lipp, H P; Schwegler, H

    1980-10-20

    Addition of the non-ionic detergent Nonidet P-40 (NP-40) to horseradish peroxidase (HRP) injected in small quantities into barrelfields of mouse somatosensory cortex results in a significant increase of labeled neurons in the contralateral barrelfield cortex as compared to normal HRP. Comparisons with lysolethicin as an additive to HRP show that with NP-40 neurons are labeled more reliably and spread of label is less extensive at the injection site. Using NP-40, the region of dense label spread at the injection site as revealed by the diaminobenzidine/cobalt procedure coincides rather precisely with the contralateral cortical region containing labeled neurons as visualized by tetramethylbenzidine.

  19. Detection of basal acetylcholine release in the microdialysis of rat frontal cortex by high-performance liquid chromatography using a horseradish peroxidase-osmium redox polymer electrode with pre-enzyme reactor.

    PubMed

    Kato, T; Liu, J K; Yamamoto, K; Osborne, P G; Niwa, O

    1996-06-28

    To determine the basal acetylcholine level in the dialysate of rat frontal cortex, a horseradish peroxidase-osmium redox polymer-modified glassy carbon electrode (HRP-GCE) was employed instead of the conventional platinum electrode used in high-performance liquid chromatography-electrochemical detection (HPLC-ED). In initial experiments, an oxidizable unknown compound interfered with the detection of basal acetylcholine release on HPLC-HRP-GCE. An immobilized peroxidase-choline oxidase precolumn (pre-reactor) was included in the HPLC system, to eliminate the interference from the unknown compound. This combination could detect less than 10 fmol of standard acetylcholine and basal acetylcholine levels in the dialysate from a conventional concentric design microdialysis probe, without the use of cholinesterase inhibitor, and may facilitate physiological investigation of cholinergic neuronal activity in the central nervous system.

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

    PubMed

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

    1992-01-01

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

  1. The effect of pH on horseradish peroxidase-catalyzed oxidation of melatonin: production of N1-acetyl-N2-5-methoxykynuramine versus radical-mediated degradation.

    PubMed

    Ximenes, Valdecir F; Fernandes, João Roberto; Bueno, Vânia B; Catalani, Luiz H; de Oliveira, Georgino H; Machado, Rosângela G P

    2007-04-01

    There is a growing body of evidence that melatonin and its oxidation product, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK), have anti-inflammatory properties. From a nutritional point of view, the discovery of melatonin in plant tissues emphasizes the importance of its relationship with plant peroxidases. Here we found that the pH of the reaction mixture has a profound influence in the reaction rate and products distribution when melatonin is oxidized by the plant enzyme horseradish peroxidase. At pH 5.5, 1 mm of melatonin was almost completely oxidized within 2 min, whereas only about 3% was consumed at pH 7.4. However, the relative yield of AFMK was higher in physiological pH. Radical-mediated oxidation products, including 2-hydroxymelatonin, a dimer of 2-hydroxymelatonin and O-demethylated dimer of melatonin account for the fast consumption of melatonin at pH 5.5. The higher production of AFMK at pH 7.4 was explained by the involvement of compound III of peroxidases as evidenced by spectral studies. On the other hand, the fast oxidative degradation at pH 5.5 was explained by the classic peroxidase cycle.

  2. Quantification and evaluation of kinetic bio-catalytic pathway of horseradish peroxidase in an electron mediated reaction system and its applications in plant extracts

    NASA Astrophysics Data System (ADS)

    Krishna, Honnur; Nagaraja, Padmarajaiah; Shivakumar, Anantharaman; Chamaraja, Nelligere A.; Aradhana, Narayan

    2013-02-01

    The intermolecular coupling of 2,5-dimethoxyaniline (DMA) as mediated electron transfer reaction in presence of H2O2 and peroxidase in acetate buffer of pH 4.2 resulting green colored product having maximum absorption at λmax = 740 nm was investigated by spectrophotometer. Under optimum conditions, linearity range for the quantification of H2O2 was 2.0-288.0 μM and for peroxidase were 0.59-9.46 and 0.443-9.46 nM by kinetic and fixed-time method, respectively. The catalytic efficiency and catalytic power were KeffD = 2.354 × 105 M-1 min-1 and KpowD = 4.59 × 10-4 min-1, respectively. From the plot of d(1/Do) vs d(1/Vo) and d(1/Ho) vs d(1/Vo), Michaelis-Menten constants for DMA and H2O2were found that KmD = 1458 μM and KmHO = 301 μM. Applicability of the method was tested for peroxidase activity in some plant extracts and compared with guaiacol/peroxidase system. Regarding superiority of the method, it is suggested that DMA/peroxidase system can be a better hydrogen donor for HRP assay than guaiacol system as evident from kinetic data.

  3. 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.

  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

    2015-08-05

    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.

  6. 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.

  7. 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.

  8. Ionic strength and pH effect on the Fe(III)-imidazolate bond in the heme pocket of horseradish peroxidase: an EPR and UV-visible combined approach.

    PubMed

    Laurenti, E; Suriano, G; Ghibaudi, E M; Ferrari, R P

    2000-10-01

    The effects of chloride, dihydrogenphosphate and ionic strength on the spectroscopic properties of horseradish peroxidase in aqueous solution at pH=3.0 were investigated. A red-shift (lambda=408 nm) of the Soret band was observed in the presence of 40 mM chloride; 500 mM dihydrogenphosphate or chloride brought about a blue shift of the same band (lambda=370 nm). The EPR spectrum of the native enzyme at pH 3.0 was characterized by the presence of two additional absorption bands in the region around g=6, with respect to pH 6.5. Chloride addition resulted in the loss of these features and in a lower rhombicity of the signal. A unique EPR band at g=6.0 was obtained as a result of the interaction between HRP and dihydrogenphosphate, both in the absence and presence of 40 mM Cl-. We suggest that a synergistic effect of low pH, Cl- and ionic strength is responsible for dramatic modifications of the enzyme conformation consistent with the Fe(II)-His170 bond cleavage. Dihydrogenphosphate as well as high chloride concentrations are shown to display an unspecific effect, related to ionic strength. A mechanistic explanation for the acid transition of HRP, previously observed by Smulevich et al. [Biochemistry 36 (1997) 640] and interpreted as a pure pH effect, is proposed.

  9. Resonance Raman evidence for oxygen exchange between the FeIV = O heme and bulk water during enzymic catalysis of horseradish peroxidase and its relation with the heme-linked ionization.

    PubMed Central

    Hashimoto, S; Tatsuno, Y; Kitagawa, T

    1986-01-01

    Raman spectroscopic studies of compound II of horseradish peroxidase show that the oxygen atom in the FeIV = O group of the heme is rapidly exchanged in H2O at pH 7.0 but not in an alkaline solution (pH 11.0). This conclusion is based on studies of shift in the FeIV = O stretching mode of compound II in H2(18)O; further studies show that the FeIV = O heme is hydrogen-bonded to an amino acid residue of the protein in neutral solutions but not in the alkaline solution. Deprotonation of this residue takes place with the midpoint pH at 8.8 and accordingly corresponds to the so-called heme-linked ionization. It is concluded that this hydrogen-bonded proton plays an important part in the oxygen exchange mechanism. From this it seems clear that this hydrogen-bonded proton has an essential role in the acid/base catalysis of this enzyme and that alkaline deactivation of this enzyme can be attributed to the lack of a hydrogen-bonded proton at high pH. PMID:3458206

  10. Toxic effect of terbium ion on horseradish cell.

    PubMed

    Jiang, Na; Wang, Lihong; Lu, Tianhong; Huang, Xiaohua

    2011-12-01

    The toxic effect of terbium (III) ion on the horseradish cell was investigated by scanning electron microscopy, gas chromatography, and standard biochemical methods. It was found that the activity of horseradish peroxidase in the horseradish treated with 0.2 mM terbium (III) ion decreased and led to the excessive accumulation of free radicals compared with that in the control horseradish. The excessive free radicals could oxidize unsaturated fatty acids in the horseradish cell and then increase the cell membrane lipid peroxidation of horseradish. The increase in the lipid peroxidation could lead to the destruction of the structure and function of the cell membrane and then damage of the horseradish cell. We propose that this is a possible mechanism for the toxic action of terbium in the biological systems.

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

    SciTech Connect

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

    1988-09-01

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

  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. 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

  14. Plant peroxidases. Their primary, secondary and tertiary structures, and relation to cytochrome c peroxidase.

    PubMed

    Welinder, K G

    1985-09-16

    The amino acid sequences of the 51% different horseradish peroxidase HRP C and turnip peroxidase TP 7 have previously been completed by us, but the three-dimensional structures are unknown. Recently the amino acid sequence and the crystal structure of yeast cytochrome c peroxidase have appeared. The three known apoperoxidases consist of 300 +/- 8 amino acid residues. The sequences have now been aligned and show 18% and 16% identity only, between the yeast peroxidase and plant peroxidase HRP C and TP 7, respectively. We show that different structural tests all support similar protein folds in plant peroxidases and yeast peroxidase and, therefore, a common evolutionary origin. The following tests support this thesis: (a) predicted helices in the plant peroxidases follow the complex pattern observed in the crystal structure of cytochrome c peroxidase; (b) their hydropathic profiles are similar and agree with observed buried and exposed peptide chain in cytochrome c peroxidase; (c) half-cystines which are distant in the amino acid sequence of plant peroxidases become spatial neighbours when fitted into the cytochrome c peroxidase model; (d) the two-domain structure proposed from limited proteolysis of apoperoxidase HRP C is observed in the crystal structure of cytochrome c peroxidase. The similarities and differences of the plant and yeast peroxidases and the reactive side chains of a plant peroxidase active site are described. The characteristics of Ca2+-binding sequences, derived from several superfamilies, are applied to predict the Ca2+-binding sequences in plant peroxidases.

  15. 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.

  16. 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.

  17. 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

  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. 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.

  20. Chemiluminescence lateral flow immunoassay based on Pt nanoparticle with peroxidase activity.

    PubMed

    Park, Jong-Min; Jung, Ha-Wook; Chang, Young Wook; Kim, Hyung-Seok; Kang, Min-Jung; Pyun, Jae-Chul

    2015-01-01

    A lateral flow immunoassay (LF-immunoassay) with an enhanced sensitivity and thermostability was developed by using Pt nanoparticles with a peroxidase activity. The Pt nanoparticles were synthesized by citrate reduction method, and the peroxidase activity of Pt nanoparticles was optimized by adjusting reaction conditions. The peroxidase activity was estimated by using Michaelis-Menten kinetics model with TMB as a chromogenic substrate. The kinetics parameters of KM and Vmax were calculated and compared with horseradish peroxidase (HRP). The thermal stability of the Pt nanoparticles was compared with horseradish peroxidase (HRP) according to the storage temperature and long-term storage period. The feasibility of lateral flow immunoassay with a chemiluminescent signal band was demonstrated by the detection of human chorionic gonadotropin (hCG) as a model analyte, and the sensitivity was determined to be improved by as much as 1000-fold compared to the conventional rapid test based on colored gold-colloids.

  1. Immobilization of Peroxidase onto Magnetite Modified Polyaniline

    PubMed Central

    Barbosa, Eduardo Fernandes; Molina, Fernando Javier; Lopes, Flavio Marques; García-Ruíz, Pedro Antonio; Caramori, Samantha Salomão; Fernandes, Kátia Flávia

    2012-01-01

    The present study describes the immobilization of horseradish peroxidase (HRP) on magnetite-modified polyaniline (PANImG) activated with glutaraldehyde. After the optimization of the methodology, the immobilization of HRP on PANImG produced the same yield (25%) obtained for PANIG with an efficiency of 100% (active protein). The optimum pH for immobilization was displaced by the effect of the partition of protons produced in the microenvironment by the magnetite. The tests of repeated use have shown that PANImG-HRP can be used for 13 cycles with maintenance of 50% of the initial activity. PMID:22489198

  2. 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.

  3. 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...

  4. Inhibition of Heme Peroxidases by Melamine

    PubMed Central

    Vanachayangkul, Pattaraporn; Tolleson, William H.

    2012-01-01

    In 2008 melamine-contaminated infant formula and dairy products in China led to over 50,000 hospitalizations of children due to renal injuries. In North America during 2007 and in Asia during 2004, melamine-contaminated pet food products resulted in numerous pet deaths due to renal failure. Animal studies have confirmed the potent renal toxicity of melamine combined with cyanuric acid. We showed previously that the solubility of melamine cyanurate is low at physiologic pH and ionic strength, provoking us to speculate how toxic levels of these compounds could be transported through the circulation without crystallizing until passing into the renal filtrate. We hypothesized that melamine might be sequestered by heme proteins, which could interfere with heme enzyme activity. Four heme peroxidase enzymes were selected for study: horseradish peroxidase (HRP), lactoperoxidase (LPO), and cyclooxygenase-1 and -2 (COX-1 and -2). Melamine exhibited noncompetitive inhibition of HRP (Ki  9.5 ± 0.7 mM), and LPO showed a mixed model of inhibition (Ki  14.5 ± 4.7 mM). The inhibition of HRP and LPO was confirmed using a chemiluminescent peroxidase assay. Melamine also exhibited COX-1 inhibition, but inhibition of COX-2 was not detected. Thus, our results demonstrate that melamine inhibits the activity of three heme peroxidases. PMID:22852071

  5. Palm tree peroxidases.

    PubMed

    Sakharov, I Yu

    2004-08-01

    Over the years novel plant peroxidases have been isolated from palm trees leaves. Some molecular and catalytic properties of palm peroxidases have been studied. The substrate specificity of palm peroxidases is distinct from the specificity of other plant peroxidases. Palm peroxidases show extremely high stability under acidic and alkaline conditions and high thermal stability. Moreover, these enzymes are more stable with respect to hydrogen peroxide treatment than other peroxidases. Due to their extremely high stability, palm peroxidases have been used successfully in the development of new bioanalytical tests, the construction of improved biosensors, and in polymer synthesis.

  6. Reactions of the class II peroxidases, lignin peroxidase and Arthromyces ramosus peroxidase, with hydrogen peroxide. Catalase-like activity, compound III formation, and enzyme inactivation.

    PubMed

    Hiner, Alexander N P; Hernández-Ruiz, Josefa; Rodríguez-López, José Neptuno; García-Cánovas, Francisco; Brisset, Nigel C; Smith, Andrew T; Arnao, Marino B; Acosta, Manuel

    2002-07-26

    The reactions of the fungal enzymes Arthromyces ramosus peroxidase (ARP) and Phanerochaete chrysosporium lignin peroxidase (LiP) with hydrogen peroxide (H(2)O(2)) have been studied. Both enzymes exhibited catalase activity with hyperbolic H(2)O(2) concentration dependence (K(m) approximately 8-10 mm, k(cat) approximately 1-3 s(-1)). The catalase and peroxidase activities of LiP were inhibited within 10 min and those of ARP in 1 h. The inactivation constants were calculated using two independent methods; LiP, k(i) approximately 19 x 10(-3) s(-1); ARP, k(i) approximately 1.6 x 10(-3) s(-1). Compound III (oxyperoxidase) was detected as the majority species after the addition of H(2)O(2) to LiP or ARP, and its formation was accompanied by loss of enzyme activity. A reaction scheme is presented which rationalizes the turnover and inactivation of LiP and ARP with H(2)O(2). A similar model is applicable to horseradish peroxidase. The scheme links catalase and compound III forming catalytic pathways and inactivation at the level of the [compound I.H(2)O(2)] complex. Inactivation does not occur from compound III. All peroxidases studied to date are sensitive to inactivation by H(2)O(2), and it is suggested that the model will be generally applicable to peroxidases of the plant, fungal, and prokaryotic superfamily.

  7. 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

  8. Use of additives to enhance the removal of phenols from water treated with horseradish and hydrogen peroxide.

    PubMed

    Tonegawa, Masami; Dec, Jerzy; Bollag, Jean-Marc

    2003-01-01

    Use of additives, such as polyethylene glycol (PEG), selected surfactants, chitosan gel, or activated carbon, has been shown to enhance enzymatic treatment of water polluted with organic compounds. In this study, additives were used to facilitate the removal of 2,4-dichlorophenol (2,4-DCP) from water using minced horseradish (Armoracia rusticana P. Gaertn. et al.) as a carrier of peroxidase activity. The specific objectives of the study were to (i) enhance the pollutant removal activity of minced horseradish by the addition of PEG and other additives (e.g., Tween 20, Triton X-100, and rhamnolipid); (ii) eliminate colored reaction products by the addition of chitosan; and (iii) eliminate color by amending treated water with activated carbon. The disappearance of 2,4-DCP in horseradish-treated water samples amended with PEG or various surfactants (75-90%) was greatly increased over that observed in nonamended samples (29%). The effect of PEG depended on its average molecular weight. As indicated by visible spectrophotometry, enclosing horseradish pieces between two sealed chitosan films completely eliminated colored reaction products; however, the decolorization was accompanied by a reduction in 2,4-DCP removal (from 95 to 60%). On the other hand, commercially available activated carbon completely removed colored reaction products from the treated water without reducing the removal efficiency. Based on the results obtained, it can be concluded that the use of additives may considerably improve the quality of wastewater treated by plant materials.

  9. 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.

  10. 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

  11. Peroxidase(s) in environment protection.

    PubMed

    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.

  12. 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

  13. [Prospects for leprosy treatment via complexation of rifampicin witH iodide and horse-radish root].

    PubMed

    Maslov, A K; Khivrina, S A

    2007-01-01

    A model of leprosy was used to study the therapeutic effect of horse-radish root (HRR) containing peroxidase in combination with rifampicin (RFP) and potassium iodide (PI) as compared to routine combined therapy with RFP and diaminodiphenylsulfonum. Therapy with HRR and iodide showed the best antimicrobial effect than the routine combined therapy. A combination of RFP, HRR, and PI increased the activity of neutrophilic myeliperoxidase produced an anti-inflammatory activity and caused no persistent anemia or toxic effect on the murine liver.

  14. Compounds I of catalase and horse radish peroxidase: pi-cation radicals.

    PubMed

    Dolphin, D; Forman, A; Borg, D C; Fajer, J; Felton, R H

    1971-03-01

    Two-electron oxidation of cobaltous octaethylporphyrin [Co(II)(Et)(8)P] yields a stable pi-cation radical [Co(III)(Et)(8)P](2+.), the optical spectrum of which exhibits spectral changes dependent upon the nature of the counterion. Comparison of these spectra with those of Compounds I of horseradish peroxidase and catalase leads us to propose that these Compounds I contain a pi-cation radical of the heme prosthetic group. This proposal explains the oxidation level, optical spectra, and stability of the primary compounds without recourse to properties such as stoichiometric mixtures of special porphyrins, stable Fe(V) porphyrins, or unique conformers of heme porphyrins. Explanations are advanced to account for the missing electron spin resonance signal of Compound I of horseradish peroxidase.

  15. 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.

  16. Interaction of metals with peroxidase--mediated luminol-enhanced, chemiluminescence (PLmCL).

    PubMed

    Coteur, G; Dubois, P

    2004-01-01

    The peroxidase-mediated luminol-enhanced chemiluminescence (PLmCL) method has been used to study the in vitro effect of contaminants such as heavy metals on the reactive oxygen species production by immunocytes. We were interested to know whether metals could directly affect peroxidase-mediated luminescence, taking horseradish peroxidase (HRP) as a model enzyme, since this could contribute to the inhibition of immunocyte LmCL. Copper inhibited PLmCL in a dose-dependent manner, while cadmium, iron, silver and lead only partly decreased the signal in the concentration range tested. In contrast, zinc enhanced the signal at high concentrations. Eventually, chromium, mercury and aluminium did not affect PLmCL. It is suggested that these effects reflect the ability of the metals to interact with the active site of the peroxidase. These results demonstrate that such interactions have to be considered when interpreting the effects of metals on immunocytes using the LmCL method.

  17. Kinetics of phenolic polymerization catalyzed by peroxidase in organic media

    SciTech Connect

    Xu, Y.P.; Huang, G.L; Yu, Y.T.

    1995-07-05

    Phenolic polymerization was carried out by enzymatic catalysis in organic media, and its kinetics was studied by using high-pressure liquid chromatography (HPLC). Phenols and aromatic amines with electron-withdrawing groups could hardly be polymerized by HRP catalysis, but phenols and aromatic amines with electron-donating groups could easily by polymerized. The reaction rate of either the para-substituted substrate or meta-substituted substrate was higher than that of ortho-substituted substrate. When ortho-position of hydroxy group of phenols was occupied by an electron-donating group and if another electron-donating group occupied para-position of hydroxy group, the reaction rate increased. Horseradish peroxidase and lactoperoxidase could easily catalyze the polymerization, but chloroperoxidase and laccase failed to yield polymers. Metallic ions such as Mn{sup 2+}, Fe{sup 2+}, or Fe{sup 3+}, and Cu{sup 2+} could poison horseradish peroxidase to various extents, but ions such as Co{sup 2+}, Cd{sup 2+}, Zn{sup 2+}, and K{sup +} were not found to inhibit the reaction.

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

    SciTech Connect

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

    1986-03-01

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

  19. Anterograde transport of horseradish peroxidase in the nigrostriatal pathway after neostriatal kainic acid lesions.

    PubMed

    Walker, P D; McAllister, J P

    1986-08-01

    We used the anterograde transport of HRP to analyze the nigrostriatal pathway after intrastriatal injections of kainic acid. A total volume of 1 microliter kainic acid (3 nM) was injected unilaterally into the neostriatum of adult rats. After 5, 10, or 35 days, HRP was injected into the ipsilateral substantia nigra. Sections stained for Nissl substance revealed that kainic acid damaged as much as three-quarters of the neostriatum. Lesion sites were characterized by gliosis and the absence of neurons. Alternate sections processed for HRP histochemistry and analyzed with bright- and dark-field microscopy revealed labeled axons and terminals in the lesion site. These findings were consistent in all three time periods. Much of the labeling was similar to that seen in neostriatal of control animals. However, the normal homogeneous pattern of the nigrostriatal terminal field was disrupted in all experimental groups, illustrated by changes in some labeling characteristics in the lesion site. These findings provide morphologic evidence for the preservation of much of the nigrostriatal pathway but indicate that some axons and their terminals may be altered after kainic acid injection.

  20. Peroxidase and fluorescein isothiocyanate as antibody markers. A quantitative comparison of two peroxidase conjugates prepared with glutaraldehyde or periodate anda fluorescein conjugate.

    PubMed

    Broorsma, D M; Steefkerk, J G; Kors, N

    1976-09-01

    Batches of rabbit anti-human immunoglobulin G antibodies were labeled either with horseradish peroxidase, using the two-step glutaraldehyde method or the periodate method, or with fluorescein isothiocyanate (FITC). The peroxidase conjugates were isolated by chromatography using two different gel types. The five types of conjugates thus obtained were standardized to the same amount of rabbit immunoglobulin G. The antibody activity, as estimated by means of single radial immunodiffusion and passive hemagglutination, and the enzyme activity, determined with orthodianisidine, were compared. The ultimate dilutions and absolute amounts of the five conjugates giving positive reactions were determined in direct and indirect immunohistochemical tests, using both cryostat sections of skin and the agarose bead model system. It appeared that during the peroxidase conjugation procedures there was a considerable loss of abtibody and enzyme activity, whereas in the FITC conjugation procedure the antibody activity remained intact. Neverthe less, peroxidase conjugates prepared with glutaraldehyde still gave positive staining reactions in equal or somewhat higher dilutions than the fluorescin conjugate did. The peroxidase conjugates prepared with periodate could not be diluted to the same extent. For the detection of antibodies by indirect immunohistochemical methods, the peroxidase conjugate, prepared with glutaraldehyde, was comparable to the FITC conjugate. The peroxidase conjugate, prepared with periodate, was less effective.

  1. 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.

  2. 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.

  3. Mechanism of reaction of chlorite with mammalian heme peroxidases

    PubMed Central

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

    2014-01-01

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

  4. The role of stigma peroxidases in flowering plants: insights from further characterization of a stigma-specific peroxidase (SSP) from Senecio squalidus (Asteraceae).

    PubMed

    McInnis, Stephanie M; Emery, David C; Porter, Robert; Desikan, Radhika; Hancock, John T; Hiscock, Simon J

    2006-01-01

    Angiosperm stigmas have long been known to exhibit high levels of peroxidase activity when they are mature and most receptive to pollen but the biological function of stigma peroxidases is not known. A novel stigma-specific class III peroxidase gene, SSP (stigma-specific peroxidase) expressed exclusively in the stigmas of Senecio squalidus L. (Asteraceae) has recently been identified. Expression of SSP is confined to the specialized secretory cells (papillae) that compose the stigma epidermis. The literature on stigma peroxidases and hypotheses on their function(s) is reviewed here before further characterization of SSP and an attempt to determine its function are described. It is shown that SSP is localized to cytoplasmic regions of stigmatic papillae and also to the surface of these cells, possibly as a component of the pellicle, a thin layer of condensed protein typical of "dry" stigmas. Enzyme assays on recombinant SSP showed it to be a peroxidase with a preference for diphenolic substrates (ABTS and TMB) and a pH optimum of approximately 4.5. In such assays the peroxidase activity of SSP was low when compared with horseradish peroxidase. To explore the function of SSP and other stigmatic peroxidases, levels of reactive oxygen species (ROS) in stigmas of S. squalidus were investigated. Relatively large amounts of ROS, principally H(2)O(2), were detected in S. squalidus stigmas where most ROS/H(2)O(2) was localized to the stigmatic papillae, the location of SSP. These observations are discussed in the context of possible functions for SSP, other peroxidases, and ROS in the stigmas of angiosperms.

