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Sample records for agglutinin-horseradish peroxidase wga-hrp

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

    PubMed

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

    1988-12-01

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

  2. 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.

  3. Common fur and mystacial vibrissae parallel sensory pathways: /sup 14/C 2-deoxyglucose and WGA-HRP studies in the rat

    SciTech Connect

    Sharp, F.R.; Gonzalez, M.F.; Morgan, C.W.; Morton, M.T.; Sharp, J.W.

    1988-04-15

    Stimulation of mystacial vibrissae in rows A,B, and C increased (14C) 2-deoxyglucose (2DG) uptake in spinal trigeminal nucleus pars caudalis (Sp5c) mostly in ventral portions of laminae III-IV with less activation of II and V. Stimulation of common fur above the whiskers mainly activated lamina II, with less activation in deeper layers. The patterns of activation were compatible with an inverted head, onion skin Sp5c somatotopy. Wheatgerm Agglutinin-Horseradish Peroxidase (WGA-HRP) injections into common fur between mystacial vibrissae rows A-B and B-C led to anterograde transganglionic labeling only of Sp5c, mainly of lamina II with less label in layer V, and very sparse label in III and IV. WGA-HRP skin injections appear to primarily label small fibers, which along with larger fibers, were metabolically activated during common fur stimulation. Mystacial vibrissae stimulation increased 2DG uptake in ventral ipsilateral spinal trigeminal nuclei pars interpolaris (Sp5i) and oralis (Sp5o) and principal trigeminal sensory nucleus (Pr5). Common fur stimulation above the whiskers slightly increased 2DG uptake in ventral Sp5i, Sp5o, and possibly Pr5. The most dorsal aspect of the ventroposteromedial (VPM) nucleus of thalamus was activated contralateral to whisker stimulation. Stimulation of the common fur dorsal to the whiskers activated a region of dorsal VPM caudal to the VPM region activated during whisker stimulation. This is consistent with previous data showing that ventral whiskers and portions of the face are represented rostrally in VPM, and more dorsal whiskers and dorsal portions of the face are represented progressively more caudally in VPM. Mystacial vibrissae stimulation activated the contralateral primary sensory SI barrelfield cortex and a separate region in the second somatosensory SII cortex.

  4. 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.

  5. Afferent projections to the Bötzinger complex from the upper cervical cord and other respiratory related structures in the brainstem in cats: retrograde WGA-HRP tracing.

    PubMed

    Gang, S; Sato, Y; Kohama, I; Aoki, M

    1995-12-01

    Following injection of WGA-HRP (30-60 nl, 5%) into the Bötzinger complex (Böt.c), a group of expiratory neurons in the vicinity of the retrofacial nucleus, a number of labeled neurons were observed, predominantly ipsilaterally, in the intermediate zone of the upper cervical cord at the C1 and C2 segments, the retrotrapezoid nucleus (RTN) in the ventrolateral medulla and the parabrachial-Kölliker-Fuse nuclear complex in the rostral pons. In addition, clusters of labeled cells were also observed in and around the solitary tract nucleus, nuclei ambiguus and retroambiguus, and nucleus raphe magnus. Control injections into the magnocellular tegmental field adjacent to the Böt.c resulted in a diffuse distribution of labeled neurons in the reticular formation. These results demonstrate that the Böt.c receives convergent monosynaptic axonal projections from the upper cervical spinal cord, the pontine pneumotaxic area, the RTN and several other respiratory related structures in the medulla. PMID:8786271

  6. Innervation of the lacrimal gland in the cynomolgous monkey: a retrograde tracing study.

    PubMed Central

    van der Werf, F; Baljet, B; Prins, M; Otto, J A

    1996-01-01

    Retrograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) was used to study the localisation of neurons that innervate the lacrimal gland of the cynomolgous monkey. WGA-HRP-labelled neurons were localised in the ipsilateral trigeminal, superior cervical and ciliary ganglia and in the ipsilateral and contralateral pterygopalatine ganglia. In the trigeminal ganglion WGA-HRP-labelled somata were found in the ophthalmic part (18%) and the maxillary part (5%). Identification of labelled neurons in the ciliary and pterygopalatine ganglia indicates a dual parasympathetic innvervation of the lacrimal gland. There is no known pathway to account for the contralateral location or pterygopalatine neurons. These novel findings are incorporated in a concept of a neural control mechanism for the lacrimal gland. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 6 Fig. 8 Fig. 9 Fig. 10 Fig. 11 PMID:8763476

  7. Anatomical evidence for red nucleus projections to motoneuronal cell groups in the spinal cord of the monkey

    NASA Technical Reports Server (NTRS)

    Holstege, Gert; Blok, Bertil F.; Ralston, Diane Daly

    1988-01-01

    In four rhesus monkeys wheat germ agglutinin-horseradish peroxidase (WGA-HRP) injections were made in the mesencephalic tegmentum. In three cases with injections involving the red nucleus (RN), rubrospinal fibers descended mainly contralaterally to terminate in laminae V, VI and dorsal VII of the spinal cord and in the lateral motoneuronal cell groups at the level of the cervical and lumbosacral enlargements. In all four cases the area of the interstitial nucleus of Cajal (INC) was injected, which resulted in labeled interstitiospinal fibers in the medial part of the ipsilateral ventral funiculus of the spinal cord. The results indicate that there is no major qualitative difference between the mesencephalic (RN and INC) and motor cortical projections to the spinal cord.

  8. Caudal medullary pathways to lumbosacral motoneuronal cell groups in the cat: evidence for direct projections possibly representing the final common pathway for lordosis.

    PubMed

    Vanderhorst, V G; Holstege, G

    1995-08-28

    The nucleus retroambiguus (NRA) projects to distinct brainstem and cervical and thoracic cord motoneuronal cell groups. The present paper describes NRA projections to distinct motoneuronal cell groups in the lumbar enlargement. Lumbosacral injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) were made to localize and quantify the retrogradely labeled neurons in the caudal medullary lateral tegmentum. These injections were combined with spinal hemisections to distinguish between neurons having ipsi-or contralaterally descending axons. The NRA-lumbosacral fibers descend almost exclusively contralaterally, but neurons in areas surrounding the NRA project mainly ipsilaterally. In an anterograde tracing study, injections of WGA-HRP or tritiated leucine were made in the region of the NRA to determine the NRA targets in the lumbosarcral cord. Hemisections in C2 made it possible to distinguish between NRA projections and projections from neurons in the adjoining lateral tegmentum. The results show delicate NRA projections to distinct lumbosacral motoneuronal cell groups innervating specific hindlimb muscles (iliopsoas, adductors, and hamstrings) as well as axial muscles (medial longissimus and proximal tail muscles). The projection is bilateral, with a contralateral predominance. Ipsilaterally terminating fibers are derived from NRA neurons whose axons cross the midline at the level of the obex, descend through the contralateral spinal white matter, and recross at the level of termination. A conceptual description is presented in which the periaqueductal gray-NRA-lumbosacral projections form the final common pathway for lordosis in the cat. PMID:7499541

  9. Unilateral innervation of guinea pig vallate taste buds as determined by glossopharyngeal neurectomy and HRP neural tracing.

    PubMed Central

    Huang, Y J; Lu, K S

    1996-01-01

    The innervation pattern by primary afferent nerve fibres and the neurotrophic effect on taste cells were investigated in the guinea pig vallate taste bud by means of glossopharyngeal neurectomy and horseradish peroxidase (HRP) or wheat germ agglutinin-horseradish peroxidase (WGA-HRP) tracing. In the glossopharyngeal neurectomy study, taste buds in the vallate papillae of adult guinea pigs were denervated by unilateral resection of the right glossopharyngeal nerve. Denervated animals were killed on days 1, 3 and 5 and weeks 1-9, 12 and 24 postneurectomy. The results showed that, on the denervated side, the taste buds decreased significantly in number during the 1st 2 wk, and disappeared completely by wk 3; no mature taste buds were present even 24 wk after neurectomy. This suggests that the vallate taste buds disappear in the absence of the glossopharyngeal nerve. In the neural tracing study, HRP or WGA-HRP was injected into the proximal end of the right glossopharyngeal nerve, near the jugular foramen. After a survival time of 24 h, the vallate papillae were sectioned and examined by light and electron microscopy. Light microscopy revealed that the HRP or WGA-HRP-labelled fibres innervated the vallate taste buds of the injected side. Most of the taste cells in the buds were labelled with HRP or WGA-HRP reaction products from the basal to the apical region. At the ultrastructural level, the reaction products were confined to the cytoplasm of the labelled cells, which were identified as type I, II and III cells, but not basal cells. Labelled intragemmal nerve profiles were seen among the taste cells. No synapse formation was seen with nerve profiles abutting on type I and II cells, whereas on certain type III cells, typical synapses were formed. We conclude that both the right and left vallate papilla in the guinea pig are unilaterally innervated by the glossopharyngeal nerve without cross-innervation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID

  10. Origin of primary sensory neurons innervating the buccal stretch receptor.

    PubMed

    Yamamoto, T; Onozuka, M; Nagasaki, S; Watanabe, K; Ozono, S

    1999-01-01

    The primary sensory neurons innervating mechanoreceptors in oro-facial regions have their cell bodies in either the trigeminal ganglion or the mesencephalic nucleus of the trigeminal nerve. The buccal stretch receptor (BSR), a type of mechanoreceptor in the jaw of rodents, has recently been recognized as signaling the position of the mandible. The location of the primary afferent neurons innervating this receptor is unknown. To investigate the cell bodies of the BSR afferent neurons in rats, we applied wheat germ agglutinin-horseradish peroxidase (WGA-HRP) to the proximal stump of the severed nerve branch of the buccal nerve that supplied the BSR. HRP-labeled cell bodies were observed in the posterolateral portion of the ipsilateral trigeminal ganglion. None was found in the contralateral trigeminal ganglion or in the brainstem. All labeled cell bodies were oval or round and closely resembled pseudo-unipolar neurons. The mean diameter of the labeled somata ranged between 25.5 and 52.5 microm, with small (< or = 30 microm), medium (from 31 to 40 microm), and large somata (> or = 41 microm) accounting for 8.8%, 54.9%, and 36.3%, respectively. Among the myelinated nerve fibers in the branch in which WGA-HRP was applied, 78.5% terminated in the BSR and had larger fiber diameters than the rest, indicating that most of the medium and large HRP-labeled cell bodies were BSR afferents. From these results and the ontogenetic origin of this receptor, it is suggested that the BSR differentiated from the mechanoreceptors in the oral mucosa or the fascia of masticatory muscles. PMID:10065945

  11. Characterization of glutamatergic neurons in the rat atrial intrinsic cardiac ganglia that project to the cardiac ventricular wall.

    PubMed

    Wang, Ting; Miller, Kenneth E

    2016-08-01

    The intrinsic cardiac nervous system modulates cardiac function by acting as an integration site for regulating autonomic efferent cardiac output. This intrinsic system is proposed to be composed of a short cardio-cardiac feedback control loop within the cardiac innervation hierarchy. For example, electrophysiological studies have postulated the presence of sensory neurons in intrinsic cardiac ganglia (ICG) for regional cardiac control. There is still a knowledge gap, however, about the anatomical location and neurochemical phenotype of sensory neurons inside ICG. In the present study, rat ICG neurons were characterized neurochemically with immunohistochemistry using glutamatergic markers: vesicular glutamate transporters 1 and 2 (VGLUT1; VGLUT2), and glutaminase (GLS), the enzyme essential for glutamate production. Glutamatergic neurons (VGLUT1/VGLUT2/GLS) in the ICG that have axons to the ventricles were identified by retrograde tracing of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) injected in the ventricular wall. Co-labeling of VGLUT1, VGLUT2, and GLS with the vesicular acetylcholine transporter (VAChT) was used to evaluate the relationship between post-ganglionic autonomic neurons and glutamatergic neurons. Sequential labeling of VGLUT1 and VGLUT2 in adjacent tissue sections was used to evaluate the co-localization of VGLUT1 and VGLUT2 in ICG neurons. Our studies yielded the following results: (1) ICG contain glutamatergic neurons with GLS for glutamate production and VGLUT1 and 2 for transport of glutamate into synaptic vesicles; (2) atrial ICG contain neurons that project to ventricle walls and these neurons are glutamatergic; (3) many glutamatergic ICG neurons also were cholinergic, expressing VAChT; (4) VGLUT1 and VGLUT2 co-localization occurred in ICG neurons with variation of their protein expression level. Investigation of both glutamatergic and cholinergic ICG neurons could help in better understanding the function of the intrinsic cardiac

  12. Heterologous Expression of Peroxidases

    NASA Astrophysics Data System (ADS)

    de Weert, Sandra; Lokman, B. Christien

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

  13. Redox Potential of Peroxidases

    NASA Astrophysics Data System (ADS)

    Ayala, Marcela

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

  14. 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

  15. Nanostructures for peroxidases.

    PubMed

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

    2015-01-01

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

  16. 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

  17. 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.

  18. 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

  19. Central Pupillary Light Reflex Circuits in the Cat: I. The Olivary Pretectal Nucleus

    PubMed Central

    Sun, Wensi; May, Paul J.

    2014-01-01

    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 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. PMID:24706328

  20. Novel Applications of Peroxidase

    NASA Astrophysics Data System (ADS)

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

    1997-02-01

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

  1. 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.

  2. Magnetic resonance imaging of neuronal connections in the macaque monkey.

    PubMed

    Saleem, Kadharbatcha S; Pauls, Jon M; Augath, Mark; Trinath, Torsten; Prause, Burkhard A; Hashikawa, Tsutomu; Logothetis, Nikos K

    2002-05-30

    Recently, an MRI-detectable, neuronal tract-tracing method in living animals was introduced that exploits the anterograde transport of manganese (Mn2+). We present the results of experiments simultaneously tracing manganese chloride and wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) to evaluate the specificity of the former by tracing the neuronal connections of the basal ganglia of the monkey. Mn2+ and WGA-HRP yielded remarkably similar and highly specific projection patterns. By showing the sequential transport of Mn2+ from striatum to pallidum-substantia nigra and then to thalamus, we demonstrated MRI visualization of transport across at least one synapse in the CNS of the primate. Transsynaptic tract tracing in living primates will allow chronic studies of development and plasticity and provide valuable anatomical information for fMRI and electrophysiological experiments in primates. PMID:12062017

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

    PubMed

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

    2014-11-01

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

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

    PubMed Central

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

    1987-01-01

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

  5. Lignin-degrading peroxidases of Phanerochaete chrysosporium.

    PubMed

    Cai, D; Tien, M

    1993-07-01

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

  6. 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.

  7. Screening Actinomycetes for Extracellular Peroxidase Activity

    PubMed Central

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

    1996-01-01

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

  8. Catalase and glutathione peroxidase mimics

    PubMed Central

    Day, Brian J.

    2009-01-01

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

  9. Independent evolution of four heme peroxidase superfamilies.

    PubMed

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

    2015-05-15

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

  10. (Characterization of lignin peroxidases from Phanerochaete)

    SciTech Connect

    Not Available

    1990-11-14

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

  11. 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.

  12. 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.

  13. 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.

  14. Soybean peroxidase as an industrial catalyst

    SciTech Connect

    Pokora, A.R.

    1995-12-01

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

  15. Recents patents in the use of peroxidases.

    PubMed

    Alvarado, Berenize; Torres, Eduardo

    2009-01-01

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

  16. Disulfide bonds and glycosylation in fungal peroxidases.

    PubMed

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

    1995-01-15

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

  17. 21 CFR 864.7675 - Leukocyte peroxidase test.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

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

  18. 21 CFR 864.7675 - Leukocyte peroxidase test.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  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... 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...

  20. 21 CFR 864.7675 - Leukocyte peroxidase test.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

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

  1. 21 CFR 864.7675 - Leukocyte peroxidase test.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Leukocyte peroxidase test. 864.7675 Section 864... 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...

  2. Growth-associated protein 43 immunoreactivity in the superficial dorsal horn of the rat spinal cord is localized in atrophic C-fiber, and not in sprouted A-fiber, central terminals after peripheral nerve injury.

    PubMed

    Doubell, T P; Woolf, C J

    1997-09-15

    Peripheral nerve injury induces the up-regulation in dorsal root ganglion cells of growth-associated protein 43 (GAP-43) and its transport to the superficial laminae of the dorsal horn of the spinal cord, where it is located primarily in unmyelinated axons and growth-cone like structures. Peripheral nerve injury also induces the central terminals of axotomized myelinated axons to sprout and form novel synaptic contacts in lamina II of the dorsal horn. To investigate whether the sprouting of A-fiber central terminals into lamina II is the consequence of GAP-43 incorporation into their terminal membranes, we have used an ultrastructural analysis with double labelling to identify the localization of GAP-43 immunoreactivity. Transganglionic transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was used to identify C-fiber terminals. Transganglionic transport of the B fragment of cholera toxin conjugated to horseradish peroxidase (B-HRP) was used to label A-fiber sciatic nerve central terminals in combination with GAP-43 immunocytochemistry. GAP-43 was found to colocalize only with WGA-HRP- and not with B-HRP-labelled synapses or axons. In addition, many single-labelled GAP-43 synapses were observed. Many of the WGA-HRP-labelled terminals that were characterized by degenerative changes were GAP-43 immunoreactive. Our results indicate that peripheral nerve injury induces novel synapse formation of A fibers in lamina II but that up-regulated levels of GAP-43 are present mainly in other axon projections to the superficial dorsal horn. PMID:9303528

  3. (Molecular characteristics of the lignin forming peroxidase)

    SciTech Connect

    Lagrimini, L.M.

    1990-01-01

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

  4. Modelling a Peroxidase-based Optical Biosensor

    PubMed Central

    Baronas, Romas; Gaidamauskaite, Evelina; Kulys, Juozas

    2007-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  7. 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…

  8. Microfluidic peroxidase biochip for polyphenol synthesis.

    PubMed

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

    2003-03-01

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

  9. 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...

  10. Creation of a Thermally Tolerant Peroxidase.

    PubMed

    Watanabe, Y; Nakajima, H

    2016-01-01

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

  11. Peroxidase gene discovery from the horseradish transcriptome

    PubMed Central

    2014-01-01

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

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

    SciTech Connect

    Not Available

    1992-01-01

    Lignin peroxidases were investigated with respect to enzyme kinetics and NMR spectroscopy of the heme domain. MN peroxidases were studied with respect to the role of oxalate in enzyme activity, the NMR spectroscopy of the heme domain. Gene expression of both lignin and MN peroxidases were examined as well as expression of site-directed mutants aimed at scale up production of these enzymes.

  13. PEROXIDASE GENE POLYMORPHISM IN BUFFALOGRASS AND OTHER GRASSES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant peroxidases are a family of related proteins possessing highly conserved domains. Degenerate oligonucleotide primers based on these conserved domains can be used to amplify DNA sequences coding for peroxidases from plants with unsequenced genomes. Polymorphisms in peroxidase genes among buffa...

  14. NADH peroxidase: kinetic mechanism and nucleotide specificity

    SciTech Connect

    Stoll, V.S.; Blanchard, J.S.

    1987-05-01

    NADH peroxidase is a flavoprotein reductase isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide dependent reduction of hydrogen peroxide to water. Initial velocity, product and dead-end inhibition studies have been performed and all support a ping-pong kinetic mechanism. Further support for the ping-pong nature of the kinetic mechanism are the hydrogen peroxide independent transhydrogenase activity of the enzyme, measured either with thio-NAD or with radiolabeled NAD (isotope exchange studies). Kinetic parameters will be presented for a number of reduced pyridine nucleotide analogs. Analogs which have been modified in the adenine ring exhibit much higher K/sub m/'s relative to their adenine analogs. NADH peroxidase catalyzes the stereo-specific removal of the 4S hydrogen of NADH and primary deuterium kinetic isotope effects have been determined for a number of these substrates with 4S-deuterated molecules. There is a strong correlation between their steady-state K/sub m/ and /sup D/V/K. Small values for /sup D/V are interpreted as supporting rate-limitation in the oxidative half-reaction. These data will be discussed in terms of a kinetic and chemical mechanism proposed for NADH peroxidase.

  15. 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

  16. Redox thermodynamics of lactoperoxidase and eosinophil peroxidase.

    PubMed

    Battistuzzi, Gianantonio; Bellei, Marzia; Vlasits, Jutta; Banerjee, Srijib; Furtmüller, Paul G; Sola, Marco; Obinger, Christian

    2010-02-01

    Eosinophil peroxidase (EPO) and lactoperoxidase (LPO) are important constituents of the innate immune system of mammals. These heme enzymes belong to the peroxidase-cyclooxygenase superfamily and catalyze the oxidation of thiocyanate, bromide and nitrite to hypothiocyanate, hypobromous acid and nitrogen dioxide that are toxic for invading pathogens. In order to gain a better understanding of the observed differences in substrate specificity and oxidation capacity in relation to heme and protein structure, a comprehensive spectro-electrochemical investigation was performed. The reduction potential (E degrees ') of the Fe(III)/Fe(II) couple of EPO and LPO was determined to be -126mV and -176mV, respectively (25 degrees C, pH 7.0). Variable temperature experiments show that EPO and LPO feature different reduction thermodynamics. In particular, reduction of ferric EPO is enthalpically and entropically disfavored, whereas in LPO the entropic term, which selectively stabilizes the oxidized form, prevails on the enthalpic term that favors reduction of Fe(III). The data are discussed with respect to the architecture of the heme cavity and the substrate channel. Comparison with published data for myeloperoxidase demonstrates the effect of heme to protein linkages and heme distortion on the redox chemistry of mammalian peroxidases and in consequence on the enzymatic properties of these physiologically important oxidoreductases. PMID:19944669

  17. Further characterization of human eosinophil peroxidase.

    PubMed Central

    Olsen, R L; Syse, K; Little, C; Christensen, T B

    1985-01-01

    The large and the small subunits (Mr 50 000 and 10 500 respectively) of human eosinophil peroxidase were isolated by gel filtration under reducing conditions. The subunits were very strongly associated but not apparently cross-linked by disulphide bridges. During storage, the large subunit tended to form aggregates, which required reduction to dissociate them. Amino acid analysis of the performic acid-treated large subunit showed the presence of 19 cysteic acid residues. The small subunit of eosinophil peroxidase had the same Mr value as the small subunit of myeloperoxidase. However, although these subunits have very similar amino acid compositions, they showed different patterns of peptide fragmentation after CNBr treatment. The carbohydrate of eosinophil peroxidase seemed associated exclusively with the large subunit and comprised mannose (4.5%, w/w) and N-acetylglucosamine (0.8%, w/w). The far-u.v.c.d. spectrum of the enzyme indicated the presence of relatively little ordered secondary structure. Images Fig. 3. PMID:4052025

  18. 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.

  19. 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

  20. 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.

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

    PubMed

    Riquelme, A; Cardemil, L

    1995-05-01

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

  2. Transporter protein and drug-conjugated gold nanoparticles capable of bypassing the blood-brain barrier.

    PubMed

    Zhang, Yanhua; Walker, Janelle Buttry; Minic, Zeljka; Liu, Fangchao; Goshgarian, Harry; Mao, Guangzhao

    2016-01-01

    Drug delivery to the central nervous system (CNS) is challenging due to the inability of many drugs to cross the blood-brain barrier (BBB). Here, we show that wheat germ agglutinin horse radish peroxidase (WGA-HRP) chemically conjugated to gold nanoparticles (AuNPs) can be transported to the spinal cord and brainstem following intramuscular injection into the diaphragm of rats. We synthesized and determined the size and chemical composition of a three-part nanoconjugate consisting of WGA-HRP, AuNPs, and drugs for the treatment of diaphragm paralysis associated with high cervical spinal cord injury (SCI). Upon injection into the diaphragm muscle of rats, we show that the nanoconjugate is capable of delivering the drug at a much lower dose than the unconjugated drug injected systemically to effectively induce respiratory recovery in rats following SCI. This study not only demonstrates a promising strategy to deliver drugs to the CNS bypassing the BBB but also contributes a potential nanotherapy for the treatment of respiratory muscle paralysis resulted from cervical SCI. PMID:27180729

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

    PubMed

    Chickering, J G; Sokoloff, A J

    1996-01-01

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

  4. Transporter protein and drug-conjugated gold nanoparticles capable of bypassing the blood-brain barrier

    PubMed Central

    Zhang, Yanhua; Walker, Janelle Buttry; Minic, Zeljka; Liu, Fangchao; Goshgarian, Harry; Mao, Guangzhao

    2016-01-01

    Drug delivery to the central nervous system (CNS) is challenging due to the inability of many drugs to cross the blood-brain barrier (BBB). Here, we show that wheat germ agglutinin horse radish peroxidase (WGA-HRP) chemically conjugated to gold nanoparticles (AuNPs) can be transported to the spinal cord and brainstem following intramuscular injection into the diaphragm of rats. We synthesized and determined the size and chemical composition of a three-part nanoconjugate consisting of WGA-HRP, AuNPs, and drugs for the treatment of diaphragm paralysis associated with high cervical spinal cord injury (SCI). Upon injection into the diaphragm muscle of rats, we show that the nanoconjugate is capable of delivering the drug at a much lower dose than the unconjugated drug injected systemically to effectively induce respiratory recovery in rats following SCI. This study not only demonstrates a promising strategy to deliver drugs to the CNS bypassing the BBB but also contributes a potential nanotherapy for the treatment of respiratory muscle paralysis resulted from cervical SCI. PMID:27180729

  5. 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.

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

    PubMed Central

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

    2012-01-01

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

  7. Cytochrome c peroxidase from Methylococcus capsulatus Bath.

    PubMed

    Zahn, J A; Arciero, D M; Hooper, A B; Coats, J R; DiSpirito, A A

    1997-11-01

    A bacterial cytochrome c peroxidase was purified from the obligate methanotroph Methylococcus capsulatus Bath in either the fully oxidized or the half reduced form depending on the purification procedure. The cytochrome was a homo-dimer with a subunit mol mass of 35.8 kDa and an isoelectric point of 4.5. At physiological temperatures, the enzyme contained one high-spin, low-potential (Em7 = -254 mV) and one low-spin, high-potential (Em7 = +432 mM ) heme. The low-potential heme center exhibited a spin-state transition from the penta-coordinated, high-spin configuration to a low-spin configuration upon cooling the enzyme to cryogenic temperatures. Using M. capsulatus Bath ferrocytochrome c555 as the electron donor, the KM and Vmax for peroxide reduction were 510 +/- 100 nM and 425 +/- 22 mol ferrocytochrome c555 oxidized min-1 (mole cytochrome c peroxidase)-1, respectively. PMID:9325424

  8. Horseradish peroxidase catalyzed hydroxylations: mechanistic studies.

    PubMed

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

    1986-05-20

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

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

    PubMed

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

    2015-10-15

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

  10. Peroxidase Isozymes from Meloidogyne spp. and Their Origin

    PubMed Central

    Starr, J. L.

    1979-01-01

    Two peroxidase isozymes (Ef 0.43 and 0.53) were detected by electrophoretic analysis in homogenates of Meloidogyne arenaria, M. hapla, M. javanica, and M. incognita females reared on tomato. No peroxidase isozymes were detected electrophoretically in homogenates of adult males, preparasitic larvae, or eggs. Peroxidase isozymes from females reared on bean, eggplant, or tobacco differed from those from females reared on tomato. Bean and eggplant populations had a single peroxidase isozyme each, respectively Ef 0.21 and 0.28. No peroxidase isozymes were detected in tobacco populations under the conditions used, although total activity assays did reveal low levels of peroxidase activity in homogenates of tobacco populations. The peroxidase isozymes detected in females reared on tomato or bean appear similar to the peroxidase isozymes present in root-knot galls, adjacent ungalled roots, and roots from uninoculated plants of the corresponding hosts. The probability is discussed that most of the peroxittase activity associated with Meloidogyne spp. females is of host origin. PMID:19305520

  11. Heterogeneity and regulation of manganese peroxidases from Phanerochaete chrysosporium.

    PubMed Central

    Pease, E A; Tien, M

    1992-01-01

    Lignin and Mn peroxidases are two families of isozymes produced by the lignin-degrading fungus Phanerochaete chrysosporium under nutrient nitrogen or carbon limitation. We purified to homogeneity the three major Mn peroxidase isozymes, H3 (pI = 4.9), H4 (pI = 4.5), and H5 (pI = 4.2). Amino-terminal sequencing of these isozymes demonstrates that they are encoded by different genes. We also analyzed the regulation of these isozymes in carbon- and nitrogen-limited cultures and found not only that the lignin and Mn peroxidases are differentially regulated but also that differential regulation occurs within the Mn peroxidase isozyme family. The isozyme profile and the time at which each isozyme appears in secondary metabolism differ in both nitrogen- and carbon-limited cultures. Each isozyme also responded differently to the addition of a putative inducer, divalent Mn. The stability of the Mn peroxidases in carbon- and nitrogen-limited cultures was also characterized after cycloheximide addition. The Mn peroxidases are more stable in carbon-limited cultures than in nitrogen-limited cultures. They are also more stable than the lignin peroxidases. These data collectively suggest that the Mn peroxidase isozymes serve different functions in lignin biodegradation. Images PMID:1592808

  12. Accelerating peroxidase mimicking nanozymes using DNA

    NASA Astrophysics Data System (ADS)

    Liu, Biwu; Liu, Juewen

    2015-08-01

    DNA-capped iron oxide nanoparticles are nearly 10-fold more active as a peroxidase mimic for TMB oxidation than naked nanoparticles. To understand the mechanism, the effect of DNA length and sequence is systematically studied, and other types of polymers are also compared. This rate enhancement is more obvious with longer DNA and, in particular, poly-cytosine. Among the various polymer coatings tested, DNA offers the highest rate enhancement. A similar acceleration is also observed for nanoceria. On the other hand, when the positively charged TMB substrate is replaced by the negatively charged ABTS, DNA inhibits oxidation. Therefore, the negatively charged phosphate backbone and bases of DNA can increase TMB binding by the iron oxide nanoparticles, thus facilitating the oxidation reaction in the presence of hydrogen peroxide.DNA-capped iron oxide nanoparticles are nearly 10-fold more active as a peroxidase mimic for TMB oxidation than naked nanoparticles. To understand the mechanism, the effect of DNA length and sequence is systematically studied, and other types of polymers are also compared. This rate enhancement is more obvious with longer DNA and, in particular, poly-cytosine. Among the various polymer coatings tested, DNA offers the highest rate enhancement. A similar acceleration is also observed for nanoceria. On the other hand, when the positively charged TMB substrate is replaced by the negatively charged ABTS, DNA inhibits oxidation. Therefore, the negatively charged phosphate backbone and bases of DNA can increase TMB binding by the iron oxide nanoparticles, thus facilitating the oxidation reaction in the presence of hydrogen peroxide. Electronic supplementary information (ESI) available: Methods, TEM, UV-vis and DLS data. See DOI: 10.1039/c5nr04176g

  13. Luffa aegyptiaca (Gourd) Fruit Juice as a Source of Peroxidase

    PubMed Central

    Yadav, R. S. S.; Yadav, K. S.; Yadav, H. S.

    2011-01-01

    Peroxidases have turned out to be potential biocatalyst for a variety of organic reactions. The research work reported in this communication was done with the objective of finding a convenient rich source of peroxidase which could be used as a biocatalyst for organic synthetic reactions. The studies made have shown that Luffa aegyptiaca (gourd) fruit juice contains peroxidase activity of the order of 180 enzyme unit/mL. The Km values of this peroxidase for the substrates guaiacol and hydrogen peroxide were 2.0 and 0.2 mM, respectively. The pH and temperature optima were 6.5 and 60°C, respectively. Like other peroxidases, it followed double displacement type mechanism. Sodium azide inhibited the enzyme competitively with Ki value of 3.35 mM. PMID:21804936

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

    PubMed

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

    2016-01-01

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

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. Horseradish peroxidase catalyzed nitric oxide formation from hydroxyurea.

    PubMed

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

    2002-04-01

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

  20. Stabilization of lignin peroxidases in white rot fungi by tryptophan.

    PubMed Central

    Collins, P J; Field, J A; Teunissen, P; Dobson, A D

    1997-01-01

    Supplementation of various cultures of white rot fungi with tryptophan was found to have a large stimulatory effect on lignin peroxidase activity levels. This enhancement was greater than that observed in the presence of the lignin peroxidase recycling agent veratryl alcohol. Using reverse transcription-PCR, we found that tryptophan does not act to induce lignin peroxidase expression at the level of gene transcription. Instead, the activity enhancement observed is likely to result from the protective effect of tryptophan against H2O2 inactivation. In experiments using a partially purified lignin peroxidase preparation, tryptophan and its derivative indole were determined to function in the same way as veratryl alcohol in converting compound II, an oxidized form of lignin peroxidase, to ferric enzyme, thereby completing the catalytic cycle. Furthermore, tryptophan was found to be a better substrate for lignin peroxidase than veratryl alcohol. Inclusion of either tryptophan or indole enhanced the oxidation of the azo dyes methyl orange and Eriochrome blue black. Stimulation of azo dye oxidations by veratryl alcohol has previously been shown to be due to its enzyme recycling function. Our data allow us to propose that tryptophan stabilizes lignin peroxidase by acting as a reductant for the enzyme. PMID:9212404

  1. 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.

  2. 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.

  3. Graft copolymers with immobilized peroxidase for organic synthesis

    NASA Astrophysics Data System (ADS)

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

    1999-07-01

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

  4. Redundancy among manganese peroxidases in Pleurotus ostreatus.

    PubMed

    Salame, Tomer M; Knop, Doriv; Levinson, Dana; Yarden, Oded; Hadar, Yitzhak

    2013-04-01

    Manganese peroxidases (MnPs) are key players in the ligninolytic system of white rot fungi. In Pleurotus ostreatus (the oyster mushroom) these enzymes are encoded by a gene family comprising nine members, mnp1 to -9 (mnp genes). Mn(2+) amendment to P. ostreatus cultures results in enhanced degradation of recalcitrant compounds (such as the azo dye orange II) and lignin. In Mn(2+)-amended glucose-peptone medium, mnp3, mnp4, and mnp9 were the most highly expressed mnp genes. After 7 days of incubation, the time point at which the greatest capacity for orange II decolorization was observed, mnp3 expression and the presence of MnP3 in the extracellular culture fluids were predominant. To determine the significance of MnP3 for ligninolytic functionality in Mn(2+)-sufficient cultures, mnp3 was inactivated via the Δku80 strain-based P. ostreatus gene-targeting system. In Mn(2+)-sufficient medium, inactivation of mnp3 did not significantly affect expression of nontargeted MnPs or their genes, nor did it considerably diminish the fungal Mn(2+)-mediated orange II decolorization capacity, despite the significant reduction in total MnP activity. Similarly, inactivation of either mnp4 or mnp9 did not affect orange II decolorization ability. These results indicate functional redundancy within the P. ostreatus MnP gene family, enabling compensation upon deficiency of one of its members. PMID:23377936

  5. Immobilization of horseradish peroxidase onto kaolin.

    PubMed

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

    2016-03-01

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

  6. 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

  7. 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.

  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. 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.

  10. Stimuli-responsive peroxidase mimicking at a smart graphene interface.

    PubMed

    Liu, Meng; Zhao, Huimin; Chen, Shuo; Yu, Hongtao; Quan, Xie

    2012-07-18

    A synergistic graphene-based catalyst was engineered by the in situ growth of "naked" Au-nanoparticles (NPs) on graphene sheets. The catalyst exhibits excellent switchable peroxidase-like activity in response to specific DNA. PMID:22673613

  11. Cell wall bound anionic peroxidases from asparagus byproducts.

    PubMed

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

    2014-10-01

    Asparagus byproducts are a good source of cationic soluble peroxidases (CAP) useful for the bioremediation of phenol-contaminated wastewaters. In this study, cell wall bound peroxidases (POD) from the same byproducts have been purified and characterized. The covalent forms of POD represent >90% of the total cell wall bound POD. Isoelectric focusing showed that whereas the covalent fraction is constituted primarily by anionic isoenzymes, the ionic fraction is a mixture of anionic, neutral, and cationic isoenzymes. Covalently bound peroxidases were purified by means of ion exchange chromatography and affinity chromatography. In vitro detoxification studies showed that although CAP are more effective for the removal of 4-CP and 2,4-DCP, anionic asparagus peroxidase (AAP) is a better option for the removal of hydroxytyrosol (HT), the main phenol present in olive mill wastewaters. PMID:25195693

  12. Grafting of Functional Molecules: Insights into Peroxidase-Derived Materials

    NASA Astrophysics Data System (ADS)

    Nyanhongo, Gibson S.; Prasetyo, Endry Nugroho; Kudanga, Tukayi; Guebitz, Georg

    An insight into the progress made in applying heme peroxidases in grafting processes, starting from the production of simple resins to more complex polymers, is presented. The refinement of the different reaction conditions (solvents, concentrations of the reactants) and careful study of the reaction mechanisms have been instrumental in advancing enzymatic grafting processes. A number of processes described here show how peroxidase mediated catalysis could provide a new strategy as an alternative to conventional energy intensive procedures mediated by chemical catalysts.

  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. PMID:26291534

  14. Serine incorporation into the selenocysteine moiety of glutathione peroxidase

    SciTech Connect

    Sunde, R.A.; Evenson, J.K.

    1987-01-15

    The selenium in mammalian glutathione peroxidase is present as a selenocysteine ((Se)Cys) moiety incorporated into the peptide backbone 41-47 residues from the N-terminal end. To study the origin of the skeleton of the (Se)Cys moiety, we perfused isolated rat liver with /sup 14/C- or /sup 3/H-labeled amino acids for 4 h, purified the GSH peroxidase, derivatized the (Se)Cys in GSH peroxidase to carboxymethylselenocysteine ((Se)Cys(Cm)), and determined the amino acid specific activity. Perfusion with (/sup 14/C)cystine resulted in (/sup 14/C)cystine incorporation into GSH peroxidase without labeling (Se)Cys(Cm), indicating that cysteine is not a direct precursor for (Se)Cys. (/sup 14/C)Serine perfusion labeled serine, glycine (the serine hydroxymethyltransferase product), and (Se)Cys(Cm) in purified GSH peroxidase, whereas (3-3H)serine perfusion only labeled serine and (Se)Cys(Cm), thus demonstrating that the (Se)Cys in GSH peroxidase is derived from serine. The similar specific activities of serine and (Se)Cys(Cm) strongly suggest that the precursor pool of serine used for (Se) Cys synthesis is the same or similar to the serine pool used for acylation of seryl-tRNAs.

  15. Engineering the proximal heme cavity of catalase-peroxidase.

    PubMed

    Jakopitsch, Christa; Regelsberger, Günther; Furtmüller, Paul Georg; Rüker, Florian; Peschek, Günter A; Obinger, Christian

    2002-07-25

    Catalase-peroxidases (KatGs) are prokaryotic heme peroxidases with homology to yeast cytochrome c peroxidase (CCP) and plant ascorbate peroxidases (APXs). KatGs, CCP and APXs contain identical amino acid triads in the heme pocket (distal Arg/Trp/His and proximal His/Trp/Asp), but differ dramatically in their reactivities towards hydrogen peroxide and various one-electron donors. Only KatGs have high catalase activity in addition to a peroxidase activity of broad specificity. Here, we investigated the effect of mutating the conserved proximal triad on KatG catalysis. With the exception of W341F, all variants (H290Q, W341A, D402N, D402E) exhibited a catalase activity <1% of wild-type KatG and spectral properties indicating alterations in heme coordination and spin states. Generally, the peroxidase activity was much less effected by these mutations. Compared with wild-type KatG the W341F variant had a catalase and halogenation activity of about 40% and an even increased overall peroxidase activity. This variant, for the first time, allowed to monitor the hydrogen peroxide mediated transitions of ferric KatG to compound I and back to the resting enzyme. Compound I reduction by aromatic one-electron donors (o-dianisidine, pyrogallol, aniline) was not influenced by exchanging Trp by Phe. The findings are discussed in comparison with the data known from CCP and APX and a reaction mechanism for the multifunctional activity of the W341F variant is suggested. PMID:12121764

  16. 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.

  17. Peroxidase activation of cytoglobin by anionic phospholipids: Mechanisms and consequences.

    PubMed

    Tejero, Jesús; Kapralov, Alexandr A; Baumgartner, Matthew P; Sparacino-Watkins, Courtney E; Anthonymutu, Tamil S; Vlasova, Irina I; Camacho, Carlos J; Gladwin, Mark T; Bayir, Hülya; Kagan, Valerian E

    2016-05-01

    Cytoglobin (Cygb) is a hexa-coordinated hemoprotein with yet to be defined physiological functions. The iron coordination and spin state of the Cygb heme group are sensitive to oxidation of two cysteine residues (Cys38/Cys83) and/or the binding of free fatty acids. However, the roles of redox vs lipid regulators of Cygb's structural rearrangements in the context of the protein peroxidase competence are not known. Searching for physiologically relevant lipid regulators of Cygb, here we report that anionic phospholipids, particularly phosphatidylinositolphosphates, affect structural organization of the protein and modulate its iron state and peroxidase activity both conjointly and/or independently of cysteine oxidation. Thus, different anionic lipids can operate in cysteine-dependent and cysteine-independent ways as inducers of the peroxidase activity. We establish that Cygb's peroxidase activity can be utilized for the catalysis of peroxidation of anionic phospholipids (including phosphatidylinositolphosphates) yielding mono-oxygenated molecular species. Combined with the computational simulations we propose a bipartite lipid binding model that rationalizes the modes of interactions with phospholipids, the effects on structural re-arrangements and the peroxidase activity of the hemoprotein. PMID:26928591

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

    PubMed

    Veitch, Nigel C

    2004-02-01

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

  19. Peroxidase extraction from jicama skin peels for phenol removal

    NASA Astrophysics Data System (ADS)

    Chiong, T.; Lau, S. Y.; Khor, E. H.; Danquah, M. K.

    2016-06-01

    Phenol and its derivatives exist in various types of industrial effluents, and are known to be harmful to aquatic lives even at low concentrations. Conventional treatment technologies for phenol removal are challenged with long retention time, high energy consumption and process cost. Enzymatic treatment has emerged as an alternative technology for phenol removal from wastewater. These enzymes interact with aromatic compounds including phenols in the presence of hydrogen peroxide, forming free radicals which polymerize spontaneously to produce insoluble phenolic polymers. This work aims to extract peroxidase from agricultural wastes materials and establish its application for phenol removal. Peroxidase was extracted from jicama skin peels under varying extraction conditions of pH, sample-to-buffer ratio (w/v %) and temperature. Experimental results showed that extraction process conducted at pH 10, 40% w/v and 25oC demonstrated a peroxidase activity of 0.79 U/mL. Elevated temperatures slightly enhanced the peroxidase activities. Jicama peroxidase extracted at optimum extraction conditions demonstrated a phenol removal efficiency of 87.5% at pH 7. Phenol removal efficiency was ∼ 97% in the range of 30 - 40oC, and H2O2 dosage has to be kept below 100 mM for maximum removal under phenol concentration tested.

  20. Diverse functions and reactions of class III peroxidases.

    PubMed

    Shigeto, Jun; Tsutsumi, Yuji

    2016-03-01

    Higher plants contain plant-specific peroxidases (class III peroxidase; Prxs) that exist as large multigene families. Reverse genetic studies to characterize the function of each Prx have revealed that Prxs are involved in lignification, cell elongation, stress defense and seed germination. However, the underlying mechanisms associated with plant phenotypes following genetic engineering of Prx genes are not fully understood. This is because Prxs can function as catalytic enzymes that oxidize phenolic compounds while consuming hydrogen peroxide and/or as generators of reactive oxygen species. Moreover, biochemical efforts to characterize Prxs responsible for lignin polymerization have revealed specialized activities of Prxs. In conclusion, not only spatiotemporal regulation of gene expression and protein distribution, but also differentiated oxidation properties of each Prx define the function of this class of peroxidases. PMID:26542837

  1. Purification, crystallization and preliminary crystallographic analysis of banyan peroxidase

    PubMed Central

    Sharma, Anurag; Palm, Gottfried J.; Kumari, Moni; Panjikar, Santosh; Jagannadham, M. V.; Hinrichs, Winfried

    2012-01-01

    Plant peroxidases are extensively used in a wide range of biotechnological applications owing to their high environmental and thermal stability. A new peroxidase, named banyan peroxidase, was purified from the latex of Ficus benghalensis and crystallized. X-ray diffraction data were collected from native crystals and from bromide and xenon derivatives to resolutions of up to 1.66 Å in the trigonal space group P3221, with unit-cell parameters a = b = 73.1, c = 164.6 Å. The anomalous signal of the intrinsic iron and calcium ions was sufficient for structure solution by SAD, although the sequence is not yet known. PMID:22869125

  2. Nucleotide sequence of the tobacco (Nicotiana tabacum) anionic peroxidase gene

    SciTech Connect

    Diaz-De-Leon, F.; Klotz, K.L.; Lagrimini, L.M. )

    1993-03-01

    Peroxidases have been implicated in numerous physiological processes including lignification (Grisebach, 1981), wound-healing (Espelie et al., 1986), phenol oxidation (Lagrimini, 1991), pathogen defense (Ye et al., 1990), and the regulation of cell elongation through the formation of interchain covalent bonds between various cell wall polymers (Fry, 1986; Goldberg et al., 1986; Bradley et al., 1992). However, a complete description of peroxidase action in vivo is not available because of the vast number of potential substrates and the existence of multiple isoenzymes. The tobacco anionic peroxidase is one of the better-characterized isoenzymes. This enzyme has been shown to oxidize a number of significant plant secondary compounds in vitro including cinnamyl alcohols, phenolic acids, and indole-3-acetic acid (Maeder, 1980; Lagrimini, 1991). A cDNA encoding the enzyme has been obtained, and this enzyme was shown to be expressed at the highest levels in lignifying tissues (xylem and tracheary elements) and also in epidermal tissue (Lagrimini et al., 1987). It was shown at this time that there were four distinct copies of the anionic peroxidase gene in tobacco (Nicotiana tabacum). A tobacco genomic DNA library was constructed in the [lambda]-phase EMBL3, from which two unique peroxidase genes were sequenced. One of these clones, [lambda]POD1, was designated as a pseudogene when the exonic sequences were found to differ from the cDNA sequences by 1%, and several frame shifts in the coding sequences indicated a dysfunctional gene (the authors' unpublished results). The other clone, [lambda]POD3, described in this manuscript, was designated as the functional tobacco anionic peroxidase gene because of 100% homology with the cDNA. Significant structural elements include an AS-2 box indicated in shoot-specific expression (Lam and Chua, 1989), a TATA box, and two intervening sequences. 10 refs., 1 tab.

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

    PubMed

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

    2004-10-01

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

  4. Suicide inactivation of peroxidase from Chamaerops excelsa palm tree leaves.

    PubMed

    Cuadrado, Nazaret Hidalgo; Zhadan, Galina G; Roig, Manuel G; Shnyrov, Valery L

    2011-12-01

    The concentration and time-dependences and the mechanism of the inactivation of Chamaerops excelsa peroxidase (CEP) by hydrogen peroxide were studied kinetically with four co-substrates (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), guaiacol, o-dianisidine and o-phenylenediamine). The turnover number (r) of H(2)O(2) required to complete the inactivation of the enzyme varied for the different substrates, the enzyme most resistant to inactivation (r=4844) with ABTS being the most useful substrate for biotechnological applications, opening a new avenue of enquiry with this peroxidase. PMID:21925205

  5. Absence of transsynaptic transport in cerebello-thalamo-cortical path of the rat.

    PubMed

    Petrovic-Minic, B; Mojsilovic-Petrovic, J; Holm, S; Mogensen, J; Divac, I

    1994-01-01

    Attempts to visualize the cerebello-thalamo-cortical path in the rat were made with different approaches. We tried (1) double labelling with somatopetal tracing from the motor cortex and somatofugal from the cerebellar nuclei, (2) transmembrane labelling by depositing biocytin or wheat germ agglutinin (WGA) into the motor cortex or cerebellum. WGA was either iodinated with 125I or conjugated with horseradish peroxidase (HRP). The double labelling technique showed an overlap of the tracers in the same thalamic region but no evidence of transsynaptic transport in either direction was obtained. Our results indicate a difference in the organization of this system in primates and rodents, since transsynaptic labelling in the cerebellar nuclei after injections of WGA-HRP conjugate in the monkey motor cortex has been found. PMID:7517613

  6. Immobilization of peroxidase on SPEU film via radiation grafting

    NASA Astrophysics Data System (ADS)

    Hongfei, Ha; Guanghui, Wang; Jilan, Wu

    The acrylic acid or acrylamide were grafted via radiation onto segmented polyetherurethane (SPEU) film which is a kind of biocompatible material. Then the Horse radish peroxidase was immobilized on the grafted SPEU film through chemical binding. Some quantitative relationships between the percent graft and the activity, amount of immobilized enzyme were given. The properties and application of obtained biomaterial was studied as well.

  7. Peroxidase activity in cotton cell culture infected with Verticillium dahliae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In our studies with cotton, we have shown that the plant’s induced anionic peroxidases bind to chitin, which is a component of the cell wall of the plant pathogenic fungus Verticillium dahliae. In binding to the cell wall surface, they disrupt the integrity of the pathogen’s cell wall. Thus, these...

  8. TiO(2) nanotube arrays: intrinsic peroxidase mimetics.

    PubMed

    Zhang, Lingling; Han, Lei; Hu, Peng; Wang, Li; Dong, Shaojun

    2013-11-18

    TiO2 nanotube arrays (NTA), prepared by potentiostatic anodization, were discovered to possess an intrinsic peroxidase-like activity. The colorimetric and electrochemical assays both demonstrated their excellent catalytic activity towards H2O2 reduction. On this basis, a simple and inexpensive electrochemical biosensor for glucose detection was developed. PMID:24084751

  9. Structural and spectroscopic characterisation of a heme peroxidase from sorghum.

    PubMed

    Nnamchi, Chukwudi I; Parkin, Gary; Efimov, Igor; Basran, Jaswir; Kwon, Hanna; Svistunenko, Dimitri A; Agirre, Jon; Okolo, Bartholomew N; Moneke, Anene; Nwanguma, Bennett C; Moody, Peter C E; Raven, Emma L

    2016-03-01

    A cationic class III peroxidase from Sorghum bicolor was purified to homogeneity. The enzyme contains a high-spin heme, as evidenced by UV-visible spectroscopy and EPR. Steady state oxidation of guaiacol was demonstrated and the enzyme was shown to have higher activity in the presence of calcium ions. A Fe(III)/Fe(II) reduction potential of -266 mV vs NHE was determined. Stopped-flow experiments with H2O2 showed formation of a typical peroxidase Compound I species, which converts to Compound II in the presence of calcium. A crystal structure of the enzyme is reported, the first for a sorghum peroxidase. The structure reveals an active site that is analogous to those for other class I heme peroxidase, and a substrate binding site (assigned as arising from binding of indole-3-acetic acid) at the γ-heme edge. Metal binding sites are observed in the structure on the distal (assigned as a Na(+) ion) and proximal (assigned as a Ca(2+)) sides of the heme, which is consistent with the Ca(2+)-dependence of the steady state and pre-steady state kinetics. It is probably the case that the structural integrity (and, thus, the catalytic activity) of the sorghum enzyme is dependent on metal ion incorporation at these positions. PMID:26666777

  10. 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

  11. Towards uncovering the roles of switchgrass peroxidases in plant processes

    PubMed Central

    Saathoff, Aaron J.; Donze, Teresa; Palmer, Nathan A.; Bradshaw, Jeff; Heng-Moss, Tiffany; Twigg, Paul; Tobias, Christian M.; Lagrimini, Mark; Sarath, Gautam

    2013-01-01

    Herbaceous perennial plants selected as potential biofuel feedstocks had been understudied at the genomic and functional genomic levels. Recent investments, primarily by the U.S. Department of Energy, have led to the development of a number of molecular resources for bioenergy grasses, such as the partially annotated genome for switchgrass (Panicum virgatum L.), and some related diploid species. In its current version, the switchgrass genome contains 65,878 gene models arising from the A and B genomes of this tetraploid grass. The availability of these gene sequences provides a framework to exploit transcriptomic data obtained from next-generation sequencing platforms to address questions of biological importance. One such question pertains to discovery of genes and proteins important for biotic and abiotic stress responses, and how these components might affect biomass quality and stress response in plants engineered for a specific end purpose. It can be expected that production of switchgrass on marginal lands will expose plants to diverse stresses, including herbivory by insects. Class III plant peroxidases have been implicated in many developmental responses such as lignification and in the adaptive responses of plants to insect feeding. Here, we have analyzed the class III peroxidases encoded by the switchgrass genome, and have mined available transcriptomic datasets to develop a first understanding of the expression profiles of the class III peroxidases in different plant tissues. Lastly, we have identified switchgrass peroxidases that appear to be orthologs of enzymes shown to play key roles in lignification and plant defense responses to hemipterans. PMID:23802005

  12. Removal of phenolic compounds from wastewaters using soybean peroxidase

    SciTech Connect

    Wright, H.; Nicell, J.A.

    1996-11-01

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

  13. Removal of chlorophenols from wastewater by immobilized horseradish peroxidase

    SciTech Connect

    Tatsumi, Kenji; Wada, Shinji; Ichikawa, Hiroyasu

    1996-07-05

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

  14. Broccoli processing wastes as a source of peroxidase.

    PubMed

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

    2007-12-12

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

  15. 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.

  16. Degradation of textile dyes mediated by plant peroxidases.

    PubMed

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

    2002-01-01

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

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

    PubMed

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

    2002-12-01

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

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

    EPA Science Inventory

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

  19. Variation in Yield of Near-isogenic Soybean Lines for High and Low Seed Coat Peroxidase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Peroxidase is an enzyme present in soybean [Glycine max (L.) Merrill] seed coats and is characterized as either high (dominant allele) or low (recessive allele) activity. Cultivar Cutler 71 is a mixture of high and low seed coat peroxidase genotypes. Mechanical mixtures of 1 high: 1 low peroxidase...

  20. Identification of IAA-oxidase in peroxidase isozymes from cotton plant leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    One of the functions of plant peroxidase is to regulate the indole acetic acid (IAA) hormonal level by oxidizing it to inactive 3-methyleneoxyindole. IAA-binding proteins and plant peroxidase revealed five structurally similar fragments. We have isolated peroxidase isozymes with IAA-oxidase activit...

  1. Lignin-degrading Peroxidases from Genome of Selective Ligninolytic Fungus Ceriporiopsis subvermispora*

    PubMed Central

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

    2012-01-01

    The white-rot fungus Ceriporiopsis subvermispora delignifies lignocellulose with high selectivity, but until now it has appeared to lack the specialized peroxidases, termed lignin peroxidases (LiPs) and versatile peroxidases (VPs), that are generally thought important for ligninolysis. We screened the recently sequenced C. subvermispora genome for genes that encode peroxidases with a potential ligninolytic role. A total of 26 peroxidase genes was apparent after a structural-functional classification based on homology modeling and a search for diagnostic catalytic amino acid residues. In addition to revealing the presence of nine heme-thiolate peroxidase superfamily members and the unexpected absence of the dye-decolorizing peroxidase superfamily, the search showed that the C. subvermispora genome encodes 16 class II enzymes in the plant-fungal-bacterial peroxidase superfamily, where LiPs and VPs are classified. The 16 encoded enzymes include 13 putative manganese peroxidases and one generic peroxidase but most notably two peroxidases containing the catalytic tryptophan characteristic of LiPs and VPs. We expressed these two enzymes in Escherichia coli and determined their substrate specificities on typical LiP/VP substrates, including nonphenolic lignin model monomers and dimers, as well as synthetic lignin. The results show that the two newly discovered C. subvermispora peroxidases are functionally competent LiPs and also suggest that they are phylogenetically and catalytically intermediate between classical LiPs and VPs. These results offer new insight into selective lignin degradation by C. subvermispora. PMID:22437835

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

    SciTech Connect

    Not Available

    1992-12-31

    Lignin peroxidases were investigated with respect to enzyme kinetics and NMR spectroscopy of the heme domain. MN peroxidases were studied with respect to the role of oxalate in enzyme activity, the NMR spectroscopy of the heme domain. Gene expression of both lignin and MN peroxidases were examined as well as expression of site-directed mutants aimed at scale up production of these enzymes.

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

    PubMed

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

    2001-09-18

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

  4. Data on the catalytic mechanism of thiol peroxidase mimics.

    PubMed

    Zadehvakili, B; Giles, N M; Fawcett, J P; Giles, G I

    2016-09-01

    We have recently reported SAR data describing the pharmacological activity of a series of phenyl alkyl selenides and tellurides which catalyse the oxidation of thiols by hydrogen peroxide (H2O2), "The design of redox active thiol peroxidase mimics: dihydrolipoic acid recognition correlates with cytotoxicity and prooxidant action" B. Zadehvakili, S.M. McNeill, J.P. Fawcett, G.I. Giles (2016) [1]. This thiol peroxidase (TPx) activity is potentially useful for a number of therapeutic applications, as it can alter the outcome of oxidative stress related pathologies and modify redox signalling. This article presents data describing the molecular changes that occur to a TPx mimic upon exposure to H2O2, and then the thiol mercaptoethanol, as characterised by UV-vis spectroscopy and HPLC retention time. PMID:27331089

  5. Thermal denaturation and regeneration of japanese-radish peroxidase.

    PubMed

    Tamura, Y; Morita, Y

    1975-09-01

    Thermal denaturation of Japanese-radish peroxidase [EC 1.11.1.7] was investigated with respect to its spectrophotometric properties and effect on the enzymatic activity. Inactivation of the peroxidase occurred at temperatures higher than 60degrees and involved three processes, i.e., dissociation of protohemin from the holoperoxidase, a conformation change in the apperoxidase, and the modification or degradation of protohemin. The splitting process of protohemin from holoperoxidase as followed by the change in the absorption spectrum at high temperatures coincided with the degrease in the activity, and it was found to be at least biphasic. The regeneration of peroxidase on cooling to room temperature was essentially reversible at neutral pH, while at pH 5 and pH 9 these processes were irreversible. The irreversibility at acidic pH was mainly due to an irreversible change in the conformation of the apoenzyme. The difference spectrum of heat-treated apoperoxidase exhibited a denaturation blueshift with negative maxima at 287 and 294 nm, and the total protein fluorescence quantum yield. qprotein, increased by 20% compared to that of the untreated apoenzyme. On the other hand, the irreversibility at alkaline pH was largely attributable to the modification of protohemin. Apoperoxidase was more resistnat to heat denaturation but the modification or degradation of protohemin in heated enzyme was greater at alkaline pH than at acidic pH. The pyridine-ferrohemochrome spectrum of peroxidase exhibited slight shifts of the maxima of the alpha-band to shorter wavelength on heat treatment, and the paper chromatogram showed the presence of a new derivative other than protohemin. The modified product is probably (2(4)-vinyl-4(2)-hydroxyethyldeuterohemin. PMID:5412

  6. Prokaryotic origins of the non-animal peroxidase superfamily and organelle-mediated transmission to eukaryotes.

    PubMed

    Passardi, Filippo; Bakalovic, Nenad; Teixeira, Felipe Karam; Margis-Pinheiro, Marcia; Penel, Claude; Dunand, Christophe

    2007-05-01

    Members of the superfamily of plant, fungal, and bacterial peroxidases are known to be present in a wide variety of living organisms. Extensive searching within sequencing projects identified organisms containing sequences of this superfamily. Class I peroxidases, cytochrome c peroxidase (CcP), ascorbate peroxidase (APx), and catalase peroxidase (CP), are known to be present in bacteria, fungi, and plants, but have now been found in various protists. CcP sequences were detected in most mitochondria-possessing organisms except for green plants, which possess only ascorbate peroxidases. APx sequences had previously been observed only in green plants but were also found in chloroplastic protists, which acquired chloroplasts by secondary endosymbiosis. CP sequences that are known to be present in prokaryotes and in Ascomycetes were also detected in some Basidiomycetes and occasionally in some protists. Class II peroxidases are involved in lignin biodegradation and are found only in the Homobasidiomycetes. In fact class II peroxidases were identified in only three orders, although degenerate forms were found in different Pezizomycota orders. Class III peroxidases are specific for higher plants, and their evolution is thought to be related to the emergence of the land plants. We have found, however, that class III peroxidases are present in some green algae, which predate land colonization. The presence of peroxidases in all major phyla (except vertebrates) makes them powerful marker genes for understanding the early evolutionary events that led to the appearance of the ancestors of each eukaryotic group. PMID:17355904

  7. Mechanistic study of a diazo dye degradation by Soybean Peroxidase

    PubMed Central

    2013-01-01

    Background Enzyme based remediation of wastewater is emerging as a novel, efficient and environmentally-friendlier approach. However, studies showing detailed mechanisms of enzyme mediated degradation of organic pollutants are not widely published. Results The present report describes a detailed study on the use of Soybean Peroxidase to efficiently degrade Trypan Blue, a diazo dye. In addition to examining various parameters that can affect the dye degradation ability of the enzyme, such as enzyme and H2O2 concentration, reaction pH and temperature, we carried out a detailed mechanistic study of Trypan Blue degradation. HPLC-DAD and LC-MS/MS studies were carried out to confirm dye degradation and analyze the intermediate metabolites and develop a detailed mechanistic dye degradation pathway. Conclusion We report that Soybean peroxidase causes Trypan Blue degradation via symmetrical azo bond cleavage and subsequent radical-initiated ring opening of the metabolites. Interestingly, our results also show that no high molecular weight polymers were produced during the peroxidase-H2O2 mediated degradation of the phenolic Trypan Blue. PMID:23711110

  8. Kinetic mechanism and nucleotide specificity of NADH peroxidase

    SciTech Connect

    Stoll, V.S.; Blanchard, J.S.