  5. Hydrogen peroxide-independent generation of superoxide by plant peroxidase: hypotheses and supportive data employing ferrous ion as a model stimulus

    PubMed Central

    Kimura, Makoto; Umemoto, Yosuke; Kawano, Tomonori

    2014-01-01

    When plants are threaten by microbial attacks or treated with elicitors, alkalization of extracellular space is often induced and thus pH-dependent extracellular peroxidase-mediated oxidative burst reportedly takes place, especially at the site of microbial challenge. However, direct stimulus involved in activation of peroxidase-catalyzed oxidative burst has not been identified to date. Here, we would like to propose a likely role for free ferrous ion in reduction of ferric native peroxidase into ferrous enzyme intermediate which readily produces superoxide anion via mechanism involving Compound III, especially under alkaline condition, thus, possibly contributing to the plant defense mechanism. Through spectroscopic and chemiluminescence (CL) analyses of reactions catalyzed by horseradish peroxidase (HRP), the present study proposed that plant peroxidase-catalyzed production of superoxide anion can be stimulated in the absence of conventional peroxidase substrates but in the presence of free ferrous ion. PMID:25071789

  6. 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

  7. Degradation of textile dyes mediated by plant peroxidases.

    PubMed

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

    2002-01-01

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

  8. Hemoglobins Likely Function as Peroxidase in Blood Clam Tegillarca granosa Hemocytes.

    PubMed

    Wang, Sufang; Yu, Xiaopei; Lin, Zhihua; Zhang, Shunqin; Xue, Liangyi; Xue, Qinggang; Bao, Yongbo

    2017-01-01

    Hemoglobins are a group of respiratory proteins principally functioning in transport of oxygen and carbon dioxide in red blood cells of all vertebrates and some invertebrates. The blood clam T. granosa is one of the few invertebrates that have hemoglobin-containing red hemocytes. In the present research, the peroxidase activity of T. granosa hemoglobins (Tg-Hbs) was characterized and the associated mechanism of action was deciphered via structural comparison with other known peroxidases. We detected that purified Tg-Hbs catalyzed the oxidation of phenolic compounds in the presence of exogenous H2O2. Tg-Hbs peroxidase activity reached the maximum at pH 5 and 35°C and was inhibited by Fe(2+), Cu(2+), SDS, urea, and sodium azide. Tg-Hbs shared few similarities in amino acid sequence and overall structural characteristics with known peroxidases. However, the predicted structure at their heme pocket was highly similar to that of horseradish peroxidase (HRP) and myeloperoxidase (MPO). This research represented the first systemic characterization of hemoglobin as a peroxidase.

  9. Hemoglobins Likely Function as Peroxidase in Blood Clam Tegillarca granosa Hemocytes

    PubMed Central

    Yu, Xiaopei; Lin, Zhihua; Xue, Liangyi

    2017-01-01

    Hemoglobins are a group of respiratory proteins principally functioning in transport of oxygen and carbon dioxide in red blood cells of all vertebrates and some invertebrates. The blood clam T. granosa is one of the few invertebrates that have hemoglobin-containing red hemocytes. In the present research, the peroxidase activity of T. granosa hemoglobins (Tg-Hbs) was characterized and the associated mechanism of action was deciphered via structural comparison with other known peroxidases. We detected that purified Tg-Hbs catalyzed the oxidation of phenolic compounds in the presence of exogenous H2O2. Tg-Hbs peroxidase activity reached the maximum at pH 5 and 35°C and was inhibited by Fe2+, Cu2+, SDS, urea, and sodium azide. Tg-Hbs shared few similarities in amino acid sequence and overall structural characteristics with known peroxidases. However, the predicted structure at their heme pocket was highly similar to that of horseradish peroxidase (HRP) and myeloperoxidase (MPO). This research represented the first systemic characterization of hemoglobin as a peroxidase. PMID:28182094

  10. Electroenzymatic degradation of azo dye using an immobilized peroxidase enzyme.

    PubMed

    Kim, Gha-Young; Lee, Ki-Beom; Cho, Seung-Hee; Shim, Joonmok; Moon, Seung-Hyeon

    2005-11-11

    Azo dyes are largely resistant to biodegradation and persist in conventional wastewater treatment processes. Combining enzymatic catalysis and the electrochemical generation of hydrogen peroxide (H2O2), an electroenzymatic process was developed, which is a potential alternative to traditional processes. In this study, an electroenzymatic method that uses an immobilized horseradish peroxidase enzyme (HRP), was investigated to degrade orange II (azo dye) within a two-compartment packed-bed flow reactor. To evaluate the electroenzymatic degradation of orange II, electrolytic experiments were carried out with 0.42 U/mL HRP at -0.5 V. It was found that removal of orange II was partly due to its adsorption to the graphite felt. The overall application of the electroenzymatic led to a greater degradation rate than the use of electrolysis alone. Also the by-products formed were found to consist primarily of an aromatic amine, sulfanilic acid, and unknown compounds.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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

    2014-04-04

    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.

  17. 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.

  18. Permeability alteration of sarcolemmal membrane in catecholamine-induced cardiac muscle cell injury. In vivo studies with fine structural diffusion tracer horse radish peroxidase.

    PubMed

    Boutet, M; Hüttner, I; Rona, G

    1976-05-01

    Cardiac muscle cell injury was produced in male Sprague-Dawley rats weighing 300 to 450 gm. with catecholamines, norepinephrine, and isoproterenol; sarcolemmal membrane alteration was tested in vivo using the extracellular macromolecular tracer, horseradish peroxidase. Norepinephrine was administered in continuous intravenous infusion in a dose of 4 to 6 mug. per 100 gm. of body weight per minute, whereas isoproterenol was given as a single subcutaneous injection in a dose of 8.5 mg. per 100 gm. of body weight. Horseradish peroxidase was injected intravenously and localized in the right ventricular myocardium following 6 and 30 minutes of circulation time by light and electron microscopy. As early as 10 minutes after norepinephrine infusion, horseradish peroxidase appeared within cardiac muscle cells possessing normal fine structure. Selective deposition of the tracer on normal and altered myofilaments was noted. Similar observations were made in the isoproterenol model at 60 to 90 minutes. The results indicate that sarcolemmal membrane permeability alteration is an early event in catecholamine-induced cardiac muscle injury. The possible functional significance of the findings is discussed.

  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. 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.

  2. Hemin-block copolymer micelle as an artificial peroxidase and its applications in chromogenic detection and biocatalysis.

    PubMed

    Qu, Rui; Shen, Liangliang; Chai, Zhihua; Jing, Chen; Zhang, Yufeng; An, Yingli; Shi, Linqi

    2014-01-01

    Following an inspiration from the fine structure of natural peroxidases, such as horseradish peroxidase (HRP), an artificial peroxidase was constructed through the self-assembly of diblock copolymers and hemin, which formed a functional micelle with peroxidase-like activity. The pyridine moiety in block copolymer poly(ethylene glycol)-block-poly(4-vinylpyridine) (PEG-b-P4VP) can coordinate with hemin, and thus hemin is present in a five-coordinate complex with an open site for binding substrates, which mimics the microenvironment of heme in natural peroxidases. The amphiphilic core-shell structure of the micelle and the coordination interaction of the polymer to the hemin inhibit the formation of hemin μ-oxo dimers, and thereby enhance the stability of hemin in the water phase. Hemin-micelles exhibited excellent catalytic performance in the oxidation of phenolic and azo compounds by H2O2. In comparison with natural peroxidases, hemin-micelles have higher catalytic activity and better stability over wide temperature and pH ranges. Hemin-micelles can be used as a detection system for H2O2 with chromogenic substrates, and they anticipate the possibility of constructing new biocatalysts tailored to specific functions.

  3. 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.

  4. 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.

  5. Hyaluronate - Peanut Agglutinin Conjugates for Target-Specific Bioimaging of Colon Cancer.

    PubMed

    Beack, Songeun; Cho, Minsoo; Kim, Young-Eun; Ahn, G-One; Hahn, Sei Kwang

    2017-03-27

    Colon cancer is one of the most common death-related cancers in the world. For treating the colon cancer, it is crucial to detect and remove malignant lesions in the early time. Here, we developed hyaluronate (HA) - peanut agglutinin (PNA) conjugates for bioimaging of colon cancer. The HA-PNA conjugates were successfully synthesized by the coupling reaction between aldehyde modified HA and the N-terminal amine group of PNA. For diagnostic imaging, rhodamine B (RhoB) was chemically conjugated onto PNA in HA-PNA conjugates. After intraluminal injection of HA-PNA-RhoB conjugates into tumor-bearing mice, small-sized colon cancers could be effectively visualized by ex vivo IVIS imaging and two-photon microscopy. Taken together, we could confirm the feasibility of HA-PNA-RhoB conjugates as a bioimaging agent for detecting colon cancers. [Keywords] Hyaluronate; Peanut agglutinin; Colon cancer; Two-photon imaging; Diagnostics.

  6. 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

  7. Substrate oxidation sites in versatile peroxidase and other basidiomycete peroxidases.

    PubMed

    Ruiz-Dueñas, Francisco J; Morales, María; García, Eva; Miki, Yuta; Martínez, María Jesús; Martínez, Angel T

    2009-01-01

    Versatile peroxidase (VP) is defined by its capabilities to oxidize the typical substrates of other basidiomycete peroxidases: (i) Mn(2+), the manganese peroxidase (MnP) substrate (Mn(3+) being able to oxidize phenols and initiate lipid peroxidation reactions); (ii) veratryl alcohol (VA), the typical lignin peroxidase (LiP) substrate; and (iii) simple phenols, which are the substrates of Coprinopsis cinerea peroxidase (CIP). Crystallographic, spectroscopic, directed mutagenesis, and kinetic studies showed that these 'hybrid' properties are due to the coexistence in a single protein of different catalytic sites reminiscent of those present in the other basidiomycete peroxidase families. Crystal structures of wild and recombinant VP, and kinetics of mutated variants, revealed certain differences in its Mn-oxidation site compared with MnP. These result in efficient Mn(2+) oxidation in the presence of only two of the three acidic residues forming its binding site. On the other hand, a solvent-exposed tryptophan is the catalytically-active residue in VA oxidation, initiating an electron transfer pathway to haem (two other putative pathways were discarded by mutagenesis). Formation of a tryptophanyl radical after VP activation by peroxide was detected using electron paramagnetic resonance. This was the first time that a protein radical was directly demonstrated in a ligninolytic peroxidase. In contrast with LiP, the VP catalytic tryptophan is not beta-hydroxylated under hydrogen peroxide excess. It was also shown that the tryptophan environment affected catalysis, its modification introducing some LiP properties in VP. Moreover, some phenols and dyes are oxidized by VP at the edge of the main haem access channel, as found in CIP. Finally, the biotechnological interest of VP is discussed.

  8. 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.

  9. 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.

  10. Enhanced peroxidase-like activity of MoS2/graphene oxide hybrid with light irradiation for glucose detection.

    PubMed

    Peng, Jian; Weng, Jian

    2017-03-15

    Construction of novel enzyme-free mimetic is very important in improving the sensitivity of biosensor. Here, an intrinsic peroxidase-like activity of MoS2 and graphene oxide (MoS2/GO) hybrid is demonstrated and the hybrid is also used to detect glucose with high sensitivity. Firstly, the peroxidase-like activity of hydrid is compared with that of two components alone, the mixture of two components and horseradish peroxidase. The results show that the hydrid has highest catalytic activity and the Michaelis constant of this hybrid is 4.35 times lower and the maximal reaction velocity is 3.34 times higher than those of horseradish peroxidase, respectively. Electrochemical technologies are used to investigate the enhancing mechanism. The results show that the excellently catalytic performance could be attributed to the fast electron transfer on the surface of MoS2/GO and the synergistic interaction of two components. Secondly, the effect of visible light and near-infrared light on the peroxidase-like activity of hybrid is also investigated. The results show that the limit of detection for H2O2 can be reduced from 10nM to 2.5nM with visible light. Thirdly, the hybrid is further used to detect glucose in serum with and without light. The results show that the hybrid has high selectivity and sensitivity for glucose detection in serum and the limit of detection for glucose is reduced from 0.83μM to 65nM with visible light. Therefore, the hybrid may have a potential application in glucose detection in serum with high sensitivity and selectivity.

  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. 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.

  13. 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. --------------------------------------------------------------------------------

  14. 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

  15. 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.

  16. 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.

  17. The effect of bilirubin photoisomers on unbound-bilirubin concentrations estimated by the peroxidase method.

    PubMed Central

    Itoh, S; Yamakawa, T; Onishi, S; Isobe, K; Manabe, M; Sasaki, K

    1986-01-01

    Unbound bilirubin is oxidized to nearly colourless substances in the presence of H2O2 or ethyl hydroperoxide and horseradish peroxidase. To predict the risk of kernicterus (degenerated yellow pigmentation of nerve cells), this principle has been widely utilized for estimating the concentration of unbound bilirubin in hyperbilirubinaemic serum. However, the serum contains polar geometric photoisomers of bilirubin. Therefore, to clarify the effect of bilirubin photoisomer concentrations on unbound-bilirubin concentration, the concentration of bilirubin and its photoisomer and of unbound bilirubin in samples obtained from experiments in vivo and in vitro were simultaneously and individually estimated by h.p.l.c. and the peroxidase method. During photoirradiation, both in vivo and in vitro, the serum polar (ZE)-bilirubin IX alpha concentration increased remarkably, but unbound-bilirubin values were not affected at all. However, during experiments in vitro, unbound bilirubin concentrations increased only when concentrations of the rather polar (EZ)- and (EE)-cyclobilirubin IX alpha increased considerably in a human serum albumin-bilirubin solution irradiated with blue light. Thus it is concluded that unbound-bilirubin concentrations, and consequently the initial rate of the peroxidase reaction, is not accelerated by the increase in either (ZE)-bilirubin or (EZ)-cyclobilirubin concentration within the clinically observed range. PMID:3545181

  18. 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

    2001-02-05

    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.

  19. Recombinant production of a peroxidase-protein G fusion protein in Pichia pastoris.

    PubMed

    Krainer, Florian Wolfgang; Darnhofer, Barbara; Birner-Gruenberger, Ruth; Glieder, Anton

    2016-02-10

    Streptococcal protein G (SpG) binds immunoglobulin G from a broad range of mammalian species with high affinity. Chemical conjugations of SpG to the reporter enzyme horseradish peroxidase (HRP) are commonly used in immunohistochemical applications. However, commercial HRP preparations are typically isolated from horseradish roots as varying mixtures of HRP isoenzymes with different biochemical properties, and chemical conjugation procedures lead to heterogeneous HRP-SpG preparations, partially including inactivated enzyme. A recombinant process allows the production of a well-defined HRP isoenzyme fused to SpG at constant 1:1 stoichiometry in a single step without the need for laborious chemical conjugation. By using state-of-the-art biotechnological tools, we produced a recombinant HRP-SpG fusion protein in Pichia pastoris in bioreactor cultivations. Purified HRP-SpG was tested successfully for functional binding of antibodies from different mammalian serums. Recombinant production of this novel well-defined fusion protein follows quality-by-design principles and facilitates the production of more reliable and cost-effective diagnostic kits.

  20. Purification and basic biochemical characterization of 19 recombinant plant peroxidase isoenzymes produced in Pichia pastoris☆

    PubMed Central

    Krainer, Florian W.; Pletzenauer, Robert; Rossetti, Laura; Herwig, Christoph; Glieder, Anton; Spadiut, Oliver

    2014-01-01

    The plant enzyme horseradish peroxidase (HRP) is used in several important industrial and medical applications, of which especially biosensors and diagnostic kits describe an emerging field. Although there is an increasing demand for high amounts of pure enzyme preparations, HRP is still isolated from the plant as a mixture of different isoenzymes with different biochemical properties. Based on a recent next generation sequencing approach of the horseradish transcriptome, we produced 19 individual HRP isoenzymes recombinantly in the yeast Pichia pastoris. After optimizing a previously reported 2-step purification strategy for the recombinant isoenzyme HRP C1A by substituting an unfavorable size exclusion chromatography step with an anion exchange step using a monolithic column, we purified the 19 HRP isoenzymes with varying success. Subsequent basic biochemical characterization revealed differences in catalytic activity, substrate specificity and thermal stability of the purified HRP preparations. The preparations of the isoenzymes HRP A2A and HRP A2B were found to be highly interesting candidates for future applications in diagnostic kits with increased sensitivity. PMID:24342173

  1. Analysis of peroxidase activity of rice (Oryza sativa) recombinant hemoglobin 1: implications for in vivo function of hexacoordinate non-symbiotic hemoglobins in plants.

    PubMed

    Violante-Mota, Fernando; Tellechea, Edurne; Moran, Jose F; Sarath, Gautam; Arredondo-Peter, Raúl

    2010-01-01

    In plants, it has been proposed that hexacoordinate (class 1) non-symbiotic Hbs (nsHb-1) function in vivo as peroxidases. However, little is known about peroxidase activity of nsHb-1. We evaluated the peroxidase activity of rice recombinant Hb1 (a nsHb-1) by using the guaiacol/H2O2 system at pH 6.0 and compared it to that from horseradish peroxidase (HRP). Results showed that the affinity of rice Hb1 for H2O2 was 86-times lower than that of HRP (K(m)=23.3 and 0.27 mM, respectively) and that the catalytic efficiency of rice Hb1 for the oxidation of guaiacol using H2O2 as electron donor was 2838-times lower than that of HRP (k(cat)/K(m)=15.8 and 44,833 mM(-1) min(-1), respectively). Also, results from this work showed that rice Hb1 is not chemically modified and binds CO after incubation with high H2O2 concentration, and that it poorly protects recombinant Escherichia coli from H2O2 stress. These observations indicate that rice Hb1 inefficiently scavenges H2O2 as compared to a typical plant peroxidase, thus indicating that non-symbiotic Hbs are unlikely to function as peroxidases in planta.

  2. 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.

  3. 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.

  4. Intracisternal granules in the adipokinetic cells of locusts are not degraded and apparently function as supplementary stores of secretory material.

    PubMed

    Harthoorn, L F; Diederen, J H; Oudejans, R C; Verstegen, M M; Vullings, H G; Van der Horst, D J

    2000-01-01

    The intracisternal granules in locust adipokinetic cells appear to represent accumulations of secretory material within cisternae of the rough endoplasmic reticulum. An important question is whether these granules are destined for degradation or represent stores of (pro)hormones. Two strategies were used to answer this question. First, cytochemistry was applied to elucidate the properties of intracisternal granules. The endocytic tracers horseradish peroxidase and wheat-germ agglutinin-conjugated horseradish peroxidase were used to facilitate the identification of endocytic, autophagic, and lysosomal organelles, which may be involved in the degradation of intracisternal granules. No intracisternal granules could be found within autophagosomes, and granules fused with endocytic and lysosomal organelles were not observed, nor could tracer be found within the granules. The lysosomal enzyme acid phosphatase was absent from the granules. Second, biochemical analysis of the content of intracisternal granules revealed that these granules contain prohormones as well as hormones. Prohormones were present in relatively higher amounts compared with ordinary secretory granules. Since the intracisternal granules in locust adipokinetic cells are not degraded and contain intact (pro)hormones it is concluded that they function as supplementary stores of secretory material.

  5. 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

  6. 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.

  7. Neuronal transport of acid hydrolases and peroxidase within the lysosomal system or organelles: involvement of agranular reticulum-like cisterns.

    PubMed

    Broadwell, R D; Oliver, C; Brightman, M W

    1980-04-01

    Neurosecretory neurons of the hyperosmotically stressed hypothalamo-neurohypophysial system have been a useful model with which to demonstrate interrelationships among perikaryal lysosomes, agranular reticulum-like cisterns, endocytotic vacuoles, and the axoplasmic transport of acid hydrolases and horseradish peroxidase. Supraoptic neurons from normal mice and mice given 2% salt water to drink for 5--8 days have been studied using enzyme cytochemical techniques for peroxidase and lysosomal acid hydrolases. Peroxidase-labeling of these neurons was accomplished by intravenous injection or cerebral ventriculocisternal perfusion of the protein as previously reported (Broadwell and Brightman, '79). Compared to normal controls, supraoptic cell bodies from hyperosmotically stimulated mice contained elevated concentrations of peroxidase-labeled dense bodies demonstrated to be secondary lysosomes and acid hydrolase-positive and peroxidase-positive cisterns either attached or unattached to secondary lysosomes. These cisterns were smooth-surfaced and 400--1,000 A wide. Their morphology was similar to that of the agranular reticulum. Some of the cisterns contained both peroxidase and acid hydrolase activities. The cisterns probably represent an elongated form of lysosome and, therefore, are not elements of the agranular reticulum per se. By virtue of their direct connections with perikaryal secondary lysosomes, these cisterns may provide the route by which acid hydrolases and exogenous macromolecules can leave perikaryal secondary lysosomes for anterograde flow down the axon. Very few smooth-surfaced cisterns were involved in the retrograde transport of peroxidase within pituitary stalk axons from normal and salt-treated mice injected intravenously with peroxidase. Peroxidase undergoing retrograde transport was predominantly in endocytotic structures such as vacuoles and cup-shaped organelles, which deliver this exogenous macromolecule directly to secondary lysosomes for

  8. 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.

  9. 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.

  10. 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.

  11. DyP-type peroxidases comprise a novel heme peroxidase family.

    PubMed

    Sugano, Y

    2009-04-01

    Dye-decolorizing peroxidase (DyP) is produced by a basidiomycete (Thanatephorus cucumeris Dec 1) and is a member of a novel heme peroxidase family (DyP-type peroxidase family) that appears to be distinct from general peroxidases. Thus far, 80 putative members of this family have been registered in the PeroxiBase database (http://peroxibase.isbsib.ch/) and more than 400 homologous proteins have been detected via PSI-BLAST search. Although few studies have characterized the function and structure of these proteins, they appear to be bifunctional enzymes with hydrolase or oxygenase, as well as typical peroxidase activities. DyP-type peroxidase family suggests an ancient root compared with other general peroxidases because of their widespread distribution in the living world. In this review, firstly, an outline of the characteristics of DyP from T. cucumeris is presented and then interesting characteristics of the DyP-type peroxidase family are discussed.

  12. 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.

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

    PubMed

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

    2015-03-15

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

  14. Facile method to synthesize dopamine-capped mixed ferrite nanoparticles and their peroxidase-like activity

    NASA Astrophysics Data System (ADS)

    Mumtaz, Shazia; Wang, Li-Sheng; Abdullah, Muhammad; Zajif Hussain, Syed; Iqbal, Zafar; Rotello, Vincent M.; Hussain, Irshad

    2017-03-01

    A facile single-step strategy to prepare stable and water-dispersible dopamine-functionalized ultra-small mixed ferrite nanoparticles MFe2O4-DOPA (where M is a bivalent metal atom i.e. Fe, Co Cu, Mn and Ni) at room temperature is described. The nanoparticles formed have narrow size distribution as indicated by their characterization using transmission electron microscopy (TEM) and dynamic light scattering. The surface chemistry of these nanoparticles was probed by FTIR spectroscopy indicating their successful capping with dopamine ligands, which was further confirmed using zetapotential measurements and thermogravimetric analysis. The comparative horseradish peroxidase (HRP)—like activity of these cationic mixed ferrites nanoparticles was studied at pH 4.6 using a negatively-charged 2, 2‧-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) as a chromogenic substrate in the presence of hydrogen peroxide. A time-dependent relative peroxidase-like activity follows the following order CoFe2O4-DOPA  >  MnFe2O4-DOPA  >  CuFe2O4-DOPA  >  NiFe2O4-DOPA  >  Fe3O4-DOPA. This diversity in HRP-like activity may be attributed to the different redox properties of ferrite nanoparticles when doped with M (Fe, Co Cu, Mn and Ni).

  15. Kinetic characterization of the oxidation of esculetin by polyphenol oxidase and peroxidase.

    PubMed

    Munoz-Munoz, Joseph Louis; Garcia-Molina, Francis; Varon, Raymond; Rodriguez-Lopez, Joseph Neptune; Garcia-Canovas, Francis; Tudela, Joseph

    2007-02-01

    Esculetin has been described as an inhibitor of tyrosinase and polyphenol oxidase and, therefore, of melanogenesis. In this work, we demonstrate that esculetin is not an inhibitor but a substrate of mushroom polyphenol oxidase (PPO) and horseradish peroxidase (POD), enzymes which oxidize esculetin, generating its o-quinone. Since o-quinones are very unstable, the usual way of determining the enzymatic activity (slope of recordings) is difficult. For this reason, we developed a chronometric method to characterize the kinetics of this substrate, based on measurements of the lag period in the presence of micromolar concentrations of ascorbic acid. The catalytic constant determined was of the same order for both enzymes. However, polyphenol oxidase showed greater affinity (a lower Michaelis constant) than peroxidase for esculetin. The affinity of PPO and POD towards oxygen and hydrogen peroxide was very high, suggesting the possible catalysis of both enzymes in the presence of low physiological concentrations of these oxidizing substrates. Taking into consideration optimum pHs of 4.5 and 7 for POD and PPO respectively, and the acidic pHs of melanosomes, the studies were carried out at pH 4.5 and 7. The in vivo pH might be responsible for the stronger effect of these enzymes on L-tyrosine and L-3,4-dihydroxyphenylanaline (L-DOPA) (towards melanogenesis) and on cumarins such as esculetin towards an alternative oxidative pathway.

  16. Reactivity of chemical respiratory allergens in the Peroxidase Peptide Reactivity Assay.

    PubMed

    Lalko, J F; Dearman, R J; Gerberick, G F; Troutman, J A; Api, A M; Kimber, I

    2013-03-01

    Sensitizing chemicals are commonly associated primarily with either skin or respiratory sensitization. In the Direct Peptide Reactivity Assay (DPRA), when compared with skin sensitizers, respiratory allergens have been demonstrated to selectively react with lysine rather than cysteine. The Peroxidase Peptide Reactivity Assay (PPRA) has been developed as a refinement to the DPRA. The PPRA incorporates dose-response analyses, mass spectroscopy for peptide detection and a horseradish peroxidase-hydrogen peroxide enzymatic system, increasing the potential to identify pro-haptens. In the investigations reported here, the PPRA was evaluated to determine whether it provides advantages for the identification of respiratory allergens. Twenty respiratory sensitizers, including five predicted to be pre-/pro-haptens were evaluated. The PPRA performed similarly to the DPRA with respect to identifying inherently reactive respiratory sensitizers. However, three respiratory sensitizers predicted to be pre-/pro-haptens (chlorhexidine, ethylenediamine and piperazine) were non-reactive and the general selectivity of the respiratory allergens for lysine was lost in the PPRA. Overall, the data indicate that the PPRA does not provide an advantage over the DPRA for discriminating allergens as either contact or respiratory sensitizers. Nevertheless, the PPRA provides a number of refinements to the DPRA that allow for an enhanced characterization of reactivity for both classes of chemical allergens.

  17. Actinobacterial peroxidases: an unexplored resource for biocatalysis.

    PubMed

    le Roes-Hill, Marilize; Khan, Nuraan; Burton, Stephanie Gail

    2011-07-01

    Peroxidases are redox enzymes that can be found in all forms of life where they play diverse roles. It is therefore not surprising that they can also be applied in a wide range of industrial applications. Peroxidases have been extensively studied with particular emphasis on those isolated from fungi and plants. In general, peroxidases can be grouped into haem-containing and non-haem-containing peroxidases, each containing protein families that share sequence similarity. The order Actinomycetales comprises a large group of bacteria that are often exploited for their diverse metabolic capabilities, and with recent increases in the number of sequenced genomes, it has become clear that this metabolically diverse group of organisms also represents a large resource for redox enzymes. It is therefore surprising that, to date, no review article has been written on the wide range of peroxidases found within the actinobacteria. In this review article, we focus on the different types of peroxidases found in actinobacteria, their natural role in these organisms and how they compare with the more well-described peroxidases. Finally, we also focus on work remaining to be done in this research field in order for peroxidases from actinobacteria to be applied in industrial processes.

  18. 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.

  19. 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.

  20. 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

  1. Secretory granule formation and membrane recycling by the trans-Golgi network in adipokinetic cells of Locusta migratoria in relation to flight and rest.

    PubMed

    Diederen, J H; Vullings, H G

    1995-03-01

    The influence of flight activity on the formation of secretory granules and the concomitant membrane recycling by the trans-Golgi network in the peptidergic neurosecretory adipokinetic cells of Locusta migratoria was investigated by means of ultrastructural morphometric methods. The patterns of labelling of the trans-Golgi network by the exogenous adsorptive endocytotic tracer wheat-germ agglutinin-conjugated horse-radish peroxidase and by the endogenous marker enzyme acid phosphatase were used as parameters and were measured by an automatic image analysis system. The results show that endocytosed fragments of plasma membrane with bound peroxidase label were transported to the trans-Golgi network and used to build new secretory granules. The amounts of peroxidase and especially of acid phosphatase within the trans-Golgi network showed a strong tendency to be smaller in flight-stimulated cells than in non-stimulated cells. The amounts of acid phosphatase in the immature secretory granules originating from the trans-Golgi network were significantly smaller in stimulated cells. The number of immature secretory granules positive for acid phosphatase tended to be higher in stimulated cells. Thus, flight stimulation of adipokinetic cells for 1 h influences the functioning of the trans-Golgi network; this most probably results in a slight enhancement of the production of secretory granules by the trans-Golgi network.

  2. Comparative activity of peroxidase-antibody conjugates with periodate and glutaraldehyde coupling according to an enzyme immunoassay.

    PubMed

    Tresca, J P; Ricoux, R; Pontet, M; Engler, R

    1995-01-01

    Horseradish peroxidase is often used as an antibody-coupled enzyme and several procedures have been developed to obtain IgG-peroxidase conjugates. The most widely used are coupling with periodate or glutaraldehyde. To compare the efficiency of these methods, the authors conducted periodate coupling or glutaraldehyde coupling in one or two steps, using the same batches of peroxidase, C-reactive protein (CRP) and anti-CRP monoclonal antibodies to develop a specially sensitive Elisa for CRP. Comparison of immunoenzymatic activities showed that periodate-mediated conjugation was much more efficient, because the activity of the coupling products was about 100 times greater than that of the products obtained after one or two-step conjugation with glutaraldehyde. The lower coupling efficiency observed with glutaraldehyde was not due to inactivation of the coupling agent or to a possible decrease in the affinity of the conjugates for CRP due to the coupling procedure. The differences in efficiency can be ascribed to the fact that periodate induced more coupling sites than glutaraldehyde. Periodate is therefore a better coupling agent for preparing conjugates to be used in Elisa or related techniques, in which conjugate size does not hinder accessibility to the antigen.

  3. 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.

  4. 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.

  5. Controlling thermo-reversibility of gelatin gels through a peroxidase-catalyzed reaction under mild conditions for mammalian cells.

    PubMed

    Sakai, Shinji; Moriyama, Kousuke; Kawakami, Koei

    2011-01-01

    A variety of cross-linking methods is used for obtaining gelatin gels having a tolerance to thermo-reversible gel-sol transition at physiological temperature. In this paper, we investigated the applicability of horseradish peroxidase-catalyzed cross-linking of tyrosine residues originally contained in native gelatin molecules for preparing such gelatin gels. The gelatin gels obtained through exposure to the enzymatic reaction showed a higher resistance to thermo-reversibility at 37°C than gels obtained through a thermally-induced gelation alone. In addition, the resistance property to thermo-reversible gel-sol transition was tunable by controlling enzymatic reaction conditions: higher peroxidase concentration and thermally-induced pre-gelation accomplished by cooling the gelatin solution prior to the enzymatic reaction produced gels with higher resistance to thermo-reversibility. Fibroblast cells enclosed in the gelatin gels obtained through the enzymatic reaction with thermally-induced pre-gelation showed 93% viability. These results demonstrate the feasibility of peroxidase-catalyzed reaction for obtaining gelatin gels having a tolerance to thermo-reversible gel-to-sol transition at physiological temperature toward applications in biomedical and biopharmaceutical fields.

  6. 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.

  7. 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

    2016-06-05

    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.

  8. 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.

  9. 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.

  10. 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.

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

    PubMed Central

    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. PMID:26810073

  12. Recents patents in the use of peroxidases.

    PubMed

    Alvarado, Berenize; Torres, Eduardo

    2009-01-01

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

  13. 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.

  14. Substrates and products of eosinophil peroxidase.

    PubMed Central

    van Dalen, C J; Kettle, A J

    2001-01-01

    Eosinophil peroxidase has been implicated in promoting oxidative tissue damage in a variety of inflammatory conditions, including asthma. It uses H(2)O(2) to oxidize chloride, bromide and thiocyanate to their respective hypohalous acids. The aim of this study was to establish which oxidants eosinophil peroxidase produces under physiological conditions. By measuring rates of H(2)O(2) utilization by the enzyme at neutral pH, we determined the catalytic rate constants for bromide and thiocyanate as 248 and 223 s(-1) and the Michaelis constants as 0.5 and 0.15 mM respectively. On the basis of these values thiocyanate is preferred 2.8-fold over bromide as a substrate for eosinophil peroxidase. Eosinophil peroxidase catalysed substantive oxidation of chloride only below pH 6.5. We found that when eosinophil peroxidase or myeloperoxidase oxidized thiocyanate, another product besides hypothiocyanite was formed; it also converted methionine into methionine sulphoxide. During the oxidation of thiocyanate, the peroxidases were present as their compound II forms. Compound II did not form when GSH was included to scavenge hypothiocyanite. We propose that the unidentified oxidant was derived from a radical species produced by the one-electron oxidation of hypothiocyanite. We conclude that at plasma concentrations of bromide (20-120 microM) and thiocyanate (20-100 microM), hypobromous acid and oxidation products of thiocyanate are produced by eosinophil peroxidase. Hypochlorous acid is likely to be produced only when substrates preferred over chloride are depleted. Thiocyanate should be considered to augment peroxidase-mediated toxicity because these enzymes can convert relatively benign hypothiocyanite into a stronger oxidant. PMID:11485572

  15. Engineering the active site of ascorbate peroxidase.

    PubMed

    Lloyd Raven, E; Celik, A; Cullis, P M; Sangar, R; Sutcliffe, M J

    2001-05-01

    Understanding the catalytic versatility of haem enzymes, and in particular the relationships that exist between different classes of haem-containing proteins and the mechanisms by which the apo-protein structure controls chemical reactivity, presents a major experimental and theoretical challenge. These issues are discussed in the general context of peroxidase and cytochrome P450 chemistry, and specific issues relating to the catalytic chemistry of ascorbate peroxidase are highlighted.

  16. 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.

  17. Synthesis of conducting polyelectrolyte complexes of polyaniline and poly(2-acrylamido-3-methyl-1-propanesulfonic acid) catalyzed by pH-stable palm tree peroxidase.

    PubMed

    Caramyshev, Alexei V; Evtushenko, Evgeny G; Ivanov, Viktor F; Barceló, Alfonso Ros; Roig, Manuel G; Shnyrov, Valery L; van Huystee, Robert B; Kurochkin, Iliya N; Vorobiev, Andrey Kh; Sakharov, Ivan Yu

    2005-01-01

    Comparison of the stability of five plant peroxidases (horseradish, royal palm tree leaf, soybean, and cationic and anionic peanut peroxidases) was carried out under acidic conditions favorable for synthesis of polyelectrolyte complexes of polyaniline (PANI). It demonstrates that palm tree peroxidase has the highest stability. Using this peroxidase as a catalyst, the enzymatic synthesis of polyelectrolyte complexes of PANI and poly(2-acrylamido-3-methyl-1-propanesulfonic acid) (PAMPS) was developed. The template polymerization of aniline was carried out in aqueous buffer at pH 2.8. Varying the concentrations of aniline, PAMPS, and hydrogen peroxide as reagents, favorable conditions for production of PANI were determined. UV-vis-NIR absorption and EPR demonstrated that PAMPS and PANI formed the electroactive complex similar to PANI doped traditionally using low molecular weight sulfonic acids. The effect of pH on conformational variability of the complex was evaluated by UV-vis spectroscopy. Atomic force microscopy showed that a size of the particles of the PANI-PAMPS complexes varied between 10 and 25 nm, depending on a concentration of PAMPS in the complex. The dc conductivity of the complexes depends also on the content of PAMPS, the higher conductivity being for the complexes containing the lower content of the polymeric template.

  18. 21 CFR 864.7675 - Leukocyte peroxidase test.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  19. Wheat germ agglutinin-conjugated liposomes incorporated with cardiolipin to improve neuronal survival in Alzheimer’s disease treatment

    PubMed Central

    Kuo, Yung-Chih; Lin, Che-Yu; Li, Jay-Shake; Lou, Yung-I

    2017-01-01

    Curcumin (CRM) and nerve growth factor (NGF) were entrapped in liposomes (LIP) with surface wheat germ agglutinin (WGA) to downregulate the phosphorylation of kinases in Alzheimer’s disease (AD) therapy. Cardiolipin (CL)-conjugated LIP carrying CRM (CRM-CL/LIP) and also carrying NGF (NGF-CL/LIP) were used with AD models of SK-N-MC cells and Wistar rats after an insult with β-amyloid peptide (Aβ). We found that CRM-CL/LIP inhibited the expression of phosphorylated p38 (p-p38), phosphorylated c-Jun N-terminal kinase (p-JNK), and p-tau protein at serine 202 and prevented neurodegeneration of SK-N-MC cells. In addition, NGF-CL/LIP could enhance the quantities of p-neurotrophic tyrosine kinase receptor type 1 and p-extracellular signal-regulated kinase 5 for neuronal rescue. Moreover, WGA-grafted CRM-CL/LIP and WGA-grafted NGF-CL/LIP significantly improved the permeation of CRM and NGF across the blood–brain barrier, reduced Aβ plaque deposition and the malondialdehyde level, and increased the percentage of normal neurons and cholinergic activity in the hippocampus of AD rats. Based on the marker expressions and in vivo evidence, current LIP carriers can be promising drug delivery systems to protect nervous tissue against Aβ-induced apoptosis in the brain during the clinical management of AD. PMID:28280340

  20. 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.

  1. [Determination of isothiocyanates and related compounds in mustard extract and horseradish extract used as natural food additives].

    PubMed

    Uematsu, Yoko; Hirata, Keiko; Suzuki, Kumi; Iida, Kenji; Ueta, Tadahiko; Kamata, Kunihiro

    2002-02-01

    Amounts of isothiocyanates and related compounds in a mustard extract and a horseradish extract for food additive use were determined by GC, after confirmation of the identity of GC peaks by GC/MS. Amounts of allyl isothiocyanate, which included that of allyl thiocyanate, because most of the allyl thiocyanate detected in the sample was assumed to have been formed from allyl isothiocyanate during GC analysis, were 97.6% and 85.4%, in the mustard extract and the horseradish extract, respectively. Total amounts of the identified isothiocyanates in the mustard extract and the horseradish extract were 98.5% and 95.4%, respectively. Allyl cyanide, a degradation product of allyl isothiocyanate, was found in the mustard extract and the horseradish extract at the levels of 0.57% and 1.73%, respectively. beta-Phenylethyl cyanide, a possible degradation product of beta-phenylethyl isothiocyanate, and allyl sulfides were found in the horseradish extract, at the levels of 0.13% and 0.46%, respectively. Allylamine, which is another degradation product of allyl isothiocyanate, was determined after acetylation, and was found in the mustard extract and the horseradish extract at the levels of 8 micrograms/g and 67 micrograms/g, respectively.

  2. Intrinsic Peroxidase-like Activity of Ficin

    PubMed Central

    Yang, Yufang; Shen, Dongjun; Long, Yijuan; Xie, Zhixiong; Zheng, Huzhi

    2017-01-01

    Ficin is classified as a sulfhydryl protease isolated from the latex of fig trees. In most cases, a particular enzyme fits a few types of substrate and catalyzes one type of reaction. In this investigation, we found sufficient proofs for the intrinsic peroxidase-like activity of ficin and designed experiments to examine its effectiveness in a variety of scenarios. Ficin can transform peroxidase substrates to colored products in the existence of H2O2. Our results also indicate that the active sites of peroxidase-like activity of ficin are different from that of protease, which reveals that one enzyme may catalyze more than one kind of substrate to perform different types of reactions. On the basis of these findings, H2O2 releasing from MCF-7 cells was detected successfully. Our findings support a wider application of ficin in biochemistry and open up the possibility of utilizing ficin as enzymatic mimics in biotechnology and environmental monitoring. PMID:28224979

  3. Intrinsic Peroxidase-like Activity of Ficin

    NASA Astrophysics Data System (ADS)

    Yang, Yufang; Shen, Dongjun; Long, Yijuan; Xie, Zhixiong; Zheng, Huzhi

    2017-02-01

    Ficin is classified as a sulfhydryl protease isolated from the latex of fig trees. In most cases, a particular enzyme fits a few types of substrate and catalyzes one type of reaction. In this investigation, we found sufficient proofs for the intrinsic peroxidase-like activity of ficin and designed experiments to examine its effectiveness in a variety of scenarios. Ficin can transform peroxidase substrates to colored products in the existence of H2O2. Our results also indicate that the active sites of peroxidase-like activity of ficin are different from that of protease, which reveals that one enzyme may catalyze more than one kind of substrate to perform different types of reactions. On the basis of these findings, H2O2 releasing from MCF-7 cells was detected successfully. Our findings support a wider application of ficin in biochemistry and open up the possibility of utilizing ficin as enzymatic mimics in biotechnology and environmental monitoring.

  4. 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

  5. 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).

  6. The effect of exogenous peroxidase on the evolution of murine leprosy.

    PubMed

    Rojas-Espinosa, Oscar; Wek-Rodríguez, Kendy; Arce-Paredes, Patricia

    2002-09-01

    Mycobacterium lepraemurium (MLM) is a successful parasite of murine macrophages; in vitro, this microorganism infects macrophages without triggering these cells' ability to produce either the reactive oxygen intermediaries (ROI) or the reactive nitrogen intermediaries (RNI), and ends up lodging within these cells, that, in addition, do not contain myeloperoxidase (MPO). In this study, we analyzed the effect of exogenous peroxidase on the evolution of murine leprosy. Bacilli were intraperitoneally injected, either alone (MLM) or precoated with horseradish peroxidase (MLM-PO), into two different groups of mice. At two-week intervals, the groups were blood-sampled to measure the levels of antibodies to protein- or lipid-MLM antigens. The extent of the disease was also assessed by looking at the histopathologic changes that occurred both in the liver and the spleen of the infected animals. We found that the animals injected with MLM-PO developed a disease that evolved at a slower pace than the disease that occurred in the animals injected with intact MLM. The difference between groups, both in terms of antibody levels and histological changes, was clearly evident at the intermediate stages of the disease (2 to 2.5 months), but was not so obvious at the more advanced stage of 3 months. Several possibilities to explain how the PO-coated bacilli might have regained their infectiousness are discussed. Lowering the infective dose of MLM and MLM-PO from 5 x 10(7) bacilli to 5 x 10(6) bacilli would, probably, have resulted in a different outcome of the disease: more extended in the MLM-group than in the MLM-PO group.

  7. Enhanced killing of Acanthamoeba cysts with a plant peroxidase-hydrogen peroxide-halide antimicrobial system.

    PubMed

    Hughes, Reanne; Andrew, Peter W; Kilvington, Simon

    2003-05-01

    The activity of H(2)O(2) against the resistant cyst stage of the pathogenic free-living amoeba Acanthamoeba was enhanced by the addition of KI and either horseradish peroxidase or soybean peroxidase or, to a lesser degree, lactoperoxidase. This resulted in an increase in the cysticidal activity of 3% (wt/vol) H(2)O(2), and there was >3-log killing in 2 h, compared with the 6 h required for comparable results with the peroxide solution alone (P < 0.05). With 2% H(2)O(2), enhancement was observed at all time points (P < 0.05), and total killing of the cyst inoculum occurred at 4 h, compared with 6 h for the peroxide alone. The activity of sublethal 1% H(2)O(2) was enhanced to give 3-log killing after 8 h of exposure (P < 0.05). No enhancement was obtained when KCl or catalase was used as a substitute in the reaction mixtures. The H(2)O(2) was not neutralized in the enhanced system during the experiments. However, in the presence of a platinum disk used to neutralize H(2)O(2) in contact lens care systems, the enhanced 2% H(2)O(2) system gave 2.8-log killing after 6 h or total cyst killing by 8 h, and total neutralization of the H(2)O(2) occurred by 4 h. In contrast, 2% H(2)O(2) alone resulted in <0.8-log killing of cysts in the presence of the platinum disk due to rapid (<1 h) neutralization of the peroxide. Our observations could result in significant improvement in the efficacy of H(2)O(2) contact lens disinfection systems against Acanthamoeba cysts and prevention of acanthamoeba keratitis.

  8. Enhanced Killing of Acanthamoeba Cysts with a Plant Peroxidase-Hydrogen Peroxide-Halide Antimicrobial System

    PubMed Central

    Hughes, Reanne; Andrew, Peter W.; Kilvington, Simon

    2003-01-01

    The activity of H2O2 against the resistant cyst stage of the pathogenic free-living amoeba Acanthamoeba was enhanced by the addition of KI and either horseradish peroxidase or soybean peroxidase or, to a lesser degree, lactoperoxidase. This resulted in an increase in the cysticidal activity of 3% (wt/vol) H2O2, and there was >3-log killing in 2 h, compared with the 6 h required for comparable results with the peroxide solution alone (P < 0.05). With 2% H2O2, enhancement was observed at all time points (P < 0.05), and total killing of the cyst inoculum occurred at 4 h, compared with 6 h for the peroxide alone. The activity of sublethal 1% H2O2 was enhanced to give 3-log killing after 8 h of exposure (P < 0.05). No enhancement was obtained when KCl or catalase was used as a substitute in the reaction mixtures. The H2O2 was not neutralized in the enhanced system during the experiments. However, in the presence of a platinum disk used to neutralize H2O2 in contact lens care systems, the enhanced 2% H2O2 system gave 2.8-log killing after 6 h or total cyst killing by 8 h, and total neutralization of the H2O2 occurred by 4 h. In contrast, 2% H2O2 alone resulted in <0.8-log killing of cysts in the presence of the platinum disk due to rapid (<1 h) neutralization of the peroxide. Our observations could result in significant improvement in the efficacy of H2O2 contact lens disinfection systems against Acanthamoeba cysts and prevention of acanthamoeba keratitis. PMID:12732522

  9. 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

  10. 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.

  11. Determination of asulam by fast stopped-flow chemiluminescence inhibition of luminol/peroxidase.

    PubMed

    García Sánchez, F; Navas Díaz, A; Delgado Téllez, C; Algarra, M

    2008-10-19

    An efficient, sensitive and fast stopped-flow method has been developed to determine asulam in water, based on its inhibition effect on the horseradish peroxidase-luminol-hydrogen peroxide chemiluminescence reaction, (HRP-luminol-H(2)O(2)). Ultra fast data acquisition (0.20s) facilitates excellent selectivity because no interferences from concomitants in the matrix act in such short time scale. The precision as repeatability (expressed as relative standard deviation, n=10) was 0.4% at a 40 pM level. The detection limit was 1.5 pM (0.35 ng/L) and 7.15 pM in pure and raw water, respectively. The calibration data over the range 5-60 pM present a correlation coefficient of r=0.9993. The proposed method has been applied to determine asulam in water samples by using solid-phase extraction (SPE). Mean recovery value was 98.1+/-2% at 50 pM level.

  12. Peroxidase-catalyzed-3-(glutathion-S-yl)-p,p'-biphenol formation.

    PubMed

    McGirr, L G; Subrahmanyam, V V; Moore, G A; O'Brien, P J

    1986-10-15

    Oxidation of p,p'-biphenol with horseradish peroxidase (HRP)-hydrogen peroxide in the presence of bovine serum albumin or with bone marrow cell homogenate-hydrogen peroxide resulted in the formation of reactive products that conjugate with protein. Glutathione prevented the protein binding. Glutathione readily reacted with p,p'-biphenoquinone, the principal oxidation product of p,p'-biphenol in the HRP-hydrogen peroxide system and resulted in the formation of several glutathione conjugates, p,p'-biphenol and small amounts of oxidized glutathione. The major glutathione conjugate was identified as 3-(glutathion-S-yl)-p,p'-biphenol by high field nuclear magnetic resonance and fast atom bombardment mass spectrometry. The same conjugate was formed in the bone marrow homogenate-hydrogen peroxide system. p,p'-Biphenoquinone reduction by glutathione to p,p'-biphenol without glutathione oxidation was explained by the rapid reduction of p,p'-biphenoquinone by 3-(glutathion-S-yl)-p,p'-biphenol.

  13. 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.

  14. An optical sensor for pesticide determination based on the autoindicating optical properties of peroxidase.

    PubMed

    de Marcos, Susana; Callizo, Esther; Mateos, Elena; Galbán, Javier

    2014-05-01

    During the enzymatic reaction of the heme-protein Horseradish peroxidase (HRP) with hydrogen peroxide there are changes in the molecular absorption spectra of HRP and its different oxidation states which can be used for quantitative determination of the substrate. One of these intermediate oxidation states is the HRPII, with iron as an oxyferryl. This compound is assumed to be responsible for the organophosphate pesticide degradation in the Fenton reaction. In this work, the enzymatic HRP-H₂O₂ reaction has been studied, based on the effect of different pesticides on the mechanism reaction; these modifications have been used for the quantitative determination of pesticides. A mathematical model has been developed relating to the analytical signal with the pesticide concentration. Three organophosphate pesticides (diazinon, trichlorfon and tetrachlorvinphos) and one sulfamide (dichlofluanid) have been used to demonstrate the viability of the methodology and the accomplishment fulfillment of the model. Tetrachlorvinphos was chosen as the pesticide model to develop the optical sensor film for continuous pesticide determination, consisting of HRP immobilized in a polyacrylamide gel. The sensor can be used for at least 15 days and responds linearly to tetrachlorvinphos concentrations in the range from 4.0 × 10(-7) to 4.0 × 10(-6)mol L(-1). The main advantage of the methodology is its reversibility in contrast to the irreversible Fenton reaction. The HRP-H2O2 methodology has been used to measure the pesticides in a waste water sample spiked with tetrachlorvinphos.

  15. TAML activator-based amperometric analytical devices as alternatives to peroxidase biosensors.

    PubMed

    Ryabov, Alexander D; Cerón-Camacho, Ricardo; Saavedra-Díaz, Omar; Denardo, Matthew A; Ghosh, Anindya; Le Lagadec, Ronan; Collins, Terrence J

    2012-11-06

    The ferric TAML catalysts [Fe{C(6)H(2)-1,2-( NCOCMe(2)NCO)(2)CMe(2)}(OH(2))](-) (1) with counterions Na(+) (a) and PPh(4)(+) (b) function similar to horseradish peroxidase in the mediated electron transfer relays, which constitute a basis for amperometric biosensors. The mediators are mono- and bis-cyclometalated Ru and Os compounds of the type of [M(C∼N)(x)(N∼N)(3-x)](m+) with x = 1 and 2 (N∼N = 2,2'-bipyridine, (-)C∼N = 2-phenylpyridinato). Cyclic voltammograms of the Ru and Os compounds are not affected by 1a though cathodic currents increase drastically in the presence of hydrogen peroxide. The reduction potentials of [M(C∼N)(x)(N∼N)(3-x)](m+) complexes vary with both the nature of metal (Ru or Os) and the number of cyclometalated ligands x (1 or 2) and therefore the potential of working electrode can be set in the range of from -0.1 to +0.6 V versus the normal hydrogen electrode (NHE). A prototype of a biosensor for H(2)O(2) is described, in which the 1b catalyst and [Os(C∼N)(2)(N∼N)](+) mediator were coimmobilized on the surface of the glassy carbon electrode using a polymeric coating.

  16. 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.