    1988-02-01

    NADH peroxidase is a flavoprotein isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide-dependent reduction of hydrogen peroxide to water. Initial velocity, product, and dead-end inhibition studies have been performed at pH 7.5 and support a ping-pong kinetic mechanism. In the absence of hydrogen peroxide, both transhydrogenation between NADH and thioNAD, and isotope exchange between (/sup 14/C)NADH and NAD, have been demonstrated, although in both these experiments, the maximal velocity of nucleotide exchange was less than 1.5% the maximal velocity of the peroxidatic reaction. We propose that NADH binds tightly to both oxidized and two-electron reduced enzyme. NADH oxidation proceeds stereospecifically with the transfer of the 4S hydrogen to enzyme, and then, via exchange, to water. No primary tritium kinetic isotope effect was observed, and no statistically significant primary deuterium kinetic isotope effects on V/K were determined, although primary deuterium kinetic isotope effects on V were observed in the presence and absence of sodium acetate. NADH peroxidase thus shares with other flavoprotein reductases striking kinetic, spectroscopic, and stereochemical similarities. On this basis, we propose a chemical mechanism for the peroxide cleaving reaction catalyzed by NADH peroxidase which involves the obligate formation of a flavinperoxide, and peroxo bond cleavage by nucleophilic attack by enzymatic dithiols.

  9. Relative Binding Affinities of Monolignols to Horseradish Peroxidase.

    PubMed

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

    2016-08-11

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

  10. Detoxification of pesticides aqueous solution using horseradish peroxidase.

    PubMed

    El-Said, Saad Mohamed

    2013-03-15

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

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

    SciTech Connect

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

    1991-08-15

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

  12. 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.

  13. The role of ascorbate peroxidase, guaiacol peroxidase, and polysaccharides in cassava (Manihot esculenta Crantz) roots under postharvest physiological deterioration.

    PubMed

    Uarrota, Virgílio Gavicho; Moresco, Rodolfo; Schmidt, Eder Carlos; Bouzon, Zenilda Laurita; Nunes, Eduardo da Costa; Neubert, Enilto de Oliveira; Peruch, Luiz Augusto Martins; Rocha, Miguel; Maraschin, Marcelo

    2016-04-15

    This study aimed to investigate the role of ascorbate peroxidase (APX), guaiacol peroxidase (GPX), polysaccharides, and protein contents associated with the early events of postharvest physiological deterioration (PPD) in cassava roots. Increases in APX and GPX activity, as well as total protein contents occurred from 3 to 5 days of storage and were correlated with the delay of PPD. Cassava samples stained with Periodic Acid-Schiff (PAS) highlighted the presence of starch and cellulose. Degradation of starch granules during PPD was also detected. Slight metachromatic reaction with toluidine blue is indicative of increasing of acidic polysaccharides and may play an important role in PPD delay. Principal component analysis (PCA) classified samples according to their levels of enzymatic activity based on the decision tree model which showed GPX and total protein amounts to be correlated with PPD. The Oriental (ORI) cultivar was more susceptible to PPD. PMID:26617011

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

    PubMed

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

    1993-05-01

    The location of vagal preganglionic neurones (VPN) has been determined in nine ferrets (Mustela putorius furo) and seven mink (M. vison) using neuronal tract-tracing techniques employing horseradish peroxidase (HRP) and wheat-germ agglutinin conjugated HRP (WGA-HRP) mixtures injected into the nodose ganglion of the vagus nerve. Labelled VPN were located ipsilaterally in the dorsal motor nucleus of the vagus (DmnX), nucleus ambiguus (nA), and reticular formation (rf) of the medulla oblongata. In four of the ferrets, labelled VPN were also identified in the nucleus dorsomedialis (ndm) and the nucleus of the spinal accessory nerve (nspa). In a single mink a few labelled cells were observed in the ndm but no labelled VPN were found in the nspa. Labelling of afferent components of the vagus nerve was seen in two ferrets and two mink with the best labelling obtained following an injection of an HRP/WGA-HRP mixture into the nodose ganglion. Labelled afferents were observed to cross the ipsilateral spinal trigeminal tract (SpV) before entering the tractus solitarius (TS) in regions separate from the motor axons which exit the medulla in separate fasicles. Sensory terminal fields were identified bilaterally in the nucleus of the tractus solitarius (nTS) in both species and bilaterally in the area postrema (ap) of the ferret; however, the contralateral labelling was sparse in comparison to the densely labelled ipsilateral nTS/ap. Maximal terminal labelling was seen in regions just rostral and caudal to obex in both species. PMID:7686926

  15. An evaluation of retrograde tracing methods for the identification of chemically distinct cochlear efferent neurons.

    PubMed

    Vetter, D E; Mugnaini, E

    1990-07-01

    We have compared retrograde labelling of rat olivocochlear neurons after unilateral cochlear injections of wheatgerm agglutinin conjugated horseradish peroxidase (WGA-HRP) and free HRP. After cochlear injection of WGA-HRP, labelling of nerve cell bodies in the brainstem can be explained not only as conventional retrograde labelling resulting from uptake by efferent nerve terminals synapsing on or near hair cells, but also as spurious labelling originating from tracer leakage, through the periotic duct and over the eighth nerve sheaths, into the cerebral-spinal fluid. Depending on the length of survival time, spurious labelling can involve small portions of the nucleus of the trapezoid body or the entire auditory brainstem and other non-auditory centers. On the contrary, moderate amounts of free HRP delivered to the cochlea do not lead to spurious labelling. With free HRP as the tracer of choice, we found that cochlear efferent cells were located not only in the ipsilateral LSO body and bilaterally within MVPO and RPO as already described by White and Warr, but also surrounding the ipsilateral LSO and in the ipsilateral LVPO. The allocation of these newly described olivocochlear neurons to the medial large cell or lateral small cell system is uncertain because they are located laterally in the brainstem and project ipsilaterally but are large spherical to fusiform or multipolar cells. A zinc salicylate-formol fixative and a metal intensified DAB reaction were found to be effective in visualizing retrogradely transported HRP in neurons and allowed immunocytochemical staining of the same sections with antisera to glutamic acid decarboxylase and choline acetyltransferase. This double label protocol can be used to produce a neurochemical map of the OC systems. PMID:1702612

  16. Nociceptive Afferents to the Premotor Neurons That Send Axons Simultaneously to the Facial and Hypoglossal Motoneurons by Means of Axon Collaterals

    PubMed Central

    Dong, Yulin; Li, Jinlian; Zhang, Fuxing; Li, Yunqing

    2011-01-01

    It is well known that the brainstem premotor neurons of the facial nucleus and hypoglossal nucleus coordinate orofacial nociceptive reflex (ONR) responses. However, whether the brainstem PNs receive the nociceptive projection directly from the caudal spinal trigeminal nucleus is still kept unclear. Our present study focuses on the distribution of premotor neurons in the ONR pathways of rats and the collateral projection of the premotor neurons which are involved in the brainstem local pathways of the orofacial nociceptive reflexes of rat. Retrograde tracer Fluoro-gold (FG) or FG/tetramethylrhodamine-dextran amine (TMR-DA) were injected into the VII or/and XII, and anterograde tracer biotinylated dextran amine (BDA) was injected into the caudal spinal trigeminal nucleus (Vc). The tracing studies indicated that FG-labeled neurons receiving BDA-labeled fibers from the Vc were mainly distributed bilaterally in the parvicellular reticular formation (PCRt), dorsal and ventral medullary reticular formation (MdD, MdV), supratrigeminal nucleus (Vsup) and parabrachial nucleus (PBN) with an ipsilateral dominance. Some FG/TMR-DA double-labeled premotor neurons, which were observed bilaterally in the PCRt, MdD, dorsal part of the MdV, peri-motor nucleus regions, contacted with BDA-labeled axonal terminals and expressed c-fos protein-like immunoreactivity which induced by subcutaneous injection of formalin into the lip. After retrograde tracer wheat germ agglutinated horseradish peroxidase (WGA-HRP) was injected into VII or XII and BDA into Vc, electron microscopic study revealed that some BDA-labeled axonal terminals made mainly asymmetric synapses on the dendritic and somatic profiles of WGA-HRP-labeled premotor neurons. These data indicate that some premotor neurons could integrate the orofacial nociceptive input from the Vc and transfer these signals simultaneously to different brainstem motonuclei by axonal collaterals. PMID:21980505

  17. Nociceptive afferents to the premotor neurons that send axons simultaneously to the facial and hypoglossal motoneurons by means of axon collaterals.

    PubMed

    Dong, Yulin; Li, Jinlian; Zhang, Fuxing; Li, Yunqing

    2011-01-01

    It is well known that the brainstem premotor neurons of the facial nucleus and hypoglossal nucleus coordinate orofacial nociceptive reflex (ONR) responses. However, whether the brainstem PNs receive the nociceptive projection directly from the caudal spinal trigeminal nucleus is still kept unclear. Our present study focuses on the distribution of premotor neurons in the ONR pathways of rats and the collateral projection of the premotor neurons which are involved in the brainstem local pathways of the orofacial nociceptive reflexes of rat. Retrograde tracer Fluoro-gold (FG) or FG/tetramethylrhodamine-dextran amine (TMR-DA) were injected into the VII or/and XII, and anterograde tracer biotinylated dextran amine (BDA) was injected into the caudal spinal trigeminal nucleus (Vc). The tracing studies indicated that FG-labeled neurons receiving BDA-labeled fibers from the Vc were mainly distributed bilaterally in the parvicellular reticular formation (PCRt), dorsal and ventral medullary reticular formation (MdD, MdV), supratrigeminal nucleus (Vsup) and parabrachial nucleus (PBN) with an ipsilateral dominance. Some FG/TMR-DA double-labeled premotor neurons, which were observed bilaterally in the PCRt, MdD, dorsal part of the MdV, peri-motor nucleus regions, contacted with BDA-labeled axonal terminals and expressed c-fos protein-like immunoreactivity which induced by subcutaneous injection of formalin into the lip. After retrograde tracer wheat germ agglutinated horseradish peroxidase (WGA-HRP) was injected into VII or XII and BDA into Vc, electron microscopic study revealed that some BDA-labeled axonal terminals made mainly asymmetric synapses on the dendritic and somatic profiles of WGA-HRP-labeled premotor neurons. These data indicate that some premotor neurons could integrate the orofacial nociceptive input from the Vc and transfer these signals simultaneously to different brainstem motonuclei by axonal collaterals. PMID:21980505

  18. Tracing neural circuits in vivo with Mn-enhanced MRI.

    PubMed

    Murayama, Yusuke; Weber, Bruno; Saleem, Kadharbatcha S; Augath, Mark; Logothetis, Nikos K

    2006-05-01

    The application of MRI-visible paramagnetic tracers to reveal in vivo connectivity can provide important subject-specific information for multisite, multielectrode intracortical recordings in combined behavioral and physiology experiments. To establish the use of such tracers in the nonhuman primate, we recently compared the specificity of the anterograde tracer Mn2+ with that of wheat-germ-agglutinin conjugated to horseradish peroxidase (WGA-HRP) in experiments tracing the neuronal connections of the basal ganglia of the monkey. It was shown that Mn2+ and WGA-HRP yield the same projection patterns and that the former tracer crosses at least two synapses, for it could be found in thalamus following injections into the striatum. Here we provide evidence that Mn2+ reaches the cortex following striatum injections and, thus, is transferred even further than previously shown. In other words, used as a paramagnetic MRI tracer, Mn2+ can permit the visualization of neural networks covering at least four processing stages. Moreover, unilateral intravitreal injections show that Mn2+ is sufficiently synapse specific to permit visualization of the lamina of the dorsal lateral geniculate nucleus (dLGN). Interestingly, the transfer rate of the substance reflected the well-known axonal size differences between the parvocellular and magnocellular layers of dLGN. After intravitreal injections, we were able to demonstrate transfer of Mn2+ into several subcortical and cortical areas, including the inferotemporal cortex. The specificity of the transsynaptic transfer of manganese that we report here indicates the value of this tracer for chronic studies of development and plasticity, as well as for studies of brain pathology. PMID:16677940

  19. Comparative analysis of lignin peroxidase and manganese peroxidase activity on coniferous and deciduous wood using ToF-SIMS.

    PubMed

    MacDonald, Jacqueline; Goacher, Robyn E; Abou-Zaid, Mamdouh; Master, Emma R

    2016-09-01

    White-rot fungi are distinguished by their ability to efficiently degrade lignin via lignin-modifying type II peroxidases, including manganese peroxidase (MnP) and lignin peroxidase (LiP). In the present study, time-of flight secondary ion mass spectrometry (ToF-SIMS) was used to evaluate lignin modification in three coniferous and three deciduous wood preparations following treatment with commercial preparations of LiP and MnP from two different white-rot fungi. Percent modification of lignin was calculated as a loss of intact methoxylated lignin over nonfunctionalized aromatic rings, which is consistent with oxidative cleavage of methoxy moieties within the lignin structure. Exposure to MnP resulted in greater modification of lignin in coniferous compared to deciduous wood (28 vs. 18 % modification of lignin); and greater modification of G-lignin compared to S-lignin within the deciduous wood samples (21 vs. 12 %). In contrast, exposure to LiP resulted in similar percent modification of lignin in all wood samples (21 vs 22 %), and of G- and S-lignin within the deciduous wood (22 vs. 23 %). These findings suggest that the selected MnP and LiP may particularly benefit delignification of coniferous and deciduous wood, respectively. Moreover, the current analysis further demonstrates the utility of ToF-SIMS for characterizing enzymatic modification of lignin in wood fibre along with potential advantages over UV and HPCL-MS detection of solubilized delignification products. PMID:27138198

  20. Heterologous Expression of Pleurotus eryngii Peroxidase Confirms Its Ability To Oxidize Mn2+ and Different Aromatic Substrates

    PubMed Central

    Ruiz-Dueñas, Francisco Javier; Martínez, María Jesús; Martínez, Ángel T.

    1999-01-01

    A versatile ligninolytic peroxidase has been cloned from Pleurotus eryngii and its allelic variant MnPL2 expressed in Aspergillus nidulans, with properties similar to those of the mature enzyme from P. eryngii. These include the ability to oxidize Mn2+ and aromatic substrates, confirming that this is a new peroxidase type sharing catalytic properties of lignin peroxidase and manganese peroxidase. PMID:10508113

  1. Identification of an orthologous clade of peroxidases that respond to feeding by greenbugs (Schizaphis graminum) in c4 grasses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Scully et al. FY15 – C4 grass peroxidases and greenbug herbivory TECHNICAL ABSTRACT The role of peroxidases in response to green bug-feeding has been well characterized in several plant species, but information linking specific peroxidases to resistance and the identification of peroxidases that re...

  2. Peroxidase synthesis and activity in the interaction of soybean with Phytophthora megasperma f. sp. glycinea (Pmg)

    SciTech Connect

    Chibbar, R.N.; Esnault, R.; Lee, D.; van Huystee, R.B.; Ward, E.W.B.

    1986-04-01

    Changes, in peroxidase (EC1.11.1.7) have been reported following infection. However, determinations of biosynthesis of quantities of the peroxidase protein molecule have not been madeexclamation In this study hypocotyl of soybean seedlings (Glycine max; cv Harosoy, susceptible; cv Harosoy 63, resistant) were inoculated with zoospores of Pmg. Incorporation of /sup 35/S-methionine (supplied with inoculum) in TCA precipitates was measured. Peroxidase synthesis was measured by immuno precipitation using antibodies against a cationic and an anionic peroxidase derived from peanut cells. Specific peroxidase activity increased rapidly from 5 to 9 h following infection in the resistant reaction but not in the susceptible reaction or the water controls. There was increased synthesis of the anionic peroxidase but not of the cationic peroxidase in the resistant reaction. The anionic peroxidase did not increase in the susceptible until 15 h. The ratio of peroxidase synthesis to total protein synthesis decreased in inoculated tissues compared to control. Peroxidase synthesis is, therefore, a relative minor host response to infection.

  3. 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

  4. Fluoride inhibits the antimicrobial peroxidase systems in human whole saliva.

    PubMed

    Hannuksela, S; Tenovuo, J; Roger, V; Lenander-Lumikari, M; Ekstrand, J

    1994-01-01

    Fluoride (F-) ions at concentrations present in vivo at the plaque/enamel interface (0.05-10 mM) inhibited the activities of lactoperoxidase (LP), myeloperoxidase (MP) and total salivary peroxidase (TSP) in a pH- and dose-dependent way. The inhibition was observed only at pH < or = 6.5 and with F- concentrations > or = 0.1 mM. At pH 5.5 LP activity was inhibited by 85% and MP by 34% with 10 mM F-. TSP activity was also inhibited only at low pH (5.5) by approximately 25%. Furthermore, the generation of the actual antimicrobial agent in vivo, hypothiocyanite (HOSCN/OSCN-), of the oral peroxidase systems was inhibited by F-, again at low pH (5.0-5.5) both in buffer (by 45%) and in saliva (by 15%). This inhibition was observed only with the highest F- concentrations studied (5-10 mM). Fluoridated toothpaste (with 0.10 or 0.14% F) mixed with saliva did not inhibit TSP or HOSCN/OSCN- generation. This may have been due to the 'buffering' effect of toothpaste which did not allow salivary pH to drop below 5.9. We conclude that the F- ions in acidic fluoride products, e.g. in gels or varnishes (but not in toothpastes), may have the potential to locally inhibit the generation of a nonimmune host defense factor, HOSCN/OSCN/SCN-, produced by oral peroxidase systems. The possible clinical significance of this finding remains to be shown. PMID:7850846

  5. Antifungal Properties of Haem Peroxidase from Acorus calamus

    PubMed Central

    GHOSH, MODHUMITA

    2006-01-01

    • Background and Aims Plants have evolved a number of inducible defence mechanisms against pathogen attack, including synthesis of pathogenesis-related proteins. The aim of the study was to purify and characterize antifungal protein from leaves of Acorus calamus. • Methods Leaf proteins from A. calamus were fractionated by cation exchange chromatography and gel filtration and the fraction inhibiting the hyphal extension of phytopathogens was characterized. The temperature stability and pH optima of the protein were determined and its presence was localized in the leaf tissues. • Key Results The purified protein was identified as a class III haem peroxidase with a molecular weight of approx. 32 kDa and pI of 7·93. The temperature stability of the enzyme was observed from 5 °C to 60 °C with a temperature optimum of 36 °C. Maximum enzyme activity was registered at pH 5·5. The pH and temperature optima were corroborated with the antifungal activity of the enzyme. The enzyme was localized in the leaf epidermal cells and lumen tissues of xylem, characteristic of class III peroxidases. The toxic nature of the enzyme which inhibited hyphal growth was demonstrated against phytopathogens such as Macrophomina phaseolina, Fusarium moniliforme and Trichosporium vesiculosum. Microscopic observations revealed distortion in the hyphal structure with stunted growth, increased volume and extensive hyphal branching. • Conclusions This study indicates that peroxidases may have a role to play in host defence by inhibiting the hyphal extension of invading pathogens. PMID:17056613

  6. FTIR study of horseradish peroxidase in reverse micelles.

    PubMed

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

    2001-04-20

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

  7. Synthesis and properties of lignin peroxidase from Streptomyces viridosporus T7A

    SciTech Connect

    Lodha, S.J.; Korus, R.A.; Crawford, D.L.

    1991-12-31

    The production of lignin peroxidase by Streptomyces viridosporus T7A was studied in shake flasks and under aerobic conditions in a 7.5-L batch fermentor. Lignin peroxidase synthesis was found to be strongly affected by catabolite repression. Lignin peroxidase was a non-growth-associated, secondary metabolite. The maximum lignin peroxidase activity was 0.064 U/mL at 36 h. In order to maximize lignin peroxidase activity, optimal conditions were determined. The optimal incubation temperature, pH, and substrate (2,4-dichlorophenol) concentration for the enzyme assays were 45{degrees}C, 6, and 3 m-M, respectively. Stability of lignin peroxidase was determined at 37, 45, and 60{degrees}C, and over the pH range 4-9.

  8. Purification and characterization of a cationic peroxidase Cs in Raphanus sativus.

    PubMed

    Kim, Soung Soo; Lee, Dong Ju

    2005-06-01

    A short distance migrating cationic peroxidase from Korean radish seeds (Raphanus sativus) was detected. Cationic peroxidase Cs was purified to apparent homogeneity and characterized. The molecular mass of the purified cationic peroxidase Cs was estimated to be about 44 kDa on SDS-PAGE. After reconstitution of apoperoxidase Cs with protohemin, the absorption spectra revealed a new peak in the Soret region around 400 nm, which is typical in a classical type III peroxidase family. The optimum pH of peroxidase activity for o-dianisidine oxidation was observed at pH 7.0. Kinetic studies revealed that the reconstituted cationic peroxidase Cs has Km values of 1.18 mM and of 1.27 mM for o-dianisidine and H2O2, respectively. The cationic peroxidase Cs showed the peroxidase activities for native substrates, such as coumaric acid, ferulic acid, and scopoletin. This result suggested that cationic peroxidase Cs plays an important role in plant cell wall formation during seed germination. PMID:16008083

  9. A structural and functional perspective of DyP-type peroxidase family.

    PubMed

    Yoshida, Toru; Sugano, Yasushi

    2015-05-15

    Dye-decolorizing peroxidase from the basidiomycete Bjerkandera adusta Dec 1 (DyP) is a heme peroxidase. This name reflects its ability to degrade several anthraquinone dyes. The substrate specificity, the amino acid sequence, and the tertiary structure of DyP are different from those of the other heme peroxidase (super)families. Therefore, many proteins showing the similar amino acid sequences to that of DyP are called DyP-type peroxidase which is a new family of heme peroxidase identified in 2007. In fact, all structures of this family show a similar structure fold. However, this family includes many proteins whose amino acid sequence identity to DyP is lower than 15% and/or whose catalytic efficiency (kcat/Km) is a few orders of magnitude less than that of DyP. A protein showing an activity different from peroxidase activity (dechelatase activity) has been also reported. In addition, the precise physiological roles of DyP-type peroxidases are unknown. These facts raise a question of whether calling this family DyP-type peroxidase is suitable. Here, we review the differences and similarities of structure and function among this family and propose the reasonable new classification of DyP-type peroxidase family, that is, class P, I and V. In this contribution, we discuss the adequacy of this family name. PMID:25655348

  10. 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.

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

    PubMed Central

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

    1997-01-01

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

  12. 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

  13. Improving the pH-stability of Versatile Peroxidase by Comparative Structural Analysis with a Naturally-Stable Manganese Peroxidase.

    PubMed

    Sáez-Jiménez, Verónica; Fernández-Fueyo, Elena; Medrano, Francisco Javier; Romero, Antonio; Martínez, Angel T; Ruiz-Dueñas, Francisco J

    2015-01-01

    Versatile peroxidase (VP) from the white-rot fungus Pleurotus eryngii is a high redox potential peroxidase of biotechnological interest able to oxidize a wide range of recalcitrant substrates including lignin, phenolic and non-phenolic aromatic compounds and dyes. However, the relatively low stability towards pH of this and other fungal peroxidases is a drawback for their industrial application. A strategy based on the comparative analysis of the crystal structures of VP and the highly pH-stable manganese peroxidase (MnP4) from Pleurotus ostreatus was followed to improve the VP pH stability. Several interactions, including hydrogen bonds and salt bridges, and charged residues exposed to the solvent were identified as putatively contributing to the pH stability of MnP4. The eight amino acid residues responsible for these interactions and seven surface basic residues were introduced into VP by directed mutagenesis. Furthermore, two cysteines were also included to explore the effect of an extra disulfide bond stabilizing the distal Ca2+ region. Three of the four designed variants were crystallized and new interactions were confirmed, being correlated with the observed improvement in pH stability. The extra hydrogen bonds and salt bridges stabilized the heme pocket at acidic and neutral pH as revealed by UV-visible spectroscopy. They led to a VP variant that retained a significant percentage of the initial activity at both pH 3.5 (61% after 24 h) and pH 7 (55% after 120 h) compared with the native enzyme, which was almost completely inactivated. The introduction of extra solvent-exposed basic residues and an additional disulfide bond into the above variant further improved the stability at acidic pH (85% residual activity at pH 3.5 after 24 h when introduced separately, and 64% at pH 3 when introduced together). The analysis of the results provides a rational explanation to the pH stability improvement achieved. PMID:26496708

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

    PubMed

    Shrivastav, Tulsidas G

    2003-01-01

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

  15. Manganese peroxidase gene transcription in Phanerochaete chrysosporium: Activation by manganese

    SciTech Connect

    Brown, J.A.; Alic, M. Gold, M.H. )

    1991-07-01

    The expression of manganese peroxidase in nitrogen-limited cultures of Phanerochaete chrysosporium is dependent on Mn, and initial work suggested that Mn regulates transcription of the mnp gene. In this study, using Northern (RNA) blot analysis of kinetic, dose-response, and inhibitor experiments, the authors demonstrate unequivocally that Mn regulates mnp gene transcription. The amount of mnp mRNA in cells of 4-day-old nitrogen-limited cultures is a direct function of the concentration of Mn in the culture medium up to a maximum of 180 {mu}M. Addition of Mn to nitrogen-limited Mn-deficient secondary metabolic (4-, 5-, and 6-day-old) cultures results in the appearance of mnp mRNA within 40 min. The appearance of this message is completely inhibited by the RNA synthesis inhibitor dactinomycin but not by the protein synthesis inhibitor cycloheximide. Furthermore, the amount of mnp mRNA produced is a direct function of the concentration of added Mn. In contrast, addition of Mn to low-nitrogen Mn-deficient 2- or 3-day-old cultures does not result in the appearance of mnp mRNA. Manganese peroxidase protein is detected by specific immunoprecipitation of the in vitro translation products of poly(A) RNA isolated from Mn-supplemented (but nor from Mn-deficient) cells. All of these results demonstrate that Mn, the substrate for the enzyme, regulates mnp gene transcription via a growth-stage-specific and concentration-dependent mechanism.