  17. 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…

  18. The study of ascorbate peroxidase, catalase and peroxidase during in vitro regeneration of Argyrolobium roseum.

    PubMed

    Habib, Darima; Chaudhary, Muhammad Fayyaz; Zia, Muhammad

    2014-01-01

    Here, we demonstrate the micropropagation protocol of Argyrolobium roseum (Camb.), an endangered herb exhibiting anti-diabetic and immune-suppressant properties, and antioxidant enzymes pattern is evaluated. Maximum callogenic response (60 %) was observed from leaf explant at 1.0 mg L(-1) 1-nephthalene acetic acid (NAA) and 0.5 mg L(-1) 6-benzyl aminopurine (BA) in Murashige and Skoog (MS) medium using hypocotyl and root explants (48 % each). Addition of AgNO3 and PVP in the culture medium led to an increase in callogenic response up to 86 % from leaf explant and 72 % from hypocotyl and root explants. The best shooting response was observed in the presence of NAA, while maximum shoot length and number of shoots were achieved based on BA-supplemented MS medium. The regenerated shoots were rooted and successfully acclimatized under greenhouse conditions. Catalase and peroxidase enzymes showed ascending pattern during in vitro plant development from seed while ascorbate peroxidase showed descending pattern. Totally reverse response of these enzymes was observed during callus induction from three different explants. During shoot induction, catalase and peroxidase increased at high rate while there was a mild reduction in ascorbate peroxidase activity. Catalase and peroxidase continuously increased; on the other hand, ascorbate peroxidase activity decreased during root development and acclimatization states. The protocol described here can be employed for the mass propagation and genetic transformation of this rare herb. This study also highlights the importance and role of ascorbate peroxidase, catalase, and peroxidase in the establishment of A. roseum in vitro culture through callogenesis and organogenesis.

  19. 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.

  20. The co-catalytic effect of chlorpromazine on peroxidase-mediated oxidation of melatonin: enhanced production of N1-acetyl-N2-formyl-5-methoxykynuramine.

    PubMed

    Ximenes, Valdecir F; Rodrigues, Ana Paula; Cabello, Cláudio; de Menezes, Manoel L; Fernandes, João Roberto

    2008-03-01

    Accumulating evidence points to relationships between increased production of reactive oxygen or decreased antioxidant protection in schizophrenic patients. Chlorpromazine (CPZ), which remains a benchmark treatment for people with schizophrenia, has been described as a pro-oxidant compound. Because the antioxidant compound melatonin exerts protective effects against CPZ-induced liver disease in rats, in this investigation, our main objective was to study the effect of CPZ as a co-catalyst of peroxidase-mediated oxidation of melatonin. We found that melatonin was an excellent reductor agent of preformed CPZ cation radical (CPZ(*+)). The addition of CPZ during the horseradish peroxidase (HRP)-catalyzed oxidation of melatonin provoked a significant increase in the rate of oxidation and production of N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK). Similar results were obtained using myeloperoxidase. The effect of CPZ on melatonin oxidation was rather higher at alkaline pH. At pH 9.0, the efficiency of oxidation of melatonin was 15 times higher and the production of AFMK was 30 times higher as compared with the assays in the absence of CPZ. We suggest that CPZ is able to exacerbate the rate of oxidation of melatonin by an electron transfer mechanism where CPZ(*+), generated during the peroxidase-catalyzed oxidation, is able to efficiently oxidize melatonin.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  2. Layer I of striate cortex of Tupaia glis and Galago senegalensis: projections from thalamus and claustrum revealed by retrograde transport of horseradish peroxidase.

    PubMed

    Carey, R G; Fitzpatrick, D; Diamond, I T

    1979-08-01

    We have examined the origin of the subcortical projections to the superficial layers of the striate cortex in Tupaia glis and Galago senegalensis by using the retrograde transport of HRP. Crystals of HRP were laid directly on the moist pial surface of the cortex which had been gently pricked with a small glass pipette. The diffusion of HRP was limited to layers I and II by restricting the length of time that the HRP was in contact with the surface. Following the application of HRP to the striate cortex, labeled cells were found in restricted regions of the lateral geniculate body of both species. Layers 4 and 5 of galago and layer 3 of tree shrew contained dense clusters of labeled cells. Labeled neurons were also found in the zones between the layers of the lateral geniculate body in both species and these cells were always in register with the labeled cells within the layers. In galago, curved columns of labeled cells were observed in the inferior and superior subdivisions of the pulvinar nucleus. These columns were arranged in the shape of two arcs, joined at the fiber bundle which separates the two subdivisions. The position of the bands in the pulvinar nucleus varied with the locus of the application in the striate cortex. While no labeled cells were seen in the body of the pulvinar nucleus of tree shrew, small labeled neurons were found in the external medullary lamina forming the capsule of the pulvinar nucleus. These cells were continuous with a larger population of labeled cells in the lateral intermediate nucleus. In both species, labeled cells were also found in the intralaminar nuclei (particularly the paracentral nucleus) and in the dorsal-caudal portion of the claustrum. In the claustrum, few unlabeled neurons were present within the zone containing labeled cells. In conclusion, layer I os striate cortex appears to be the site of convergence of several projection systems originating from principal and intralaminar thalamic nuclei as well as the claustrum. The significance of this overlap is discussed in terms of the total cortical extent of each system.

  3. 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

  4. 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.

  5. 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.

  6. The biological effects of vanadyl curcumin and vanadyl diacetylcurcumin complexes: the effect on structure, function and oxidative stability of the peroxidase enzyme, antibacterial activity and cytotoxic effect.

    PubMed

    Hamidi, Akram; Hassani, Leila; Mohammadi, Fakhrossadat; Jahangoshayi, Parisa; Mohammadi, Khosro

    2016-12-01

    Curcumin has multiple pharmacological effects, but it has poor stability. Complexation of curcumin with metals improves its stability. Here, the effects of vanadyl curcumin and vanadyl diacetylcurcumin on the function and structure of horseradish peroxidase enzyme were evaluated by spectroscopic techniques. Cytotoxic effect of the complexes was also assessed on MCF-7 breast cancer, bladder and LNCaP prostate carcinoma cell line. The results showed that the complexes improve catalytic activity of HRP, and also increase its tolerance against the oxidative condition. The result also indicated that the affinity of HRP for hydrogen peroxide substrate decreases, while the affinity increases for phenol substrate. Circular dichroism and fluorescence spectroscopies showed that compactness of the enzyme structure around the catalytic heme group and the distance between the heme group and tryptophan residue decreases after the binding. The antibacterial and cytotoxic results indicated that the complexes have anticancer potential, but they have no considerable antibacterial activity.

  7. 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

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

    PubMed

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

    2014-05-13

    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. 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.

  10. 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.

  11. Direct synaptic connections between superior colliculus afferents and thalamo-insular projection neurons in the feline suprageniculate nucleus: a double-labeling study with WGA-HRP and kainic acid.

    PubMed

    Hoshino, Kaeko; Horie, Masao; Nagy, Attila; Berényi, Antal; Benedek, György; Norita, Masao

    2010-01-01

    The suprageniculate nucleus (Sg) of the feline thalamus, which subserves largely unimodal sensory and orientation behavior, receives input from the deep layers of the superior colliculus (SC), and projects to the suprasylvian cortical areas, such as the anterior ectosylvian visual area and the insular visual area (IVA), which contain visually responsive neurons. Through a double tract-tracing procedure involving the injection of wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) into the IVA and the injection of kainic acid into the SC, this study sought to determine the nature of the synaptic relationship between the SC afferents and the thalamo-cortical projection neurons. WGA-HRP injections labeled numerous neurons in the Sg, while kainic acid injections destroyed many tectothalamic terminals in the Sg. The distributions of the WGA-HRP-labeled neurons and the degenerated axon terminals overlapped in the dorsal part of the Sg. Electron microscopic observations demonstrated that the degenerated axon terminals made synaptic contacts with the dendrites of the WGA-HRP-labeled neurons in this overlapping region of the Sg. These results provide the first anatomical evidence that the Sg may play a role in the key relay of visual information from the SC to the IVA, within an identified extrageniculo-cortical pathway.

  12. 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.

  13. Ultrastructural evidence for a direct excitatory pathway from the nucleus retroambiguus to lateral longissimus and quadratus lumborum motoneurons in the female golden hamster.

    PubMed

    Gerrits, Peter O; Mouton, Leonora J; de Weerd, Henk; Georgiadis, Janniko R; Krukerink, Marco; Holstege, Gert

    2004-12-20

    During mating, the female golden hamster displays a stereotyped specific receptive posture, characterized by lordosis of the back, elevation of the tail, and extension of the legs. Muscles involved in this posture are thought to be iliopsoas, cutaneus trunci, lateral longissimus (LL), and quadratus lumborum (QL). Lesion studies in rats suggest that mating behavior is controlled by the mesencephalic periaqueductal gray (PAG). The PAG does not project directly to the motoneurons innervating the muscles involved in mating, but is thought to make use of the nucleus retroambiguus (NRA) as relay. The NRA is located ventrolaterally in the most caudal medulla, and projects directly to iliopsoas and cutaneus trunci motoneuronal cell groups. The question is whether this is also true for LL and QL muscles. Retrograde HRP tracing experiments revealed that LL and QL motoneurons are located medially in the ventral horn of the T12-L6 and T13-L4 segments, respectively. A subsequent ultrastructural study combined wheatgerm agglutinin-conjugated horseradish peroxidase injections in the NRA with cholera-toxin B-subunit injections in LL and QL muscles. The results revealed monosynaptic contacts between anterogradely labeled NRA-fiber terminals with retrogradely labeled dendrites of both LL and QL motoneurons. Almost all these terminals had asymmetrical synapses and contained spherical vesicles, suggesting an excitatory function of this NRA-motoneuronal pathway. These results correspond with the hypothesis that in hamster the PAG-NRA-motoneuronal projection not only involves motoneurons of iliopsoas and cutaneus trunci but also of LL and QL.

  14. Afferent and efferent connections of the nucleus rotundus demonstrated by WGA-HRP in the chick.

    PubMed

    Hu, M; Naito, J; Chen, Y; Ohmori, Y; Fukuta, K

    2003-12-01

    Organization of the fibre connections in the chick nucleus rotundus (Rt) was investigated by an axonal tracing method using wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). After an injection of WGA-HRP into the Rt, labelled neurones were observed in the striatum griseum centrale (SGC) in both sides of the tectum (TO) and in the ipsilateral nucleus subpretectalis/nucleus interstito-pretecto-subpretectalis (SP/IPS). Labelled fibres and terminals were also found in the ipsilateral ectostriatum (Ect). These fibre connections were topographically organized rostrocaudally. In the TO-Rt projection, the rostral and the dorsocaudal parts of the Rt received afferents from the superficial part of the SGC, the middle part of the Rt received afferents from the intermediate part of the SGC, and the ventrocaudal part of the Rt received mainly fibres from the deep part of the SGC. These topographic projections were accompanied by a considerable number of diffuse projections to the thalamic regions surrounding the Rt. In addition, the rostral and middle caudal parts of the Rt received afferents from the lateral and medial parts of the SP/IPS, respectively, and respective parts of the Rt sent efferents to the lateral and medial parts of the Ect.

  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. DyP, a unique dye-decolorizing peroxidase, represents a novel heme peroxidase family: ASP171 replaces the distal histidine of classical peroxidases.

    PubMed

    Sugano, Yasushi; Muramatsu, Riichi; Ichiyanagi, Atsushi; Sato, Takao; Shoda, Makoto

    2007-12-14

    DyP, a unique dye-decolorizing enzyme from the fungus Thanatephorus cucumeris Dec 1, has been classified as a peroxidase but lacks homology to almost all other known plant peroxidases. The primary structure of DyP shows moderate sequence homology to only two known proteins: the peroxide-dependent phenol oxidase, TAP, and the hypothetical peroxidase, cpop21. Here, we show the first crystal structure of DyP and reveal that this protein has a unique tertiary structure with a distal heme region that differs from that of most other peroxidases. DyP lacks an important histidine residue known to assist in the formation of a Fe4+ oxoferryl center and a porphyrin-based cation radical intermediate (compound I) during the action of ubiquitous peroxidases. Instead, our tertiary structural and spectrophotometric analyses of DyP suggest that an aspartic acid and an arginine are involved in the formation of compound I. Sequence analysis reveals that the important aspartic acid and arginine mentioned above and histidine of the heme ligand are conserved among DyP, TAP, and cpop21, and structural and phylogenetic analyses confirmed that these three enzymes do not belong to any other families of peroxidase. These findings, which strongly suggest that DyP is a representative heme peroxidase from a novel family, should facilitate the identification of additional new family members and accelerate the classification of this novel peroxidase family.

  17. Evaluation of an Aqueous Extract from Horseradish Root (Armoracia rusticana Radix) against Lipopolysaccharide-Induced Cellular Inflammation Reaction

    PubMed Central

    Herz, Corinna; Tran, Hoai Thi Thu; Márton, Melinda-Rita; Maul, Ronald; Schreiner, Monika

    2017-01-01

    Horseradish (Armoracia rusticana) is a perennial crop and its root is used in condiments. Traditionally, horseradish root is used to treat bacterial infections of the respiratory tract and urinary bladder. The antiphlogistic activity, determined in activated primary human peripheral blood mononuclear cells (PBMC), was evaluated for an aqueous extract and its subfractions, separated by HPLC. Compound analysis was done by UHPLC-QToF/MS and GC-MS. The aqueous extract concentration-dependently inhibited the anti-inflammatory response to lipopolysaccharide (LPS) in terms of TNF-α release at ≥37 μg/mL. Further, the cyclooxygenase as well as lipoxygenase pathway was blocked by the extract as demonstrated by inhibition of COX-2 protein expression and PGE2 synthesis at ≥4 μg/mL and leukotriene LTB4 release. Mechanistic studies revealed that inhibition of ERK1/2 and c-Jun activation preceded COX-2 suppression upon plant extract treatment in the presence of LPS. Chemical analysis identified target compounds with a medium polarity as relevant for the observed bioactivity. Importantly, allyl isothiocyanate, which is quite well known for its anti-inflammatory capacity and as the principal pungent constituent in horseradish roots, was not relevant for the observations. The results suggest that horseradish root exerts an antiphlogistic activity in human immune cells by regulation of the COX and LOX pathway via MAPK signalling. PMID:28182113

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

    PubMed Central

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

    2013-01-01

    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. PMID:22966760

  20. 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).

  1. 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.

  2. 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.

  3. Protein-Metal Organic Framework Hybrid Composites with Intrinsic Peroxidase-like Activity as a Colorimetric Biosensing Platform.

    PubMed

    Yin, Yuqing; Gao, Chen Ling; Xiao, Qi; Lin, Guo; Lin, Zian; Cai, Zongwei; Yang, Huang-Hao

    2016-10-04

    Artificial enzyme mimetics have received considerable attention because natural enzymes have some significant drawbacks, including enzyme autolysis, low catalytic activity, poor recovery and low stability to environmental changes. Herein, we demonstrated a facile approach for one-pot synthesis of hemeprotein-metal organic framework hybrid composites (H-MOFs) by using bovine hemoglobin (BHb) and zeolitic imidazolate framework-8 (ZIF-8) as a model reaction system. Surprisingly, the new hybrid composites exhibits 423% increase in peroxidase-like catalytic activity compared to free BHb. Taking advantages of the unique pore structure of H-MOFs with high catalytic property, a H-MOFs-based colorimetric biosensing platform was newly constructed and applied for the fast and sensitive detection of hydrogen peroxide (H2O2) and phenol. The corresponding detection limits as low as 1.0 μM for each analyte with wide linear ranges (0-800 μM for H2O2 and 0-200 μM for phenol) were obtained by naked-eye visualization. Significantly, sensitive and selective method for visual assay of trace H2O2 in cell and phenol in sewage was achieved with this platform. The stability of H-MOFs was also examined and excellent reproducibility and recyclability without losing in its activity were observed. In addition, the general applicability of H-MOFs was also investigated by using other hemeproteins (horseradish peroxidase, and myoglobin) and the corresponding catalytic activities were 291% and 273% enhancement, respectively. This present work not only expands the application of MOFs, but also provides an alternative technique for biological and environmental sample assay.

  4. Feeding on poplar leaves by caterpillars potentiates foliar peroxidase action in their guts and increases plant resistance.

    PubMed

    Barbehenn, Raymond; Dukatz, Chris; Holt, Chris; Reese, Austin; Martiskainen, Olli; Salminen, Juha-Pekka; Yip, Lynn; Tran, Lan; Constabel, C Peter

    2010-12-01

    Peroxidases (PODs) are believed to act as induced and constitutive defenses in plants against leaf-feeding insects. However, little work has examined the mode of action of PODs against insects. Putative mechanisms include the production of potentially antinutritive and/or toxic semiquinone free radicals and quinones (from the oxidation of phenolics), as well as increased leaf toughness. In this study, transgenic hybrid poplar saplings (Populus tremula × Populus alba) overexpressing horseradish peroxidase (HRP) were produced to examine the impact of elevated HRP levels on the performance and gut biochemistry of Lymantria dispar caterpillars. HRP-overexpressing poplars were more resistant to L. dispar than wild-type (WT) poplars when the level of a phenolic substrate of HRP (chlorogenic acid) was increased, but only when leaves had prior feeding damage. Damaged (induced) leaves produced increased amounts of hydrogen peroxide, which was used by HRP to increase the production of semiquinone radicals in the midguts of larvae. The decreased growth rates of larvae that fed on induced HRP-overexpressing poplars resulted from post-ingestive mechanisms, consistent with the action of HRP in their midguts. The toughness of HRP-overexpressing leaves was not significantly greater than that of WT leaves, whether or not they were induced. When leaves were coated with ellagitannins, induced HRP leaves also produced elevated levels of semiquinone radicals in the midgut. Decreased larval performance on induced HRP leaves in this case was due to post-ingestive mechanisms as well as decreased consumption. The results of this study provide the first demonstration that a POD is able to oxidize phenolics within an insect herbivore's gut, and further clarifies the chemical conditions that must be present for PODs to function as antiherbivore defenses.

  5. Interactions of soil-derived dissolved organic matter with phenol in peroxidase-catalyzed oxidative coupling reactions.

    PubMed

    Huang, Qingguo; Weber, Walter J

    2004-01-01

    The influence of dissolved soil organic matter (DSOM) derived from three geosorbents of different chemical composition and diagenetic history on the horseradish peroxidase (HRP) catalyzed oxidative coupling reactions of phenol was investigated. Phenol conversion and precipitate-product formation were measured, respectively, by HPLC and radiolabeled species analysis. Fourier transform infrared (FTIR) spectroscopy and capillary electrophoresis (CE) were used to characterize the products of enzymatic coupling, and the acute toxicities of the soluble products were determined by Microtox assay. Phenol conversion and precipitate formation were both significantly influenced by cross-coupling of phenol with dissolved organic matter, particularly in the cases of the more reactive and soluble DSOMs derived from two diagenetically "young" humic-type geosorbents. FTIR and CE characterizations indicate that enzymatic cross-coupling in these two cases leads to incorporation of phenol in DSOM macromolecules, yielding nontoxic soluble products. Conversely, cross-coupling appears to proceed in parallel with self-coupling in the presence of the relatively inert and more hydrophobic DSOM derived from a diagenetically "old" kerogen-type shale material. The products formed in this system have lower solubility and precipitate more readily, although their soluble forms tend to be more toxic than those formed by dominant cross-coupling reactions in the humic-type DSOM solutions. Several of the findings reported may be critically important with respect to feasibility evaluations and the engineering design of associated remediation schemes.

  6. 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.

  7. Hemin-micelles immobilized in alginate hydrogels as artificial enzymes with peroxidase-like activity and substrate selectivity.

    PubMed

    Qu, Rui; Shi, Hejin; Wang, Ruolin; Cheng, Tangjian; Ma, Rujiang; An, Yingli; Shi, Linqi

    2017-02-28

    Artificial enzymes are widely investigated to mimic the active center and the recognition center of natural enzymes. The active center is responsible for the catalytic activity of enzymes, and the recognition center provides enzymes with specificity. Most of the previous studies on artificial enzymes preferred to solve the problem of activity rather than specificity due to the complexity of the enzyme structures related to substrate recognition. Inspired by the multilevel structures of enzymes and the unique net-structures of hydrogels, hemin-micelles immobilized in alginate hydrogels (HM-AH) were constructed by multistep self-assembly. The hemin-micelle was the active center and mimicked the microenvironment of the catalytic site in horseradish peroxidase (HRP). The alginate hydrogel further enhanced the catalytic activity and stability of hemin-micelles and endowed the artificial enzymes with a catalytic capability in harsh water conditions and non-polar organic solvents. The hydrogel also served as the recognition center, which exhibited substrate selectivity owing to the diffusivity differentiations of substrates in hydrogel fibers. It is the first example of constructing a micelle-hydrogel complex system as an artificial enzyme with both catalytic activity and substrate selectivity by the method of multistep self-assembly.

  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. Non-specific pinocytosis by human endothelial cells cultured as multicellular aggregates: uptake of lucifer yellow and horse radish peroxidase.

    PubMed

    Catizone, A; Chiantore, M V; Andreola, F; Coletti, D; Medolago Albani, L; Alescio, T

    1996-12-01

    We have analyzed the pattern of time-dependent and concentration-dependent incorporation of Lucifer Yellow CH (LY) and Horseradish Peroxidase (HRP) by human umbilical vein endothelial cells cultured on a non-adhesive substratum, where they they become organized into stable, multicellular aggregates. The data were compared with those previously obtained from low-density cultures of non-growing endothelial cells adherent to plastic. While the linear trend of the incorporation kinetics is preserved, the rate of uptake with both time and concentrations is highly dependent on the culture conditions, namely typology of cell-cell and cell-substrate interactions. An at least two-fold increase of the rate of uptake was observed with both markers in the aggregated cells. The extracellular concentration of LY required to saturate the binding capacity of the cell surface shifts from approximately 0.25 mg/ml, with the adherent cells, to approximately 0.5 mg/ml in the aggregated cells; the rate of uptake of three different forms of HRP shows, besides a sharp quantitative increase, also qualitative variations, testified by differential changes of their incorporation rates. These results are entirely consistent with the assumption that the association of the endothelial cells into multicellular aggregates increases the rate of pinocytic uptake by modifying the physicochemical properties of the cell surface, thereby increasing its differential affinity for the extracellular markers.

  10. Degradation of pentachlorophenol by a novel peroxidase-catalyzed process in the presence of reduced nicotinamide adenine dinucleotide.

    PubMed

    Li, Haitao; Li, Yuping; Cao, Hongbin; Li, Xingang; Zhang, Yi

    2011-03-01

    A novel horseradish peroxidase (HRP)-catalyzed H₂O₂ process in the presence of reduced nicotinamide adenine dinucleotide (NADH) was applied to remove aqueous pentachlorophenol (PCP). Parameters (pH, H₂O₂ concentration, HRP activity and NADH dosage) on PCP removal were investigated. It was found that initial 0.05mM PCP was removed by 98% in HRP-NADH-H₂O₂ system at pH 5.0 and 30°C for 1h. Addition of O₂ in HRP-NADH-H₂O₂ system enhanced the removal rate of PCP due to promoting hydroxyl radicals (.OH) and superoxide anion radical (.O₂⁻) generation, which were confirmed by electron paramagnetic resonance (EPR)-spin trapping method. PCP removal efficiency decreased when .O₂⁻ and H₂O₂ were scavenged by superoxide dismutase and catalase in HRP-NADH-O₂ system, indicating that .OH/.O₂⁻ played a great role in the degradation of PCP. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that octachlorinated dibenzodioxin (OCDD) in residual solution was reduced after treated by the HRP-NADH-O₂ process, resulting in lower toxicity of treated solution than conventional enzymatic process. Two enzymatic-catalysis pathways were proposed for PCP removal in HRP-NADH-H₂O₂/O₂ system: (i) OH/.O₂⁻ free radical oxidation (ii) conventional phenoxy polymerization.

  11. Differential peroxidase activities in three different crops upon insect feeding

    PubMed Central

    Singh, Harpal; Dixit, Sameer; Verma, Praveen Chandra; Singh, Pradhyumna Kumar

    2013-01-01

    Peroxidases are the ubiquitous enzyme and reported to be present in all living genera. They catalyses reduction of peroxide and generate reactive oxygen species. In the present study we demonstrated that insect infestation induces peroxidase activity in sap and total soluble protein (TSP) of plant leaves. Three important crop plants viz. tomato, cowpea and cotton were used for this study. After infestation of chewing insect, Peroxidase activity in the sap and TSP of all the studied plants were enhanced in the range of 1.6 to 3.14 fold. Similar observations were also obtained with feeding of sap sucking insects, in which increment in peroxidase activity of sap and TSP was in the range of 1.8 to 2.53 fold. Enhanced peroxidase activity was reconfirmed by in-gel peroxidase assay. Enzyme kinetic study showed turn over efficiency of peroxidase from cotton (~101.3 min-1) was almost similar to tomato (~100.8 min-1) but higher than cowpea (~98.21min-1). MS/MS analysis of observed band showed significant similarity with the reported peroxidases in database. PMID:23857346

  12. Hydrogen peroxide-mediated inactivation of two chloroplastic peroxidases, ascorbate peroxidase and 2-cys peroxiredoxin.

    PubMed

    Kitajima, Sakihito

    2008-01-01

    Reactive oxygen species (ROS), such as the superoxide anion and hydrogen peroxide, are generated by the photosystems because photoexcited electrons are often generated in excess of requirements for CO2 fixation and used for reducing molecular oxygen, even under normal environmental conditions. Moreover, ROS generation is increased in chloroplasts if plants are subjected to stresses, such as drought, high salinity and chilling. Chloroplast-localized isoforms of ascorbate peroxidase and possibly peroxiredoxins assume the principal role of scavenging hydrogen peroxide. However, in vitro studies revealed that both types of peroxidases are easily damaged by hydrogen peroxide and lose their catalytic activities. This is one contributing factor for cellular damage that occurs under severe oxidative stress. In this review, I describe mechanisms of hydrogen peroxide-mediated inactivation of these two enzymes and discuss a reason why they became susceptible to damage by hydrogen peroxide.

  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. 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.