  16. Homologous expression of recombinant lignin peroxidase in Phanerochaete chrysosporium

    SciTech Connect

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

    1999-04-01

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

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

    PubMed Central

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

    1986-01-01

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

  18. 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

  19. Peroxidase-catalysed interfacial adhesion of aquatic caddisworm silk.

    PubMed

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

    2015-11-01

    Casemaker caddisfly (Hesperophylax occidentalis) larvae use adhesive silk fibres to construct protective shelters under water. The silk comprises a distinct peripheral coating on a viscoelastic fibre core. Caddisworm silk peroxinectin (csPxt), a haem-peroxidase, was shown to be glycosylated by lectin affinity chromatography and tandem mass spectrometry. Using high-resolution H2O2 and peroxidase-dependent silver ion reduction and nanoparticle deposition, imaged by electron microscopy, csPxt activity was shown to be localized in the peripheral layer of drawn silk fibres. CsPxt catalyses dityrosine cross-linking within the adhesive peripheral layer post-draw, initiated perhaps by H2O2 generated by a silk gland-specific superoxide dismutase 3 (csSOD3) from environmental reactive oxygen species present in natural water. CsSOD3 was also shown to be a glycoprotein and is likely localized in the peripheral layer. Using a synthetic fluorescent phenolic copolymer and confocal microscopy, it was shown that csPxt catalyses oxidative cross-linking to external polyphenolic compounds capable of diffusive interpenetration into the fuzzy peripheral coating, including humic acid, a natural surface-active polyphenol. The results provide evidence of enzyme-mediated covalent cross-linking of a natural bioadhesive to polyphenol conditioned interfaces as a mechanism of permanent adhesion underwater. PMID:26490632

  20. Enzymatic degradation of Congo Red by turnip (Brassica rapa) peroxidase.

    PubMed

    Ahmedi, Afaf; Abouseoud, Mahmoud; Couvert, Annabelle; Amrane, Abdeltif

    2012-01-01

    The enzyme peroxidase is known for its capacity to remove phenolic compounds and aromatic amines from aqueous solutions and also to decolourize textile effluents. This study aims at evaluating the potential of a turnip (Brassica rapa) peroxidase (TP) preparation in the discolouration of textile azo dyes and effluents. An azo dye, Congo Red (CR), was used as a model pollutant for treatment by the enzyme. The effects of various operating conditions like pH value, temperature, initial dye and hydrogen peroxide concentrations, contact time, and enzyme concentration were evaluated. The optimal conditions for maximal colour removal were at pH 2.0, 40 degrees C, 50 mM hydrogen peroxide, 50 mg/l CR dye, and TP activity of 0.45 U/ml within 10 min of incubation time. Analysis of the by-products from the enzymatic treatment by UV-Vis and IR spectroscopy showed no residual compounds in the aqueous phase and a precipitate of polymeric nature. PMID:23016283

  1. Roles of Lignin Peroxidase and Manganese Peroxidase from Phanerochaete chrysosporium in the Decolorization of Olive Mill Wastewaters

    PubMed Central

    Sayadi, S.; Ellouz, R.

    1995-01-01

    The relative contributions of lignin peroxidase (LiP) and manganese peroxidase (MnP) to the decolorization of olive mill wastewaters (OMW) by Phanerochaete chrysosporium were investigated. A relatively low level (25%) of OMW decolorization was found with P. chrysosporium which was grown in a medium with a high Mn(II) concentration and in which a high level of MnP (0.65 (mu)M) was produced. In contrast, a high degree of OMW decolorization (more than 70%) was observed with P. chrysosporium which was grown in a medium with a low Mn(II) concentration but which resulted in a high level of LiP activity (0.3 (mu)M). In this culture medium, increasing the Mn(II) concentration resulted in decreased levels of OMW decolorization and LiP activity. Decolorization by reconstituted cultures of P. chrysosporium was found to be more enhanced by the addition of isolated LiP than by the addition of isolated MnP. The highest OMW decolorization levels were obtained at low initial chemical oxygen demands combined with high levels of extracellular LiP. These data, plus the positive effect of veratryl alcohol on OMW decolorization and LiP activity, indicate that culture conditions which yield high levels of LiP activity lead to high levels of OMW decolorization. PMID:16534959

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

    PubMed Central

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

    1991-01-01

    The role of lignin peroxidases (LIPs) and manganese peroxidases (MNPs) of Phanerochaete chrysosporium in decolorizing kraft bleach plant effluent (BPE) was investigated. Negligible BPE decolorization was exhibited by a per mutant, which lacks the ability to produce both the LIPs and the MNPs. Also, little decolorization was seen when the wild type was grown in high-nitrogen medium, in which the production of LIPs and MNPs is blocked. A lip mutant of P. chrysosporium, which produces MNPs but not LIPs, showed about 80% of the activity exhibited by the wild type, indicating that the MNPs play an important role in BPE decolorization. When P. chrysosporium was grown in a medium with 100 ppm of Mn(II), high levels of MNPs but no LIPs were produced, and this culture also exhibited high rates of BPE decolorization, lending further support to the idea that MNPs play a key role in BPE decolorization. When P. chrysosporium was grown in a medium with no Mn(II), high levels of LIPs but negligible levels of MNPs were produced and the rate and extent of BPE decolorization by such cultures were quite low, indicating that LIPs play a relatively minor role in BPE decolorization. Furthermore, high rates of BPE decolorization were seen on days 3 and 4 of incubation, when the cultures exhibit high levels of MNP activity but little or no LIP activity. These results indicate that MNPs play a relatively more important role than LIPs in BPE decolorization by P. chrysosporium. Images PMID:1768105

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

    PubMed

    Chen, S X; Schopfer, P

    1999-03-01

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

  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. 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.

  6. Selective oxidation of enzyme extracts for improved quantification of peroxidase activity.

    PubMed

    Jiang, Shu; Penner, Michael H

    2015-05-01

    Natural components endogenous to plant material extracts often interfere with traditional peroxidase assays by reducing the oxidized product generated as a result of the peroxidase-catalyzed reaction. This leads to an underestimation of peroxidase activity when the oxidized product provides the signal for enzyme activity quantification. This article describes a relatively simple way to alleviate complications arising due to the presence of such confounding compounds. The method is based on using 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as the reducing substrate. The oxidized product of the reaction is ABTS(+), the accumulation of which can be followed spectrophotometrically. It is shown here that one can selectively inactivate the endogenous compounds that confound the peroxidase assay by treating the enzyme preparation with the oxidized product itself, ABTS(+), prior to initiating the quantification assay. This approach is selective for those compounds likely to interfere with peroxidase quantification. The presented method is shown to alleviate the complications associated with lag phases typical of plant extract peroxidase assays and, thus, to more accurately reflect total peroxidase activity. The presented assay is expected to be applicable to the wide range of biological systems for which the determination of peroxidase activity is desired. PMID:25640588

  7. 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...

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

    SciTech Connect

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

    1989-04-18

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

  9. Interference by morpholine ethanesulfonic acid (MES) and related buffers in phenolic oxidation by peroxidase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    While characterizing the kinetic parameters of apoplastic phenolic oxidation by peroxidase, we found anomalies caused by the 4-morpholine ethanesulfonic acid (MES) buffer being used. In the presence of MES, certain phenolics appeared not to be oxidized by peroxidase, yet the oxidant, H2O2, was uti...

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

    PubMed

    Di Risio, Sabina; Yan, Ning

    2010-09-01

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

  11. 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

  12. 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.

  13. 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

  14. 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

  15. Cytochrome c peroxidase activity of heme bound amyloid β peptides.

    PubMed

    Seal, Manas; Ghosh, Chandradeep; Basu, Olivia; Dey, Somdatta Ghosh

    2016-09-01

    Heme bound amyloid β (Aβ) peptides, which have been associated with Alzheimer's disease (AD), can catalytically oxidize ferrocytochrome c (Cyt c(II)) in the presence of hydrogen peroxide (H2O2). The rate of catalytic oxidation of Cyt(II) c has been found to be dependent on several factors, such as concentration of heme(III)-Aβ, Cyt(II) c, H2O2, pH, ionic strength of the solution, and peptide chain length of Aβ. The above features resemble the naturally occurring enzyme cytochrome c peroxidase (CCP) which is known to catalytically oxidize Cyt(II) c in the presence of H2O2. In the absence of heme(III)-Aβ, the oxidation of Cyt(II) c is not catalytic. Thus, heme-Aβ complex behaves as CCP. PMID:27270708

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

    PubMed

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

    2014-03-01

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

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

    PubMed Central

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

    2013-01-01

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

  18. Storage of Heparinised Canine Whole Blood for the Measurement of Glutathione Peroxidase Activity.

    PubMed

    van Zelst, Mariëlle; Hesta, Myriam; Gray, Kerry; Janssens, Geert P J

    2016-08-01

    Glutathione peroxidase activity is used as a biomarker of selenium status in dogs. Freshly collected blood samples are usually measured, due to the lack of knowledge on the effect of storing the samples. This study investigated if the analysis of glutathione peroxidase activity in whole blood collected from dogs was affected by storage of between 5 and 164 days. Results indicated that glutathione peroxidase activity was more variable in the freshly analysed samples compared to the stored samples. Although the mean differences between fresh and stored samples were not always equal to zero, this is thought to be caused by the variability of reagent preparation rather than by storage, as no consistent increase or decrease in glutathione peroxidase activity was found. Therefore, it can be concluded that heparinised dog blood samples can be successfully stored up to 164 days before analysis of glutathione peroxidase activity. PMID:26701335

  19. Effect of methylmercury on the activity of glutathione peroxidase in rat liver

    SciTech Connect

    Hirota, Y.

    1986-09-01

    The effect of methylmercury on the activity of glutathione peroxidase in rat liver was studied in vivo. A daily dose of 10mg methylmercuric chloride/kg body weight was administered subcutaneously to 15 male Wistar rats for 10 days, and the glutathione peroxidase activity in the liver was measured to compare with the control activity. A marked decrease was observed in the glutathione peroxidase activity in the experimental animals, which measured as low as 40% in comparison to that in the control animals. It can be speculated that the inhibition of glutathione peroxidase activity plays a significant role in the development of mercury toxicity and that the protective effect of selenium and vitamin E on the mercury intoxication might be partly due to preserving the glutathione peroxidase activity in the antioxidative defense mechanisms.

  20. Proton NMR investigation of the heme active site structure of an engineered cytochrome c peroxidase that mimics manganese peroxidase.

    PubMed

    Wang, X; Lu, Y

    1999-07-13

    The heme active site structure of an engineered cytochrome c peroxidase [MnCcP; see Yeung, B. K., et al. (1997) Chem. Biol. 4, 215-221] that closely mimics manganese peroxidase (MnP) has been characterized by both one- and two-dimensional NMR spectroscopy. All hyperfine-shifted resonances from the heme pocket as well as resonances from catalytically relevant amino acid residues in the congested diamagnetic envelope have been assigned. From the NMR spectral assignment and the line broadening pattern of specific protons in NOESY spectra of MnCcP, the location of the engineered Mn(II) center is firmly identified. Furthermore, we found that the creation of the Mn(II)-binding site in CcP resulted in no detectable structural changes on the distal heme pocket of the protein. However, notable structural changes are observed at the proximal side of the heme cavity. Both CepsilonH shift of the proximal histidine and (15)N shift of the bound C(15)N(-) suggest a weaker heme Fe(III)-N(His) bond in MnCcP compared to WtCcP. Our results indicate that the engineered Mn(II)-binding site in CcP resulted in not only a similar Mn(II)-binding affinity and improved MnP activity, but also weakened the Fe(III)-N(His) bond strength of the template protein CcP so that its bond strength is similar to that of the target protein MnP. The results presented here help elucidate the impact of designing a metal-binding site on both the local and global structure of the enzyme, and provide a structural basis for engineering the next generation of MnCcP that mimics MnP more closely. PMID:10413489

  1. 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

  2. Measurement of tomato plant gene expression on a genomic scale for tomato plants that over express peroxidase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We examined the gene expression of transgenic tomato plants that over-express the plant defense peroxidase in comparison to the control tomato plants with normal levels of peroxidase. In general, jasmonate-related plant defenses such as putative protease inhibitors were suppressed in peroxidase-rel...

  3. 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.

  4. Hevea brasiliensis cell suspension peroxidase: purification, characterization and application for dye decolorization

    PubMed Central

    2013-01-01

    Peroxidases are oxidoreductase enzymes produced by most organisms. In this study, a peroxidase was purified from Hevea brasiliensis cell suspension by using anion exchange chromatography (DEAE-Sepharose), affinity chromatography (Con A-agarose) and preparative SDS-PAGE. The obtained enzyme appeared as a single band on SDS-PAGE with molecular mass of 70 kDa. Surprisingly, this purified peroxidase also had polyphenol oxidase activity. However, the biochemical characteristics were only studied in term of peroxidase because similar experiments in term of polyphenol oxidase have been reported in our pervious publication. The optimal pH of the purified peroxidase was 5.0 and its activity was retained at pH values between 5.0–10.0. The enzyme was heat stable over a wide range of temperatures (0–60°C), and less than 50% of its activity was lost at 70°C after incubation for 30 min. The enzyme was completely inhibited by β-mercaptoethanol and strongly inhibited by NaN3; in addition, its properties indicated that it was a heme containing glycoprotein. This peroxidase could decolorize many dyes; aniline blue, bromocresol purple, brilliant green, crystal violet, fuchsin, malachite green, methyl green, methyl violet and water blue. The stability against high temperature and extreme pH supported that the enzyme could be a potential peroxidase source for special industrial applications. PMID:23402438

  5. Site-specific Effects of DUOX1-Related Peroxidase on Intercellular Apoptosis Signaling.

    PubMed

    Heinzelmann, Sonja; Bauer, Georg

    2015-11-01

    Intercellular apoptosis-inducing HOCl signaling is known as an interplay between superoxide anions/H₂O₂ of transformed target cells and dual oxidase 1 (DUOX1)-related peroxidase that is released from neighboring non-transformed or transformed effector cells. Effector cells are dispensable when the release of the peroxidase domain of DUOX1 from target cells is prevented through inhibition of matrix metalloproteinase (MMP) activity. Membrane-associated peroxidase is then co-localized to NADPH oxidase 1 (NOX1) and establishes HOCl signaling specifically in transformed cells, using the same biochemical pathways as classical intercellular HOCl signaling. Membrane-associated peroxidase protects against exogenous HOCl through reversal of the peroxidase reaction. In addition, membrane-associated peroxidase protects against NO/peroxynitrite signaling as it oxidates NO and decomposes peroxynitrite. The protective function of membrane-associated peroxidase (in the absence of MMP) is analogous to that of catalase, whereas the destructive effect of the enzyme, i.e. the synthesis of HOCl, is independent of its localization and of MMP activity. PMID:26504019

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

    PubMed

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

    2015-11-01

    Peroxidases are heme-containing enzymes released by activated immune cells at sites of inflammation. To-date their functional role in human health has mainly been limited to providing a mechanism for oxidative defence against invading bacteria and other pathogenic microorganisms. Our laboratory has recently identified a new functional role for peroxidase enzymes in stimulating fibroblast migration and collagen biosynthesis, offering a new insight into the causative association between inflammation and the pro-fibrogenic events that mediate tissue repair and regeneration. Peroxidases are found at elevated levels within and near blood vessels however, their direct involvement in angiogenesis has never been reported. Here we report for the first time that myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are readily internalised by human umbilical vein endothelial cells (HUVEC) where they promote cellular proliferation, migration, invasion, and stimulate angiogenesis both in vitro and in vivo. These pro-angiogenic effects were attenuated using the specific peroxidase inhibitor 4-ABAH, indicating the enzyme's catalytic activity is essential in mediating this response. Mechanistically, we provide evidence that MPO and EPO regulate endothelial FAK, Akt, p38 MAPK, ERK1/2 phosphorylation and stabilisation of HIF-2α, culminating in transcriptional regulation of key angiogenesis pathways. These findings uncover for the first time an important and previously unsuspected role for peroxidases as drivers of angiogenesis, and suggest that peroxidase inhibitors may have therapeutic potential for the treatment of angiogenesis related diseases driven by inflammation. PMID:26386352

  7. Is Peroxiredoxin II's peroxidase activity strongly inhibited in human erythrocytes?

    PubMed

    Benfeitas, Rui; Selvaggio, Gianluca; Antunes, Fernando; Coelho, Pedro; Salvador, Armindo

    2014-10-01

    H2O2 elimination in human erythrocytes is mainly carried out by catalase (Cat), glutathione peroxidase (GPx1) and the more recently discovered peroxiredoxin 2 (Prx2). However, the contribution of Prx2 to H2O2 consumption is still unclear. Prx2's high reactivity with H2O2 (kPrx2=10×10(7) M(-1)s(-1), kCat =7×10(7) M(-1)s(-1), kGPx1 =4×10(7) M(-1)s(-1)) and high abundance ([Prx2]= 570µM, [Cat]= 32µM, [GPx1]= 1µM) suggest that under low H2O2 supply rates it should consume >99% of the H2O2. However, extensive evidence indicates that in intact erythrocytes Prx2 contributes no more than Cat to H2O2 consumption. In order for this to be attained, Prx2's effective rate constant with H2O2would have to be just ~10(5) M(-1)s(-1), much lower than that determined in multiple experiments with the purified proteins. Nevertheless, nearly all Prx2 is oxidized within 1min of exposing erythrocytes to a H2O2 bolus, which is inconsistent with an irreversible inhibition. A mathematical model of the H2O2 metabolism in human erythrocytes [Benfeitas et al. (2014) Free Radic. Biol. Med.] where Prx2 either has a low kPrx2 or is subject to a strong (>99%) but readily reversible inhibition achieves quantitative agreement with detailed experimental observations of the responses of the redox status of Prx2 in human erythrocytes and suggests functional advantages of this design (see companion abstract). By contrast, a variant where Prx2 is fully active with kPrx2=10(8) M(-1)s(-1) shows important qualitative discrepancies. Altogether, these results suggest that Prx2's peroxidase activity is strongly inhibited in human erythrocytes. We acknowledge fellowship SFRH/BD/51199/2010, grants PEst-C/SAU/LA0001/2013-2014, PEst-OE/QUI/UI0612/2013, PEst-OE/QUI/UI0313/2014, and FCOMP-01-0124-FEDER-020978 (PTDC/QUI-BIQ/119657/2010) co-financed by FEDER through the COMPETE program and by FCT. PMID:26461310

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

    PubMed

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

    2014-11-01

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

  9. 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.

  10. Peroxidase activity in Aloe barbadensis commercial gel: probable role in skin protection.

    PubMed

    Esteban, A; Zapata, J M; Casano, L; Martín, M; Sabater, B

    2000-12-01

    A basic peroxidase (EC 1.11.1.7) (pl around 9.0) has been identified in commercial gel of Aloe barbadensis. In vivo, the activity is localised in the vascular system of inner aqueous leaf parenchyma. Some relevant properties of this basic peroxidase of Aloe have been investigated in leaf extract and in commercial gel where it is notably stable. The acid optimum pH (5.0) for activity and the low KM for H2O2 (0.14 mM) suggest that, when topically applied, Aloe peroxidase may scavenge H2O2 in skin surface. PMID:11199129

  11. Identification of a copper-sensitive ascorbate peroxidase in the unicellular green alga Selenastrum capricornutum.

    PubMed

    Sauser, K R; Liu, J K; Wong, T Y

    1997-07-01

    Extracts from the unicellular green alga Selenastrum capricornutum exhibit high superoxide dismutase activity, but only traces of catalase activity. The excess hydrogen peroxide (H2O2) generated by the superoxide dismutase in S. capricornutum may be degraded by a unique peroxidase. This peroxidase has a high specificity for ascorbate as its electron donor. The enzyme has an optimum pH at 8, is insensitive to cyanide and is inhibited by oxine. Addition of low concentrations of copper to algal cultures stimulates the peroxidase activity threefold. This enzymatic system could be used as a sensitive bioindicator for copper in fresh water. PMID:9243795

  12. The multihued palette of dye-decolorizing peroxidases.

    PubMed

    Singh, Rahul; Eltis, Lindsay D

    2015-05-15

    Dye-decolorizing peroxidases (DyPs; EC 1.11.1.19) are heme enzymes that comprise a family of the dimeric α+β barrel structural superfamily of proteins. The first DyP, identified relatively recently in the fungus Bjerkandera adusta, was characterized for its ability to catalyze the decolorization of anthraquinone-based industrial dyes. These enzymes are now known to be present in all three domains of life, but do not appear to occur in plants or animals. They are involved in a range of physiological processes, although in many cases their roles remain unknown. This has not prevented the development of their biocatalytic potential, which includes the transformation of lignin. This review highlights the functional diversity of DyPs in the light of phylogenetic, structural and biochemical data. The phylogenetic analysis reveals the existence of at least five classes of DyPs. Their potential physiological roles are discussed based in part on synteny analyses. Finally, the considerable biotechnological potential of DyPs is summarized. PMID:25743546

  13. Identification of S-RNase and peroxidase in petunia nectar.

    PubMed

    Hillwig, Melissa S; Kanobe, Charles; Thornburg, Robert W; Macintosh, Gustavo C

    2011-05-01

    Previous SDS PAGE gel analysis of the floral nectars from petunia and tobacco plants revealed significant differences in the protein patterns. Petunia floral nectar was shown to contain a number of RNase activities by in gel RNase activity assay. To identify these proteins in more detail, the bands with RNase activity were excised from gel and subjected to trypsin digestion followed by LC-MS/MS analysis. This analysis revealed that S-RNases accumulate in nectar from Petunia hybrida, where they should carry out a biological function different from self-pollen rejection. In addition, other proteins were identified by the LC-MS/MS analysis. These proteins include a peroxidase, an endochitinase, and a putative fructokinase. Each of these proteins contained a secretory signal sequence that marked them as potential nectar proteins. We developed RT-PCR assays for each of these five proteins and demonstrated that each of these proteins was expressed in the petunia floral nectary. A discussion of the role of these proteins in antimicrobial activity in nectar is presented. PMID:21093100

  14. Biodegradation of Single-Walled Carbon Nanotubes by Eosinophil Peroxidase

    PubMed Central

    Andón, Fernando T.; Kapralov, Alexandr A.; Yanamala, Naveena; Feng, Weihong; Baygan, Arjang; Chambers, Benedict J.; Hultenby, Kjell; Ye, Fei; Toprak, Muhammet S.; Brandner, Birgit D.; Fornara, Andrea; Klein-Seetharaman, Judith; Kotchey, Gregg P.; Star, Alexander; Shvedova, Anna A.

    2014-01-01

    Eosinophil peroxidase (EPO) is one of the major oxidant-producing enzymes during inflammatory states in the human lung. The degradation of single-walled carbon nanotubes (SWCNTs) upon incubation with human EPO and H2O2 is reported. Biodegradation of SWCNTs is higher in the presence of NaBr, but neither EPO alone nor H2O2 alone caused the degradation of nanotubes. Molecular modeling reveals two binding sites for SWCNTs on EPO, one located at the proximal side (same side as the catalytic site) and the other on the distal side of EPO. The oxidized groups on SWCNTs in both cases are stabilized by electrostatic interactions with positively charged residues. Biodegradation of SWCNTs can also be executed in an ex vivo culture system using primary murine eosinophils stimulated to undergo degranulation. Biodegradation is proven by a range of methods including transmission electron microscopy, UV-visible-NIR spectroscopy, Raman spectroscopy, and confocal Raman imaging. Thus, human EPO (in vitro) and ex vivo activated eosinophils mediate biodegradation of SWCNTs: an observation that is relevant to pulmonary responses to these materials. PMID:23447468

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

    PubMed

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

    2013-09-01

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

  16. Aflatoxin detoxification by manganese peroxidase purified from Pleurotus ostreatus

    PubMed Central

    Yehia, Ramy Sayed

    2014-01-01

    Manganese peroxidase (MnP) was produced from white rot edible mushroom Pleurotus ostreatus on the culture filtrate. The enzyme was purified to homogeneity using (NH4)2SO4 precipitation, DEAE-Sepharose and Sephadex G-100 column chromatography. The final enzyme activity achieved 81 U mL−1, specific activity 78 U mg−1 with purification fold of 130 and recovery 1.2% of the crude enzyme. SDS-PAGE indicated that the pure enzyme have a molecular mass of approximately 42 kDa. The optimum pH was between 4–5 and the optimum temperature was 25 °C. The pure MnP activity was enhanced by Mn2+, Cu2+, Ca2+ and K+ and inhibited by Hg+2 and Cd+2. H2O2 at 5 mM enhanced MnP activity while at 10 mM inhibited it significantly. The MnP-cDNA encoding gene was sequenced and determined (GenBank accession no. AB698450.1). The MnP-cDNA was found to consist of 497 bp in an Open Reading Frame (ORF) encoding 165 amino acids. MnP from P. ostreatus could detoxify aflatoxin B1 (AFB1) depending on enzyme concentration and incubation period. The highest detoxification power (90%) was observed after 48 h incubation at 1.5 U mL−1 enzyme activities. PMID:24948923

  17. Immobilization of horseradish peroxidase on modified chitosan beads.

    PubMed

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

    2010-04-01

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

  18. Colorimetric peroxidase mimetic assay for uranyl detection in sea water.

    PubMed

    Zhang, Dingyuan; Chen, Zhuo; Omar, Haneen; Deng, Lin; Khashab, Niveen M

    2015-03-01

    Uranyl (UO2(2+)) is a form of uranium in aqueous solution that represents the greatest risk to human health because of its bioavailability. Different sensing techniques have been used with very sensitive detection limits especially the recently reported uranyl-specific DNAzymes systems. However, to the best of our knowledge, few efficient detection methods have been reported for uranyl sensing in seawater. Herein, gold nanoclusters (AuNCs) are employed in an efficient spectroscopic method to detect uranyl ion (UO2(2+)) with a detection limit of 1.86 μM. In the absence of UO2(2+), the BSA-stabilized AuNCs (BSA-AuNCs) showed an intrinsic peroxidase-like activity. In the presence of UO2(2+), this activity can be efficiently restrained. The preliminary quenching mechanism and selectivity of UO2(2+) was also investigated and compared with other ions. This design strategy could be useful in understanding the binding affinity of protein-stabilized AuNCs to UO2(2+) and consequently prompt the recycling of UO2(2+) from seawater. PMID:25658750

  19. Calcium promotes activity and confers heat stability on plant peroxidases

    PubMed Central

    Plieth, Christoph; Vollbehr, Sonja

    2012-01-01

    In this paper we demonstrate how peroxidase (PO) activities and their heat stability correlate with the availability of free Ca2+ ions. Calcium ions work as a molecular switch for PO activity and exert a protective function, rendering POs heat stable. The concentration ranges of these two activities differ markedly. POs are activated by µM Ca2+ concentration ranges, whereas heat stabilization is observed in the nM range. This suggests the existence of different Ca2+ binding sites. The heat stability of POs depends on the source plant species. Terrestrial plants have POs that exhibit higher temperature stability than those POs from limnic and marine plants. Different POs from a single species can differ in terms of heat stability. The abundance of different POs within a plant is dependent on age and developmental stage. The heat stability of a PO does not necessarily correlate with the maximum temperature the source species is usually exposed to in its natural habitat. This raises questions on the role of POs in the heat tolerance of plants. Consequently, detailed investigations are needed to identify and characterize individual POs, with regard to their genetic origin, subcellular expression, tissue abundance, developmental emergence and their functions in innate and acquired heat tolerance. PMID:22580695

  20. Atherogenic diets exacerbate colitis in mice deficient in glutathione peroxidase

    PubMed Central

    Gao, Qiang; Esworthy, R. Steven; Kim, Byung-Wook; Synold, Timothy W.; Smith, David D.; Chu, Fong-Fong

    2010-01-01

    The pro-inflammatory effect of high-fat diet has been observed beyond the cardiovascular system, but there is little evidence to support its role in triggering inflammatory bowel disease. GPx1/2-double knockout (DKO) mice deficient in two intracellular glutathione peroxidases, GPx1 and GPx2, on a C57BL/6 (B6) background, have mild ileocolitis on a conventional chow. We fed B6 DKO mice two atherogenic diets to test the dietary effect on atherosclerosis and ileocolitis. Both atherogenic diets have high cholesterol, the Chol+/CA diet has cholic acid (CA) and the Chol+ diet has no CA. The Chol+/CA diet induced severe colitis, but not ileitis, in the DKO mice compared with Chol+ and a Chol- control diet. On the Chol+/CA diet, the wild-type (WT) mice had similar levels of aortic lesions and hypercholesterolemia as DKO mice did, but had no intestinal pathology. The diet-associated inflammatory responses in the DKO mice included increase of colonic pro-inflammatory serum amyloid A 3 expression, plasma lipopolysaccharide and TNF-α levels. The Chol+/CA diet has lowered the expression of unfolded protein response genes, ATF6, CHOP, unspliced XbpU and Grp78/Bip, in WT and DKO mice on the Chol- diet. Thus, we conclude that cholesterol diet weakens colon unfolded protein response, which can aggravate spontaneous colitis leading to gut barrier breakdown. GPx has no impact on atherosclerosis without ultra-hypercholesterolemia. PMID:20848490

  1. Eosinophil peroxidase-dependent hydroxyl radical generation by human eosinophils.

    PubMed

    McCormick, M L; Roeder, T L; Railsback, M A; Britigan, B E

    1994-11-11

    Eosinophil production of superoxide (O2-.) and hydrogen peroxide (H2O2) is important in host defense. The present study assessed the potential of eosinophils to generate another potent cytotoxic species, the hydroxyl radical (.OH). .OH formation by phorbol myristate acetate (PMA)-stimulated eosinophils was demonstrated using an alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone/ethanol spin trapping system. Additionally, .OH was spin trapped following the addition of purified eosinophil peroxidase (EPO) to a cell-free O2-./H2O2 generating systems. Effects of superoxide dismutase, catalase, azide, aminotriazole, chloride-depleted buffer, and extensive metal chelation were consistent with .OH formation via the reaction of O2-. and EPO-generated hypohalous acid. Under chloride-depleted conditions, physiologic concentrations of Br- increased .OH formation by both PMA-stimulated eosinophils and the cell-free EPO system. Physiologic concentrations of SCN-, however, did not increase .OH formation, and in the presence of both Br- and SCN-, .OH formation was similar to SCN- only. Eosinophils appear to form .OH via an EPO-dependent mechanism, the magnitude of which varies with the availability of various EPO substrates. Given the highly reactive nature of this radical and the ability of EPO to adhere to cell membranes, even small amounts of .OH formed at such sites could contribute to eosinophil-mediated cytotoxicity. PMID:7961724

  2. Activation of lignin peroxidase in organic media by reversed micelles.

    PubMed

    Kimura, Masayuki; Michizoe, Junji; Oakazaki, Shin-Ya; Furusaki, Shintaro; Goto, Masahiro; Tanaka, Hiroo; Wariishi, Hiroyuki

    2004-11-20

    Activation of lignin peroxidase (LIP) in an organic solvent by reversed micelles was investigated. Bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT) was used as a surfactant to form a reversed micelle. Lyophilized LIP from an optimized aqueous solution exhibited no enzymatic activity in any organic solvents examined in this study; however, LIP was catalytically active by being entrapped in the AOT reversed micellar solution. LIP activity in the reversed micelle was enhanced by optimizing either the preparation or the operation conditions, such as water content and pH in water pools of the reversed micelle and the reaction temperature. Stable activity was obtained in isooctane because of the stability of the reversed micelle. The optimal pH was 5 in the reversed micellar system, which shifted from pH 3 in the aqueous solution. The degradation reaction of several environmental pollutants was attempted using LIP hosted in the AOT reversed micelle. Degradation achieved after a 1-h reaction reached 81%, 50%, and 22% for p-nonylphenol, bisphenol A, and 2,4-dichlorophenol, respectively. This is the first report on the utilization of LIP in organic media. PMID:15459910

  3. Oxidation of pharmaceutically active compounds by a ligninolytic fungal peroxidase.

    PubMed

    Eibes, Gemma; Debernardi, Gianfranco; Feijoo, Gumersindo; Moreira, M Teresa; Lema, Juan M

    2011-06-01

    Pharmaceuticals are an important group of emerging pollutants with increasing interest due to their rising consumption and the evidence for ecotoxicological effects associated to trace amounts in aquatic environments. In this paper, we assessed the potential degradation of a series of pharmaceuticals: antibiotics (sulfamethoxazole), antidepressives (citalopram hydrobromide and fluoxetine hydrochloride), antiepileptics (carbamazepine), anti-inflammatory drugs (diclofenac and naproxen) and estrogen hormones (estrone, 17β-estradiol, 17α-ethinylestradiol) by means of a versatile peroxidase (VP) from the ligninolytic fungus Bjerkandera adusta. The effects of the reaction conditions: VP activity, organic acid concentration and H(2)O(2) addition rate, on the kinetics of the VP based oxidation system were evaluated. Diclofenac and estrogens were completely degraded after only 5-25 min even with a very low VP activity (10 U l(-1)). High degradation percentages (80%) were achieved for sulfamethoxazole and naproxen. Low or undetectable removal yields were observed for citalopram (up to 18%), fluoxetine (lower than 10%) and carbamazepine (not degraded). PMID:20972884

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

    PubMed

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

    2016-09-10

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

  5. A catalytic approach to estimate the redox potential of heme-peroxidases

    SciTech Connect

    Ayala, Marcela . E-mail: maa@ibt.unam.mx; Roman, Rosa; Vazquez-Duhalt, Rafael

    2007-06-08

    The redox potential of heme-peroxidases varies according to a combination of structural components within the active site and its vicinities. For each peroxidase, this redox potential imposes a thermodynamic threshold to the range of oxidizable substrates. However, the instability of enzymatic intermediates during the catalytic cycle precludes the use of direct voltammetry to measure the redox potential of most peroxidases. Here we describe a novel approach to estimate the redox potential of peroxidases, which directly depends on the catalytic performance of the activated enzyme. Selected p-substituted phenols are used as substrates for the estimations. The results obtained with this catalytic approach correlate well with the oxidative capacity predicted by the redox potential of the Fe(III)/Fe(II) couple.

  6. Inhibition of Peroxidase Activity of Cytochrome c: De Novo Compound Discovery and Validation

    PubMed Central

    Bakan, Ahmet; Kapralov, Alexandr A.; Bayir, Hulya; Hu, Feizhou; Kagan, Valerian E.

    2015-01-01

    Cytochrome c (cyt c) release from mitochondria is accepted to be the point of no return for eliciting a cascade of interactions that lead to apoptosis. A strategy for containing sustained apoptosis is to reduce the mitochondrial permeability pore opening. Pore opening is enhanced by peroxidase activity of cyt c gained upon its complexation with cardiolipin in the presence of reactive oxygen species. Blocking access to the heme group has been proposed as an effective intervention method for reducing, if not eliminating, the peroxidase activity of cyt c. In the present study, using a combination of druggability simulations, pharmacophore modeling, virtual screening, and in vitro fluorescence measurements to probe peroxidase activity, we identified three repurposable drugs and seven compounds that are validated to effectively inhibit the peroxidase activity of cyt c. PMID:26078313

  7. IMPROVED TOLERANCE TO ENVIRONMENTALLY INDUCED OXIDATIVE STRESSES IN TRANSGENIC TOMATO OVEREXPRESSING ASCORBATE PEROXIDASE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We studied the effect on oxidative stress resistance of overexpressing, in transgenic tomato (Lycopersicon esculentum) plants, a cytosolic ascorbate peroxidase (APX) gene derived from pea (Pisum sativum). Transformants were selected using Kanamycin resistance and confirmed by PCR, Southern and Nort...

  8. Degradation of disperse dye from textile effluent by free and immobilized Cucurbita pepo peroxidase

    NASA Astrophysics Data System (ADS)

    Boucherit, N.; Abouseoud, M.; Adour, L.

    2012-06-01

    Disperse dyes constitute the largest group of dyes used in local textile industry. This work evaluates the potential of the Cucurbita peroxidase(C-peroxidase) extracted from courgette in the decolourization of disperse dye in free and immobilized form. The optimal conditions for immobilization of C-peroxidase in Ca-alginate were identified. The immobilization was optimized at 2%(w/v) of sodium alginate and 0.2 M of calcium chloride. After optimization of treatment parameters, the results indicate that at pH 2, dye concentration: 80 mg/L(for FCP) and 180 mg/L(for ICP), H2O2 dose: 0,02M (for FCP) and 0,12M(for ICP), the decolourization by free and immobilized C-peroxidase were 72.02% and 69.71 % respectively. The degradation pathway and the metabolic products formed after the degradation were also predicted using UV-vis spectroscopy analysis.

  9. Amino Acid Sequence of Anionic Peroxidase from the Windmill Palm Tree Trachycarpus fortunei

    PubMed Central

    2015-01-01

    Palm peroxidases are extremely stable and have uncommon substrate specificity. This study was designed to fill in the knowledge gap about the structures of a peroxidase from the windmill palm tree Trachycarpus fortunei. The complete amino acid sequence and partial glycosylation were determined by MALDI-top-down sequencing of native windmill palm tree peroxidase (WPTP), MALDI-TOF/TOF MS/MS of WPTP tryptic peptides, and cDNA sequencing. The propeptide of WPTP contained N- and C-terminal signal sequences which contained 21 and 17 amino acid residues, respectively. Mature WPTP was 306 amino acids in length, and its carbohydrate content ranged from 21% to 29%. Comparison to closely related royal palm tree peroxidase revealed structural features that may explain differences in their substrate specificity. The results can be used to guide engineering of WPTP and its novel applications. PMID:25383699

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

    PubMed Central

    Kimura, Makoto; Kawano, Tomonori

    2015-01-01

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

  11. Influence of organophosphorus pesticides on peroxidase and chlorination activity of human myeloperoxidase.

    PubMed

    Lazarević-Pašti, Tamara; Momić, Tatjana; Radojević, Miloš M; Vasić, Vesna

    2013-09-01

    Inhibitory effects of five organophosphorus pesticides (diazinon, malathion, chlorpyrifos, azinphos-methyl and phorate) and their oxo-analogs on human myeloperoxidase (MPO) activity were investigated. While inspecting separately peroxidase and chlorination activity, it was observed that investigated OPs affect peroxidase activity, but not chlorination activity. Among investigated pesticides, malathion and malaoxon have showed the highest power to inhibit MPO peroxidase activity with IC50 values of the order of 3×10(-7) and 5×10(-9) M, respectively. It was proposed that inhibition trend is rendered by molecular structure which invokes steric hindrance for OPs interaction with MPO active center responsible for peroxidase activity. In addition, it was concluded that physiological function of MPO is not affected by any of the investigated OPs. PMID:25149236

  12. Manganese regulation of manganese peroxidase expression and lignin degradation by the white rot fungus Dichomitus squalens

    SciTech Connect

    Perie, F.; Gold, M.H. )

    1991-08-01

    Extracellular manganese peroxidase and laccase activities were detected in cultures of Dichomitus squalens (Polyporus anceps) under conditions favoring lignin degradation. In contrast, neither extracellular lignin peroxidase nor aryl alcohol oxidase activity was detected in cultures grown under a wide variety of conditions. The mineralization of {sup 14}C-ring-, -side chain-, and -methoxy-labeled synthetic guaiacyl lignins by D. squalens and the expression of extracellular manganese peroxidase were dependent on the presence of Mn(II), suggesting that manganese peroxidase is an important component of this organism's lignin degradation system. The expression of laccase activity was independent of manganese. In contrast to previous findings with Phanero-chaete chrysosporium, lignin degradation by D. squalens proceeded in the cultures containing excess carbon and nitrogen.

  13. Determination of estrogenic/antiestrogenic potential of antifertility substances using rat uterine peroxidase assay.

    PubMed

    Johri, R K; Pahwa, G S; Sharma, S C; Zutshi, U

    1991-11-01

    The effect of three compounds (clomiphene citrate, centchroman, embelin) and plant-derived methanolic extracts (Abutilon indicum and Butea monosperma) was studied on uterotropic and uterine peroxidase activities in ovariectomized rats. It was observed that these two parameters were highly correlated in response to treatment with these test materials and also to estradiol. It was suggested that the uterine peroxidase assay could be utilized as a biochemical parameter in the screening of new antifertility agents for their estrogenic/antiestrogenic properties. PMID:1665776

  14. Effects of experimental hypogravity on peroxidase and cell wall constituents in the dwarf marigold

    NASA Technical Reports Server (NTRS)

    Siegel, S.; Speitel, T.; Shiraki, D.; Fukumoto, J.

    1977-01-01

    Dwarf marigolds grown from seed under experimental hypogravity are modified in lignin content, hemicellulose composition and peroxidase activity. The two conditions used, clinostats and flotation, induced changes differing in magnitude but qualitatively similar. Most responses on clinostats required correction for vertical axis rotational effects, thus limiting the value of these instruments in free-fall simulation. These findings extend earlier observations suggesting that increased peroxidase and decreased lignin are characteristic of growth under experimental hypogravity.

  15. Effects of experimental hypogravity on peroxidase and cell wall constituents in the dwarf marigold

    NASA Technical Reports Server (NTRS)

    Siegel, S.; Speitel, T.; Shiraki, D.; Fukumoto, J.

    1978-01-01

    Dwarf Marigolds grown from seed under experimental hypogravity are modified in lignin content, hemicellulose composition, and peroxidase activity. The two conditions used, clinostats and flotation, induced changes differing in magnitude but qualitatively similar. Most responses on clinostats required corrections for vertical axis rotational effects, thus limiting the value of these instruments in free-fall simulation. These findings extend earlier observations suggesting that increased peroxidase and decreased lignin are characteristic of growth under experimental hypogravity.

  16. The horseradish peroxidase technique for cell lineage studies.

    PubMed

    Peralta, M; Denaro, F J

    2003-12-01

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

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

    PubMed Central

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

    2014-01-01

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

  18. A supramolecular microgel glutathione peroxidase mimic with temperature responsive activity.

    PubMed

    Yin, Yanzhen; Jiao, Shufei; Lang, Chao; Liu, Junqiu

    2014-05-21

    Glutathione peroxidase (GPx) protects cells from oxidative damage by scavenging surplus reactive oxygen species (ROS). Commonly, an appropriate amount of ROS acts as a signal molecule in the metabolism. A smart artificial GPx exhibits adjustable catalytic activity, which can potentially reduce the amount of ROS to an appropriate degree and maintain its important physiological functions in metabolism. To construct an optimum and excellent smart artificial GPx, a novel supramolecular microgel artificial GPx (SM-Te) was prepared based on the supramolecular host-guest interaction employing the tellurium-containing guest molecule (ADA-Te-ADA) and the cyclodextrin-containing host block copolymer (poly(N-isopropylacrylamide)-b-[polyacrylamides-co-poly(6-o-(triethylene glycol monoacrylate ether)-β-cyclodextrin)], PPAM-CD) as building blocks. Subsequently, based on these building blocks, SM-Te was constructed and the formation of its self-assembled structure was confirmed by dynamic light scattering, NMR, SEM, TEM, etc. Typically, benefitting from the temperature responsive properties of the PNIPAM scaffold, SM-Te also exhibited similar temperature responsive behaviour. Importantly, the GPx catalytic rates of SM-Te displayed a noticeable temperature responsive characteristic. Moreover, SM-Te exhibited the typical saturation kinetics behaviour of a real enzyme catalyst. It was proved that the changes of the hydrophobic microenvironment and the pore size in the supramolecular microgel network of SM-Te played significant roles in altering the temperature responsive catalytic behaviour. The successful construction of SM-Te not only overcomes the insurmountable disadvantages existing in previous covalent bond crosslinked microgel artificial GPx but also bodes well for the development of novel intelligent antioxidant drugs. PMID:24652520

  19. Recombinant horseradish peroxidase variants for targeted cancer treatment.

    PubMed

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

    2016-06-01

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

  20. Function of glutathione peroxidases in legume root nodules.

    PubMed

    Matamoros, Manuel A; Saiz, Ana; Peñuelas, Maria; Bustos-Sanmamed, Pilar; Mulet, Jose M; Barja, Maria V; Rouhier, Nicolas; Moore, Marten; James, Euan K; Dietz, Karl-Josef; Becana, Manuel

    2015-05-01

    Glutathione peroxidases (Gpxs) are antioxidant enzymes not studied so far in legume nodules, despite the fact that reactive oxygen species are produced at different steps of the symbiosis. The function of two Gpxs that are highly expressed in nodules of the model legume Lotus japonicus was examined. Gene expression analysis, enzymatic and nitrosylation assays, yeast cell complementation, in situ mRNA hybridization, immunoelectron microscopy, and LjGpx-green fluorescent protein (GFP) fusions were used to characterize the enzymes and to localize each transcript and isoform in nodules. The LjGpx1 and LjGpx3 genes encode thioredoxin-dependent phospholipid hydroperoxidases and are differentially regulated in response to nitric oxide (NO) and hormones. LjGpx1 and LjGpx3 are nitrosylated in vitro or in plants treated with S-nitrosoglutathione (GSNO). Consistent with the modification of the peroxidatic cysteine of LjGpx3, in vitro assays demonstrated that this modification results in enzyme inhibition. The enzymes are highly expressed in the infected zone, but the LjGpx3 mRNA is also detected in the cortex and vascular bundles. LjGpx1 is localized to the plastids and nuclei, and LjGpx3 to the cytosol and endoplasmic reticulum. Based on yeast complementation experiments, both enzymes protect against oxidative stress, salt stress, and membrane damage. It is concluded that both LjGpxs perform major antioxidative functions in nodules, preventing lipid peroxidation and other oxidative processes at different subcellular sites of vascular and infected cells. The enzymes are probably involved in hormone and NO signalling, and may be regulated through nitrosylation of the peroxidatic cysteine essential for catalytic function. PMID:25740929

  1. Function of glutathione peroxidases in legume root nodules

    PubMed Central

    Matamoros, Manuel A.; Saiz, Ana; Peñuelas, Maria; Bustos-Sanmamed, Pilar; Mulet, Jose M.; Barja, Maria V.; Rouhier, Nicolas; Moore, Marten; James, Euan K.; Dietz, Karl-Josef; Becana, Manuel

    2015-01-01

    Glutathione peroxidases (Gpxs) are antioxidant enzymes not studied so far in legume nodules, despite the fact that reactive oxygen species are produced at different steps of the symbiosis. The function of two Gpxs that are highly expressed in nodules of the model legume Lotus japonicus was examined. Gene expression analysis, enzymatic and nitrosylation assays, yeast cell complementation, in situ mRNA hybridization, immunoelectron microscopy, and LjGpx-green fluorescent protein (GFP) fusions were used to characterize the enzymes and to localize each transcript and isoform in nodules. The LjGpx1 and LjGpx3 genes encode thioredoxin-dependent phospholipid hydroperoxidases and are differentially regulated in response to nitric oxide (NO) and hormones. LjGpx1 and LjGpx3 are nitrosylated in vitro or in plants treated with S-nitrosoglutathione (GSNO). Consistent with the modification of the peroxidatic cysteine of LjGpx3, in vitro assays demonstrated that this modification results in enzyme inhibition. The enzymes are highly expressed in the infected zone, but the LjGpx3 mRNA is also detected in the cortex and vascular bundles. LjGpx1 is localized to the plastids and nuclei, and LjGpx3 to the cytosol and endoplasmic reticulum. Based on yeast complementation experiments, both enzymes protect against oxidative stress, salt stress, and membrane damage. It is concluded that both LjGpxs perform major antioxidative functions in nodules, preventing lipid peroxidation and other oxidative processes at different subcellular sites of vascular and infected cells. The enzymes are probably involved in hormone and NO signalling, and may be regulated through nitrosylation of the peroxidatic cysteine essential for catalytic function. PMID:25740929

  2. Characterization of manganese peroxidases from the hyperlignolytic fungus IZU-154.

    PubMed Central

    Matsubara, M; Suzuki, J; Deguchi, T; Miura, M; Kitaoka, Y

    1996-01-01

    Four isozymes of manganese peroxidase (MnP) were identified in the culture fluid of the hyperlignolytic fungus IZU-154 under nitrogen starvation conditions. One of them was purified and characterized kinetically. The specific activity and Kcat/K(m) value of the MnP from IZU-154 were 1.6 times higher than those of the MnP from a typical lignin-degrading fungus, Phanerochaete chrysosporium. Two cDNAs encoding MnP isozymes from IZU-154 were isolated. The coding sequence of the two cDNAs, IZ-MnP1 cDNA and IZ-MnP2 cDNA, were 1,152 (384 amino acids) and 1,155 (385 amino acids) bp in length, respectively. They exhibit 96.2% identity at the nucleotide level and 95.1% identity at the amino acid level. Southern blot analysis indicated that two MnP isozyme genes exist in IZU-154 genomic DNA. The primary structures of two MnPs from IZU-154 were similar to those of MnPs from P. chrysosporium. The amino acid sequences including the important residues identified in MnPs from P. chrysosporium, such as the manganese-binding residues, the calcium-binding residues, the disulfide bonds, and the N-glycosylation site, were conserved in the two deduced IZ-MnPs. However, several discrepancies were found in the context around the distal histidine residue between MnP from IZU-154 and MnP from P. chrysosporium, which likely led to the difference in the kinetic parameters for MnP function. PMID:8899997

  3. Direct interaction of lignin and lignin peroxidase from Phanerochaete chrysosporium.

    PubMed

    Johjima, T; Itoh, N; Kabuto, M; Tokimura, F; Nakagawa, T; Wariishi, H; Tanaka, H

    1999-03-01

    Binding properties of lignin peroxidase (LiP) from the basidiomycete Phanerochaete chrysosporium against a synthetic lignin (dehydrogenated polymerizate, DHP) were studied with a resonant mirror biosensor. Among several ligninolytic enzymes, only LiP specifically binds to DHP. Kinetic analysis revealed that the binding was reversible, and that the dissociation equilibrium constant was 330 microM. The LiP-DHP interaction was controlled by the ionization group with a pKa of 5.3, strongly suggesting that a specific amino acid residue plays a role in lignin binding. A one-electron transfer from DHP to oxidized intermediates LiP compounds I and II (LiPI and LiPII) was characterized by using a stopped-flow technique, showing that binding interactions of DHP with LiPI and LiPII led to saturation kinetics. The dissociation equilibrium constants for LiPI-DHP and LiPII-DHP interactions were calculated to be 350 and 250 microM, and the first-order rate constants for electron transfer from DHP to LiPI and to LiPII were calculated to be 46 and 16 s-1, respectively. These kinetic and spectral studies strongly suggest that LiP is capable of oxidizing lignin directly at the protein surface by a long-range electron transfer process. A close look at the crystal structure suggested that LiP possesses His-239 as a possible lignin-binding site on the surface, which is linked to Asp-238. This Asp residue is hydrogen-bonded to the proximal His-176. This His-Asp...proximal-His motif would be a possible electron transfer route to oxidize polymeric lignin. PMID:10051582

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

    PubMed Central

    Smeller, László; Fidy, Judit

    2002-01-01

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

  5. Nitration of veratryl alcohol by lignin peroxidase and tetranitromethane.

    PubMed

    Sheng, D; Joshi, D K; Gold, M H

    1998-04-01

    Lignin peroxidase (LiP), from Phanerochaete chrysosporium, in the presence of H2O2 and tetranitromethane (TNM), oxidizes veratryl (3,4-dimethoxybenzyl) alcohol (VA) (I) to veratraldehyde (IV), 4,5-dimethoxy-2-nitrobenzyl alcohol (V), and 3,4-dimethoxy-nitrobenzene (VI). The formation of these products is explained by a mechanism involving the one-electron oxidation of VA by LiP to produce the corresponding cation radical, which loses a proton to generate the benzylic radical. The latter reduces TNM to generate the trinitromethane anion (VIII) and the nitrogen dioxide radical (.NO2). .NO2 couples with the VA cation radical, and the subsequent loss of a proton leads to V. Alternatively, the attack of .NO2 at C-1 of the VA cation radical, followed by aromatization and loss of formaldehyde (VII), yields VI. Isotopic labeling experiments confirm that V is generated by the reaction of .NO2 with the VA cation radical, rather than with the benzylic radical. The nitration of two other LiP substrates, 1,4-dimethoxybenzene (II) and tyrosine (III), also was examined. Product analysis of reactions conducted in the presence of H2O2 with these substrates indicated less nitrated product was formed from 1,4-dimethoxybenzene and no nitrated product was formed from tyrosine. However, significant amounts of nitrated products were formed from 1,4-dimethoxybenzene and tyrosine when glucose and glucose oxidase were used as an H2O2 source. These results suggest that a reductant, either the veratryl alcohol benzylic radical or superoxide, is required in the reaction to reduce TNM to generate .NO2. These results provide further evidence for the formation of the VA cation radical and the first chemical evidence for the formation of the VA benzylic radical in LiP-catalyzed reactions. PMID:9521824

  6. Glutathione peroxidase-1 protects from CD95-induced apoptosis.

    PubMed

    Gouaze, Valerie; Andrieu-Abadie, Nathalie; Cuvillier, Olivier; Malagarie-Cazenave, Sophie; Frisach, Marie-Francoise; Mirault, Marc-Edouard; Levade, Thierry

    2002-11-01

    Through the induction of apoptosis, CD95 plays a crucial role in the immune response and the elimination of cancer cells. Ligation of CD95 receptor activates a complex signaling network that appears to implicate the generation of reactive oxygen species (ROS). This study investigated the place of ROS production in CD95-mediated apoptosis and the role of the antioxidant enzyme glutathione peroxidase-1 (GPx1). Anti-CD95 antibodies triggered an early generation of ROS in human breast cancer T47D cells that was blocked by overexpression of GPx1 and inhibition of initiator caspase activation. Enforced expression of GPx1 also resulted in inhibition of CD95-induced effector caspase activation, DNA fragmentation, and apoptotic cell death. Resistance to CD95-mediated apoptosis was not due to an increased expression of anti-apoptotic molecules and could be reversed by glutathione-depleting agents. In addition, whereas the anti-apoptotic protein Bcl-xL prevented CD95-induced apoptosis in MCF-7 cells, it did not inhibit the early ROS production. Moreover, Bcl-xL but not GPx1 overexpression could suppress the staurosporine-induced late generation of ROS and subsequent cell death. Altogether, these findings suggest that GPx1 functions upstream of the mitochondrial events to inhibit the early ROS production and apoptosis induced by CD95 ligation. Finally, transgenic mice overexpressing GPx1 were partially protected from the lethal effect of anti-CD95, underlying the importance of peroxide formation (and GPx1) in CD95-triggered apoptosis. PMID:12221075

  7. Detection of Lignin Peroxidase and Xylanase by Immunocytochemical Labeling in Wood Decayed by Basidiomycetes †

    PubMed Central

    Blanchette, R. A.; Abad, A. R.; Farrell, R. L.; Leathers, T. D.

    1989-01-01

    The white rot fungi used in this study caused two different forms of degradation. Phanerochaete chrysosporium, strain BKM-F-1767, and Phellinus pini caused a preferential removal of lignin from birch wood, whereas Trametes (Coriolus) versicolor caused a nonselective attack of all cell wall components. Use of polyclonal antisera to H8 lignin peroxidase and monoclonal antisera to H2 lignin peroxidase followed by immunogold labeling with protein A-gold or protein G-gold, respectively, showed lignin peroxidase extra-and intracellularly to fungal hyphae and within the delignified cell walls after 12 weeks of laboratory decay. Lignin peroxidase was localized at sites within the cell wall where electron-dense areas of the lignified cell wall layers remained. In wood decayed by Trametes versicolor, lignin peroxidase was located primarily along the surface of eroded cell walls. No lignin peroxidase was evident in brown-rotted wood, but slight labeling occurred within hyphal cells. Use of polyclonal antisera to xylanase followed by immunogold labeling showed intense labeling on fungal hyphae and surrounding slime layers and within the woody cell wall, where evidence of degradation was apparent. Colloidal-gold-labeled xylanase was prevalent in wood decayed by all fungi used in this study. Areas of the wood with early stages of cell wall decay had the greatest concentration of gold particles, while little labeling occurred in cells in advanced stages of decay by brown or white rot fungi. Images PMID:16347939

  8. Differences in peroxidase localization of rabbit peritoneal macrophages after surface adherence.

    PubMed Central

    Bodel, P. T.; Nichols, B. A.; Bainton, D. F.

    1978-01-01

    Unlike resident peritoneal macrophages, which contain peroxidase in the rough endoplasmic reticulum (RER) and perinuclear cisternae (PN), macrophages elicited into the rabbit peritoneal cavity by various stimulants lack the enzyme. Since we had previously found that such peroxidase reactivity rapidly appears in the RER and PN of blood monocytes after surface adherence in vitro, we wondered whether the enzyme could be similarly produced in elicited macrophages by adherence. Cells from peritoneal exudates (96 hours after endotoxin injection) were harvested, suspended in culture medium, and allowed to adhere to fibrin-coated or plastic surfaces. Following culture for various intervals, they were fixed, incubated for peroxidase, and examined by electron microscopy. We observed that these elicited cells, which initially contained no cytochemically detectable peroxidase, acquired peroxidatic activity in the RER and PN within 2 hours after adherence in culture. Thus macrophages, like blood monocytes, may rapidly acquire peroxidase reactivity as a consequence of plasma membrane: external surface interaction. In view of this finding, it would seem unwise to use peroxidase localization as the basis for advocating the existence of two separate lines of peritoneal macrophages, as has been proposed by previous investigators. Images Figure 2 Figure 3 Figure 1 PMID:645814

  9. Peroxidase Enzymes Regulate Collagen Biosynthesis and Matrix Mineralization by Cultured Human Osteoblasts.

    PubMed

    DeNichilo, Mark O; Shoubridge, Alexandra J; Panagopoulos, Vasilios; Liapis, Vasilios; Zysk, Aneta; Zinonos, Irene; Hay, Shelley; Atkins, Gerald J; Findlay, David M; Evdokiou, Andreas

    2016-03-01

    The early recruitment of inflammatory cells to sites of bone fracture and trauma is a critical determinant in successful fracture healing. Released by infiltrating inflammatory cells, myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are heme-containing enzymes, whose functional involvement in bone repair has mainly been studied in the context of providing a mechanism for oxidative defense against invading microorganisms. We report here novel findings that show peroxidase enzymes have the capacity to stimulate osteoblastic cells to secrete collagen I protein and generate a mineralized extracellular matrix in vitro. Mechanistic studies conducted using cultured osteoblasts show that peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl hydroxylase-dependent manner, which does not require ascorbic acid. Our studies demonstrate that osteoblasts rapidly bind and internalize both MPO and EPO, and the catalytic activity of these peroxidase enzymes is essential to support collagen I biosynthesis and subsequent release of collagen by osteoblasts. We show that EPO is capable of regulating osteogenic gene expression and matrix mineralization in culture, suggesting that peroxidase enzymes may play an important role not only in normal bone repair, but also in the progression of pathological states where infiltrating inflammatory cells are known to deposit peroxidases. PMID:26643175

  10. Partial purification and characterization of a peroxidase from neonatal rat skin

    SciTech Connect

    Strohm, B.H.

    1987-01-01

    Peroxidase activity was partially purified from neonatal CFN rat skin. The membrane-bound peroxidase activity was extracted with 0.5 M calcium chloride and was monitored spectrophotometrically at 470 nm with 2-methoxyphenol and hydrogen peroxide as substrates. Subcellular distribution studies indicated the specific activity to be highest and comparable in the 800 {times} g and 8000 {times} g pellets, lowest in the 100,000 {times} g pellet, and absent in the 100,000 {times} g supernatant. The peroxidase activity was partially purified by affinity chromatography on concanavaline-A-sepharose 4-B and by gel filtration using Bio-gel P-150. The apparent molecular weight of the native enzyme as determined by Bio-gel P-200 gel filtration was approximately 42,500 {plus minus} 2,300 daltons. Peroxidase activity increased linearly with increases in protein concentration, time, and guaiacol concentration. Activity was inhibited approximately 75% by 0.1 mM potassium cyanide or 0.05 mM sodium azide. Pyrogallol, hydroquinone, p-cresol, catechol, benzidine, 3,3{prime}-dimethoxybenzidine, tetramethylbenzidine, and p-phenylenediamine were active as substrates for rat skin peroxidase. Rat skin peroxidase was also shown to mediate non-extractable binding of ({sup 3}H)-benzo(a)pyrene-7,8-dihydrodiol and ({sup 3}H)-2-aminofluorene to DNA and protein.