  15. fPoxDB: fungal peroxidase database for comparative genomics

    PubMed Central

    2014-01-01

    Background Peroxidases are a group of oxidoreductases which mediate electron transfer from hydrogen peroxide (H2O2) and organic peroxide to various electron acceptors. They possess a broad spectrum of impact on industry and fungal biology. There are numerous industrial applications using peroxidases, such as to catalyse highly reactive pollutants and to breakdown lignin for recycling of carbon sources. Moreover, genes encoding peroxidases play important roles in fungal pathogenicity in both humans and plants. For better understanding of fungal peroxidases at the genome-level, a novel genomics platform is required. To this end, Fungal Peroxidase Database (fPoxDB; http://peroxidase.riceblast.snu.ac.kr/) has been developed to provide such a genomics platform for this important gene family. Description In order to identify and classify fungal peroxidases, 24 sequence profiles were built and applied on 331 genomes including 216 from fungi and Oomycetes. In addition, NoxR, which is known to regulate NADPH oxidases (NoxA and NoxB) in fungi, was also added to the pipeline. Collectively, 6,113 genes were predicted to encode 25 gene families, presenting well-separated distribution along the taxonomy. For instance, the genes encoding lignin peroxidase, manganese peroxidase, and versatile peroxidase were concentrated in the rot-causing basidiomycetes, reflecting their ligninolytic capability. As a genomics platform, fPoxDB provides diverse analysis resources, such as gene family predictions based on fungal sequence profiles, pre-computed results of eight bioinformatics programs, similarity search tools, a multiple sequence alignment tool, domain analysis functions, and taxonomic distribution summary, some of which are not available in the previously developed peroxidase resource. In addition, fPoxDB is interconnected with other family web systems, providing extended analysis opportunities. Conclusions fPoxDB is a fungi-oriented genomics platform for peroxidases. The sequence

  16. Flow system for the automatic screening of the effect of phenolic compounds on the luminol-hydrogen peroxide-peroxidase chemiluminescence system.

    PubMed

    Araujo, André R T S; Maya, Fernando; Saraiva, M Lúcia M F S; Lima, José L F C; Estela, José M; Cerdà, Víctor

    2011-01-01

    In this work, an automated flow-based procedure for the screening of the effect of the different phenolic compounds on the chemiluminescence (CL) luminol-hydrogen peroxide-horseradish peroxidase (HRP) system is presented. This procedure involves the combination of multisyringe flow injection analysis (MFSIA) and sequential injection analysis (SIA) techniques and exploits the ability of the different subgroups of phenols, such as cholorophenols, nitrophenols, methylphenols and polyphenols, to enhance or inhibit the described CL system. The implementation of this reaction in the SIA-MSFIA system enabled favourable and precise conditions to evaluate the effect of phenolic compounds, as it involves an in-line reaction between the phenolic derivative, hydrogen peroxide and peroxidase and subsequent oxidized HRP intermediates generation prior to the fast reaction with the chemiluminogenic reagent. Several studies were then performed with the aim of establishing the appropriate flow system configuration and reaction conditions. It was shown that phenol and chlorophenols produce an enhanced CL response and nitrophenols, methylphenols and polyphenols are inhibitors within the range of concentrations studied (1-100 mg/L). Based on these studies, the developed method was applied to the determination of total polyphenol and phenol content in wine/grape seeds and water samples, respectively, and the results obtained showed good agreement with those furnished by the corresponding Folin-Ciocalteu and 4-aminoantipyrine reference methods. The developed approach is further pursued by designing an automated generic tool for performing studies of peroxidase-catalysed CL reactions of luminol focused on the detection of compounds that will affect the rate of those reactions.

  17. 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.

  18. Limits of Versatility of Versatile Peroxidase

    PubMed Central

    Knop, Doriv; Levinson, Dana; Makovitzki, Arik; Agami, Avi; Lerer, Elad; Mimran, Avishai; Yarden, Oded

    2016-01-01

    ABSTRACT Although Mn2+ is the most abundant substrate of versatile peroxidases (VPs), repression of Pleurotus ostreatus vp1 expression occurred in Mn2+-sufficient medium. This seems to be a biological contradiction. The aim of this study was to explore the mechanism of direct oxidation by VP1 under Mn2+-deficient conditions, as it was found to be the predominant enzyme during fungal growth in the presence of synthetic and natural substrates. The native VP1 was purified and characterized using three substrates, Mn2+, Orange II (OII), and Reactive Black 5 (RB5), each oxidized by a different active site in the enzyme. While the pH optimum for Mn2+ oxidation is 5, the optimum pH for direct oxidation of both dyes was found to be 3. Indeed, effective in vivo decolorization occurred in media without addition of Mn2+ only under acidic conditions. We have determined that Mn2+ inhibits in vitro the direct oxidation of both OII and RB5 while RB5 stabilizes both Mn2+ and OII oxidation. Furthermore, OII was found to inhibit the oxidation of both Mn2+ and RB5. In addition, we could demonstrate that VP1 can cleave OII in two different modes. Under Mn2+-mediated oxidation conditions, VP1 was able to cleave the azo bond only in asymmetric mode, while under the optimum conditions for direct oxidation (absence of Mn2+ at pH 3) both symmetric and asymmetric cleavages occurred. We concluded that the oxidation mechanism of aromatic compounds by VP1 is controlled by Mn2+ and pH levels both in the growth medium and in the reaction mixture. IMPORTANCE VP1 is a member of the ligninolytic heme peroxidase gene family of the white rot fungus Pleurotus ostreatus and plays a fundamental role in biodegradation. This enzyme exhibits a versatile nature, as it can oxidize different substrates under altered environmental conditions. VPs are highly interesting enzymes due to the fact that they contain unique active sites that are responsible for direct oxidation of various aromatic compounds

  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. 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.

  1. Thyroid peroxidase activity in human nodular goiters.

    PubMed

    Moura, E G; Rosenthal, D; Carvalho-Guimarães, D P

    1989-01-01

    1. Thyroid peroxidase (TPO, iodide-oxidation) activity was evaluated in nodular and paranodular tissue samples from 27 patients with nodular goiter (19 "cold" and 8 "hot" nodules), and compared to 11 diffuse toxic goiter and 9 normal thyroid tissue samples. 2. In terms of U/g digitonin solubilized protein, TPO activity was increased in hot nodules (P less than 0.05), although not as much as in diffuse toxic goiters (P less than 0.01). 3. The mean TPO activity of tissues paranodular to a cold nodule was not different from that of normal thyroids. 4. Both the highest and the lowest TPO activities were found in cold nodules, but their mean value did not differ from those of their paranodular tissues or normal thyroids. 5. Inter-tissue variability was significantly increased (P less than 0.01) in cold nodules and in tissues paranodular to a hot nodule. 6. These data show that heterogeneity both within and among tissues contributes to the wide range of TPO activity detected in nodular goiters.

  2. Cellular glutathione peroxidase deficiency and endothelial dysfunction.

    PubMed

    Forgione, Marc A; Weiss, Norbert; Heydrick, Stanley; Cap, André; Klings, Elizabeth S; Bierl, Charlene; Eberhardt, Robert T; Farber, Harrison W; Loscalzo, Joseph

    2002-04-01

    Cellular glutathione peroxidase (GPx-1) is the most abundant intracellular isoform of the GPx antioxidant enzyme family. In this study, we hypothesized that GPx-1 deficiency directly induces an increase in vascular oxidant stress, with resulting endothelial dysfunction. We studied vascular function in a murine model of homozygous deficiency of GPx-1 (GPx-1(-/-)). Mesenteric arterioles of GPx-1(-/-) mice demonstrated paradoxical vasoconstriction to beta-methacholine and bradykinin, whereas wild-type (WT) mice showed dose-dependent vasodilation in response to both agonists. One week of treatment of GPx-1(-/-) mice with L-2-oxothiazolidine-4-carboxylic acid (OTC), which increases intracellular thiol pools, resulted in restoration of normal vascular reactivity in the mesenteric bed of GPx-1(-/-) mice. We observed an increase of the isoprostane iPF(2alpha)-III, a marker of oxidant stress, in the plasma and aortas of GPx-1(-/-) mice compared with WT mice, which returned toward normal after OTC treatment. Aortic sections from GPx-1(-/-) mice showed increased binding of an anti-3-nitrotyrosine antibody in the absence of frank vascular lesions. These findings demonstrate that homozygous deficiency of GPx-1 leads to impaired endothelium-dependent vasodilator function presumably due to a decrease in bioavailable nitric oxide and to increased vascular oxidant stress. These vascular abnormalities can be attenuated by increasing bioavailable intracellular thiol pools.

  3. Redundancy among Manganese Peroxidases in Pleurotus ostreatus

    PubMed Central

    Salame, Tomer M.; Knop, Doriv; Levinson, Dana; Yarden, Oded

    2013-01-01

    Manganese peroxidases (MnPs) are key players in the ligninolytic system of white rot fungi. In Pleurotus ostreatus (the oyster mushroom) these enzymes are encoded by a gene family comprising nine members, mnp1 to -9 (mnp genes). Mn2+ amendment to P. ostreatus cultures results in enhanced degradation of recalcitrant compounds (such as the azo dye orange II) and lignin. In Mn2+-amended glucose-peptone medium, mnp3, mnp4, and mnp9 were the most highly expressed mnp genes. After 7 days of incubation, the time point at which the greatest capacity for orange II decolorization was observed, mnp3 expression and the presence of MnP3 in the extracellular culture fluids were predominant. To determine the significance of MnP3 for ligninolytic functionality in Mn2+-sufficient cultures, mnp3 was inactivated via the Δku80 strain-based P. ostreatus gene-targeting system. In Mn2+-sufficient medium, inactivation of mnp3 did not significantly affect expression of nontargeted MnPs or their genes, nor did it considerably diminish the fungal Mn2+-mediated orange II decolorization capacity, despite the significant reduction in total MnP activity. Similarly, inactivation of either mnp4 or mnp9 did not affect orange II decolorization ability. These results indicate functional redundancy within the P. ostreatus MnP gene family, enabling compensation upon deficiency of one of its members. PMID:23377936

  4. 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.

  5. Molecular characterization of lignin peroxidase from the white-rot basidiomycete Trametes cervina: a novel fungal peroxidase.

    PubMed

    Miki, Yuta; Ichinose, Hirofumi; Wariishi, Hiroyuki

    2010-03-01

    The lignin peroxidase (LiP) from Trametes cervina was cloned, characterized, and identified as a novel fungal peroxidase. The sequence of T. cervina LiP encodes the essential amino acids for shaping the heme cavity and calcium-binding sites, which are conserved in plant and fungal peroxidases. However, a sequence homology analysis showed that T. cervina LiP has two unique features: it lacks the conserved tryptophan residue corresponding to the substrate-oxidation site (Trp171) of Phanerochaete chrysosporium LiP and it has a tyrosine residue (Tyr181) that has never been reported in other lignin peroxidases. A tertiary model of T. cervina LiP showed that Tyr181 sterically adjacent to the 6-propionate group of heme is surrounded by acidic amino acids and is exposed to the exterior. These attributes indicate that Tyr181 could be a T. cervina LiP substrate-oxidation site. A phylogenetic analysis showed that T. cervina LiP does not cluster with any other fungal peroxidases, suggesting that it is a unique molecule that is evolutionarily distant from other peroxidases. Thus, we concluded that T. cervina LiP could be a novel secreted peroxidase, among those produced by fungi, with a new oxidation mechanism probably involving Tyr181.

  6. 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

    2014-10-08

    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.

  7. 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.

  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. Heme electron transfer in peroxidases: the propionate e-pathway.

    PubMed

    Guallar, Victor

    2008-10-23

    Computational modeling offers a new insight about the electron transfer pathway in heme peroxidases. Available crystal structures have revealed an intriguing arrangement of the heme propionate side chains in heme-heme and heme-substrate complexes. By means of mixed quantum mechanical/molecular mechanics calculations, we study the involvement of these propionate groups into the substrate oxidation in ascorbate peroxidase and into the heme to heme electron transfer in bacterial cytochrome c peroxidase. By selectively turning on/off different quantum regions, we obtain the electron transfer pathway which directly involves the porphyrin ring and the heme propionates. Furthermore, in ascorbate peroxidase the presence of the substrate appears to be crucial for the activation of the electron transfer channel. The results might represent a general motif for electron transfer from/to the heme group and change our view for the propionate side chains as simple electrostatic binding anchors. We name the new mechanism "the propionate e-pathway".

  10. 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

  11. Conversion of adamsite (phenarsarzin chloride) by fungal manganese peroxidase.

    PubMed

    Haas, R; Tsivunchyk, O; Steinbach, K; von Löw, E; Scheibner, K; Hofrichter, M

    2004-02-01

    Fungal manganese peroxidase was found to convert the persistent chemical warfare agent adamsite (phenarsarzin chloride) in a cell-free reaction mixture containing sodium malonate, Mn(2+) ions, and reduced glutathione. The organo-arsenical compound disappeared completely within 48 h accompanied by the formation of a more polar metabolite with a clearly modified UV spectrum. Thus, As(III) in the adamsite molecule was oxidized by manganese peroxidase to As(V) which added dioxygen and released chloride.

  12. Hypocotyl Growth and Peroxidases of Bidens pilosus1

    PubMed Central

    Desbiez, Marie-Odile; Boyer, Nicole; Gaspar, Thomas

    1981-01-01

    Pricking one cotyledon of young Bidens pilosus plants induces rapid inhibition of hypocotyl growth, essentially in its middle portion. Analysis of soluble peroxidases indicates rapid changes (increase of activity) in basic isoenzymes followed by more progressive enhancement of the acidic ones. Pretreatment of the plants with lithium prevents the inhibition of elongation due to pricking as well as the peroxidase changes. The phenomenon is similar to the previously described thigmomorphogenetic process in Bryonia dioica. PMID:16661885

  13. The impact of thiol peroxidases on redox regulation.

    PubMed

    Flohé, Leopold

    2016-01-01

    The biology of glutathione peroxidases and peroxiredoxins is reviewed with emphasis on their role in metabolic regulation. Apart from their obvious function in balancing oxidative challenge, these thiol peroxidases are not only implicated in orchestrating the adaptive response to oxidative stress, but also in regulating signaling triggered by hormones, growth factors and cytokines. The mechanisms presently discussed comprise dampening of redox-sensitive regulatory processes by elimination of hydroperoxides, suppression of lipoxygenase activity, committing suicide to save H2O2 for signaling, direct binding to receptors or regulatory proteins in a peroxidase activity-independent manner, or acting as sensors for hydroperoxides and as transducers of oxidant signals. The various mechanistic proposals are discussed in the light of kinetic data, which unfortunately are scarce. Taking into account pivotal criteria of a meaningful regulatory circuit, kinetic plausibility and specificity, the mechanistic concepts implying a direct sensor/transducer function of the thiol peroxidases appear most appealing. With rate constants for the reaction with hydroperoxide of 10(5)-10(8) M(-1) s(-1), thiol peroxidases are qualified as kinetically preferred hydroperoxide sensors, and the ability of the oxidized enzymes to react with defined protein thiols lends specificity to the transduction process. The versatility of thiol peroxidases, however, allows multiple ways of interaction with regulatory pathways.

  14. Role of peroxidases in the compensation of cytosolic ascorbate peroxidase knockdown in rice plants under abiotic stress.

    PubMed

    Bonifacio, Aurenivia; Martins, Marcio O; Ribeiro, Carolina W; Fontenele, Adilton V; Carvalho, Fabricio E L; Margis-Pinheiro, Márcia; Silveira, Joaquim A G

    2011-10-01

    Current studies, particularly in Arabidopsis, have demonstrated that mutants deficient in cytosolic ascorbate peroxidases (APXs) are susceptible to the oxidative damage induced by abiotic stress. In contrast, we demonstrate here that rice mutants double silenced for cytosolic APXs (APx1/2s) up-regulated other peroxidases, making the mutants able to cope with abiotic stress, such as salt, heat, high light and methyl viologen, similar to non-transformed (NT) plants. The APx1/2s mutants exhibited an altered redox homeostasis, as indicated by increased levels of H₂O₂ and ascorbate and glutathione redox states. Both mutant and NT plants exhibited similar photosynthesis (CO₂) assimilation and photochemical efficiency) under both normal and stress conditions. Overall, the antioxidative compensatory mechanism displayed by the mutants was associated with increased expression of OsGpx genes, which resulted in higher glutathione peroxidase (GPX) activity in the cytosolic and chloroplastic fractions. The transcript levels of OsCatA and OsCatB and the activities of catalase (CAT) and guaiacol peroxidase (GPOD; type III peroxidases) were also up-regulated. None of the six studied isoforms of OsApx were up-regulated under normal growth conditions. Therefore, the deficiency in cytosolic APXs was effectively compensated for by up-regulation of other peroxidases. We propose that signalling mechanisms triggered in rice mutants could be distinct from those proposed for Arabidopsis.

  15. 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.

  16. Rescuing apoptotic neurons in Alzheimer's disease using wheat germ agglutinin-conjugated and cardiolipin-conjugated liposomes with encapsulated nerve growth factor and curcumin.

    PubMed

    Kuo, Yung-Chih; Lin, Ching-Chun

    2015-01-01

    Liposomes with cardiolipin (CL) and wheat germ agglutinin (WGA) were developed to permeate the blood-brain barrier and treat Alzheimer's disease. WGA-conjugated and CL-incorporated liposomes (WGA-CL-liposomes) were used to transport nerve growth factor (NGF) and curcumin (CUR) across a monolayer of human brain-microvascular endothelial cells regulated by human astrocytes and to protect SK-N-MC cells against apoptosis induced by β-amyloid1-42 (Aβ(1-42)) fibrils. An increase in the CL mole percentage in lipids increased the liposomal diameter, absolute zeta potential value, entrapment efficiency of NGF and CUR, release of NGF, biocompatibility, and viability of SK-N-MC cells with Aβ(1-42), but decreased the atomic ratio of nitrogen to phosphorus and release of CUR. In addition, an increase in the WGA concentration for grafting enhanced the liposomal diameter, atomic ratio of nitrogen to phosphorus, and permeability of NGF and CUR across the blood-brain barrier, but reduced the absolute zeta potential value and biocompatibility. WGA-CL-liposomes carrying NGF and CUR could be promising colloidal delivery carriers for future clinical application in targeting the blood-brain barrier and inhibiting neurotoxicity.

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

    PubMed

    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 α (TNF-α) 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-α 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. Candida albicans biofilm on titanium: effect of peroxidase precoating

    PubMed Central

    Ahariz, Mohamed; Courtois, Philippe

    2010-01-01

    The present study aimed to document Candida albicans biofilm development on titanium and its modulation by a peroxidase-precoated material which can generate antimicrobials, such as hypoiodite or hypothiocyanite, from hydrogen peroxide, iodide, or thiocyanate. For this purpose, titanium (powder or foil) was suspended in Sabouraud liquid medium inoculated with C. albicans ATCC10231. After continuous stirring for 2–21 days at room temperature, the supernatant was monitored by turbidimetry at 600 nm and titanium washed three times in sterile Sabouraud broth. Using the tetrazolium salt MTT-formazan assay, the titanium-adherent fungal biomass was measured as 7.50 ± 0.60 × 106 blastoconidia per gram of titanium powder (n = 30) and 0.50 ± 0.04 × 106 blastoconidia per cm2 of titanium foil (n = 12). The presence of yeast on the surface of titanium was confirmed by microscopy both on fresh preparations and after calcofluor white staining. However, in the presence of peroxidase systems (lactoperoxidase with substrates such as hydrogen peroxide donor, iodide, or thiocyanate), Candida growth in both planktonic and attached phases appeared to be inhibited. Moreover, this study demonstrates the possible partition of peroxidase systems between titanium material (peroxidase-precoated) and liquid environment (containing peroxidase substrates) to limit C. albicans biofilm formation. PMID:22915919

  3. Feasible Relation between Glutathione Peroxidase and Febrile Seizure

    PubMed Central

    MAHYAR, Abolfazl; AYAZI, Parviz; DALIRANI, Reza; MOHAMMAD HOSEINI, Behzad; SAROOKHANI, Mohammad Reza; JAVADI, Amir; ESMAEILY, Shiva

    2017-01-01

    Objective We aimed to determine the relationship between serum glutathione peroxidase and febrile seizure. Materials & Methods In this case-control study, 43 children with simple febrile seizure (case group) were compared with 43 febrile children without seizure (control group) in terms of serum glutathione peroxidase level, measured by ELISA method. This study was conducted in Qazvin Children Hospital, Qazvin University of Medical Sciences in Qazvin, Iran in 2012-2013. The results were analyzed and compared in two groups. Results From 43 children 24 (53%) were male and 19 (47%) were female in children with simple febrile seizure, and 26 (60%) were male and 17 (40%) were female in febrile children without seizure (control group) (P=0.827). Serum glutathione peroxidase level was 166 U/ml (SD=107) in the case group and 141 U/ml (SD=90.5) in the control group of no significant difference. Conclusion There was no significant relationship between serum glutathione peroxidase and simple febrile seizure. Thus, it seems that glutathione peroxidase, an essential component of antioxidant system, does not play any role in the pathogenesis of simple febrile seizure. PMID:28277558

  4. 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

  5. Participation of the photosensitizer alpha-terthienyl in the peroxidase-catalyzed oxidation of indole-3-acetic acid.

    PubMed

    Brennan, T M; Lee, E; Battaglia, P R

    2000-04-01

    The plant photosensitizer alpha-terthienyl (alpha T) is toxic toward a variety of organisms, and normally requires exposure to ultraviolet-A radiation for activation and singlet molecular oxygen formation. However, some toxicity has also been reported to occur in the dark. One hypothesis that has been proposed to account for this light-independent toxicity is that the sensitizer becomes activated by energy transfer from the excited-state products of enzymatic reactions. We have investigated this hypothesis using the horseradish peroxidase (HRP)-catalyzed oxidation of indole-3-acetic acid (IAA), which generates indole-3-aldehyde in an excited triplet state. Light is emitted during the IAA/HRP reaction at acidic pH, is increased by inclusion of alpha T and is not observed with heat-denatured HRP. The rates of both the oxidation of IAA and the subsidence of light emission are more rapid in the IAA/alpha T/HRP system than with IAA and HRP alone, indicating that the presence of alpha T accelerates the reaction. Bleaching occurs at the wavelength of maximal alpha T absorbance and is promoted by the inclusion of IAA. Readdition of both IAA and alpha T to a spent reaction mixture is required to restore light emission after it has subsided, further suggesting that both are consumed in the reaction. We were unable to detect measurable quantities of singlet molecular oxygen formation in this system. These results do not support the energy transfer hypothesis, but instead are more compatible with a model proposed by Krylov and Chebotareva [Krylov, S. N. and A. B. Chebotareva (1993) FEBS Lett. 324, 6-8] for the co-oxidation of IAA and xanthene dyes.

  6. Assay of methylglyoxal and glyoxal and control of peroxidase interference.

    PubMed

    Thornalley, Paul J; Rabbani, Naila

    2014-04-01

    Methylglyoxal and glyoxal are endogenous α-oxoaldehyde metabolites and substrates of the glyoxalase system. These and related α-oxoaldehydes are often determined in cell, tissue and body fluid samples by derivatization with 1,2-diaminobenzene and similar compounds. Peroxidase activity in physiological tissues is a potential interference in estimation of methylglyoxal and glyoxal as it catalyses the conversion of 1,2-diaminobenzene into trace amounts of these dicarbonyl metabolites. Residual peroxidase activity in deproteinized extracts is found to cause significant interference in methylglyoxal and glyoxal estimations. This interference is blocked by the addition of sodium azide in the derivatizing buffer. Estimates of methylglyoxal concentration thereby obtained are in keeping with those predicted by systems modelling of methylglyoxal glycation kinetics in situ. Blocking sample peroxidase activity is important to avoid overestimation in the measurement of glyoxal and methylglyoxal. A dicarbonyl assay protocol resistant to interferences is described in the present article.

  7. Two distinct groups of fungal catalase/peroxidases.

    PubMed

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

    2009-08-01

    Catalase/peroxidases (KatGs) are bifunctional haem b-containing (Class I) peroxidases with overwhelming catalase activity and substantial peroxidase activity with various one-electron donors. These unique oxidoreductases evolved in ancestral bacteria revealing a complex gene-duplicated structure. Besides being found in numerous bacteria of all phyla, katG genes were also detected in genomes of lower eukaryotes, most prominently of sac and club fungi. Phylogenetic analysis demonstrates the occurrence of two distinct groups of fungal KatGs that differ in localization, structural and functional properties. Analysis of lateral gene transfer of bacterial katGs into fungal genomes reveals that the most probable progenitor was a katG from a bacteroidetes predecessor. The putative physiological role(s) of both fungal KatG groups is discussed with respect to known structure-function relationships in bacterial KatGs and is related with the acquisition of (phyto)pathogenicity in fungi.

  8. Glutathione peroxidase in early and advanced Parkinson's disease.

    PubMed Central

    Johannsen, P; Velander, G; Mai, J; Thorling, E B; Dupont, E

    1991-01-01

    A defective antioxidant scavenging system plays a major role in one of the theories of the pathogenesis of Parkinson's disease. The aim of this study was to investigate whether there is a general difference in antioxidant activity between early and advanced cases of Parkinson's disease. Twenty five recently diagnosed patients, without any clinical fluctuations (group A), and 25 patients in a late phase of the disease with severe fluctuations in response to levodopa therapy (group B) were included in the study. Erythrocyte glutathione peroxidase was determined as a measure of antioxidant activity and significantly lower values were found in group B than in group A. Regression analyses in groups A and B showed significant correlation between glutathione peroxidase and duration of disease, but not between glutathione peroxidase and age of patients. Images PMID:1940936

  9. Diverse functions and reactions of class III peroxidases.

    PubMed

    Shigeto, Jun; Tsutsumi, Yuji

    2016-03-01

    Higher plants contain plant-specific peroxidases (class III peroxidase; Prxs) that exist as large multigene families. Reverse genetic studies to characterize the function of each Prx have revealed that Prxs are involved in lignification, cell elongation, stress defense and seed germination. However, the underlying mechanisms associated with plant phenotypes following genetic engineering of Prx genes are not fully understood. This is because Prxs can function as catalytic enzymes that oxidize phenolic compounds while consuming hydrogen peroxide and/or as generators of reactive oxygen species. Moreover, biochemical efforts to characterize Prxs responsible for lignin polymerization have revealed specialized activities of Prxs. In conclusion, not only spatiotemporal regulation of gene expression and protein distribution, but also differentiated oxidation properties of each Prx define the function of this class of peroxidases.