  11. Peroxidase-dependent apoplastic oxidative burst in Arabidopsis required for pathogen resistance.

    PubMed

    Bindschedler, Laurence V; Dewdney, Julia; Blee, Kris A; Stone, Julie M; Asai, Tsuneaki; Plotnikov, Julia; Denoux, Carine; Hayes, Tezni; Gerrish, Chris; Davies, Dewi R; Ausubel, Frederick M; Bolwell, G Paul

    2006-09-01

    The oxidative burst is an early response to pathogen attack leading to the production of reactive oxygen species (ROS) including hydrogen peroxide. Two major mechanisms involving either NADPH oxidases or peroxidases that may exist singly or in combination in different plant species have been proposed for the generation of ROS. We identified an Arabidopsis thaliana azide-sensitive but diphenylene iodonium-insensitive apoplastic oxidative burst that generates H(2)O(2) in response to a Fusarium oxysporum cell-wall preparation. Transgenic Arabidopsis plants expressing an anti-sense cDNA encoding a type III peroxidase, French bean peroxidase type 1 (FBP1) exhibited an impaired oxidative burst and were more susceptible than wild-type plants to both fungal and bacterial pathogens. Transcriptional profiling and RT-PCR analysis showed that the anti-sense (FBP1) transgenic plants had reduced levels of specific peroxidase-encoding mRNAs, including mRNAs corresponding to Arabidopsis genes At3g49120 (AtPCb) and At3g49110 (AtPCa) that encode two class III peroxidases with a high degree of homology to FBP1. These data indicate that peroxidases play a significant role in generating H(2)O(2) during the Arabidopsis defense response and in conferring resistance to a wide range of pathogens. PMID:16889645

  12. Mode of binding of the antithyroid drug propylthiouracil to mammalian haem peroxidases.

    PubMed

    Singh, R P; Singh, A; Kushwaha, G S; Singh, A K; Kaur, P; Sharma, S; Singh, T P

    2015-03-01

    The mammalian haem peroxidase superfamily consists of myeloperoxidase (MPO), lactoperoxidase (LPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). These enzymes catalyze a number of oxidative reactions of inorganic substrates such as Cl(-), Br(-), I(-) and SCN(-) as well as of various organic aromatic compounds. To date, only structures of MPO and LPO are known. The substrate-binding sites in these enzymes are located on the distal haem side. Propylthiouracil (PTU) is a potent antithyroid drug that acts by inhibiting the function of TPO. It has also been shown to inhibit the action of LPO. However, its mode of binding to mammalian haem peroxidases is not yet known. In order to determine the mode of its binding to peroxidases, the structure of the complex of LPO with PTU has been determined. It showed that PTU binds to LPO in the substrate-binding site on the distal haem side. The IC50 values for the inhibition of LPO and TPO by PTU are 47 and 30 µM, respectively. A comparision of the residues surrounding the substrate-binding site on the distal haem side in LPO with those in TPO showed that all of the residues were identical except for Ala114 (LPO numbering scheme), which is replaced by Thr205 (TPO numbering scheme) in TPO. A threonine residue in place of alanine in the substrate-binding site may affect the affinity of PTU for peroxidases. PMID:25760705

  13. The peroxidase-mediated biodegradation of petroleum hydrocarbons in a H2O2-induced SBR using in-situ production of peroxidase: Biodegradation experiments and bacterial identification.

    PubMed

    Shekoohiyan, Sakine; Moussavi, Gholamreza; Naddafi, Kazem

    2016-08-01

    A bacterial peroxidase-mediated oxidizing process was developed for biodegrading total petroleum hydrocarbons (TPH) in a sequencing batch reactor (SBR). Almost complete biodegradation (>99%) of high TPH concentrations (4g/L) was attained in the bioreactor with a low amount (0.6mM) of H2O2 at a reaction time of 22h. A specific TPH biodegradation rate as high as 44.3mgTPH/gbiomass×h was obtained with this process. The reaction times required for complete biodegradation of TPH concentrations of 1, 2, 3, and 4g/L were 21, 22, 28, and 30h, respectively. The catalytic activity of hydrocarbon catalyzing peroxidase was determined to be 1.48U/mL biomass. The biodegradation of TPH in seawater was similar to that in fresh media (no salt). A mixture of bacteria capable of peroxidase synthesis and hydrocarbon biodegradation including Pseudomonas spp. and Bacillus spp. were identified in the bioreactor. The GC/MS analysis of the effluent indicated that all classes of hydrocarbons could be well-degraded in the H2O2-induced SBR. Accordingly, the peroxidase-mediated process is a promising method for efficiently biodegrading concentrated TPH-laden saline wastewater. PMID:27060866

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

    PubMed

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

    2015-07-20

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

  15. The ligninolytic peroxidases in the genus Pleurotus: divergence in activities, expression, and potential applications.

    PubMed

    Knop, Doriv; Yarden, Oded; Hadar, Yitzhak

    2015-02-01

    Mushrooms of the genus Pleurotus are comprised of cultivated edible ligninolytic fungi with medicinal properties and a wide array of biotechnological and environmental applications. Like other white-rot fungi (WRF), they are able to grow on a variety of lignocellulosic biomass substrates and degrade both natural and anthropogenic aromatic compounds. This is due to the presence of the non-specific oxidative enzymatic systems, which are mainly consisted of lacasses, versatile peroxidases (VPs), and short manganese peroxidases (short-MnPs). Additional, less studied, peroxidase are dye-decolorizing peroxidases (DyPs) and heme-thiolate peroxidases (HTPs). During the past two decades, substantial information has accumulated concerning the biochemistry, structure and function of the Pleurotus ligninolytic peroxidases, which are considered to play a key role in many biodegradation processes. The production of these enzymes is dependent on growth media composition, pH, and temperature as well as the growth phase of the fungus. Mn(2+) concentration differentially affects the expression of the different genes. It also severs as a preferred substrate for these preoxidases. Recently, sequencing of the Pleurotus ostreatus genome was completed, and a comprehensive picture of the ligninolytic peroxidase gene family, consisting of three VPs and six short-MnPs, has been established. Similar enzymes were also discovered and studied in other Pleurotus species. In addition, progress has been made in the development of molecular tools for targeted gene replacement, RNAi-based gene silencing and overexpression of genes of interest. These advances increase the fundamental understanding of the ligninolytic system and provide the opportunity for harnessing the unique attributes of these WRF for applied purposes. PMID:25503316

  16. 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. PMID:25660651

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

    PubMed

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

    2010-02-01

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

  18. Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples.

    PubMed

    Flemmig, Jörg; Schwarz, Pauline; Bäcker, Ingo; Leichsenring, Anna; Lange, Franziska; Arnhold, Jürgen

    2016-01-01

    In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 µl makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments. Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes. Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases. PMID:27501318

  19. 2,4,6-Trichlorophenol mediated increases in extracellular peroxidase activity in three species of Lemnaceae.

    PubMed

    Biswas, Dilip K; Scannell, Gillian; Akhmetov, Nurlan; Fitzpatrick, Dara; Jansen, Marcel A K

    2010-11-01

    Chlorinated phenols, or chlorophenols, are persistent priority pollutants that are widespread in the environment. Class III peroxidases are well-characterised plant enzymes that can catalyse the oxidative dechlorination of chlorophenols. Expression of these enzymes by plants is commonly associated with plant stress, therefore limiting scope for phytoremediation. In this study, we have quantitatively compared peroxidase activity and phytotoxicity as a function of 2,4,6-trichlorophenol (TCP) concentration in three species of Lemnaceae; Lemna minor, Lemna gibba and Landoltia punctata. Effects of TCP on the growth rates of the three species differed considerably with L. punctata being the most tolerant species. TCP also affected photosynthetic parameters, causing a decrease in open photosystem II reaction centres (qP) and, in L. punctata only, a decrease in non-photochemical quenching (qN). In parallel, TCP exposure resulted in increased peroxidase activity in all three species. Peroxidase activity in L. minor and L. gibba displayed an inverse relationship with biomass accumulation, i.e. the more growth reduction the more peroxidase activity. In contrast, induction of peroxidase activity in L. punctata was bi-phasic, with a TCP-induced activity peak at concentrations that had no major effect on growth, and further induction under phytotoxic concentrations. The mechanism by which L. punctata recognises and responds to low concentrations of an anthropogenic compound, in the absence of wide-ranging stress, remains enigmatic. However, we conclude that this "window" of peroxidase production in the absence of major growth inhibition offers potential for the development of sustainable, peroxidise-mediated phytoremediation systems. PMID:20810175

  20. Michaelis-Menten Kinetics and the Activation Energy Relate Soil Peroxidase Kinetics to the Lignin Chemistry

    NASA Astrophysics Data System (ADS)

    Triebwasser-Freese, D.; Tharayil, N.; Preston, C. M.; Gerard, P.

    2013-12-01

    Recently, it has been suggested that lignin exhibit a turnover rate of less than 6 years, suggesting that the enzymatic mechanisms mediating the decay of lignin are less understood. One factor that could be affecting the mean residence time of lignin in the soil is the catalytic efficiency of soil oxidoreductase enzymes. We characterized the spatial and seasonal transitions in the Michaelis-Menten kinetics and activation energy of the soil oxidoreductase enzyme, peroxidase, across three ecosystems of differing litter chemistries- pine, deciduous forest, and a cultivated field- and associate it to the soil lignin chemistries. To interpret the combined effect of Vmax and Km, the two parameters were integrated into one term which we defined as the catalytic efficiency. Generally, the peroxidases in pine soils exhibited the highest Vmax and Km, resulting in the lowest catalytic efficiency, followed by that in the deciduous soils. Meanwhile, the agricultural soils which exhibited the lowest Vmax and Km contained the highest catalytic efficiency of peroxidase. Through linear regression analysis of the kinetic parameters to the soil lignin chemistry, we discerned that the catalytic efficiency term best associated to the lignin monomer ratios (C/V, P/V, and SCV/V). The Activation Energy of peroxidase varied by depth, and seasons across the ecosystems. However, the Activation Energy of peroxidase did not relate to the lignin chemistry or quantity. Collectively, our results show that although the peroxidase Vmax and Km in the phenolic-poor soils are low, the degradation efficiency of peroxidases in this soils can be equivalent or exceed that of phenolic-rich soils. This study, through the characterization of Michaelis-Menten kinetics, provides a new insight into the mechanisms that could moderate the decomposition of lignin in soils.

  1. Lignin Peroxidase Activity Is Not Important in Biological Bleaching and Delignification of Unbleached Kraft Pulp by Trametes versicolor

    PubMed Central

    Archibald, Frederick S.

    1992-01-01

    The discovery in 1983 of fungal lignin peroxidases able to catalyze the oxidation of nonphenolic aromatic lignin model compounds and release some CO2 from lignin has been seen as a major advance in understanding how fungi degrade lignin. Recently, the fungus Trametes versicolor was shown to be capable of substantial decolorization and delignification of unbleached industrial kraft pulps over 2 to 5 days. The role, if any, of lignin peroxidase in this biobleaching was therefore examined. Several different assays indicated that T. versicolor can produce and secrete peroxidase proteins, but only under certain culture conditions. However, work employing a new lignin peroxidase inhibitor (metavanadate ions) and a new lignin peroxidase assay using the dye azure B indicated that secreted lignin peroxidases do not play a role in the T. versicolor pulp-bleaching system. Oxidative activity capable of degrading 2-keto-4-methiolbutyric acid (KMB) appeared unique to ligninolytic fungi and always accompanied pulp biobleaching. PMID:16348775

  2. Activity and isoforms of peroxidases, lignin and anatomy, during adventitious rooting in cuttings of Ebenus cretica L.

    PubMed

    Syros, Thomas; Yupsanis, Traianos; Zafiriadis, Helias; Economou, Athanasios

    2004-01-01

    Adventitious rooting of Ebenus cretica cuttings was studied in order to examine a) the rooting ability of different genotypes in relation to electrophoretic patterns of peroxidases. b) the activity and electrophoretic patterns of soluble and wall ionically bound peroxidases, the lignin content and anatomical changes in the control and IBA treated cuttings of and genotypes in the course of adventitious root formation. In addition, a fraction of soluble cationic peroxidases was separated by gel filtration chromatography from the total soluble peroxidases of a genotype. No rooting occurred in cuttings without IBA-treatment. In both genotypes, electrophoretic patterns of soluble anionic peroxidases revealed two common peroxidase isoforms, while a fast-migrating anionic peroxidase isoform (A3) appeared only in genotypes. Both genotypes showed similar patterns of soluble, as well as wall ionically bound cationic peroxidase isoforms. The number of isoforms was unchanged during the rooting process (induction, initiation and expression phase) but an increase in peroxidase activity (initiation phase) followed by decrease has been found in IBA-treated cuttings. During initiation phase the lignin content was almost similar to that on day 0 in genotype while it was reduced at by about 50% in genotype at the respective time. Microscopic observations revealed anatomical differences between genotypes. According to this study, the and genotypes display differences in anatomy, lignin content, activity of soluble peroxidases and the electrophoretic patterns of soluble anionic peroxidase isoforms. The A3-anionic peroxidase isoform could be used as biochemical marker to distinguish and genotypes of E. cretica and seems to be correlated to lignin synthesis in rooting process. PMID:15002666

  3. Activity and Isoenzyme Profile of Peroxidase as Affected by Microgravity Stress

    NASA Astrophysics Data System (ADS)

    Sarnatska, V. V.; Gladun, H. O.; Padalko, S. F.

    2008-06-01

    To investigate microgravity (clinorotation) effect on activity and isoenzyme pattern of peroxidase the culture of primary explants of potato tubers with normal activation of proliferation in vitro, explants inoculated with Agrobacterium tumefaciens(A.t.), where crown-gall tumors were formed and dormant potato tubers were used. Substantial decrease of total peroxidase activity after one day-clinorotation of potato explants, normal and inoculated with A.t., was revealed. Seven day- clinorotation resulted in the decreased peroxidase activity in normal clinorotated explants, while peroxidase activity in clinorotated explants, inoculated with A.t., returned to the level of its stationary control. When peroxidase of potato explants was analyzed by PAGE, the result obtained show the decrease in activity of one electrophoretic fractions with low migrating mobility and two fractions with moderate mobility in clinorotated explants, normal and with crown gall, as compared with the ones in stationary conditions. The decrease in activity of these fractions under microgravity was less pronounced in explants with crown-galls.

  4. Phenol-Oxidizing Peroxidases Contribute to the Protection of Plants from Ultraviolet Radiation Stress1

    PubMed Central

    Jansen, Marcel A.K.; van den Noort, Ria E.; Tan, M.Y. Adillah; Prinsen, Els; Lagrimini, L. Mark; Thorneley, Roger N.F.

    2001-01-01

    We have studied the mechanism of UV protection in two duckweed species (Lemnaceae) by exploiting the UV sensitivity of photosystem II as an in situ sensor for radiation stress. A UV-tolerant Spirodela punctata G.F.W. Meyer ecotype had significantly higher indole-3-acetic acid (IAA) levels than a UV-sensitive ecotype. Parallel work on Lemna gibba mutants suggested that UV tolerance is linked to IAA degradation rather than to levels of free or conjugated IAA. This linkage is consistent with a role for class III phenolic peroxidases, which have been implicated both in the degradation of IAA and the cross-linking of various UV-absorbing phenolics. Biochemical analysis revealed increased activity of a specific peroxidase isozyme in both UV-tolerant duckweed lines. The hypothesis that peroxidases play a role in UV protection was tested in a direct manner using genetically modified tobacco (Nicotiana sylvestris). It was found that increased activity of the anionic peroxidase correlated with increased tolerance to UV radiation as well as decreased levels of free auxin. We conclude that phenol-oxidizing peroxidases concurrently contribute to UV protection as well as the control of leaf and plant architecture. PMID:11457952

  5. Oxidative 4-dechlorination of polychlorinated phenols is catalyzed by extracellular fungal lignin peroxidases

    SciTech Connect

    Hammel, K.E.; Tardone, P.J.

    1988-08-23

    The extracellular lignin peroxidases (ligninases) of Phanerochaete chrysosporium catalyzed H/sub 2/O/sub 2/-dependent spectral changes in several environmentally significant polychlorinated phenols: 2,4-dichloro-, 2,4,5-trichloro-, 2,4,6-trichloro-, and pentachlorophenol. Gas chromatography/mass spectrometry of reduced and acetylated reaction products showed that, in each case, lignin peroxidase catalyzed a 4-dechlorination of the starting phenol to yield a p-benzoquinone. The oxidation of 2,4-dichlorophenol also yielded a dechlorinated coupling dimer, tentatively identified as 2-chloro-6-(2,4-dichlorophenoxy)-p-benzoquinone. Experiments on the stoichiometry of 2,4,6-trichlorophenol oxidation showed that this substrate was quantitatively dechlorinated to give the quinone and inorganic chloride. H/sub 2//sup 18/O-labeling experiments on 2,4,6-trichlorophenol oxidation demonstrated that water was the source of the new 4-oxo substituent in 2,6-di-chloro-p-benzoquinone. The results indicate a mechanism whereby lignin peroxidase oxidizes a 4-chlorinated phenol to an electrophilic intermediate, perhaps the 4-chlorocyclohexadienone cation. Nucleophilic attack by water and elimination of HCl then ensue at the 4-position, which produces the quinone. Lignin peroxidases have previously been implicated in the degradation by Phanerochaete of several nonphenolic aromatic pollutants. It appears likely from their results that these peroxidases could also catalyze the initial dechlorination of certain polychlorinated phenols in vivo.

  6. Lignin Peroxidase Oxidation of Aromatic Compounds in Systems Containing Organic Solvents

    PubMed Central

    Vazquez-Duhalt, Rafael; Westlake, Donald W. S.; Fedorak, Phillip M.

    1994-01-01

    Lignin peroxidase from Phanerochaete chrysosporium was used to study the oxidation of aromatic compounds, including polycyclic aromatic hydrocarbons and heterocyclic compounds, that are models of moieties of asphaltene molecules. The oxidations were done in systems containing water-miscible organic solvents, including methanol, isopropanol, N, N-dimethylformamide, acetonitrile, and tetrahydrofuran. Of the 20 aromatic compounds tested, 9 were oxidized by lignin peroxidase in the presence of hydrogen peroxide. These included anthracene, 1-, 2-, and 9-methylanthracenes, acenaphthene, fluoranthene, pyrene, carbazole, and dibenzothiophene. Of the compounds studied, lignin peroxidase was able to oxidize those with ionization potentials of <8 eV (measured by electron impact). The reaction products contain hydroxyl and keto groups. In one case, carbon-carbon bond cleavage, yielding anthraquinone from 9-methylanthracene, was detected. Kinetic constants and stability characteristics of lignin peroxidase were determined by using pyrene as the substrate in systems containing different amounts of organic solvent. Benzyl alkylation of lignin peroxidase improved its activity in a system containing water-miscible organic solvent but did not increase its resistance to inactivation at high solvent concentrations. PMID:16349176

  7. Lignin peroxidase oxidation of aromatic compounds in systems containing organic solvents.

    PubMed

    Vazquez-Duhalt, R; Westlake, D W; Fedorak, P M

    1994-02-01

    Lignin peroxidase from Phanerochaete chrysosporium was used to study the oxidation of aromatic compounds, including polycyclic aromatic hydrocarbons and heterocyclic compounds, that are models of moieties of asphaltene molecules. The oxidations were done in systems containing water-miscible organic solvents, including methanol, isopropanol, N, N-dimethylformamide, acetonitrile, and tetrahydrofuran. Of the 20 aromatic compounds tested, 9 were oxidized by lignin peroxidase in the presence of hydrogen peroxide. These included anthracene, 1-, 2-, and 9-methylanthracenes, acenaphthene, fluoranthene, pyrene, carbazole, and dibenzothiophene. Of the compounds studied, lignin peroxidase was able to oxidize those with ionization potentials of <8 eV (measured by electron impact). The reaction products contain hydroxyl and keto groups. In one case, carbon-carbon bond cleavage, yielding anthraquinone from 9-methylanthracene, was detected. Kinetic constants and stability characteristics of lignin peroxidase were determined by using pyrene as the substrate in systems containing different amounts of organic solvent. Benzyl alkylation of lignin peroxidase improved its activity in a system containing water-miscible organic solvent but did not increase its resistance to inactivation at high solvent concentrations. PMID:16349176

  8. Characteristics of estrogen-induced peroxidase in mouse uterine luminal fluid

    SciTech Connect

    Jellinck, P.H.; Newbold, R.R.; McLachlan, J.A. )

    1991-04-01

    Peroxidase activity in the uterine luminal fluid of mice treated with diethylstilbestrol was measured by the guaiacol assay and also by the formation of 3H2O from (2-3H)estradiol. In the radiometric assay, the generation of 3H2O and 3H-labeled water-soluble products was dependent on H2O2 (25 to 100 microM), with higher concentrations being inhibitory. Tyrosine or 2,4-dichlorophenol strongly enhanced the reaction catalyzed either by the luminal fluid peroxidase or the enzyme in the CaCl2 extract of the uterus, but decreased the formation of 3H2O from (2-3H)estradiol by lactoperoxidase in the presence of H2O2 (80 microM). NADPH, ascorbate, and cytochrome c inhibited both luminal fluid and uterine tissue peroxidase activity to the same extent, while superoxide dismutase showed a marginal activating effect. Lactoferrin, a major protein component of uterine luminal fluid, was shown not to contribute to its peroxidative activity, and such an effect by prostaglandin synthase was also ruled out. However, it was not possible to exclude eosinophil peroxidase, brought to the uterus after estrogen stimulation, as being the source of peroxidase activity in uterine luminal fluid.

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

    PubMed

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

    2004-01-01

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

  10. Purification and biochemical characterization of a heme containing peroxidase from the human parasite P. falciparum.

    PubMed

    Trivedi, Vishal; Srivastava, Kumkum; Puri, Sunil K; Maulik, Prakas R; Bandyopadhyay, Uday

    2005-05-01

    A peroxidase (30 kDa) has been purified from the human malaria parasite Plasmodium falciparum to its homogeneity. The protein is a dimer of 15 kDa subunit as evident from SDS-PAGE and MALDI-TOF mass analysis. The antibodies developed against the purified protein cross-react selectively with this protein present in parasite lysate. It is a heme containing peroxidase [R/Z value (A408/A278)=2.33] showing characteristic heme spectra with Soret peak at 408 nm and visible peaks at 536 and 572 nm. Analysis of Soret spectra in presence or absence of cyanide or azide reveals that iron of heme is in Fe-III state. Circular dichroism spectral analysis establishes that this protein contains mainly alpha-helix (60-70%). H2O2 interacts with the heme moiety of the enzyme as evidenced by optical difference spectroscopy and spectral studies indicate the formation of catalytically active peroxidase-H2O2 complex (Soret peak at 413 nm) to exhibit peroxidase activity. During the erythrocytic stages of its life cycle, the parasite is exposed to oxidative stress. As the parasite is susceptible to oxidative stress, this peroxidase may offer antioxidant role by scavenging endogenous H2O2. PMID:15802233

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

    PubMed

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

    2016-09-01

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

  12. Interprotein Coupling Enhances the Electrocatalytic Efficiency of Tobacco Peroxidase Immobilized at a Graphite Electrode.

    PubMed

    Olloqui-Sariego, José Luis; Zakharova, Galina S; Poloznikov, Andrey A; Calvente, Juan José; Hushpulian, Dmitry M; Gorton, Lo; Andreu, Rafael

    2015-11-01

    Covalent immobilization of enzymes at electrodes via amide bond formation is usually carried out by a two-step protocol, in which surface carboxylic groups are first activated with the corresponding cross-coupling reagents and then reacted with protein amine groups. Herein, it is shown that a modification of the above protocol, involving the simultaneous incubation of tobacco peroxidase and the pyrolytic graphite electrode with the cross-coupling reagents produces higher and more stable electrocatalytic currents than those obtained with either physically adsorbed enzymes or covalently immobilized enzymes according to the usual immobilization protocol. The remarkably improved electrocatalytic properties of the present peroxidase biosensor that operates in the 0.3 V ≤ E ≤ 0.8 V (vs SHE) potential range can be attributed to both an efficient electronic coupling between tobacco peroxidase and graphite and to the formation of intra- and intermolecular amide bonds that stabilize the protein structure and improve the percentage of anchoring groups that provide an adequate orientation for electron exchange with the electrode. The optimized tobacco peroxidase sensor exhibits a working concentration range of 10-900 μM, a sensitivity of 0.08 A M(-1) cm(-2) (RSD 0.05), a detection limit of 2 μM (RSD 0.09), and a good long-term stability, as long as it operates at low temperature. These parameter values are among the best reported so far for a peroxidase biosensor operating under simple direct electron transfer conditions. PMID:26437673

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

    PubMed

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

    2003-05-01

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

  14. Peroxidase 4 is involved in syringyl lignin formation in Arabidopsis thaliana.

    PubMed

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

    2015-03-01

    Syringyl lignins result from the oxidative polymerization of sinapyl alcohol in a reaction mediated by syringyl (basic) peroxidases. Several peroxidases have been identified in the genome of Arabidopsis thaliana as close homologues to ZePrx, the best characterized basic peroxidase so far, but none of these has been directly involved in lignification. We have used a knock-out mutant of AtPrx4, the closest homologue to ZePrx, to study the involvement of this basic peroxidase in the physiology of the plant under both long- and short-day light conditions. Our results suggest that AtPrx4 is involved in cell wall lignification, especially in syringyl monomer formation. The disruption of AtPrx4 causes a decrease in syringyl units proportion, but only when light conditions are optimal. Moreover, the effect of AtPrx4 disruption is age-dependent, and it is only significant when the elongation process of the stem has ceased and lignification becomes active. In conclusion, AtPrx4 emerges as a basic peroxidase regulated by day length with an important role in lignification. PMID:25506770

  15. The early development of major projections from caudal levels of the spinal cord to the brainstem and cerebellum in the gray short-tailed Brazilian opossum, Monodelphis domestica.

    PubMed

    Qin, Y Q; Wang, X M; Martin, G F

    1993-09-17

    The Brazilian short-tailed opossum, Monodelphis domestica, is born 14-15 days after copulation and is available for experimentation at stages of development corresponding to those which occur in utero in placental mammals. In the present study, we took advantage of the opossum's embryology to study the development of projections from caudal levels of the spinal cord to the brainstem and cerebellum using axonal tracing methods. In all cases, a 2-3 day survival time was used for axonal transport. When injections of Fast blue (FB) were made into caudal levels of the thoracic cord at postnatal day (PD) 1 or 2, axonal labeling could not be identified at supraspinal levels. When injections were made at PD3, however, labeled axons were found in the fasciculus gracilis at caudal medullary levels, within the ventrolateral medulla and pons, within an incipient inferior cerebellar peduncle, and within the cerebellar anlage. The dorsal root origin of at least some of the axons within the fasciculus gracilis was evidenced by the transganglionic transport of cholera toxin conjugated to horseradish peroxidase from the hindlimbs. After FB injections at PD7, a few labeled axons could be traced from the fasciculus gracilis into the nucleus gracilis and from the ventrolateral pathway to the inferior olive. Generally comparable results were obtained using wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). In cases injected with FB at PD9, the pattern of brainstem labeling was adult-like. Although labeled axons were present within the cerebellum of animals injected with FB on PD3, they were limited to the marginal zone. Axonal labeling was present within an identifiable internal granular layer in cases injected with either FB or WGA-HRP at PD16, and it appeared to be limited to specific bands which foreshadowed those seen at later stages of development and in the adult animal. In some cases, labeled axons were present within the molecular layer where they were not

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

    PubMed Central

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

    2004-01-01

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

  17. Spatial and temporal accumulation of mRNAs encoding two common lignin peroxidases in Phanerochaete chrysosporium.

    PubMed Central

    Moukha, S M; Wösten, H A; Mylius, E J; Asther, M; Wessels, J G

    1993-01-01

    Accumulation of peroxidases and their mRNAs was localized in colonies of Phanerochaete chrysosporium sandwiched between perforated polycarbonate membranes. Northern (RNA) blot analyses of colonial rings and in situ hybridizations with specific probes for manganese(II)-dependent peroxidase (MnP-1) and lignin peroxidase (LiP H8) mRNAs indicated that the expression of MnP-1 and Lip H8 genes started simultaneously in the central area of 3-day-old colonies. With time the signals for both transcripts spread to more-peripheral areas while decreasing in intensity. Furthermore, the appearance of MnP protein, as detected with specific immune serum, immediately followed accumulation of the MnP-1 mRNA transcript. However, LiP protein could be detected only some time after accumulation of LiP H8 mRNA. Images PMID:8501073

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

    PubMed

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

    2005-01-01

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

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

    SciTech Connect

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

    1999-05-01

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

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

    PubMed

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

    2000-08-15

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

  1. Dechlorination of chlorophenols using extracellular peroxidases produced by streptomyces albus ATCC 3005.

    PubMed

    Antonopoulos, V T.; Rob, A; Ball, A S.; Wilson, M T.

    2001-07-01

    Streptomyces albus ATCC 3005 was found to produce higher levels of extracellular peroxidase activity (3.420 U mg(-1)) than previously reported for any other actinomycete. Maximum peroxidase activity was obtained after 72 h of incubation at a temperature of 30 degrees C in a liquid medium (pH 7.6) containing (in w/v) 0.8% to 0.9% oat spelts xylan and 0.6% yeast extract, corresponding to a C:N ratio of around 8.4:1. Characterization of the peroxidases revealed that the optimal temperature for peroxidase activity, using the standard 2,4-dichlorophenol (2,4-DCP) assay was 53 degrees C, when the enzyme reaction was performed at pH 7.2. A study of the effect of temperature on the stability of peroxidase over time, showed that the enzyme was stable at 40 degrees C, with a half-life of 224 min, while at higher temperatures the stability and activity was reduced such that at 50 degrees C and 70 degrees C the half-life of the enzyme was 50 min and 9 min respectively. The optimum pH for the activity of the enzyme occurred between pH 8.1 and 10.4. In terms of substrate specificity, the peroxidase was able to catalyze a broad range of substrates including 2,4-DCP, L-3,4-dihydroxyphenylalanine (L-DOPA), 2,4,5-trichlorophenol and other chlorophenols in the presence of hydrogen peroxide. Ion exchange chromatography was used to confirm that the enzyme was able to release chloride ions from a range of chlorophenols. PMID:11427236

  2. Inducible peroxidases mediate nitration of anopheles midgut cells undergoing apoptosis in response to Plasmodium invasion.