  10. The effect of an aqueous horse-radish extract, applied as such or in association with caffeine, on experimental influenza in mice.

    PubMed

    Eşanu, V; Prahoveanu, E

    1985-01-01

    An aqueous horse-radish extract was repeatedly administered to mice by intranasal (i.n.) route prior to i.n. inoculation of influenza virus A/PR8/34 (H1N1). The antiviral effect of the extract--also demonstrated in vitro--was reflected by a significant decrease in the hemagglutination titers recorded in mouse lung homogenates and by a slight increase in the mean survival time of treated mice versus untreated controls. Association of horse-radish extract with caffeine led to a synergic effect only as regards the mortality rate.

  11. Inorganic chemistry of defensive peroxidases in the human oral cavity.

    PubMed

    Ashby, M T

    2008-10-01

    The innate host response system is comprised of various mechanisms for orchestrating host response to microbial infection of the oral cavity. The heterogeneity of the oral cavity and the associated microenvironments that are produced give rise to different chemistries that affect the innate defense system. One focus of this review is on how these spatial differences influence the two major defensive peroxidases of the oral cavity, salivary peroxidase (SPO) and myeloperoxidase (MPO). With hydrogen peroxide (H(2)O(2)) as an oxidant, the defensive peroxidases use inorganic ions to produce antimicrobials that are generally more effective than H(2)O(2) itself. The concentrations of the inorganic substrates are different in saliva vs. gingival crevicular fluid (GCF). Thus, in the supragingival regime, SPO and MPO work in unison for the exclusive production of hypothiocyanite (OSCN(-), a reactive inorganic species), which constantly bathes nascent plaques. In contrast, MPO is introduced to the GCF during inflammatory response, and in that environment it is capable of producing hypochlorite (OCl(-)), a chemically more powerful oxidant that is implicated in host tissue damage. A second focus of this review is on inter-person variation that may contribute to different peroxidase function. Many of these differences are attributed to dietary or smoking practices that alter the concentrations of relevant inorganic species in the oral cavity (e.g.: fluoride, F(-); cyanide, CN(-); cyanate, OCN(-); thiocyanate, SCN(-); and nitrate, NO(3)(-)). Because of the complexity of the host and microflora biology and the associated chemistry, it is difficult to establish the significance of the human peroxidase systems during the pathogenesis of oral diseases. The problem is particularly complex with respect to the gingival sulcus and periodontal pockets (where the very different defensive stratagems of GCF and saliva co-mingle). Despite this complexity, intriguing in vitro and in vivo

  12. Urchin-like (gold core)@(platinum shell) nanohybrids: A highly efficient peroxidase-mimetic system for in situ amplified colorimetric immunoassay.

    PubMed

    Gao, Zhuangqiang; Xu, Mingdi; Lu, Minghua; Chen, Guonan; Tang, Dianping

    2015-08-15

    The development of signal-amplified colorimetric immunoassay relies on the design of highly efficient signal-transduction tags. One promising route is to exploit a novel enzyme mimetic system as the signal label. Herein, we report that urchin-like (gold core)@(platinum shell) nanohybrids (Au@PtNHs) can be utilized as a highly efficient peroxidase mimetic system for in situ amplified colorimetric immunoassay of prostate-specific antigen (PSA, one kind of tumor marker). Initially, urchin-like Au@PtNHs were discovered to outperform horseradish peroxidase (HRP) by a vast margin in terms of the turnover number toward hydrogen peroxide (H2O2)-3,3',5,5'-tetramethylbenzidine (TMB) system and the stability against high temperatures and HRP inhibitors. Based on this discovery, the assay was simply carried out on a capture antibody-immobilized microplate by using the Au@PtNH-labeled detection antibody as a signal-transduction tag with a sandwich-type assay mode. The colorimetric signal stemmed from the labeled Au@PtNHs toward catalytic oxidation of TMB-H2O2 system. Experimental results indicated that the Au@PtNH-based colorimetric immunoassay could display a good colorimetric response toward PSA in the dynamic working range of 5-500 pg mL(-1) with a low detection limit of 2.9 pg mL(-1). Meanwhile, the developed immunoassay exhibited good precision and reproducibility, high specificity and acceptable accuracy for the detection of clinical serum samples. These results open up a new horizon for the development of highly sensitive, highly stable and inexpensive non-enzyme immunoassay platforms as an alternative to conventional enzyme-based immunoassay platforms.

  13. Phylogeny, topology, structure and functions of membrane-bound class III peroxidases in vascular plants.

    PubMed

    Lüthje, Sabine; Meisrimler, Claudia-Nicole; Hopff, David; Möller, Benjamin

    2011-07-01

    Peroxidases are key player in the detoxification of reactive oxygen species during cellular metabolism and oxidative stress. Membrane-bound isoenzymes have been described for peroxidase superfamilies in plants and animals. Recent studies demonstrated a location of peroxidases of the secretory pathway (class III peroxidases) at the tonoplast and the plasma membrane. Proteomic approaches using highly enriched plasma membrane preparations suggest organisation of these peroxidases in microdomains, a developmentally regulation and an induction of isoenzymes by oxidative stress. Phylogenetic relations, topology, putative structures, and physiological function of membrane-bound class III peroxidases will be discussed.

  14. 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.

  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. Determination of optimum process parameters for peroxidase-catalysed treatment of bisphenol A and application to the removal of bisphenol derivatives.

    PubMed

    Yamada, Kazunori; Ikeda, Naoya; Takano, Yoko; Kashiwada, Ayumi; Matsuda, Kiyomi; Hirata, Mitsuo

    2010-03-01

    Systematic investigations were carried out to determine the optimum process parameters such as the hydrogen peroxide (H2O2) concentration, concentration and molar mass of poly(ethylene glycol) (PEG) as an additive, pH value, temperature and enzyme dose for treatment of bisphenol A (BPA) with horseradish peroxidase (HRP). The HRP-catalysed treatment of BPA was effectively enhanced by adding PEG, and BPA was completely converted into phenoxy radicals by HRP dose of 0.10 U/cm3. The optimum conditions for HRP-catalysed treatment of BPA at 0.3 mM was determined to be 0.3 mM for H2O2 and 0.10 mg/cm3 for PEG with a molar mass of 1.0 x 10(4) in a pH 6.0 buffer at 30 degrees C. Different kinds of bisphenol derivatives were completely or effectively treated by HRP under the optimum conditions determined for treatment of BPA, although the HRP dose was further increased as necessary for some of them. The aggregation of water-insoluble oligomers generated by the enzymatic radicalization and radical coupling reaction was enhanced by decreasing the pH values to 4.0 with HCl after the enzymatic treatment, and BPA and bisphenol derivatives were removed from aqueous solutions by filtering out the oligomer precipitates.

  17. 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...

  18. 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...

  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. Mammalian heme peroxidases: from molecular mechanisms to health implications.

    PubMed

    Davies, Michael J; Hawkins, Clare L; Pattison, David I; Rees, Martin D

    2008-07-01

    A marked increase in interest has occurred over the last few years in the role that mammalian heme peroxidase enzymes, primarily myeloperoxidase, eosinophil peroxidase, and lactoperoxidase, may play in both disease prevention and human pathologies. This increased interest has been sparked by developments in our understanding of polymorphisms that control the levels of these enzymes, a greater understanding of the basic chemistry and biochemistry of the oxidants formed by these species, the development of specific biomarkers that can be used in vivo to detect damage induced by these oxidants, the detection of active forms of these peroxidases at most, if not all, sites of inflammation, and a correlation between the levels of these enzymes and a number of major human pathologies. This article reviews recent developments in our understanding of the enzymology, chemistry, biochemistry and biologic roles of mammalian peroxidases and the oxidants that they generate, the potential role of these oxidants in human disease, and the use of the levels of these enzymes in disease prognosis.

  1. The glucose oxidase-peroxidase assay for glucose

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The glucose oxidase-peroxidase assay for glucose has served as a very specific, sensitive, and repeatable assay for detection of glucose in biological samples. It has been used successfully for analysis of glucose in samples from blood and urine, to analysis of glucose released from starch or glycog...

  2. 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.

  3. Calorimetric studies of the thermal denaturation of cytochrome c peroxidase

    SciTech Connect

    Kresheck, G.C.; Erman, J.E.

    1988-04-05

    Two endotherms are observed by differential scanning calorimetry during the thermal denaturation of cytochrome c peroxidase at pH 7.0. The transition midpoint temperatures (t/sub m/) were 43.9 +- 1.4 and 63.3 +- 1.6 /sup 0/C, independent of concentration. The two endotherms were observed at all pH values between 4 and 8, with the transition temperatures varying with pH. Precipitation was observed between pH 4 and 6, and only qualitative data are presented for this region. The thermal unfolding of cytochrome c peroxidase was sensitive to the presence and ligation state of the heme. Only a single endotherm was observed for the unfolding of the apoprotein, and this transition was similar to the high-temperature transition in the holoenzyme. Addition of KCN to the holoenzyme increases the midpoint of the high-temperature transition whereas the low-temperature transition was increased upon addition of KF. Binding of the natural substrate ferricytochrome c to the enzyme increases the low-temperature transition by 4.8 +- 1.3 /sup 0/C but has no effect on the high-temperature transition at pH 7. The presence of cytochrome c peroxidase decreases the stability of cytochrome c, and both proteins appear to unfold simultaneously. The results are discussed in terms of the two domains evident in the X-ray crystallographic structure of cytochrome c peroxidase

  4. Genome Sequence of Pectobacterium carotovorum Phage PPWS1, Isolated from Japanese Horseradish [Eutrema japonicum (Miq.) Koidz] Showing Soft-Rot Symptoms.

    PubMed

    Hirata, Hisae; Kashihara, Misako; Horiike, Tokumasa; Suzuki, Tomohiro; Dohra, Hideo; Netsu, Osamu; Tsuyumu, Shinji

    2016-04-21

    ITALIC! Pectobacterium carotovorumsubsp. ITALIC! carotovorumand its lytic bacteriophage PPWS1 were isolated from a Japanese horseradish rhizome with soft rot. Sequencing of the phage genomic DNA suggested that PPWS1 is a new species of the family ITALIC! Podoviridaeand has high similarity to the bacteriophage Peat1 infectious to ITALIC! P. atrosepticum.

  5. Genome Sequence of Pectobacterium carotovorum Phage PPWS1, Isolated from Japanese Horseradish [Eutrema japonicum (Miq.) Koidz] Showing Soft-Rot Symptoms

    PubMed Central

    Kashihara, Misako; Horiike, Tokumasa; Suzuki, Tomohiro; Dohra, Hideo; Netsu, Osamu; Tsuyumu, Shinji

    2016-01-01

    Pectobacterium carotovorum subsp. carotovorum and its lytic bacteriophage PPWS1 were isolated from a Japanese horseradish rhizome with soft rot. Sequencing of the phage genomic DNA suggested that PPWS1 is a new species of the family Podoviridae and has high similarity to the bacteriophage Peat1 infectious to P. atrosepticum. PMID:27103734

  6. 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.

  7. Ethylene production and peroxidase activity in aphid-infested barley.

    PubMed

    Argandoña, V H; Chaman, M; Cardemil, L; Muñoz, O; Zúñiga, G E; Corcuera, L J

    2001-01-01

    The purpose of this work was to investigate whether ethylene is involved in the oxidative and defensive responses of barley to the aphids Schizaphis graminum (biotype C) and Rhopalophum padi. The effect of aphid infestation on ethylene production was measured in two barley cultivars (Frontera and Aramir) that differ in their susceptibility to aphids. Ethylene evolution was higher in plants infested for 16 hr than in plants infested for 4 hr in both cultivars. Under aphid infestation, the production of ethylene was higher in cv. Frontera than in Aramir, the more aphid susceptible cultivar. Ethylene production also increases with the degree of infestation. Maximum ethylene evolution was detected after 16 hr when plants were infested with 10 or more aphids. Comparing the two species of aphids, Schizaphis graminum induced more ethylene evolution than Rhopalosiphum padi. Infestation with S. graminum increased hydrogen peroxide content and total soluble peroxidase activity in cv. Frontera, with a maximum level of H2O2 observed after 20 min of infestation and the maximum in soluble peroxidase activity after 30 min of infestation. When noninfested barley seedlings from cv. Frontera were exposed to ethylene, an increase in hydrogen peroxide and in total peroxidase activity was detected at levels similar to those of infested plants from cv. Frontera. When noninfested plants were treated with 40 ppm of ethylene, the maximum levels of H2O2 and soluble peroxidase activity were at 10 and 40 min, respectively. Ethylene also increased the activity of both cell-wall-bound peroxidases types (ionically and covalently bound), comparable with infestation. These results suggest that ethylene is involved in the oxidative responses of barley plants induced by infestation.

  8. 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.

  9. Pleurotus ostreatus heme peroxidases: an in silico analysis from the genome sequence to the enzyme molecular structure.

    PubMed

    Ruiz-Dueñas, Francisco J; Fernández, Elena; Martínez, María Jesús; Martínez, Angel T

    2011-11-01

    An exhaustive screening of the Pleurotus ostreatus genome was performed to search for nucleotide sequences of heme peroxidases in this white-rot fungus, which could be useful for different biotechnological applications. After sequence identification and manual curation of the corresponding genes and cDNAs, the deduced amino acid sequences were converted into structural homology models. A comparative study of these sequences and their structural models with those of known fungal peroxidases revealed the complete inventory of heme peroxidases of this fungus. This consists of cytochrome c peroxidase and ligninolytic peroxidases, including manganese peroxidase and versatile peroxidase but not lignin peroxidase, as representative of the "classical" superfamily of plant, fungal, and bacterial peroxidases; and members of two relatively "new" peroxidase superfamilies, namely heme-thiolate peroxidases, here described for the first time in a fungus from the genus Pleurotus, and dye-decolorizing peroxidases, already known in P. ostreatus but still to be thoroughly explored and characterized.

  10. 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

  11. Chlorpromazine as permeabilizer and reagent for detection of microbial peroxidase and peroxidaselike activities.

    PubMed Central

    Galeazzi, L; Turchetti, G; Grilli, G; Groppa, G; Giunta, S

    1986-01-01

    Chlorpromazine was used to perform a test for the detection of microbial peroxidase activities. The compound acts as both a cell permeabilizer and a reagent in the procedure developed which allows the detection of peroxidase and peroxidase like reactions both semiquantitatively in whole cell determinations and quantitatively in cell-free supernatants. PMID:3539020

  12. Lignin-degrading peroxidases from genome of selective ligninolytic fungus Ceriporiopsis subvermispora.

    PubMed

    Fernández-Fueyo, Elena; Ruiz-Dueñas, Francisco J; Miki, Yuta; Martínez, María Jesús; Hammel, Kenneth E; Martínez, Angel T

    2012-05-11

    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.

  13. Peroxidase Activity in Relation to Suberization and Respiration in White Spruce (Picea glauca [Moench] Voss) Seedling Roots 1

    PubMed Central

    Johnson-Flanagan, Anne M.; Owens, John N.

    1985-01-01

    Peroxidase (EC 1.11.1.7) activity is associated with suberization during endodermal development and metacutization in roots of white spruce (Picea glauca [Moench] Voss) seedlings. Histochemical analysis indicates a relationship between suberization and peroxidase activity, but peroxidase is ubiquitous. Increased peroxidase activity results from the induction of four anodic peroxidase isozymes in addition to quantitative increases in two anodic peroxidase isozymes. Four of these polymerized eugenol. Cold temperatures induce formation of two anodic isozymes and result in suberization. The increased peroxidase activity associated with suberization is correlated to residual respiration. In an attempt to elucidate this relationship, the effect of respiratory inhibitors on respiration and peroxidase activity are compared. PMID:16664352

  14. Peripheral Muscle Targets and Central Projections of the Mesencephalic Trigeminal Nucleus in Macaque Monkeys

    PubMed Central

    Wang, Niping; May, Paul J.

    2010-01-01

    The mesencephalic trigeminal nucleus (MesV) contains the somata of primary afferent neurons that innervate muscle spindles in masticatory muscles and mechanoreceptors in the periodontal ligaments. There are conflicting reports about additional peripheral targets of MesV, such as the extraocular muscles, as well as about its central targets. In addition, only limited primate data are available. Consequently, we examined MesV projections in macaque monkeys. The retrograde tracer wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) was injected into masticatory or extraocular muscles to define the peripheral targets of the primate MesV. Numerous labeled neurons were found in ipsilateral MesV following masticatory muscle injections. The scattered distribution of labeled cells, and their presence amongst clusters of unlabeled cells, suggests the muscle representations overlap. Just a few MesV neurons were labeled after extraocular muscle injections. This correlates with the small number of muscle spindles present in macaque extraocular muscles, suggesting MesV cells supplying extraocular muscle spindles may contribute a minor component to oculomotor proprioception. To examine the central connections of MesV, biotinylated dextran amine (BDA) was injected into the spinal trigeminal nucleus (Vs). The presence of retrogradely labeled MesV cells indicated a projection to Vs from MesV. These injections also anterogradely labeled terminals that lay in close association with MesV cells, suggesting an ascending projection from Vs to MesV. Finally, a small number of MesV neurons were labeled following WGA-HRP injections into the upper cervical spinal cord. This pattern of central connections indicates MesV and Vs information is combined to guide mastication. PMID:18461596

  15. Organization of spinocerebellar projection map in three types of agranular cerebellum: Purkinje cells vs. granule cells as organizer element

    SciTech Connect

    Arsenio Nunes, M.L.; Sotelo, C.; Wehrle, R.

    1988-07-01

    The organization of the spinocerebellar projection was analysed by the anterograde axonal WGA-HRP (horseradish peroxidase-wheat germ agglutinin conjugate) tracing method in three different types of agranular cerebellar cortex either induced experimentally by X-irradiation or occurring spontaneously in weaver (wv/wv) and staggerer (sg/sg) mutant mice. The results of this study show that in the X-irradiated rat and weaver mouse, in both of which the granule cells are directly affected and die early in development, the spinal axons reproduce, with few differences, the normal spinocerebellar pattern. Conversely, in staggerer mouse, in which the Purkinje cells are intrinsically affected and granule neurons do not seem to be primarily perturbed by the staggerer gene action, the spinocerebellar organization is severely modified. These findings appear somewhat paradoxical because if granule cells, the synaptic targets of mossy spinocerebellar fibers, were necessary for the organization of spinocerebellar projection, the staggerer cerebellum would exhibit a much more normal projectional map than the weaver and the X-irradiated cerebella. It is, therefore, obvious that granule cells, and even specific synaptogenesis, are not essential for the establishment of the normal spinocerebellar topography. On the other hand, the fact that the Purkinje cells are primarily affected in the unique agranular cortex in which the spinocerebellar organization is severely modified suggests that these neurons could be the main element in the organization of the spinocerebellar projection map. This hypothesis is discussed in correlation with already-reported findings on the zonation of the cerebellar cortex by biochemically different clusters of Purkinje cells.

  16. Direct synaptic contacts on the myenteric ganglia of the rat stomach from the dorsal motor nucleus of the vagus.

    PubMed

    Hayakawa, Tetsu; Kuwahara, Sachi; Maeda, Seishi; Tanaka, Koichi; Seki, Makoto

    2006-09-20

    The myenteric ganglia regulate not only gastric motility but also secretion, because a submucous plexus is sparsely developed in the rodent stomach. We have examined whether the neurons of the dorsal motor nucleus of the vagus (DMV) have direct synaptic contacts on the myenteric ganglia and the ultrastructure of the vagal efferent terminals by using wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). The myenteric ganglia of the rat were composed of four types of neurons, i.e., small, medium-sized, large, and elongated neurons. The average numbers of axosomatic terminals per profile were 2.0 on the small neurons, 3.1 on the medium-sized neurons, 1.2 on the large neurons, and 4.2 on the elongated neuron. More than half of the axosomatic terminals contained round vesicles and formed asymmetric synaptic contacts on the small, medium-sized, and large neurons. About 80% of the axosomatic terminals on the elongated neurons contained pleomorphic vesicles and formed asymmetric synaptic contacts. When WGA-HRP was injected into the DMV, many anterogradely labeled terminals were found around the myenteric neurons. The labeled terminals were large (3.16 +/- 0.10 microm) and contacted exclusively the somata. Most of them (about 90%) contained round vesicles and formed asymmetric synaptic contacts. Serial ultrathin sections revealed that almost all neurons in a ganglion received projections from the DMV. The vagal axon terminals generally contacted the medium-sized or the elongated neurons, whereas a few labeled terminals contacted the small and the large neurons. The present results indicate that the DMV projects to all types of neurons and that their axon terminals contain mostly round synaptic vesicles and form asymmetric synaptic contacts.

  17. Projections from the cat posterior lateral hypothalamus to the ventral part of the oral pontine reticular nucleus contain a GABAergic component.

    PubMed

    De La Roza, Carmen; Martínez-Mena, Juana; Sánchez-Valle, María Elena; Reinoso-Suárez, Fernando

    2004-09-10

    The posterior lateral hypothalamus (PLH) has long been considered crucial to normal wakefulness while the ventral part of the oral pontine reticular nucleus (vRPO) is involved in the generation and maintenance of rapid eye movement (REM) sleep. However, to date, there is no information on the ultrastructure or neurotransmitter content of the hypothalamo-reticular projection. In the present study, we examined the morphology and synaptic organization of PLH terminals in the vRPO using PLH injections of biotinylated dextran amine (BDA) as well as of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Since some PLH neurons are GABAergic, we used a post-embedding immunogold technique to determine whether any anterogradely labeled terminals were GABA-immunopositive. Electron microscope analyses revealed a variety of ultrastructural features in the vRPO anterogradely labeled terminals. Although most labeled terminals (over 63%) formed symmetric synapses on vRPO somata and dendrites, others made asymmetric synapses on vRPO dendrites. The relative percentages of labeled terminals observed on large, medium and small diameter dendrites were 44.3 +/- 5.5%, 35.3 +/- 3.0% and 20.4 +/- 3.1%, respectively. Finally, post-embedding immunogold technique revealed that there are GABA-immunopositive and immunonegative components to this projection, indicating that GABA is one of the transmitters used by the PLH cells that project to the vRPO. Furthermore, most, if not all, of the GABA-labeled axon terminals formed symmetric synapsis. In conclusion, our results suggest that the PLH could modulate the physiological responses of vRPO neurons through a GABAergic pathway as well as by other inhibitory and/or excitatory pathways. Activation of the descending PLH GABAergic projection may inhibit the REM sleep-inducing neurons within the vRPO and thus contribute to the suppression of REM sleep activation during wakefulness.

  18. Topographic organization of subcortical projections to the anterior thalamic nuclei in the rat.

    PubMed

    Shibata, H

    1992-09-01

    Subcortical projections to the anterior thalamic nuclei were studied in the rat, with special reference to projections from the mammillary nuclei, by retrograde and anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase. The medial mammillary nucleus (MM) projects predominantly ipsilaterally to the entire anterior thalamic nuclei, whereas the lateral mammillary nucleus projects bilaterally to the anterodorsal nucleus (AD) of the anterior thalamic nuclei. A topographic relationship was recognized between the MM and the anterior thalamic nuclei. The dorsal region of the pars mediana of the MM projects to the interanteromedial nucleus (IAM), whereas the ventral region projects to the rostral part of the anteromedial nucleus (AM). The dorsal and the ventral regions of the pars medialis project to the dorsomedial part of the AM at its caudal and rostral levels, respectively. The dorsomedial region of the pars lateralis projects to the ventral AM. The ventrolateral region of the pars lateralis projects to the ventral part of the anteroventral nucleus (AV) in such a manner that rostral cells project rostrally and caudal cells project caudally. The pars basalis projects predominantly ipsilaterally to the dorsolateral AV and bilaterally to the AD. The rostrolateral region of the pars posterior projects to the lateral AV, whereas the medial and the caudal regions of the pars posterior project to the dorsomedial AV. The rostrodorsal part of the nucleus reticularis thalami was found to project to the anterior thalamic nuclei; cells located rostrally in this part project to the IAM and AM, whereas cells located caudodorsally project to the AV and AD. The laterodorsal tegmental nucleus projects predominantly ipsilaterally to the AV, especially to its dorsolateral part. The present study demonstrates that subdivisions of the subcortical structures are connected to the subnuclei of the anterior thalamic nuclei, with a clear-cut topography arranged in

  19. Reticulospinal neurons in the pontomedullary reticular formation of the monkey (Macaca fascicularis).

    PubMed

    Sakai, S T; Davidson, A G; Buford, J A

    2009-11-10

    Recent neurophysiological studies indicate a role for reticulospinal neurons of the pontomedullary reticular formation (PMRF) in motor preparation and goal-directed reaching in the monkey. Although the macaque monkey is an important model for such investigations, little is known regarding the organization of the PMRF in the monkey. In the present study, we investigated the distribution of reticulospinal neurons in the macaque. Bilateral injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) were made into the cervical spinal cord. A wide band of retrogradely labeled cells was found in the gigantocellular reticular nucleus (Gi) and labeled cells continued rostrally into the caudal pontine reticular nucleus (PnC) and into the oral pontine reticular nucleus (PnO). Additional retrograde tracing studies following unilateral cervical spinal cord injections of cholera toxin subunit B revealed that there were more ipsilateral (60%) than contralateral (40%) projecting cells in Gi, while an approximately 50:50 ratio contralateral to ipsilateral split was found in PnC and more contralateral projections arose from PnO. Reticulospinal neurons in PMRF ranged widely in size from over 50 microm to under 25 microm across the major somatic axis. Labeled giant cells (soma diameters greater than 50 microm) comprised a small percentage of the neurons and were found in Gi, PnC and PnO. The present results define the origins of the reticulospinal system in the monkey and provide an important foundation for future investigations of the anatomy and physiology of this system in primates.