    PubMed

    Kumar, Sanjeev; Gupta, Lalita; Han, Yeon Soo; Barillas-Mury, Carolina

    2004-12-17

    Plasmodium berghei invasion of Anopheles stephensi midgut cells causes severe damage, induces expression of nitric-oxide synthase, and leads to apoptosis. The present study indicates that invasion results in tyrosine nitration, catalyzed as a two-step reaction in which nitric-oxide synthase induction is followed by increased peroxidase activity. Ookinete invasion induced localized expression of peroxidase enzymes, which catalyzed protein nitration in vitro in the presence of nitrite and H(2)O(2). Histochemical stainings revealed that when a parasite migrates laterally and invades more than one cell, the pattern of induced peroxidase activity is similar to that observed for tyrosine nitration. In Anopheles gambiae, ookinete invasion elicited similar responses; it induced expression of 5 of the 16 peroxidase genes predicted by the genome sequence and decreased mRNA levels of one of them. One of these inducible peroxidases has a C-terminal oxidase domain homologous to the catalytic moiety of phagocyte NADPH oxidase and could provide high local levels of superoxide anion (O(2)), that when dismutated would generate the local increase in H(2)O(2) required for nitration. Chemically induced apoptosis of midgut cells also activated expression of four ookinete-induced peroxidase genes, suggesting their involvement in general apoptotic responses. The two-step nitration reaction provides a mechanism to precisely localize and circumscribe the toxic products generated by defense reactions involving nitration. The present study furthers our understanding of the biochemistry of midgut defense reactions to parasite invasion and how these may influence the efficiency of malaria transmission by anopheline mosquitoes. PMID:15456781

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

    PubMed

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

    2011-10-01

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

  4. Description of the first fungal dye-decolorizing peroxidase oxidizing manganese(II).

    PubMed

    Fernández-Fueyo, Elena; Linde, Dolores; Almendral, David; López-Lucendo, María F; Ruiz-Dueñas, Francisco J; Martínez, Angel T

    2015-11-01

    Two phylogenetically divergent genes of the new family of dye-decolorizing peroxidases (DyPs) were found during comparison of the four DyP genes identified in the Pleurotus ostreatus genome with over 200 DyP genes from other basidiomycete genomes. The heterologously expressed enzymes (Pleos-DyP1 and Pleos-DyP4, following the genome nomenclature) efficiently oxidize anthraquinoid dyes (such as Reactive Blue 19), which are characteristic DyP substrates, as well as low redox-potential dyes (such as 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) and substituted phenols. However, only Pleos-DyP4 oxidizes the high redox-potential dye Reactive Black 5, at the same time that it displays high thermal and pH stability. Unexpectedly, both enzymes also oxidize Mn(2+) to Mn(3+), albeit with very different catalytic efficiencies. Pleos-DyP4 presents a Mn(2+) turnover (56 s(-1)) nearly in the same order of the two other Mn(2+)-oxidizing peroxidase families identified in the P. ostreatus genome: manganese peroxidases (100 s(-1) average turnover) and versatile peroxidases (145 s(-1) average turnover), whose genes were also heterologously expressed. Oxidation of Mn(2+) has been reported for an Amycolatopsis DyP (24 s(-1)) and claimed for other bacterial DyPs, albeit with lower activities, but this is the first time that Mn(2+) oxidation is reported for a fungal DyP. Interestingly, Pleos-DyP4 (together with ligninolytic peroxidases) is detected in the secretome of P. ostreatus grown on different lignocellulosic substrates. It is suggested that generation of Mn(3+) oxidizers plays a role in the P. ostreatus white-rot lifestyle since three different families of Mn(2+)-oxidizing peroxidase genes are present in its genome being expressed during lignocellulose degradation. PMID:25967658

  5. [Isolation and purification of Mn-peroxidase from Azospirillum brasilense Sp245].

    PubMed

    Kupriashina, M A; Selivanov, N Iu; Nikitina, V E

    2012-01-01

    Homogenous Mn-peroxidase of a 26-fold purity grade was isolated from a culture of Azospirillum brasilense Sp245 cultivated on a medium containing 0.1 mM pyrocatechol. The molecular weight of the enzyme is 43 kD as revealed by electrophoresis in SDS-PAAG. It was shown that the use of pyrocatechol and 2,2'-azino-bis(3-ethylbenzotiazoline-6-sulfonate) at concentrations of 0.1 and I mM as inductors increased the Mn-peroxidase activity by a factor of 3. PMID:22567881

  6. Purification, crystallization and preliminary crystallographic analysis of peroxidase from the palm tree Chamaerops excelsa.

    PubMed

    Textor, Larissa C; Santos, Jademilson C; Cuadrado, Nazaret Hidalgo; Roig, Manuel G; Zhadan, Galina G; Shnyrov, Valery L; Polikarpov, Igor

    2011-12-01

    Plant peroxidases are presently used extensively in a wide range of biotechnological applications owing to their high environmental and thermal stability. As part of efforts towards the discovery of appealing new biotechnological enzymes, the peroxidase from leaves of the palm tree Chamaerops excelsa (CEP) was extracted, purified and crystallized in its native form. An X-ray diffraction data set was collected at a synchrotron source and data analysis showed that the CEP crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 70.2, b = 100.7, c = 132.3 Å. PMID:22139187

  7. 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

  8. Purification, crystallization and preliminary crystallographic analysis of peroxidase from the palm tree Chamaerops excelsa

    PubMed Central

    Textor, Larissa C.; Santos, Jademilson C.; Hidalgo Cuadrado, Nazaret; Roig, Manuel G.; Zhadan, Galina G.; Shnyrov, Valery L.; Polikarpov, Igor

    2011-01-01

    Plant peroxidases are presently used extensively in a wide range of bio­technological applications owing to their high environmental and thermal stability. As part of efforts towards the discovery of appealing new biotechnological enzymes, the peroxidase from leaves of the palm tree Chamaerops excelsa (CEP) was extracted, purified and crystallized in its native form. An X-­ray diffraction data set was collected at a synchrotron source and data analysis showed that the CEP crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 70.2, b = 100.7, c = 132.3 Å. PMID:22139187

  9. A Peroxidase/Dual Oxidase System Modulates Midgut Epithelial Immunity in Anopheles gambiae

    PubMed Central

    Kumar, Sanjeev; Molina-Cruz, Alvaro; Gupta, Lalita; Rodrigues, Janneth; Barillas-Mury, Carolina

    2012-01-01

    Extracellular matrices in diverse biological systems are crosslinked by dityrosine covalent bonds catalyzed by the peroxidase/oxidase system. We show that the Immunomodulatory Peroxidase (IMPer), an enzyme secreted by the mosquito Anopheles gambiae midgut, and dual oxidase (Duox) form a dityrosine network that decreases gut permeability to immune elicitors and protects the microbiota by preventing activation of epithelial immunity. It also provides a suitable environment for malaria parasites to develop within the midgut lumen without inducing nitric oxide synthase expression. Disruption of this barrier results in strong and effective pathogen-specific immune responses. PMID:20223948

  10. A peroxidase/dual oxidase system modulates midgut epithelial immunity in Anopheles gambiae.

    PubMed

    Kumar, Sanjeev; Molina-Cruz, Alvaro; Gupta, Lalita; Rodrigues, Janneth; Barillas-Mury, Carolina

    2010-03-26

    Extracellular matrices in diverse biological systems are cross-linked by dityrosine covalent bonds catalyzed by the peroxidase/oxidase system. We show that a peroxidase, secreted by the Anopheles gambiae midgut, and dual oxidase form a dityrosine network that decreases gut permeability to immune elicitors. This network protects the microbiota by preventing activation of epithelial immunity. It also provides a suitable environment for malaria parasites to develop within the midgut lumen without inducing nitric oxide synthase expression. Disruption of this barrier results in strong and effective pathogen-specific immune responses. PMID:20223948

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

    PubMed

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

    2011-06-15

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

  12. Identification of an orthologous clade of peroxidases that respond to feeding by greenbugs (Schizaphis graminum) in c4 grasses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Knowledge of specific peroxidases that respond to aphid herbivory is limited in C4 grasses, but could provide targets for improving defence against these pests. A sorghum (Sorghum bicolor (L.) Moench) peroxidase (SbPrx-1; Sobic.002G416700) has been previously linked to biotic stress responses, and w...

  13. Induction of peroxidases and superoxide dismutases in transformed embryogenic calli of alfalfa (Medicago sativa L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Activities of peroxidase (POD) and superoxide dismutase (SOD) enzymes were analyzed in non-regenerative transformed embryogenic lines of alfalfa (Medicago sativa L.) carrying wound-inducible oryzacystatin I (OC-I), wound-inducible oryzacystatin I antisense (OC-Ias) or hygromycin phosphotransferase (...

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

    PubMed

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

    2012-06-01

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

  15. MECHANISMS OF THE STIMULATION OF RAT UTERINE PEROXIDASE ACTIVITY BY METHOXYCHLOR

    EPA Science Inventory

    Methoxychlor (MXC), a pro-estrogenic pesticide, has adverse effects on fertility and affects the rat uterus directly via its active metabolite HPTE. terine peroxidase, a marker of estrogen action, was used to probe the mechanisms through which MXC exerts its activity on the uteru...

  16. In planta anthocyanin degradation by a vacuolar class III peroxidase in Brunfelsia calycina flowers.

    PubMed

    Zipor, Gadi; Duarte, Patrícia; Carqueijeiro, Inês; Shahar, Liat; Ovadia, Rinat; Teper-Bamnolker, Paula; Eshel, Dani; Levin, Yishai; Doron-Faigenboim, Adi; Sottomayor, Mariana; Oren-Shamir, Michal

    2015-01-01

    In contrast to detailed knowledge regarding the biosynthesis of anthocyanins, the largest group of plant pigments, little is known about their in planta degradation. It has been suggested that anthocyanin degradation is enzymatically controlled and induced when beneficial to the plant. Here we investigated the enzymatic process in Brunfelsia calycina flowers, as they changed color from purple to white. We characterized the enzymatic process by which B. calycina protein extracts degrade anthocyanins. A candidate peroxidase was partially purified and characterized and its intracellular localization was determined. The transcript sequence of this peroxidase was fully identified. A basic peroxidase, BcPrx01, is responsible for the in planta degradation of anthocyanins in B. calycina flowers. BcPrx01 has the ability to degrade complex anthocyanins, it co-localizes with these pigments in the vacuoles of petals, and both the mRNA and protein levels of BcPrx01 are greatly induced parallel to the degradation of anthocyanins. Both isoelectric focusing (IEF) gel analysis and 3D structure prediction indicated that BcPrx01 is cationic. Identification of BcPrx01 is a significant breakthrough both in the understanding of anthocyanin catabolism in plants and in the field of peroxidases, where such a consistent relationship between expression levels, in planta subcellular localization and activity has seldom been demonstrated. PMID:25256351

  17. Structure of a mitochondrial cytochrome c conformer competent for peroxidase activity

    PubMed Central

    McClelland, Levi J.; Mou, Tung-Chung; Jeakins-Cooley, Margaret E.; Sprang, Stephen R.; Bowler, Bruce E.

    2014-01-01

    At the onset of apoptosis, the peroxidation of cardiolipin at the inner mitochondrial membrane by cytochrome c requires an open coordination site on the heme. We report a 1.45-Å resolution structure of yeast iso-1-cytochrome c with the Met80 heme ligand swung out of the heme crevice and replaced by a water molecule. This conformational change requires modest adjustments to the main chain of the heme crevice loop and is facilitated by a trimethyllysine 72-to-alanine mutation. This mutation also enhances the peroxidase activity of iso-1-cytochrome c. The structure shows a buried water channel capable of facilitating peroxide access to the active site and of moving protons produced during peroxidase activity to the protein surface. Alternate positions of the side chain of Arg38 appear to mediate opening and closing of the buried water channel. In addition, two buried water molecules can adopt alternate positions that change the network of hydrogen bonds in the buried water channel. Taken together, these observations suggest that low and high proton conductivity states may mediate peroxidase function. Comparison of yeast and mammalian cytochrome c sequences, in the context of the steric factors that permit opening of the heme crevice, suggests that higher organisms have evolved to inhibit peroxidase activity, providing a more stringent barrier to the onset of apoptosis. PMID:24760830

  18. Prussian blue nanoparticles as peroxidase mimetics for sensitive colorimetric detection of hydrogen peroxide and glucose.

    PubMed

    Zhang, Weimin; Ma, Diao; Du, Jianxiu

    2014-03-01

    Prussian blue nanoparticles (PB NPs) exhibits an intrinsic peroxidase-like catalytic activity towards the hydrogen peroxide (H2O2)-mediated oxidation of classical peroxidase substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt to produce a colored product. The catalysis follows Michaelis-Menen kinetics and shows strong affinity for H2O2. Using PB NPs as a peroxidase mimetics, a colorimetric method was developed for the detection of 0.05-50.0 μM H2O2, with a detection limit of 0.031 μM. When the catalytic reaction of PB NPs was coupled with the reaction of glucose oxidation catalyzed by glucose oxidase, a sensitive and selective colorimetric method for the detection of glucose was realized. The limit of detection for glucose was determined to be as low as 0.03 μM and the linear range was from 0.1 μM to 50.0 μM. The method was successfully applied to the determination of glucose in human serum. Compared with other nanomaterials-based peroxidase mimetics, PB NPs provides 10-100 times higher sensitivity toward the detection of H2O2 and glucose. The detection platform developed showed great potential applications in varieties of physiological importance substances when merged with appropriate H2O2-producing oxidases. PMID:24468383

  19. Tissue Printing to Visualize Polyphenol Oxidase and Peroxidase in Vegetables, Fruits, and Mushrooms

    ERIC Educational Resources Information Center

    Melberg, Amanda R.; Flurkey, William H.; Inlow, Jennifer K.

    2009-01-01

    A simple tissue-printing procedure to determine the tissue location of the endogenous enzymes polyphenol oxidase and peroxidase in a variety of vegetables, fruits, and mushrooms is described. In tissue printing, cell contents from the surface of a cut section of the tissue are transferred to an adsorptive surface, commonly a nitrocellulose…

  20. SELENIUM CONCENTRATIONS AND GLULATHIONE PEROXIDASE ACTIVITIES IN A POPULATION EXPOSED TO SELENIUM VIA DRINKING WATER

    EPA Science Inventory

    Selenium concentrations in blood, urine, hair, and tap water were determined in samples obtained from individuals exposed to varying amounts of the element via water from home wells. Glutathione peroxidase activities were also determined on the blood samples. Correlations of bloo...

  1. Biochemical characterization of the suberization-associated anionic peroxidase of potato.

    PubMed

    Bernards, M A; Fleming, W D; Llewellyn, D B; Priefer, R; Yang, X; Sabatino, A; Plourde, G L

    1999-09-01

    The anionic peroxidase associated with the suberization response in potato (Solanum tuberosum L.) tubers during wound healing has been purified and partially characterized at the biochemical level. It is a 45-kD, class III (plant secretory) peroxidase that is localized to suberizing tissues and shows a preference for feruloyl (o-methoxyphenol)-substituted substrates (order of substrate preference: feruloyl > caffeoyl > p-coumaryl approximately syringyl) such as those that accumulate in tubers during wound healing. There was little influence on oxidation by side chain derivatization, although hydroxycinnamates were preferred over the corresponding hydroxycinnamyl alcohols. The substrate specificity pattern is consistent with the natural substrate incorporation into potato wound suberin. In contrast, the cationic peroxidase(s) induced in response to wound healing in potato tubers is present in both suberizing and nonsuberizing tissues and does not discriminate between hydroxycinnamates and hydroxycinnamyl alcohols. A synthetic polymer prepared using E-[8-(13)C]ferulic acid, H(2)O(2), and the purified anionic enzyme contained a significant amount of cross-linking through C-8, albeit with retention of unsaturation. PMID:10482668

  2. Graphene-Based Nanomaterials as Efficient Peroxidase Mimetic Catalysts for Biosensing Applications: An Overview.

    PubMed

    Garg, Bhaskar; Bisht, Tanuja; Ling, Yong-Chien

    2015-01-01

    "Artificial enzymes", a term coined by Breslow for enzyme mimics is an exciting and promising branch of biomimetic chemistry aiming to imitate the general and essential principles of natural enzymes using a variety of alternative materials including heterogeneous catalysts. Peroxidase enzymes represent a large family of oxidoreductases that typically catalyze biological reactions with high substrate affinity and specificity under relatively mild conditions and thus offer a wide range of practical applications in many areas of science. The increasing understanding of general principles as well as intrinsic drawbacks such as low operational stability, high cost, difficulty in purification and storage, and sensitivity of catalytic activity towards atmospheric conditions of peroxidases has triggered a dynamic field in nanotechnology, biochemical, and material science that aims at joining the better of three worlds by combining the concept adapted from nature with the processability of catalytically active graphene-based nanomaterials (G-NMs) as excellent peroxidase mimetic catalysts. This comprehensive review discusses an up-to-date synthesis, kinetics, mechanisms, and biosensing applications of a variety of G-NMs that have been explored as promising catalysts to mimic natural peroxidases. PMID:26248071

  3. Oxidation and nitration of mononitrophenols by a DyP-type peroxidase.

    PubMed

    Büttner, Enrico; Ullrich, René; Strittmatter, Eric; Piontek, Klaus; Plattner, Dietmar A; Hofrichter, Martin; Liers, Christiane

    2015-05-15

    Substantial conversion of nitrophenols, typical high-redox potential phenolic substrates, by heme peroxidases has only been reported for lignin peroxidase (LiP) so far. But also a dye-decolorizing peroxidase of Auricularia auricula-judae (AauDyP) was found to be capable of acting on (i) ortho-nitrophenol (oNP), (ii) meta-nitrophenol (mNP) and (iii) para-nitrophenol (pNP). The pH dependency for pNP oxidation showed an optimum at pH 4.5, which is typical for phenol conversion by DyPs and other heme peroxidases. In the case of oNP and pNP conversion, dinitrophenols (2,4-DNP and 2,6-DNP) were identified as products and for pNP additionally p-benzoquinone. Moreover, indications were found for the formation of random polymerization products originating from initially formed phenoxy radical intermediates. Nitration was examined using (15)N-labeled pNP and Na(14)NO2 as an additional source of nitro-groups. Products were identified by HPLC-MS, and mass-to-charge ratios were evaluated to clarify the origin of nitro-groups. The additional nitrogen in DNPs formed during enzymatic conversion was found to originate both from (15)N-pNP and (14)NO2Na. Based on these results, a hypothetical reaction scheme and a catalytically responsible confine of the enzyme's active site are postulated. PMID:25796533

  4. CLONING AND EXPRESSION OF A LIGNIN PEROXIDASE GENE FROM STREPTOMYCES VIRIDOSPORUS IN STREPTOMYCES LIVIDANS

    EPA Science Inventory

    A lignin peroxidase gene was cloned from Streptomyces viridosporus T7A into Streptomyces lividans TK64 in plasmid pIJ702. g1II-digested genomic DNA(4-10kb) of S. viridosporus was shotgun-cloned into S. lividans after insertion into the melanin (mel+) gene of pIJ702. ransformants ...

  5. Extension of polyphenolics by CWPO-C peroxidase mutant containing radical-robust surface active site.

    PubMed

    Pham, L T Mai; Kim, S Jin; Ahn, U Suk; Choi, J Weon; Song, B Keun; Kim, Y Hwan

    2014-01-01

    Expressed as insoluble forms in Escherichia coli, native cationic cell wall peroxidase (CWPO-C) from the poplar tree and mutant variants were successfully reactivated via refolding experiments and used to elucidate the previously presumed existence of an electron transfer (ET) pathway in the CWPO-C structure. Their catalytic properties were fully characterized through various analyses including steady-state kinetic, direct oxidation of lignin macromolecules and their respective stabilities during the polymerization reactions. The analysis results proved that the 74th residue on the CWPO-C surface plays an important role in catalyzing the macromolecules via supposed ET mechanism. By comparing the residual activities of wild-type CWPO-C and mutant 74W CWPO-C after 3 min, mutation of tyrosine 74 residue to tryptophan increased the radical resistance of peroxidase up to ten times dramatically while maintaining its capability to oxidize lignin macromolecules. Furthermore, extension of poly(catechin) as well as lignin macromolecules with CWPO-C Y74W mutant clearly showed that this radical-resistant peroxidase mutant can increase the molecular weight of various kinds of polyphenolics by using surface-located active site. The anti-oxidation activity of the synthesized poly(catechin) was confirmed by xanthine oxidase assay. The elucidation of a uniquely catalytic mechanism in CWPO-C may improve the applicability of the peroxidase/H2O2 catalyst to green polymer chemistry. PMID:24122664

  6. Glutathione peroxidase 4 (Gpx4) and ferroptosis: what's so special about it?

    PubMed Central

    Conrad, Marcus; Friedmann Angeli, José Pedro

    2015-01-01

    The system XC−/glutathione/glutathione peroxidase 4 (Gpx4) axis pivotally controls ferroptosis, a recently described form of regulated non-apoptotic cell death. Compelling evidence has established that this route of cell death is not only of high relevance for triggering cancer cell death, but also proves to be amenable for therapeutic intervention to halt ischemia/reperfusion-related diseases. PMID:27308484

  7. Peroxidase as the Major Protein Constituent in Areca Nut and Identification of Its Natural Substrates

    PubMed Central

    Liu, Yu-Ching; Chen, Chao-Jung; Lee, Miau-Rong; Li, Mi; Hsieh, Wen-Tsong; Chung, Jing-Gung; Ho, Heng-Chien

    2013-01-01

    Numerous reports illustrate the diverse effects of chewing the areca nut, most of which are harmful and have been shown to be associated with oral cancer. Nearly all of the studies are focused on the extract and/or low molecular weight ingredients in the areca nut. The purpose of this report is to identify the major protein component in the areca nut. After ammonium sulfate fractionation, the concentrated areca nut extract is subjected to DEAE-cellulose chromatography. A colored protein is eluted at low NaCl concentration and the apparently homogeneous eluent represents the major protein component compared to the areca nut extract. The colored protein shares partial sequence identity with the royal palm tree peroxidase and its peroxidase activity is confirmed using an established assay. In the study, the natural substrates of areca nut peroxidase are identified as catechin, epicatechin, and procyanidin B1. The two former substrates are similarly oxidized to form a 576 Da product with concomitant removal of four hydrogen atoms. Interestingly, oxidation of procyanidin B1 occurs only in the presence of catechin or epicatechin and an additional product with an 864 Da molecular mass. In addition, procyanidin B1 is identified as a peroxidase substrate for the first time. PMID:24250715

  8. Peroxidase activity as an indicator of exposure of wetland seedlings to metals

    SciTech Connect

    Sutton, H.D.; Klaine, S.J.

    1995-12-31

    The enzyme peroxidase has been found to increase quantitatively in several aquatic plant species in response to increasing exposure to various contaminants. In this study, a number of wetland species are tested for their usefulness as bioindicators of metal exposure using the peroxidase assay. Woody species tested include Liquidambar styraciflua (sweetgum), Fraxinus pennsylvanica (green ash), and Cephalanthus occidentalis (buttonbush), while herbaceous species include Saururus cernuus (lizard`s tail) and Sparganium americanum (bur-reed). The assay has been optimized for all of these species. In all cases the pH optimum has been found to be either 5.5 or 6.0 and the substrate optimum is 2.8 or 1.4mM hydrogen peroxide. There is considerable variation in baseline peroxidase activity among the species when tested under their optimal assay conditions. These species are being dosed with copper, nickel, and cadmium in order to determine whether a response elicited. Seedlings will be dosed using both petri dish culture conditions and test tubes filled with vermiculite and sand combinations. The peroxidase response will be compared to germination and root elongation endpoints. Lettuce (Lactuca saliva) and radish (Raphanus sativus) are being tested alongside the wetland species as reference organisms for which background data is available. The wetland species tested in the present study have rarely if ever been used in toxicological studies.

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

    PubMed Central

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

    1975-01-01

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

  10. Identification and Properties of Insect Resistance-Associated Maize Anionic Peroxidases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous studies with transgenic plants have indicated a tobacco anionic peroxidase can confer enhanced resistance to a variety of insects when expressed in different plant species. Tissue that expresses high levels of this enzyme often browns rapidly when damaged. When introduced biolistically, m...

  11. Aqueous synthesis of porous platinum nanotubes at room temperature and their intrinsic peroxidase-like activity.

    PubMed

    Cai, Kai; Lv, Zhicheng; Chen, Kun; Huang, Liang; Wang, Jing; Shao, Feng; Wang, Yanjun; Han, Heyou

    2013-07-11

    Platinum nanotubes (PtNTs) exhibiting high porosity were constructed by sacrificing the exterior of tellurium nanowires (TeNWs) and disintegrating the inner part spontaneously in aqueous solution at room temperature, in which the Kirkendall effect may play an important role. The present PtNTs exhibited intrinsic peroxidase-like activity in the presence of H2O2. PMID:23598712

  12. Salicylic acid-induced superoxide generation catalyzed by plant peroxidase in hydrogen peroxide-independent manner

    PubMed Central

    Kimura, Makoto; Kawano, Tomonori

    2015-01-01

    It has been reported that salicylic acid (SA) induces both immediate spike and long lasting phases of oxidative burst represented by the generation of reactive oxygen species (ROS) such as superoxide anion radical (O2•−). In general, in the earlier phase of oxidative burst, apoplastic peroxidase are likely involved and in the late phase of the oxidative burst, NADPH oxidase is likely involved. Key signaling events connecting the 2 phases of oxidative burst are calcium channel activation and protein phosphorylation events. To date, the known earliest signaling event in response to exogenously added SA is the cell wall peroxidase-catalyzed generation of O2•− in a hydrogen peroxide (H2O2)-dependent manner. However, this model is incomplete since the source of the initially required H2O2 could not be explained. Based on the recently proposed role for H2O2-independent mechanism for ROS production catalyzed by plant peroxidases (Kimura et al., 2014, Frontiers in Plant Science), we hereby propose a novel model for plant peroxidase-catalyzed oxidative burst fueled by SA. PMID:26633563

  13. 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

  14. 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...

  15. Role of anionic isoforms of peroxidase during phytopathogenic infection of plants from the family Malvaceae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An increase of the activity and the appearance of new isoforms (high- and low-molecular-weight) in the isospectrum of peroxidase were noted after the action of the phytopathogen Verticillium dahliae on plants from the family Malvaceae. The temperature was shown to have an effect on the resistance to...

  16. Thyrotropin-dependence of the distribution of peroxidase in rat thyroid gland.

    PubMed

    Matsukawa, S; Mihara, S; Hosoya, T

    1981-04-01

    After male rats were injected daily with propylthiouracil, whale thyrotropin, or thyroxine for several days, the thyroid glands were examined for their ultrastructural localization of peroxidase. The propylthiouracil treatment caused a marked increase in the number of round, peroxidase-positive vesicles in the apical and middle regions of cytoplasm, and brought about a formation of granular reaction products in the colloid lumen adjacent to the elongated microvilli. Such changes became evident after 2 days of the drug treatment and increased with the time of the effect, until the whole colloid lumen was filled with the reaction products and very elongated microvilli. The effect of exogenous thyrotropin was essentially similar, to a lesser degree, to the drug effect mentioned above. The thyroxine treatment diminished the population of the small vesicles, as usually seen in the case of normal rat thyroids. When the thyroids of thyrotropin-stimulated and unstimulated rats were homogenized and fractionated into particulates fractions and a soluble fraction, almost all the peroxidase activity was contained in the former in both cases. Based on these results, the dynamics and the physiological role of the intracellular peroxidase are discussed. PMID:7256734

  17. Abolishing activity against ascorbate in a cytosolic ascorbate peroxidase from switchgrass

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Switchgrass (Panicum virgatum L.) is being developed as a bioenergy species. Recently an early version of its genome has been released permitting a route to the cloning and analysis of key proteins. Ascorbate peroxidases (APx) are an important part of the antioxidant defense system of plant cells a...

  18. Effects of commercial selenium products on glutathione peroxidase activity and semen quality in stud boars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this study was to determine how dietary supplementation of inorganic and organic selenium affects selenium concentration and glutathione peroxidase activity in blood and sperm of sexually mature stud boars. Twenty-four boars of the Large White, Landrace, Pietrain, and Duroc breeds of opt...

  19. A Peroxidase-linked Spectrophotometric Assay for the Detection of Monoamine Oxidase Inhibitors.

    PubMed

    Zhi, Kangkang; Yang, Zhongduo; Sheng, Jie; Shu, Zongmei; Shi, Yin

    2016-01-01

    To develop a new more accurate spectrophotometric method for detecting monoamine oxidase inhibitors from plant extracts, a series of amine substrates were selected and their ability to be oxidized by monoamine oxidase was evaluated by the HPLC method and a new substrate was used to develop a peroxidase-linked spectrophotometric assay. 4-(Trifluoromethyl) benzylamine (11) was proved to be an excellent substrate for peroxidase-linked spectrophotometric assay. Therefore, a new peroxidase-linked spectrophotometric assay was set up. The principle of the method is that the MAO converts 11 into aldehyde, ammonia and hydrogen peroxide. In the presence of peroxidase, the hydrogen peroxide will oxidize 4-aminoantipyrine into oxidised 4-aminoantipyrine which can condense with vanillic acid to give a red quinoneimine dye. The production of the quinoneimine dye was detected at 490 nm by a microplate reader. The ⊿OD value between the blank group and blank negative control group in this new method is twice as much as that in Holt's method, which enables the procedure to be more accurate and avoids the produce of false positive results. The new method will be helpful for researchers to screening monoamine oxidase inhibitors from deep-color plant extracts. PMID:27610153

  20. Correlation between Glutathione Peroxidase Activity and Anthropometrical Parameters in Adolescents with Down Syndrome

    ERIC Educational Resources Information Center

    Ordonez, F. J.; Rosety-Rodriguez, M.