  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. Ulex europaeus agglutinin-I binding to dental primary afferent projections in the spinal trigeminal complex combined with double immunolabeling of substance P and GABA elements using peroxidase and colloidal gold.

    PubMed

    Matthews, M A; Hoffmann, K D; Hernandez, T V

    1989-01-01

    Ulex europaeus agglutinin I (UEA-I) is a plant lectin with an affinity for L-fucosyl residues in the chains of lactoseries oligosaccharides associated with medium- and smaller-diameter dorsal root ganglion neurons and their axonal processes. These enter Lissauer's tract and terminate within the superficial laminae of the spinal cord overlapping projections known to have a nociceptive function. This implies that the surface coatings of neuronal membranes may have a relationship with functional modalities. The present investigation further examined this concept by studying a neuronal projection with a nociceptive function to determine whether fucosyl-lactoseries residues were incorporated in its primary afferent terminals. Transganglionic transport of horseradish peroxidase (HRP) following injection into tooth pulp chambers was employed to demonstrate dental pulp terminals in the trigeminal spinal complex, while peroxidase and fluorescent tags were used concomitantly to stain for UEA-I. Double immunolabeling for substance P (SP) and gamma-aminobutyric acid (GABA) using peroxidase and colloidal gold allowed a comparison of the distribution of a known excitatory nociceptive transmitter with that of UEA-I binding in specific subnuclei. Synaptic interrelationships between UEA-I positive dental pulp primary afferent inputs and specific inhibitory terminals were also examined. SP immunoreactivity occurred in laminae I and outer lamina II (IIo) of subnucleus caudalis (Vc) and in the ventrolateral and lateral marginal region of the caudal half of subnucleus interpolaris (Vi), including the periobex area in which Vi is slightly overlapped on its lateral aspect by cellular elements of Vc. The adjacent interstitial nucleus (IN) also showed an intense immunoreactivity for this peptide antibody. UEA-I binding displayed a similar distribution pattern in both Vc and Vi, but extended into lamina IIi and the superficial part of Lamina III in Vc. Dental pulp terminals were found to

  2. Oxidative activation of benzidine and its derivatives by peroxidases.

    PubMed Central

    Josephy, P D

    1985-01-01

    Benzidine (4,4'-diaminobiphenyl) is a known human carcinogen; exposure to this substance resulted in an epidemic of bladder cancer among workers in the dye industry in Europe and North America. The chemical or enzymatic oxidation of benzidine proceeds via a racial cation detectable by electron spin resonance. Peroxidase-catalyzed oxidation of benzidine generates reactive electrophiles which readily form adducts with phenol and thiol compounds. The structures of these novel metabolites are described. Peroxidases, including prostaglandin synthase, catalyze benzidine binding to protein and nucleic acid; the nature of the resulting adducts is unknown. The relevance of these processes to benzidine carcinogenesis in vivo is the subject of research and debate. A central question remains: is benzidine activated in extra-hepatic target tissues such as bladder epithelium, or transported to these tissues following hepatic oxidative metabolism? PMID:3007087

  3. Steady state equivalence among autocatalytic peroxidase-oxidase reactions.

    PubMed

    Méndez-González, José; Femat, Ricardo

    2016-12-14

    Peroxidase-oxidase is an enzymatic reaction that can exhibit dynamical scenarios such as bistability, sustained oscillations, and Shilnikov chaos. In this work, we apply the chemical reaction network theory approach to find kinetic constants such that the associated mass action kinetics ordinary differential equations induced by three four dimensional structurally different enzymatic reaction systems can support the same steady states for several chemical species despite differences in their chemical nature.

  4. Steady state equivalence among autocatalytic peroxidase-oxidase reactions

    NASA Astrophysics Data System (ADS)

    Méndez-González, José; Femat, Ricardo

    2016-12-01

    Peroxidase-oxidase is an enzymatic reaction that can exhibit dynamical scenarios such as bistability, sustained oscillations, and Shilnikov chaos. In this work, we apply the chemical reaction network theory approach to find kinetic constants such that the associated mass action kinetics ordinary differential equations induced by three four dimensional structurally different enzymatic reaction systems can support the same steady states for several chemical species despite differences in their chemical nature.

  5. Luminol-hydrogen peroxide chemiluminescence produced by sweet potato peroxidase.

    PubMed

    Alpeeva, Inna S; Yu Sakharov, Ivan

    2007-01-01

    Anionic sweet potato peroxidase (SPP; Ipomoea batatas) was shown to efficiently catalyse luminol oxidation by hydrogen peroxide, forming a long-term chemiluminescence (CL) signal. Like other anionic plant peroxidases, SPP is able to catalyse this enzymatic reaction efficiently in the absence of any enhancer. Maximum intensity produced in SPP-catalysed oxidation of luminol was detected at pH 7.8-7.9 to be lower than that characteristic of other peroxidases (8.4-8.6). Varying the concentrations of luminol, hydrogen peroxide and Tris buffer in the reaction medium, we determined favourable conditions for SPP catalysis (100 mmol/L Tris-HCl buffer, pH 7.8, containing 5 mmol/L hydrogen peroxide and 8 mmol/L luminol). The SPP detection limit in luminol oxidation was 1.0 x 10(-14) mol/L. High sensitivity in combination with the long-term CL signal and high stability is indicative of good promise for the application of SPP in CL enzyme immunoassay.

  6. 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

  7. 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.

  8. 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.

  9. Cu-hemin metal-organic frameworks with peroxidase-like activity as peroxidase mimics for colorimetric sensing of glucose

    NASA Astrophysics Data System (ADS)

    Liu, Fenfen; He, Juan; Zeng, Mulang; Hao, Juan; Guo, Qiaohui; Song, Yonghai; Wang, Li

    2016-05-01

    In this work, a facile strategy to synthesize Cu-hemin metal-organic frameworks (MOFs) with peroxidase-like activity was reported. The prepared Cu-hemin MOFs were characterized by various techniques such as scanning electron microscopy, transmission electron microscopy, X-ray powder diffraction, Fourier transform infrared spectroscopy, UV-visible absorbance spectra, and so on. The results showed that the prepared Cu-hemin MOFs looked like a ball-flower with an average diameter of 10 μm and provided a large specific surface area. The Cu-hemin MOFs possessing peroxidase-like activity could be used to catalyze the peroxidase substrate of 3,3,5,5-tetramethylbenzidine in the presence of H2O2, which was employed to detect H2O2 quantitatively with the linear range from 1.0 μM to 1.0 mM and the detection limit was 0.42 μM. Furthermore, with the additional help of glucose oxidase, a sensitive and selective method to detect glucose was developed by using the Cu-hemin MOFs as catalyst and the linear range was from 10.0 μM to 3.0 mM and the detection limit was 6.9 μM. This work informs researchers of the advantages of MOFs for preparing biomimetic catalysts and extends the functionality of MOFs for biosensor application.

  10. 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.

  11. Alterations in Soluble Class III Peroxidases of Maize Shoots by Flooding Stress

    PubMed Central

    Meisrimler, Claudia-Nicole; Buck, Friedrich; Lüthje, Sabine

    2014-01-01

    Due to changing climate, flooding (waterlogged soils and submergence) becomes a major problem in agriculture and crop production. In the present study, the effect of waterlogging was investigated on peroxidases of maize (Zea mays L.) leaves. The plants showed typical adaptations to flooding stress, i.e., alterations in chlorophyll a/b ratios and increased basal shoot diameter. Seven peroxidase bands could be detected by first dimension modified SDS-PAGE and 10 bands by first dimension high resolution Clear Native Electrophoresis that altered in dependence on plant development and time of waterlogging. Native isoelectric focusing revealed three acidic to neutral and four alkaline guaiacol peroxidases that could be further separated by high resolution Clear Native Electrophorese in the second dimension. One neutral peroxidase (pI 7.0) appeared to be down-regulated within four hours after flooding, whereas alkaline peroxidases (pI 9.2, 8.0 and 7.8) were up-regulated after 28 or 52 h. Second dimensions revealed molecular masses of 133 kDa and 85 kDa for peroxidases at pI 8.0 and 7.8, respectively. Size exclusion chromatography revealed native molecular masses of 30–58 kDa for peroxidases identified as class III peroxidases and ascorbate peroxidases by mass spectrometry. Possible functions of these peroxidases in flooding stress will be discussed. PMID:28250383

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. Indomethacin inactivates gastric peroxidase to induce reactive-oxygen-mediated gastric mucosal injury and curcumin protects it by preventing peroxidase inactivation and scavenging reactive oxygen.

    PubMed

    Chattopadhyay, Ishita; Bandyopadhyay, Uday; Biswas, Kaushik; Maity, Pallab; Banerjee, Ranajit K

    2006-04-15

    We have investigated the mechanism of indomethacin-induced gastric ulcer caused by reactive oxygen species (ROS) and the gastroprotective effect of curcumin thereon. Curcumin dose-dependently blocks indomethacin-induced gastric lesions, showing 82% protection at 25 mg/kg. Indomethacin-induced oxidative damage by ROS as shown by increased lipid peroxidation and thiol depletion is almost completely blocked by curcumin. Indomethacin causes nearly fivefold increase in hydroxyl radical (()OH) and significant inactivation of gastric mucosal peroxidase to elevate endogenous H(2)O(2) and H(2)O(2)-derived ()OH, which is prevented by curcumin. In vitro studies indicate that indomethacin inactivates peroxidase irreversibly only in presence of H(2)O(2) by acting as a suicidal substrate. 5,5-Dimethyl-pyrroline-N-oxide (DMPO) protects the peroxidase, indicating involvement of indomethacin radical in the inactivation. Indomethacin radical was also detected in the peroxidase-indomethacin-H(2)O(2) system as DMPO adduct (a(N) = 15 G, a(beta)(H) = 16 G) by electron spin resonance spectroscopy. Curcumin protects the peroxidase in a concentration-dependent manner and consumes H(2)O(2) for its oxidation as a suitable substrate of the peroxidase, thereby blocking indomethacin oxidation. Curcumin can also scavenge ()OH in vitro. We suggest that curcumin protects gastric damage by efficient removal of H(2)O(2) and H(2)O(2) -derived ()OH by preventing peroxidase inactivation by indomethacin.

  17. 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.

  18. Formation of a tyrosine adduct involved in lignin degradation by Trametopsis cervina lignin peroxidase: a novel peroxidase activation mechanism.

    PubMed

    Miki, Yuta; Pogni, Rebecca; Acebes, Sandra; Lucas, Fátima; Fernández-Fueyo, Elena; Baratto, Maria Camilla; Fernández, María I; de los Ríos, Vivian; Ruiz-Dueñas, Francisco J; Sinicropi, Adalgisa; Basosi, Riccardo; Hammel, Kenneth E; Guallar, Victor; Martínez, Angel T

    2013-06-15

    LiP (lignin peroxidase) from Trametopsis cervina has an exposed catalytic tyrosine residue (Tyr181) instead of the tryptophan conserved in other lignin-degrading peroxidases. Pristine LiP showed a lag period in VA (veratryl alcohol) oxidation. However, VA-LiP (LiP after treatment with H2O2 and VA) lacked this lag, and H2O2-LiP (H2O2-treated LiP) was inactive. MS analyses revealed that VA-LiP includes one VA molecule covalently bound to the side chain of Tyr181, whereas H2O2-LiP contains a hydroxylated Tyr181. No adduct is formed in the Y171N variant. Molecular docking showed that VA binding is favoured by sandwich π stacking with Tyr181 and Phe89. EPR spectroscopy after peroxide activation of the pre-treated LiPs showed protein radicals other than the tyrosine radical found in pristine LiP, which were assigned to a tyrosine-VA adduct radical in VA-LiP and a dihydroxyphenyalanine radical in H2O2-LiP. Both radicals are able to oxidize large low-redox-potential substrates, but H2O2-LiP is unable to oxidize high-redox-potential substrates. Transient-state kinetics showed that the tyrosine-VA adduct strongly promotes (>100-fold) substrate oxidation by compound II, the rate-limiting step in catalysis. The novel activation mechanism is involved in ligninolysis, as demonstrated using lignin model substrates. The present paper is the first report on autocatalytic modification, resulting in functional alteration, among class II peroxidases.

  19. 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

  20. 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.

  1. 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.

  2. Design of more powerful iron-TAML peroxidase enzyme mimics.

    PubMed

    Ellis, W Chadwick; Tran, Camly T; Denardo, Matthew A; Fischer, Andreas; Ryabov, Alexander D; Collins, Terrence J

    2009-12-23

    Environmentally useful, small molecule mimics of the peroxidase enzymes must exhibit very high reactivity in water near neutral pH. Here we describe the design and structural and kinetic characterization of a second generation of iron(III)-TAML activators with unprecedented peroxidase-mimicking abilities. Iterative design has been used to remove the fluorine that led to the best performers in first-generation iron-TAMLs. The result is a superior catalyst that meets a green chemistry objective by being comprised exclusively of biochemically common elements. The rate constants for bleaching at pH 7, 9, and 11 of the model substrate, Orange II, shows that the new Fe(III)-TAML has the fastest reactivity at pH's closer to neutral of any TAML activator to date. Under appropriate conditions, the new catalyst can decolorize Orange II without loss of activity for at least 10 half-lives, attesting to its exceptional properties as an oxidizing enzyme mimic.

  3. High Conformational Stability of Secreted Eukaryotic Catalase-peroxidases

    PubMed Central

    Zámocký, Marcel; García-Fernández, Queralt; Gasselhuber, Bernhard; Jakopitsch, Christa; Furtmüller, Paul G.; Loewen, Peter C.; Fita, Ignacio; Obinger, Christian; Carpena, Xavi

    2012-01-01

    Catalase-peroxidases (KatGs) are bifunctional heme enzymes widely spread in archaea, bacteria, and lower eukaryotes. Here we present the first crystal structure (1.55 Å resolution) of an eukaryotic KatG, the extracellular or secreted enzyme from the phytopathogenic fungus Magnaporthe grisea. The heme cavity of the homodimeric enzyme is similar to prokaryotic KatGs including the unique distal +Met-Tyr-Trp adduct (where the Trp is further modified by peroxidation) and its associated mobile arginine. The structure also revealed several conspicuous peculiarities that are fully conserved in all secreted eukaryotic KatGs. Peculiarities include the wrapping at the dimer interface of the N-terminal elongations from the two subunits and cysteine residues that cross-link the two subunits. Differential scanning calorimetry and temperature- and urea-mediated unfolding followed by UV-visible, circular dichroism, and fluorescence spectroscopy combined with site-directed mutagenesis demonstrated that secreted eukaryotic KatGs have a significantly higher conformational stability as well as a different unfolding pattern when compared with intracellular eukaryotic and prokaryotic catalase-peroxidases. We discuss these properties with respect to the structure as well as the postulated roles of this metalloenzyme in host-pathogen interactions. PMID:22822072

  4. Peroxidase-catalysed interfacial adhesion of aquatic caddisworm silk

    PubMed Central

    Wang, Ching-Shuen; Pan, Huaizhong; Weerasekare, G. Mahika; Stewart, Russell J.

    2015-01-01

    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

  5. Glutathione peroxidase 4 and vitamin E cooperatively prevent hepatocellular degeneration.

    PubMed

    Carlson, Bradley A; Tobe, Ryuta; Yefremova, Elena; Tsuji, Petra A; Hoffmann, Victoria J; Schweizer, Ulrich; Gladyshev, Vadim N; Hatfield, Dolph L; Conrad, Marcus

    2016-10-01

    The selenoenzyme glutathione peroxidase 4 (Gpx4) is an essential mammalian glutathione peroxidase, which protects cells against detrimental lipid peroxidation and governs a novel form of regulated necrotic cell death, called ferroptosis. To study the relevance of Gpx4 and of another vitally important selenoprotein, cytosolic thioredoxin reductase (Txnrd1), for liver function, mice with conditional deletion of Gpx4 in hepatocytes were studied, along with those lacking Txnrd1 and selenocysteine (Sec) tRNA (Trsp) in hepatocytes. Unlike Txnrd1- and Trsp-deficient mice, Gpx4(-/-) mice died shortly after birth and presented extensive hepatocyte degeneration. Similar to Txnrd1-deficient livers, Gpx4(-/-) livers manifested upregulation of nuclear factor (erythroid-derived)-like 2 (Nrf2) response genes. Remarkably, Gpx4(-/-) pups born from mothers fed a vitamin E-enriched diet survived, yet this protection was reversible as subsequent vitamin E deprivation caused death of Gpx4-deficient mice ~4 weeks thereafter. Abrogation of selenoprotein expression in Gpx4(-/-) mice did not result in viable mice, indicating that the combined deficiency aggravated the loss of Gpx4 in liver. By contrast, combined Trsp/Txnrd1-deficient mice were born, but had significantly shorter lifespans than either single knockout, suggesting that Txnrd1 plays an important role in supporting liver function of mice lacking Trsp. In sum our study demonstrates that the ferroptosis regulator Gpx4 is critical for hepatocyte survival and proper liver function, and that vitamin E can compensate for its loss by protecting cells against deleterious lipid peroxidation.

  6. Polyclonal antibodies mediated immobilization of a peroxidase from ammonium sulphate fractionated bitter gourd (Momordica charantia) proteins.

    PubMed

    Fatima, Aiman; Husain, Qayyum

    2007-06-01

    Polyclonal antibody bound Sepharose 4B support has been exploited for the immobilization of bitter gourd peroxidase directly from ammonium sulphate precipitated proteins. Immunoaffinity immobilized bitter gourd peroxidase exhibited high yield of immobilization. IgG-Sepharose 4B bound bitter gourd peroxidase showed a higher stability against heat, chaotropic agents (urea and guanidinium chloride), detergents (cetyl trimethyl ammonium bromide and Surf Excel), proteolytic enzyme (trypsin) and water-miscible organic solvents (propanol, THF and dioxane). The activity of immobilized bitter gourd peroxidase was significantly enhanced in the presence of cetyl trimethyl ammonium bromide and after treatment with trypsin as compared to soluble enzyme.

  7. 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.

  8. 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.

  9. 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.

  10. Platelet peroxidase deficiency in a case of myelodysplastic syndrome with myelofibrosis.

    PubMed Central

    Imbert, M; Jarry, M T; Tulliez, M; Breton-Gorius, J

    1983-01-01

    Morphological and functional abnormalities of the megakaryocytic series have been well described in myelodysplastic syndromes. Platelet peroxidase has always been demonstrated in abnormal megakaryocytes and early megakaryoblasts in such syndromes. We have studied a case of myelodysplastic syndrome with marked morphological abnormalities of megakaryocytes in which ultrastructural studies showed the coexistence of platelet peroxidase positive and platelet peroxidase negative megakaryocytes. This enzymatic deficiency was confirmed by the ultrastructural study of circulating platelets. This case appears to be the first report of a partial platelet peroxidase deficiency. It adds to the enzymatic abnormalities in myelodysplastic syndrome already described for the red cells and the granulocytic cells. Images PMID:6630573

  11. 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.

  12. Changes in ascorbate peroxidase, catalase, guaiacol peroxidase and superoxide dismutase activities in common bean (Phaseolus vulgaris) nodules under salt stress.

    PubMed

    Jebara, Salwa; Jebara, Moez; Limam, Férid; Aouani, Mohamed Elarbi

    2005-08-01

    To analyse nodular antioxidant enzyme expression in response to salt stress, Phaseolus vulgaris genotype BAT477 was inoculated with reference strain CIAT899, and treated with 50 mM NaCl. Plant growth, nodulation and nitrogen fixing activity were analysed. Results showed that: (1) all parameters, particularly in nodules, were affected by salt treatments, and (2) confirmed preferential growth allocation to roots. The ARA was significantly decreased by salt treatments. Protein dosage confirmed that nodules were more affected by salt treatment than were roots. We analysed superoxide dismutase, catalase, ascorbate peroxidase and peroxidase in nodules, roots and a free rhizobial strain. Our results indicated that SOD and CAT nodular isozymes had bacterial and root origins. The SOD expressed the same CuZn, Fe and Mn SOD isoforms in nodules and roots, whereas in free rhizobia we found only one Fe and Mn SOD. APX and POX nodule and root profiles had only root origins, as no rhizobial band was detected. Under salt stress, plant growth, nitrogen fixation and activities of antioxidant defense enzymes in nodules were affected. Thus, these enzymes appear to preserve symbiosis from stress turned out that NaCl salinity lead to a differential regulation of distinct SOD and POX isoenzyme. So their levels in nodules appeared to be consistent with a symbiotic nitrogen fixing efficiency hypothesis, and they seem to function as the molecular mechanisms underlying the nodule response to salinity.

  13. Role of manganese peroxidases and lignin peroxidases of Phanerochaete chrysosporium in the decolorization of kraft bleach plant effluent.

    PubMed

    Michel, F C; Dass, S B; Grulke, E A; Reddy, C A

    1991-08-01

    The role of lignin peroxidases (LIPs) and manganese peroxidases (MNPs) of Phanerochaete chrysosporium in decolorizing kraft bleach plant effluent (BPE) was investigated. Negligible BPE decolorization was exhibited by a per mutant, which lacks the ability to produce both the LIPs and the MNPs. Also, little decolorization was seen when the wild type was grown in high-nitrogen medium, in which the production of LIPs and MNPs is blocked. A lip mutant of P. chrysosporium, which produces MNPs but not LIPs, showed about 80% of the activity exhibited by the wild type, indicating that the MNPs play an important role in BPE decolorization. When P. chrysosporium was grown in a medium with 100 ppm of Mn(II), high levels of MNPs but no LIPs were produced, and this culture also exhibited high rates of BPE decolorization, lending further support to the idea that MNPs play a key role in BPE decolorization. When P. chrysosporium was grown in a medium with no Mn(II), high levels of LIPs but negligible levels of MNPs were produced and the rate and extent of BPE decolorization by such cultures were quite low, indicating that LIPs play a relatively minor role in BPE decolorization. Furthermore, high rates of BPE decolorization were seen on days 3 and 4 of incubation, when the cultures exhibit high levels of MNP activity but little or no LIP activity. These results indicate that MNPs play a relatively more important role than LIPs in BPE decolorization by P. chrysosporium.

  14. 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

  15. Class III peroxidases are activated in proanthocyanidin-deficient Arabidopsis thaliana seeds

    PubMed Central

    Jia, Liguo; Xu, Weifeng; Li, Wenrao; Ye, Nenghui; Liu, Rui; Shi, Lu; Bin Rahman, A. N. M. Rubaiyath; Fan, Mingshou; Zhang, Jianhua

    2013-01-01

    Background and Aims It has previously been shown that proanthocyanidins (PAs) in the seed coat of Arabidopsis thaliana have the ability to scavenge superoxide radicals (O2−). However, the physiological processess in PA-deficit seeds are not clear. It is hypothesized that there exist alternative ways in PA-deficient seeds to cope with oxidative stress. Methods The content of hydrogen peroxide (H2O2) and its relevance to the activities of superoxide dismutase (SOD), catalase (CAT) and peroxidases was investigated in both wild-type and PA-deficit mutant seeds. A biochemical staining approach was used to detect tissue localizations of peroxidase activities in PA-deficit mutant seeds. Key Results PA-deficient mutants possess significantly lower levels of H2O2 than the wild-type, despite their higher accumulation of superoxide radicals. Screening of the key antioxidant enzymes revealed that peroxidase activity was significantly over-activated in mutant seeds. This high peroxidase activity was mainly confined to the seed coat zone. Interestingly, neither ascorbate peroxidase nor glutathione peroxidase, just the guaiacol peroxidases (class III peroxidases), was specifically activated in the seed coat. However, no significant difference in peroxidase activity was observed in embryos of either mutants or the wild-type, although gene expressions of several candidate peroxidases were down-regulated in the embryos of PA-deficient seeds. Conclusions The results suggest that enhanced class III peroxidase activity in the seed coat of PA-deficient mutants is an adaptive strategy for seed development and survival. PMID:23448691

  16. Inhibition of thyroid peroxidase by Myrcia uniflora flavonoids.

    PubMed

    Ferreira, Andrea C F; Neto, Jair C; da Silva, Alba C M; Kuster, Ricardo M; Carvalho, Denise P

    2006-03-01

    Thyroid peroxidase (TPO), the key enzyme in thyroid hormone biosynthesis, is inhibited by dietary flavonoids; thus, a high consumption of plants containing inhibitory flavonoids may affect thyroid function and lead to hypothyroidism. In this work, TPO inhibition by the aqueous partition of Myrcia uniflora and its isolated compounds has been evaluated. The aqueous partition of the methanolic extract of M. uniflora is able to inhibit TPO activity in vitro. Two known flavonoids were isolated and characterized by mass spectrometry and (1)H NMR from plant extracts: mearnsitrin and myricitrin. The degree of TPO inhibition produced by the aqueous solution of the flavonoids was very high, with a 50% inhibition of the original TPO activity (IC(50)) obtained at 1.97 microM mearnsitrin and at 2.88 microM myricitrin. These results suggest that the indiscriminated consumption of M. uniflora pharmaceutical products allied to the nutritional deficiency of iodine might contribute to the development of hypothyroidism and goiter.

  17. 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

    2013-07-03

    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.

  18. 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

  19. 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.

  20. 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.

  1. Structural and Functional Features of Peroxidases with a Potential as Industrial Biocatalysts

    NASA Astrophysics Data System (ADS)

    Ruiz-Dueñas, Francisco J.; Martínez, Angel T.

    This chapter begins with a description of the main structural features of heme peroxidases representative of the two large superfamilies of plant-fungal-bacterial and animal peroxidases, and the four additional (super)families described to date. Then, we focus on several fungal peroxidases of high biotechnological potential as industrial biocatalysts. These include (1) ligninolytic peroxidases from white-rot basidiomycetes being able to oxidize high redox-potential substrates at an exposed protein radical; (2) heme-thiolate peroxidases that are structural hybrids of typical peroxidases and cytochrome P450 enzymes and, after their discovery in sooty molds, are being described in basidiomycetes with even more interesting catalytic properties, such as selective aromatic oxygenation; and (3) the so-called dye-decolorizing peroxidases that are still to be thoroughly investigated but have been identified in different basidiomycete genomes. The structural-functional description of these peroxidases includes an analysis of the heme environment and a description of their substrate oxidation sites, with the purpose of understanding their interesting catalytic properties and biotechnological potential.

  2. The kinetic properties producing the perfunctory pH profiles of catalase-peroxidases.

    PubMed

    Moore, Robert L; Powell, Luke J; Goodwin, Douglas C

    2008-06-01

    Many structure-function relationship studies performed on the catalase-peroxidase enzymes are based on limited kinetic data. To provide a more substantive understanding of catalase-peroxidase function, we undertook a more exhaustive evaluation of catalase-peroxidase catalysis as a function of pH. Kinetic parameters across a broad pH range for the catalase and peroxidase activities of E. coli catalase peroxidase (KatG) were obtained, including the separate analysis of the oxidizing and reducing substrates of the peroxidase catalytic cycle. This investigation identified ABTS-dependent inhibition of peroxidase activity, particularly at low pH, unveiling that previously reported pH optima are clearly skewed. We show that turnover and efficiency of peroxidase activity increases with decreasing pH until the protein unfolds. The data also suggest that the catalase pH optimum is more complex than it is often assumed to be. The apparent optimum is in fact the intersection of the optimum for binding (7.00) and the optimum for activity (5.75). We also report the apparent pK(a)s for binding and catalysis of catalase activity as well as approximate values for certain peroxidatic and catalatic steps.

  3. Induction of soluble and cell wall peroxidases by aphid infestation in barley.

    PubMed

    Chaman, M E; Corcuera, L J; Zúñiga, G E; Cardemil, L; Argandoña, V H

    2001-05-01

    Peroxidase enzymes have been found in soluble, ionically bound, and covalently bound forms and have been implicated in several physiological processes in plants. This paper investigates the effect of aphid infestation on soluble and bound-cell wall peroxidase activity and bound-cell wall isoform changes of barley plants. Peroxidase activity was measured in control plants and plants infested with the aphid Schizaphis graminum (Rondani). The activity of soluble peroxidases increased with time of infestation, older plants being more affected than younger ones. The increase in bound-cell wall peroxidase activity as a function of age was higher in infested than in control plants, being higher in ionically bound than in covalently bound peroxidases. When the aphids were removed from plants, the activities of both types of peroxidases decreased to control levels. Isoelectrofocusing analyses of the ionically bound peroxidases showed changes in the isoform pattern. A new isoform was induced by infestation. The activities of all covalently bound isoforms increased after infestation. The physiological implications of these changes are discussed.

  4. 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...

  5. Enzymatic activity and proteomic profile of class III peroxidases during sugarcane stem development.

    PubMed

    Cesarino, Igor; Araújo, Pedro; Sampaio Mayer, Juliana Lischka; Paes Leme, Adriana Franco; Mazzafera, Paulo

    2012-06-01

    Class III peroxidases are present as large multigene families in all land plants. This large number of genes together with the diversity of processes catalyzed by peroxidases suggests possible functional specialization of each isoform. However, assigning a precise role for each individual peroxidase gene has continued to be a major bottleneck. Here we investigated the enzyme activity and translational profile of class III peroxidases during stem development of sugarcane as a first step in the estimation of physiological functions of individual isoenzymes. Internodes at three different developmental stages (young, developing and mature) were divided into pith (inner tissue) and rind (outer tissue) fractions. The rind of mature internodes presented the highest enzymatic activity and thus could be considered the ideal tissue for the discovery of peroxidase gene function. In addition, activity staining of 2DE gels revealed different isoperoxidase profiles and protein expression regulation among different tissue fractions. In-gel tryptic digestion of excised spots followed by peptide sequencing by LC-MS/MS positively matched uncharacterized peroxidases in the sugarcane database SUCEST. Multiple spots matching the same peroxidase gene were found, which reflects the generation of more than one isoform from a particular gene by post-translational modifications. The identified sugarcane peroxidases appear to be monocot-specific sequences with no clear ortholog in dicot model plant Arabidopsis thaliana.

  6. Characterization of Peroxidase Changes in Resistant and Susceptible Warm- Season Turfgrasses Challenged by Blissus Occiduus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Peroxidases play an important role in plant stress related interactions. This research assessed the role of peroxidases in the defense response of resistant and susceptible buffalograsses [Buchloe dactyloides (Nutt.) Engelm] and zoysiagrasses (Zoysia japonica Steudel) to the western chinch bug, Bli...

  7. Participation of chitin-binding peroxidase isoforms in the wilt pathogenesis of cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Specific chitin-binding isozymes of peroxidase (POX) play an important role in pathogenesis of plant diseases caused with fungi. We studied the dynamics of peroxidase activity in two varieties of cotton (Gossypium hirsutum L.); one was a susceptible and the other resistant to the plant pathogen Vert...

  8. 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

  9. Computational Modeling of the Catalytic Cycle of Glutathione Peroxidase Nanomimic.

    PubMed

    Kheirabadi, Ramesh; Izadyar, Mohammad

    2016-12-29

    To elucidate the role of a derivative of ebselen as a mimic of the antioxidant selenoenzyme glutathione peroxidase, density functional theory and solvent-assisted proton exchange (SAPE) were applied to model the reaction mechanism in a catalytic cycle. This mimic plays the role of glutathione peroxidase through a four-step catalytic cycle. The first step is described as the oxidation of 1 in the presence of hydrogen peroxide, while selenoxide is reduced by methanthiol at the second step. In the third step of the reaction, the reduction of selenenylsulfide occurs by methanthiol, and the selenenic acid is dehydrated at the final step. Based on the kinetic parameters, step 4 is the rate-determining step (RDS) of the reaction. The bond strength of the atoms involved in the RDS is discussed with the quantum theory of atoms in molecules (QTAIM). Low value of electron density, ρ(r), and positive Laplacian values are the evidence for the covalent nature of the hydrogen bonds rupture (O30-H31, O33-H34). A change in the sign of the Laplacian, L(r), from the positive value in the reactant to a negative character at the transition state indicates the depletion of the charge density, confirming the N5-H10 and O11-Se1 bond breaking. The analysis of electron location function (ELF) and localized orbital locator (LOL) of the Se1-N5 and Se1-O11 bonds have been done by multi-WFN program. High values of ELF and LOL at the transition state regions between the Se, N, and O atoms display the bond formation. Finally, the main donor-acceptor interaction energies were analyzed using the natural bond orbital analysis for investigation of their stabilization effects on the critical bonds at the RDS.

  10. Characterization of Anionic Peroxidases in Tomato Isolines Infected by Meloidogyne incognita

    PubMed Central

    Zacheo, G.; Orlando, C.; Bleve-Zacheo, T.

    1993-01-01

    Changes in peroxidase activity during nematode infection were studied using root extracts of tomato near-isogenic lines differing in resistance to Meloidogyne incognita. Total peroxidase activity increased slightly in crude extracts of four susceptible isolines but doubled in two resistant lines, Monita and Motaci. Nematode infection enhanced levels of both p-phenylenediamine-pyrocatechol oxidase and syringaldazine oxidase 7 days after inoculation, especially in resistant lines. This elevated peroxidase activity in resistant isolines was caused by an increase in anionic peroxidase activity. These enzymes, which likely are involved in lignification, were isolated and purified from tomato isolines by ammonium sulfate precipitation, high performance ion-exchange chromatography, and gel electrophoresis. The purified anionic peroxidase extracts contained an electrophoretic band with Rf 0.51 that was present in extracts of infected but not uninfected roots. PMID:19279765

  11. Suppression of Arabidopsis peroxidase 72 alters cell wall and phenylpropanoid metabolism.

    PubMed

    Fernández-Pérez, Francisco; Pomar, Federico; Pedreño, María A; Novo-Uzal, Esther

    2015-10-01

    Class III peroxidases are glycoproteins with a major role in cell wall maturation such as lignin formation. Peroxidases are usually present in a high number of isoenzymes, which complicates to assign specific functions to individual peroxidase isoenzymes. Arabidopsis genome encodes for 73 peroxidases, among which AtPrx72 has been shown to participate in lignification. Here, we report by using knock out peroxidase mutants how the disruption of AtPrx72 causes thinner secondary walls in interfascicular fibres but not in the xylem of the stem. This effect is also age-dependent, and AtPrx72 function seems to be particularly important when lignification prevails over elongation processes. Finally, the suppression AtPrx72 leads to the down-regulation of lignin biosynthesis pathway, as well as genes and transcription factors involved in secondary wall thickening.

  12. 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.

  13. Lignin-degrading peroxidases in Polyporales: an evolutionary survey based on 10 sequenced genomes.

    PubMed

    Ruiz-Dueñas, Francisco J; Lundell, Taina; Floudas, Dimitrios; Nagy, Laszlo G; Barrasa, José M; Hibbett, David S; Martínez, Angel T

    2013-01-01

    The genomes of three representative Polyporales (Bjerkandera adusta, Phlebia brevispora and a member of the Ganoderma lucidum complex) were sequenced to expand our knowledge on the diversity of ligninolytic and related peroxidase genes in this Basidiomycota order that includes most wood-rotting fungi. The survey was completed by analyzing the heme-peroxidase genes in the already available genomes of seven more Polyporales species representing the antrodia, gelatoporia, core polyporoid and phlebioid clades. The study confirms the absence of ligninolytic peroxidase genes from the manganese peroxidase (MnP), lignin peroxidase (LiP) and versatile peroxidase (VP) families, in the brown-rot fungal genomes (all of them from the antrodia clade), which include only a limited number of predicted low redox-potential generic peroxidase (GP) genes. When members of the heme-thiolate peroxidase (HTP) and dye-decolorizing peroxidase (DyP) superfamilies (up to a total of 64 genes) also are considered, the newly sequenced B. adusta appears as the Polyporales species with the highest number of peroxidase genes due to the high expansion of both the ligninolytic peroxidase and DyP (super)families. The evolutionary relationships of the 111 genes for class-II peroxidases (from the GP, MnP, VP, LiP families) in the 10 Polyporales genomes is discussed including the existence of different MnP subfamilies and of a large and homogeneous LiP cluster, while different VPs mainly cluster with short MnPs. Finally, ancestral state reconstructions showed that a putative MnP gene, derived from a primitive GP that incorporated the Mn(II)-oxidation site, is the precursor of all the class-II ligninolytic peroxidases. Incorporation of an exposed tryptophan residue involved in oxidative degradation of lignin in a short MnP apparently resulted in evolution of the first VP. One of these ancient VPs might have lost the Mn(II)-oxidation site being at the origin of all the LiP enzymes, which are found only in

  14. Substrate oxidation by dye-decolorizing peroxidases (DyPs) from wood- and litter-degrading agaricomycetes compared to other fungal and plant heme-peroxidases.

    PubMed

    Liers, Christiane; Pecyna, Marek J; Kellner, Harald; Worrich, Anja; Zorn, Holger; Steffen, Kari T; Hofrichter, Martin; Ullrich, René

    2013-07-01

    Catalytic and physicochemical properties of representative fungal dye-decolorizing peroxidases (DyPs) of wood- (WRF) and litter-decomposing white-rot fungi (LDF) are summarized and compared, including one recombinant Mycetinis scorodonius DyP (rMscDyP; LDF), the wild-type Auricularia auricula-judae DyP (AauDyP; WRF), and two new DyPs secreted by the jelly fungi Exidia glandulosa (EglDyP; WRF) and Mycena epipterygia (MepDyP; LDF). Homogeneous preparations of these DyPs were obtained after different steps of fast protein liquid chromatography, and they increase the total number of characterized fungal DyP proteins to eight. The peptide sequences of AauDyP, MepDyP, and EglDyP showed highest homologies (52-56%) to the DyPs of M. scorodonius. Five out of the eight characterized fungal DyPs were used to evaluate their catalytic properties compared to classic fungal and plant heme peroxidases, namely lignin peroxidase of Phanerochaete chrysosporium (PchLiP; WRF), versatile peroxidase of Bjerkandera adusta (BadVP; WRF), and generic peroxidases of Coprinopsis cinerea (CiP) and Glycine max (soybean peroxidase=SBP). All DyPs tested possess unique properties regarding the stability at low pH values: 50-90% enzymatic activity remained after 4-h exposition at pH 2.5, and the oxidation of nonphenolic aromatic substrates (lignin model compounds) was optimal below pH 3. Furthermore, all DyPs efficiently oxidized recalcitrant dyes (e.g., Azure B) as well as the phenolic substrate 2,6-dimethoxyphenol. Thus, DyPs combine features of different peroxidases on the functional level and may be part of the biocatalytic system secreted by fungi for the oxidation of lignin and/or toxic aromatic compounds.

  15. Molecular characterization of the lignin-forming peroxidase: Role in growth, development and response to stress. Progress summary report, April 1, 1992--March 31, 1993

    SciTech Connect

    Lagrimini, L.M.

    1993-03-01

    This laboratory has continued its comprehensive study of the structure and function of plant peroxidases and their genes. Specifically, we are characterizing the anionic peroxidase of tobacco. During the past year we have completed the nucleotide sequence of the tobacco anionic peroxidase gene, joined the anionic peroxidase promoter to {Beta}-glucuronidase and demonstrated expression in transformed plants, measured lignin, auxin, and ethylene levels in transgenic tobacco plants over-expressing the anionic peroxidase, developed chimeric peroxidase genes to over-or under-express the anionic peroxidase in tissue specific manner in transgenic plants, and over-expressed the tobacco anionic peroxidase in transgenic tomato and sweetgum plants.

  16. Characterization of Antisense Transformed Plants Deficient in the Tobacco Anionic Peroxidase.

    PubMed Central

    Lagrimini, L. M.; Gingas, V.; Finger, F.; Rothstein, S.; Liu, TTY.

    1997-01-01

    On the basis of the biological compounds that they metabolize, plant peroxidases have long been implicated in plant growth, cell wall biogenesis, lignification, and host defenses. Transgenic tobacco (Nicotiana tabacum L.) plants that underexpress anionic peroxidase were generated using antisense RNA. The antisense RNA was found to be specific for the anionic isoenzyme and highly effective, reducing endogenous transcript levels and total peroxidase activity by as much as 1600-fold. Antisense-transformed plants appeared normal at initial observation; however, growth studies showed that plants with reduced peroxidase activity grow taller and flower sooner than control plants. In contrast, previously transformed plants overproducing anionic peroxidase were shorter and flowered later than controls. Axillary buds were more developed in antisense-transformed plants and less developed in plants overproducing this enzyme. It was found that the lignin content in leaf, stem, and root was unchanged in antisense-transformed plants, which does not support a role for anionic peroxidase in the lignification of secondary xylem vessels. However, studies of wounded tissue show some reduction in wound-induced deposition of lignin-like polymers. The data support a possible role for tobacco anionic peroxidase in host defenses but not without a reduction in growth potential. PMID:12223765

  17. Production and Preliminary Characterization of Monoclonal Antibodies against Cationic Peanut Peroxidase 1

    PubMed Central

    Hu, Chunfang; Carbonera, Daniela; van Huystee, Robert

    1987-01-01

    Ten monoclonal antibodies (McAbs) have been produced against the cationic peroxidase from peanut suspension cell culture. Eight of these antibodies were found to be of the immunoglobulin (Ig)G1 subclass and two were of IgA subclass. A combination of competitive enzyme-linked immunosorbent assay, Western blotting analysis, and direct antigen-binding assay revealed that the antibodies are directed against four different epitopes on the cationic peroxidase and the McAbs can be subdivided into four groups. Only group A inhibits peroxidase activity. Group B and D bind equally well to the native and the denatured form of cationic peroxidase, whereas the remaining McAbs react with more or less reduced affinity to the denatured antigen. Group C probably recognizes a conformation-dependent epitope. All the McAbs cross react weakly with the anionic peanut peroxidase, suggesting a structural nonidentity as well as some similarity between these two peroxidase isozymes. Cross reactivities of these McAbs with peroxidases of various plant species were also demonstrated. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:16665674

  18. Crystal structure analysis of peroxidase from the palm tree Chamaerops excelsa.

    PubMed

    Bernardes, Amanda; Textor, Larissa C; Santos, Jademilson C; Cuadrado, Nazaret Hidalgo; Kostetsky, Eduard Ya; Roig, Manuel G; Bavro, Vassiliy N; Muniz, João R C; Shnyrov, Valery L; Polikarpov, Igor

    2015-04-01

    Palm tree peroxidases are known to be very stable enzymes and the peroxidase from the Chamaerops excelsa (CEP), which has a high pH and thermal stability, is no exception. To date, the structural and molecular events underscoring such biochemical behavior have not been explored in depth. In order to identify the structural characteristics accounting for the high stability of palm tree peroxidases, we solved and refined the X-ray structure of native CEP at a resolution of 2.6 Å. The CEP structure has an overall fold typical of plant peroxidases and confirmed the conservation of characteristic structural elements such as the heme group and calcium ions. At the same time the structure revealed important modifications in the amino acid residues in the vicinity of the exposed heme edge region, involved in substrate binding, that could account for the morphological variations among palm tree peroxidases through the disruption of molecular interactions at the second binding site. These modifications could alleviate the inhibition of enzymatic activity caused by molecular interactions at the latter binding site. Comparing the CEP crystallographic model described here with other publicly available peroxidase structures allowed the identification of a noncovalent homodimer assembly held together by a number of ionic and hydrophobic interactions. We demonstrate, that this dimeric arrangement results in a more stable protein quaternary structure through stabilization of the regions that are highly dynamic in other peroxidases. In addition, we resolved five N-glycosylation sites, which might also contribute to enzyme stability and resistance against proteolytic cleavage.

  19. Probing the two-domain structure of homodimeric prokaryotic and eukaryotic catalase-peroxidases.

    PubMed

    Banerjee, Srijib; Zamocky, Marcel; Furtmüller, Paul G; Obinger, Christian

    2010-11-01

    Catalase-peroxidases (KatGs) are ancestral bifunctional heme peroxidases found in archaeons, bacteria and lower eukaryotes. In contrast to homologous cytochrome c peroxidase (CcP) and ascorbate peroxidase (APx) homodimeric KatGs have a two-domain monomeric structure with a catalytic N-terminal heme domain and a C-terminal domain of high sequence and structural similarity but without obvious function. Nevertheless, without its C-terminal counterpart the N-terminal domain exhibits neither catalase nor peroxidase activity. Except some hybrid-type proteins all other members of the peroxidase-catalase superfamily lack this C-terminal domain. In order to probe the role of the two-domain monomeric structure for conformational and thermal stability urea and temperature-dependent unfolding experiments were performed by using UV-Vis-, electronic circular dichroism- and fluorescence spectroscopy, as well as differential scanning calorimetry. Recombinant prokaryotic (cyanobacterial KatG from Synechocystis sp. PCC6803) and eukaryotic (fungal KatG from Magnaporthe grisea) were investigated. The obtained data demonstrate that the conformational and thermal stability of bifunctional KatGs is significantly lower compared to homologous monofunctional peroxidases. The N- and C-terminal domains do not unfold independently. Differences between the cyanobacterial and the fungal enzyme are relatively small. Data will be discussed with respect to known structure and function of KatG, CcP and APx.

  20. Uncovering a new role for peroxidase enzymes as drivers of angiogenesis.

    PubMed

    Panagopoulos, Vasilios; Zinonos, Irene; Leach, Damien A; Hay, Shelley J; Liapis, Vasilios; Zysk, Aneta; Ingman, Wendy V; DeNichilo, Mark O; Evdokiou, Andreas

    2015-11-01

    Peroxidases are heme-containing enzymes released by activated immune cells at sites of inflammation. To-date their functional role in human health has mainly been limited to providing a mechanism for oxidative defence against invading bacteria and other pathogenic microorganisms. Our laboratory has recently identified a new functional role for peroxidase enzymes in stimulating fibroblast migration and collagen biosynthesis, offering a new insight into the causative association between inflammation and the pro-fibrogenic events that mediate tissue repair and regeneration. Peroxidases are found at elevated levels within and near blood vessels however, their direct involvement in angiogenesis has never been reported. Here we report for the first time that myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are readily internalised by human umbilical vein endothelial cells (HUVEC) where they promote cellular proliferation, migration, invasion, and stimulate angiogenesis both in vitro and in vivo. These pro-angiogenic effects were attenuated using the specific peroxidase inhibitor 4-ABAH, indicating the enzyme's catalytic activity is essential in mediating this response. Mechanistically, we provide evidence that MPO and EPO regulate endothelial FAK, Akt, p38 MAPK, ERK1/2 phosphorylation and stabilisation of HIF-2α, culminating in transcriptional regulation of key angiogenesis pathways. These findings uncover for the first time an important and previously unsuspected role for peroxidases as drivers of angiogenesis, and suggest that peroxidase inhibitors may have therapeutic potential for the treatment of angiogenesis related diseases driven by inflammation.

  1. Localization of gastric peroxidase and its inhibition by mercaptomethylimidazole, an inducer of gastric acid secretion.

    PubMed Central

    Bandyopadhyay, U; Bhattacharyya, D K; Chatterjee, R; Banerjee, R K

    1992-01-01

    Mercaptomethylimidazole (MMI) is a potent inducer of gastric acid secretion which is associated with significant inhibition of peroxidase activity of rat gastric mucosa in vivo. A time-dependent increase in acid secretion correlates well with time-dependent decrease in the peroxidase activity. In a chamber experiment in vitro using isolated gastric mucosa, MMI stimulates acid secretion, showing an almost linear response up to 600 microM. The time-dependent increase in acid secretion is also correlated with time-dependent inhibition of the peroxidase activity. This effect is not mediated through oxidation of MMI by flavin-containing mono-oxygenase, which is absent from gastric mucosa. The peroxidase has been localized mainly in parietal cells isolated and purified from gastric mucosa by controlled digestion with collagenase followed by Percoll-density-gradient centrifugation. Peroxidase activity was further localized in the outer membrane of the purified mitochondria of the parietal cell by some membrane-impermeant reagents, indicating outward orientation of the enzyme. MMI can inhibit the peroxidase activity of both the parietal cell and its mitochondria in a concentration-dependent manner. The possible involvement of the parietal-cell peroxidase-H2O2 system in MMI-induced acid secretion may be suggested. PMID:1318028

  2. Suspension cell culture as a tool for the characterization of class III peroxidases in sugarcane.

    PubMed

    Cesarino, Igor; Araújo, Pedro; Paes Leme, Adriana Franco; Creste, Silvana; Mazzafera, Paulo

    2013-01-01

    Secreted class III peroxidases (EC 1.11.1.7) are implicated in a broad range of physiological processes throughout the plant life cycle. However, the unambiguous determination of the precise biological role of an individual class III peroxidase isoenzyme is still a difficult task due to genetic redundancy and broad substrate specificity in vitro. In addition, many difficulties are encountered during extraction and analysis of cell wall proteins. Since class III peroxidases are also secreted into the apoplast, the use of suspension cell cultures can facilitate isolation and functional characterization of individual isoforms. Here, we report on the characterization of class III peroxidases secreted in the spent medium of sugarcane suspension cell cultures. After treatment with specific inducers of cell wall lignification, peroxidases were isolated and activities assayed with guaiacol, syringaldazine and coniferyl alcohol. Enzymatic activity was not significantly different after treatments, regardless of the substrate, with the exception of methyl-jasmonate treatment, which led to a decreased guaiacol peroxidase activity. Remarkably, peroxidases isolated from the medium were capable of oxidizing syringaldazine, an analog to sinapyl alcohol, suggesting that sugarcane cultures can produce peroxidases putatively correlated to lignification. A proteomic approach using activity staining of 2-DE gels revealed a complex isoperoxidase profile, composed predominantly of cationic isoforms. Individual spots were excised and analyzed by LC-ESI-Q-TOF and homology-based search against the Sugarcane EST Database resulted in the identification of several proteins. Spatio-temporal expression pattern of selected genes was determined for validation of identified class III peroxidases that were preferentially expressed during sugarcane stem development.

  3. Phenolic mediators enhance the manganese peroxidase catalyzed oxidation of recalcitrant lignin model compounds and synthetic lignin.

    PubMed

    Nousiainen, Paula; Kontro, Jussi; Manner, Helmiina; Hatakka, Annele; Sipilä, Jussi

    2014-11-01

    Fungal oxidative enzymes, such as peroxidases and laccases, are the key catalysts in lignin biodegradation in vivo, and consequently provide an important source for industrial ligninolytic biocatalysts. Recently, it has been shown that some syringyl-type phenolics have potential as industrial co-oxidants or mediators, in laccase-catalyzed modification of lignocellulosic material. We have now studied the effect of such mediators with ligninolytic peroxidases on oxidation of the most recalcitrant lignin model compounds. We found that they are able to enhance the manganese peroxidase (MnP) catalyzed oxidation reactions of small non-phenolic compounds, veratryl alcohol and veratrylglycerol β-guaiacyl ether (adlerol), which are not usually oxidized by manganese peroxidases alone. In these experiments we compared two peroxidases from white-rot fungi, MnP from Phlebia sp. Nf b19 and versatile peroxidase (VP) from Bjerkandera adusta under two oxidation conditions: (i) the Mn(III) initiated mediated oxidation by syringyl compounds and (ii) the system involving MnP-dependent lipid peroxidation, both with production of (hydrogen) peroxides in situ to maintain the peroxidase catalytic cycle. It was found that both peroxidases produced α-carbonyl oxidation product of veratryl alcohol in clearly higher yields in reactions mediated by phenoxy radicals than in lipid-peroxyl radical system. The oxidation of adlerol, on the other hand, was more efficient in lipid-peroxidation-system. VP was more efficient than MnP in the oxidation of veratryl alcohol and showed its lignin peroxidase type activity in the reaction conditions indicated by some cleavage of Cα-Cβ-bond of adlerol. Finally, the mediator assisted oxidation conditions were applied in the oxidation of synthetic lignin (DHP) and the structural analysis of the oxidized polymers showed clear modifications in the polymer outcome, e.g. the oxidation resulted in reduced amount of aliphatic hydroxyls indicated by (31)P NMR.