    2007-01-01

    Since we have recently found that regular exercise increased erythrocyte antioxidant enzyme activities such as glutathione peroxidase (GPX) in adolescents with Down syndrome, these programs may be recommended. This study was designed to assess the role of anthropometrical parameters as easy, economic and non-invasive biomarkers of GPX. Thirty-one…

  1. A Peroxidase-linked Spectrophotometric Assay for the Detection of Monoamine Oxidase Inhibitors

    PubMed Central

    Zhi, Kangkang; Yang, Zhongduo; Sheng, Jie; Shu, Zongmei; Shi, Yin

    2016-01-01

    To develop a new more accurate spectrophotometric method for detecting monoamine oxidase inhibitors from plant extracts, a series of amine substrates were selected and their ability to be oxidized by monoamine oxidase was evaluated by the HPLC method and a new substrate was used to develop a peroxidase-linked spectrophotometric assay. 4-(Trifluoromethyl) benzylamine (11) was proved to be an excellent substrate for peroxidase-linked spectrophotometric assay. Therefore, a new peroxidase-linked spectrophotometric assay was set up. The principle of the method is that the MAO converts 11 into aldehyde, ammonia and hydrogen peroxide. In the presence of peroxidase, the hydrogen peroxide will oxidize 4-aminoantipyrine into oxidised 4-aminoantipyrine which can condense with vanillic acid to give a red quinoneimine dye. The production of the quinoneimine dye was detected at 490 nm by a microplate reader. The ⊿OD value between the blank group and blank negative control group in this new method is twice as much as that in Holt’s method, which enables the procedure to be more accurate and avoids the produce of false positive results. The new method will be helpful for researchers to screening monoamine oxidase inhibitors from deep-color plant extracts. PMID:27610153

  2. Oxidation of cinnamyl alcohols and aldehydes by a basic peroxidase from lignifying Zinnia elegans hypocotyls.

    PubMed

    Barceló, A R; Pomar, F

    2001-08-01

    The xylem of 26-day old Zinnia elegans hypocotyls synthesizes lignins derived from coniferyl alcohol and sinapyl alcohol with a G/S ratio of 43/57 in the aryl-glycerol-beta-aryl ether core, as revealed by thioacidolysis. Thioacidolysis of Z. elegans lignins also reveals the presence of coniferyl aldehyde end groups linked by beta-0-4 bonds. Both coniferyl and sinapyl alcohols, as well as coniferyl and sinapyl aldehyde, are substrates of a xylem cell wall-located strongly basic peroxidase, which is capable of oxidizing them in the absence and in the presence of hydrogen peroxide. This peroxidase shows a particular affinity for cinnamyl aldehydes with kappa(M) values in the mu(M) range, and some specificity for syringyl-type phenols. The affinity of this strongly basic peroxidase for cinnamyl alcohols and aldehydes is similar to that shown by the preceding enzymes in the lignin biosynthetic pathway (microsomal 5-hydroxylases and cinnamyl alcohol dehydrogenase), which also use cinnamyl alcohols and aldehydes as substrates, indicating that the one-way highway of construction of the lignin macromolecule has no metabolic "potholes" in which the lignin building blocks might accumulate. This fact suggests a high degree of metabolic plasticity for this basic peroxidase, which has been widely conserved during the evolution of vascular plants, making it one of the driving forces in the evolution of plant lignin heterogeneity. PMID:11430983

  3. Reducing odorous VOC emissions from swine manure using soybean peroxidase and peroxides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Air emissions from swine production facilities can cause odor nuisance issues. Peroxidase enzymes have been used to treat phenolic compounds in industrial wastewaters, but little is known about their efficacy for treating swine manure. The objective of the research was to determine the optimum app...

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

    PubMed

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

    2003-07-01

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

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

    SciTech Connect

    Lagrimini, L.M.

    1994-05-01

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

  6. Adaptive aneuploidy protects against thiol peroxidase deficiency by increasing respiration via key mitochondrial proteins.

    PubMed

    Kaya, Alaattin; Gerashchenko, Maxim V; Seim, Inge; Labarre, Jean; Toledano, Michel B; Gladyshev, Vadim N

    2015-08-25

    Aerobic respiration is a fundamental energy-generating process; however, there is cost associated with living in an oxygen-rich environment, because partially reduced oxygen species can damage cellular components. Organisms evolved enzymes that alleviate this damage and protect the intracellular milieu, most notably thiol peroxidases, which are abundant and conserved enzymes that mediate hydrogen peroxide signaling and act as the first line of defense against oxidants in nearly all living organisms. Deletion of all eight thiol peroxidase genes in yeast (∆8 strain) is not lethal, but results in slow growth and a high mutation rate. Here we characterized mechanisms that allow yeast cells to survive under conditions of thiol peroxidase deficiency. Two independent ∆8 strains increased mitochondrial content, altered mitochondrial distribution, and became dependent on respiration for growth but they were not hypersensitive to H2O2. In addition, both strains independently acquired a second copy of chromosome XI and increased expression of genes encoded by it. Survival of ∆8 cells was dependent on mitochondrial cytochrome-c peroxidase (CCP1) and UTH1, present on chromosome XI. Coexpression of these genes in ∆8 cells led to the elimination of the extra copy of chromosome XI and improved cell growth, whereas deletion of either gene was lethal. Thus, thiol peroxidase deficiency requires dosage compensation of CCP1 and UTH1 via chromosome XI aneuploidy, wherein these proteins support hydroperoxide removal with the reducing equivalents generated by the electron transport chain. To our knowledge, this is the first evidence of adaptive aneuploidy counteracting oxidative stress. PMID:26261310

  7. Purification and kinetic characterization of two peroxidases of Selaginella martensii Spring. involved in lignification.

    PubMed

    Martínez-Cortés, Teresa; Pomar, Federico; Espiñeira, José Manuel; Merino, Fuencisla; Novo-Uzal, Esther

    2012-03-01

    Two cationic peroxidases from Selaginella martensii Spring. (SmaPrx2 and SmaPrx3) were purified using a three-step protocol which includes ammonium sulfate precipitation, adsorption chromatography on phenyl sepharose and cationic exchange chromatography on SP sepharose. The molecular mass for SmaPrx2 and SmaPrx3 was calculated to be 36.3 kDa and 45.6 kDa, respectively, according to MALDI-TOF/TOF. The isoelectric points were estimated in 9.2 and 9.5 for SmaPrx2 and SmaPrx3, respectively, according to isoelectrofocusing. Both enzymes show a typical peroxidase UV-visible spectrum with a Soret peak at 403 nm for SmaPrx2 and 404 nm for SmaPrx3. The specific activities showed against several substrates and the kinetic parameters suggest SmaPrx2 and SmaPrx3 have specific roles in cell wall formation and especially in lignin biosynthesis. Several peptides from tryptic digestion of both peroxidases were identified through MALDI-TOF MS/MS. The presence in these peptides of structural determinants typical of syringyl peroxidases indicates these proteins show no structural restrictions to oxidize syringyl moieties. These data, along with the in vitro capacity of using sinapyl alcohol as substrate and the low K(m) in the μM range suggest these two peroxidases may be responsible for the oxidation of syringyl monolignols that leads to syringyl lignins biosynthesis. PMID:22305076

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    1992-01-01

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

  10. The molecular characterization of the lignin-forming peroxidase. Progress summary report, April 1, 1992--March 31, 1995

    SciTech Connect

    Lagrimini, L.M.

    1995-06-01

    My research program focuses entirely on the study of the lignin-forming peroxidase of tobacco. Ever since our cloning and sequencing of the first plant peroxidase cDNA, we have pioneered in the introduction of the tools of molecular biology to the study of plant peroxidases. A significant part of our effort has been focused on the construction and analysis of transgenic plants which either over- or under-express the tobacco anionic peroxidase. This research has not only supported the role for this enzyme in lignification, but has opened the door to our understanding of additional metabolic functions including auxin metabolism and insect defense. As you will learn, this enzyme`s role in auxin catabolism has lead to numerous phenotypes in transgenic plants. More recently, our attention has been directed towards the analysis of peroxidase gene expression. From this work we have learned that the anionic peroxidase gene is expressed at high levels in the xylem-forming cells, epidermis, and trichomes. This expression pattern supports its role lignification and hose defenses. We have also learned that this gene is down-regulated by auxin which indicates a strong relationship between auxin and the anionic peroxidase. 12 figs.

  11. Identification and structural characterization of heme binding in a novel dye-decolorizing peroxidase, TyrA.

    PubMed

    Zubieta, Chloe; Joseph, Rosanne; Krishna, S Sri; McMullan, Daniel; Kapoor, Mili; Axelrod, Herbert L; Miller, Mitchell D; Abdubek, Polat; Acosta, Claire; Astakhova, Tamara; Carlton, Dennis; Chiu, Hsiu-Ju; Clayton, Thomas; Deller, Marc C; Duan, Lian; Elias, Ylva; Elsliger, Marc-André; Feuerhelm, Julie; Grzechnik, Slawomir K; Hale, Joanna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K; Klock, Heath E; Knuth, Mark W; Kozbial, Piotr; Kumar, Abhinav; Marciano, David; Morse, Andrew T; Murphy, Kevin D; Nigoghossian, Edward; Okach, Linda; Oommachen, Silvya; Reyes, Ron; Rife, Christopher L; Schimmel, Paul; Trout, Christina V; van den Bedem, Henry; Weekes, Dana; White, Aprilfawn; Xu, Qingping; Hodgson, Keith O; Wooley, John; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

    2007-11-01

    TyrA is a member of the dye-decolorizing peroxidase (DyP) family, a new family of heme-dependent peroxidase recently identified in fungi and bacteria. Here, we report the crystal structure of TyrA in complex with iron protoporphyrin (IX) at 2.3 A. TyrA is a dimer, with each monomer exhibiting a two-domain, alpha/beta ferredoxin-like fold. Both domains contribute to the heme-binding site. Co-crystallization in the presence of an excess of iron protoporphyrin (IX) chloride allowed for the unambiguous location of the active site and the specific residues involved in heme binding. The structure reveals a Fe-His-Asp triad essential for heme positioning, as well as a novel conformation of one of the heme propionate moieties compared to plant peroxidases. Structural comparison to the canonical DyP family member, DyP from Thanatephorus cucumeris (Dec 1), demonstrates conservation of this novel heme conformation, as well as residues important for heme binding. Structural comparisons with representative members from all classes of the plant, bacterial, and fungal peroxidase superfamily demonstrate that TyrA, and by extension the DyP family, adopts a fold different from all other structurally characterized heme peroxidases. We propose that a new superfamily be added to the peroxidase classification scheme to encompass the DyP family of heme peroxidases. PMID:17654547

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

    PubMed

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

    2015-11-01

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

  13. The molecular characterization of the lignin-forming peroxidase. Progress summary report, April 1, 1989--March 31, 1992

    SciTech Connect

    Lagrimini, L.M.

    1992-04-01

    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.

  14. Identification And Structural Characterization of Heme Binding in a Novel Dye-Decolorizing Peroxidase, TyrA

    SciTech Connect

    Zubieta, C.; Joseph, R.; Krishna, S.S.; McMullan, D.; Kapoor, M.; Axelrod, H.L.; Miller, M.D.; Abdubek, P.; Acosta, C.; Astakhova, T.; Carlton, D.; Chiu, H.J.; Clayton, T.; Deller, M.C.; Duan, L.; Elias, Y.; Elsliger, M.A.; Feuerhelm, J.; Grzechnik, S.K.; Hale, J.; Han, G.W.; /JCSG /SLAC, SSRL /Burnham Inst. Med. Res. /UC, San Diego /Novartis Res. Found. /Scripps Res. Inst.

    2007-10-31

    TyrA is a member of the dye-decolorizing peroxidase (DyP) family, a new family of heme-dependent peroxidase recently identified in fungi and bacteria. Here, we report the crystal structure of TyrA in complex with iron protoporphyrin (IX) at 2.3 Angstroms. TyrA is a dimer, with each monomer exhibiting a two-domain, {alpha}/{beta} ferredoxin-like fold. Both domains contribute to the heme-binding site. Co-crystallization in the presence of an excess of iron protoporphyrin (IX) chloride allowed for the unambiguous location of the active site and the specific residues involved in heme binding. The structure reveals a Fe-His-Asp triad essential for heme positioning, as well as a novel conformation of one of the heme propionate moieties compared to plant peroxidases. Structural comparison to the canonical DyP family member, DyP from Thanatephorus cucumeris (Dec 1), demonstrates conservation of this novel heme conformation, as well as residues important for heme binding. Structural comparisons with representative members from all classes of the plant, bacterial, and fungal peroxidase superfamily demonstrate that TyrA, and by extension the DyP family, adopts a fold different from all other structurally characterized heme peroxidases. We propose that a new superfamily be added to the peroxidase classification scheme to encompass the DyP family of heme peroxidases.

  15. Identification and Comparative Analysis of H2O2-Scavenging Enzymes (Ascorbate Peroxidase and Glutathione Peroxidase) in Selected Plants Employing Bioinformatics Approaches

    PubMed Central

    Ozyigit, Ibrahim I.; Filiz, Ertugrul; Vatansever, Recep; Kurtoglu, Kuaybe Y.; Koc, Ibrahim; Öztürk, Münir X.; Anjum, Naser A.

    2016-01-01

    Among major reactive oxygen species (ROS), hydrogen peroxide (H2O2) exhibits dual roles in plant metabolism. Low levels of H2O2 modulate many biological/physiological processes in plants; whereas, its high level can cause damage to cell structures, having severe consequences. Thus, steady-state level of cellular H2O2 must be tightly regulated. Glutathione peroxidases (GPX) and ascorbate peroxidase (APX) are two major ROS-scavenging enzymes which catalyze the reduction of H2O2 in order to prevent potential H2O2-derived cellular damage. Employing bioinformatics approaches, this study presents a comparative evaluation of both GPX and APX in 18 different plant species, and provides valuable insights into the nature and complex regulation of these enzymes. Herein, (a) potential GPX and APX genes/proteins from 18 different plant species were identified, (b) their exon/intron organization were analyzed, (c) detailed information about their physicochemical properties were provided, (d) conserved motif signatures of GPX and APX were identified, (e) their phylogenetic trees and 3D models were constructed, (f) protein-protein interaction networks were generated, and finally (g) GPX and APX gene expression profiles were analyzed. Study outcomes enlightened GPX and APX as major H2O2-scavenging enzymes at their structural and functional levels, which could be used in future studies in the current direction. PMID:27047498

  16. Identification and Comparative Analysis of H2O2-Scavenging Enzymes (Ascorbate Peroxidase and Glutathione Peroxidase) in Selected Plants Employing Bioinformatics Approaches.

    PubMed

    Ozyigit, Ibrahim I; Filiz, Ertugrul; Vatansever, Recep; Kurtoglu, Kuaybe Y; Koc, Ibrahim; Öztürk, Münir X; Anjum, Naser A

    2016-01-01

    Among major reactive oxygen species (ROS), hydrogen peroxide (H2O2) exhibits dual roles in plant metabolism. Low levels of H2O2 modulate many biological/physiological processes in plants; whereas, its high level can cause damage to cell structures, having severe consequences. Thus, steady-state level of cellular H2O2 must be tightly regulated. Glutathione peroxidases (GPX) and ascorbate peroxidase (APX) are two major ROS-scavenging enzymes which catalyze the reduction of H2O2 in order to prevent potential H2O2-derived cellular damage. Employing bioinformatics approaches, this study presents a comparative evaluation of both GPX and APX in 18 different plant species, and provides valuable insights into the nature and complex regulation of these enzymes. Herein, (a) potential GPX and APX genes/proteins from 18 different plant species were identified, (b) their exon/intron organization were analyzed, (c) detailed information about their physicochemical properties were provided, (d) conserved motif signatures of GPX and APX were identified, (e) their phylogenetic trees and 3D models were constructed, (f) protein-protein interaction networks were generated, and finally (g) GPX and APX gene expression profiles were analyzed. Study outcomes enlightened GPX and APX as major H2O2-scavenging enzymes at their structural and functional levels, which could be used in future studies in the current direction. PMID:27047498

  17. Selenium-enriched Agaricus bisporus increases expression and activity of glutathione peroxidase-1 and expression of glutathione peroxidase-2 in rat colon.

    PubMed

    Maseko, Tebo; Howell, Kate; Dunshea, Frank R; Ng, Ken

    2014-03-01

    The effect of dietary supplementation with Se-enriched Agaricus bisporus on cytosolic gluthathione peroxidase-1 (GPx-1), gastrointestinal specific glutathione peroxidase-2 (GPx-2), thioredoxin reductase-1 (TrxR-1) and selenoprotein P (SeP) mRNA expression and GPx-1 enzyme activity in rat colon was examined. Rats were fed for 5weeks with control diet (0.15μg Se/g feed) or Se-enriched diet fortified with selenised mushroom (1μg Se/g feed). The mRNA expression levels were found to be significantly (P<0.01) up-regulated by 1.65-fold and 2.3-fold for GPx-1 and GPx-2, respectively, but were not significantly different for TrxR-1 and SeP between the 2 diet treatments. The up-regulation of GPx-1 mRNA expression was consistent with GPX-1 activity level, which was significantly (P<0.05) increased by 1.77-fold in rats fed with the Se-enriched diet compared to the control diet. The results showed that selenised A. bisporus can positively increase GPx-1 and GPx-2 gene expression and GPx-1 enzyme activity in rat colon. PMID:24176350

  18. MEASUREMENT OF SUPEROXIDE DISMUTASE, CATALASE, AND GLUTATHIONE PEROXIDASE IN CULTURED CELLS AND TISSUE

    PubMed Central

    Weydert, Christine J.; Cullen, Joseph J.

    2010-01-01

    Cells contain a large number of antioxidants to prevent or repair the damage caused by ROS, as well as to regulate redox-sensitive signaling pathways General protocols are described to measure the antioxidant enzyme activity of superoxide dismutase (SOD), catalase, and glutathione peroxidase. The SODs convert superoxide radical into hydrogen peroxide and molecular oxygen, while the catalase and peroxidases convert hydrogen peroxide into water. In this way, two toxic species, superoxide radical and hydrogen peroxide, are converted to the harmless product water. Western blots, activity gels and activity assays are various methods used to determine protein and activity in both cells and tissue depending on the amount of protein needed for each assay. Other techniques including immunohistochemistry and immunogold can further evaluate the levels of the various antioxidant enzymes in tissue and cells. In general, these assays require 24 to 48 hours to complete. PMID:20057381

  19. Visual detection of blood glucose based on peroxidase-like activity of WS2 nanosheets.

    PubMed

    Lin, Tianran; Zhong, Liangshuang; Song, Zhiping; Guo, Liangqia; Wu, Hanyin; Guo, Qingquan; Chen, Ying; Fu, FengFu; Chen, Guonan

    2014-12-15

    Tungsten disulfide (WS2) nanosheets were discovered to possess intrinsic peroxidase-like activity and catalyze the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) to produce a color reaction in the presence of H2O2. Based on this finding, a colorimetric method and a portable test kit for the visual detection of blood glucose have been developed by using glucose oxidase (GOx) and WS2 nanosheets-catalyzed reactions. The linear range for glucose was ranged from 5 to 300 μM (R(2)=0.999) with the detection limit of 2.9 μM. The portable test kit was successfully evaluated glucose levels in serum samples from normal persons and diabetes persons by the observable color change from pale yellow to yellow-green, blue-green. PMID:25032681

  20. The overexpression of AtPrx37, an apoplastic peroxidase, reduces growth in Arabidopsis.

    PubMed

    Pedreira, Jorge; Herrera, María Teresa; Zarra, Ignacio; Revilla, Gloria

    2011-02-01

    Understanding peroxidase function in plants is difficult because of the lack of substrate specificity, the high number of genes and their diversity in structure. In the present study, the relative expression of 22 genes coding putative peroxidases (E.C 1.11.1.x) in Arabidopsis was studied. The relative expression of AtPrx37 showed a correlation with the cessation of growth in rosette leaves as well as with the growth capacity along the flower stem. Using AtPrx37::GUS construction, its expression was associated with the vascular bundles. Furthermore, the overexpression of AtPrx37 under the control of CaMV 35S promoter rendered a dwarf phenotype with smaller plants and delayed development. The plants overexpressing AtPrx37 also showed an increase in the amount of esterified phenolic material associated with their walls. A role in the growth cessation and phenolic cross-linking during lignin deposition is postulated. PMID:21044085

  1. Graphite-like carbon nitrides as peroxidase mimetics and their applications to glucose detection.

    PubMed

    Lin, Tianran; Zhong, Liangshuang; Wang, Jing; Guo, Liangqia; Wu, Hanyin; Guo, Qingquan; Fu, FengFu; Chen, Guonan

    2014-09-15

    g-C3N4 was found to possess intrinsic peroxidase-like activity, and could catalytically oxidize 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 to produce a color reaction. Using g-C3N4 peroxidase-like catalytic activity and glucose oxidase (GOx), a colorimetric method for glucose detection in serum samples has been developed. The linear range for glucose was from 5 to 100 μM (R(2)=0.9987) and the limit of detection was as low as 1.0 μM. The proposed method was applied to detect glucose in serum samples by the naked eyes. PMID:24704762

  2. Activation of glutathione peroxidase via Nrf1 mediates genistein's protection against oxidative endothelial cell injury

    SciTech Connect

    Hernandez-Montes, Eva; Pollard, Susan E.; Vauzour, David; Jofre-Montseny, Laia; Rota, Cristina; Rimbach, Gerald; Weinberg, Peter D.; Spencer, Jeremy P.E. . E-mail: j.p.e.spencer@reading.ac.uk

    2006-08-04

    Cellular actions of isoflavones may mediate the beneficial health effects associated with high soy consumption. We have investigated protection by genistein and daidzein against oxidative stress-induced endothelial injury. Genistein but not daidzein protected endothelial cells from damage induced by oxidative stress. This protection was accompanied by decreases in intracellular glutathione levels that could be explained by the generation of glutathionyl conjugates of the oxidised genistein metabolite, 5,7,3',4'-tetrahydroxyisoflavone. Both isoflavones evoked increased protein expression of {gamma}-glutamylcysteine synthetase-heavy subunit ({gamma}-GCS-HS) and increased cytosolic accumulation and nuclear translocation of Nrf2. However, only genistein led to increases in the cytosolic accumulation and nuclear translocation of Nrf1 and the increased expression of and activity of glutathione peroxidase. These results suggest that genistein-induced protective effects depend primarily on the activation of glutathione peroxidase mediated by Nrf1 activation, and not on Nrf2 activation or increases in glutathione synthesis.

  3. [Isolation, identification of lignin-degrading bacteria and purification of lignin peroxidase].

    PubMed

    Yang, Jin-shui; Liu, Wei; Ni, Jin-ren

    2006-05-01

    Two strains that could use lignin as sole carbon source and excrete peroxidases were isolated from activated sludge. Both strains are belonged to Pseudomanas based on morphological, physio-biochenical characterizatics and homology identification of 16S rDNA sequence, in which strain PKE117 is identified as a new species, while the strain PKE225 is identified as Pseudomanas thermaerum. Crude enzyme in liquid fermentation of PKE117 was analyzed with DEAE-cellulose 32 ion-exchanger resin chromatography and Sephadex G-75 gel-filtration in proper order. The lignin peroxidase specific activity increases from 0.87 U/mg to 204.5 U/mg, purification multiple is 235.1 and callback rate is 15%. PMID:16850845

  4. Glutathione peroxidase in yeast. Presence of the enzyme and induction by oxidative conditions.

    PubMed

    Galiazzo, F; Schiesser, A; Rotilio, G

    1987-09-30

    The presence of glutathione peroxidase activity is reported for the first time for a wild type strain of Saccharomyces cerevisiae. Both forms of enzyme, i.e. that specifically active toward H2O2 alone and that decomposing also organic peroxides, were found to be present. The H2O2 specific form disappeared when cells were grown in the absence of oxygen, while the other form was much increased under the same conditions. Addition of copper to the culture greatly increased both forms. The results show that glutathione peroxidase is to be included, as an important component that is also highly responsive to oxidative environments, in the enzyme defense system of yeast against oxidative damage. PMID:3311044

  5. Peroxidase-mediated Biodegradation of Carbon Nanotubes in vitro and in vivo

    PubMed Central

    Kotchey, Gregg P.; Zhao, Yong; Kagan, Valerian E.; Star, Alexander

    2013-01-01

    As a result of their unique electronic, optical, and mechanical properties, carbon nanotubes (CNTs) have been implemented in therapeutic and imaging applications. In an idealized situation, CNTs would be disposed of after they transport their theranostic payloads. Biodegradation represents an attractive pathway for the eliminating of CNT carriers post-delivery and may be integral in catalyzing the release of the cargo from the delivery vehicle. Accordingly, recent research efforts have focused on peroxidase-driven biodegradation of CNTs. In this review, we not only summarize recent efforts to biodegrade CNTs in the test tube, in vitro, and in vivo, but also attempt to explore the fundamental parameters underlying degradation. Encouraged by the in vivo results obtained to date, we envision a future, where carbon-based nano-containers, which are specifically designed to target organs/cells, deliver their cargo, and biodegrade via peroxidase-driven mechanism, will represent an attractive therapeutic delivery option in nanomedicine. PMID:23856412

  6. Detection of the halogenating activity of heme peroxidases in leukocytes by aminophenyl fluorescein.

    PubMed

    Flemmig, J; Remmler, J; Zschaler, J; Arnhold, J

    2015-06-01

    The formation of hypochlorous and hypobromous acids by heme peroxidases is a key property of certain immune cells. These products are not only involved in defense against pathogenic microorganisms and in regulation of inflammatory processes, but contribute also to tissue damage in certain pathologies. After a short introduction about experimental approaches for the assessment of the halogenating activity in vitro and in cell suspensions, we are focusing on novel applications of fluorescent dye systems to detect the formation of hypochlorous acid (HOCl) in leukocytes. Special attention is directed to properties and applications of the non-fluorescent dye aminophenyl fluorescein that is converted by HOCl, HOBr, and other strong oxidants to fluorescein. This dye allows the detection of the halogenating activity in samples containing free myeloperoxidase and eosinophil peroxidase as well as in intact granulocytes using fluorescence spectroscopy and flow cytometry, respectively. PMID:25536418

  7. A mitochondria-targeted inhibitor of cytochrome c peroxidase mitigates radiation induced death

    PubMed Central

    Atkinson, Jeffrey; Kapralov, Alexandr A.; Yanamala, Naveena; Tyurina, Yulia Y.; Amoscato, Andrew A.; Pearce, Linda; Peterson, Jim; Huang, Zhentai; Jiang, Jianfei; Samhan-Arias, Alejandro K.; Maeda, Akihiro; Feng, Weihong; Wasserloos, Karla; Belikova, Natalia A.; Tyurin, Vladimir A.; Wang, Hong; Fletcher, Jackie; Wang, Yongsheng; Vlasova, Irina I.; Klein-Seetharaman, Judith; Stoyanovsky, Detcho A.; Bayîr, Hülya; Pitt, Bruce R.; Epperly, Michael W.; Greenberger, Joel S.; Kagan, Valerian E.

    2013-01-01

    The risk of radionuclide release in terrorist acts or exposure of healthy tissue during radiotherapy demand potent radioprotectants/radiomitigators. Ionizing radiation induces cell death by initiating the selective peroxidation of cardiolipin in mitochondria by the peroxidase activity of its complex with cytochrome c leading to release of hemoprotein into the cytosol and commitment to the apoptotic program. Here we design and synthesize mitochondria-targeted triphenylphosphonium-conjugated imidazole-substituted oleic and stearic acids which blocked peroxidase activity of cytochrome c/cardiolipin complex by specifically binding to its heme-iron. We show that both compounds inhibit pro-apoptotic oxidative events, suppress cyt c release, prevent cell death, and protect mice against lethal doses of irradiation. Significant radioprotective/radiomitigative effects of imidazole-substituted oleic acid are observed after pretreatment of mice from 1 hr before through 24 hrs after the irradiation. PMID:21988913

  8. A mitochondria-targeted inhibitor of cytochrome c peroxidase mitigates radiation-induced death.

    PubMed

    Atkinson, Jeffrey; Kapralov, Alexandr A; Yanamala, Naveena; Tyurina, Yulia Y; Amoscato, Andrew A; Pearce, Linda; Peterson, Jim; Huang, Zhentai; Jiang, Jianfei; Samhan-Arias, Alejandro K; Maeda, Akihiro; Feng, Weihong; Wasserloos, Karla; Belikova, Natalia A; Tyurin, Vladimir A; Wang, Hong; Fletcher, Jackie; Wang, Yongsheng; Vlasova, Irina I; Klein-Seetharaman, Judith; Stoyanovsky, Detcho A; Bayîr, Hülya; Pitt, Bruce R; Epperly, Michael W; Greenberger, Joel S; Kagan, Valerian E

    2011-01-01

    The risk of radionuclide release in terrorist acts or exposure of healthy tissue during radiotherapy demand potent radioprotectants/radiomitigators. Ionizing radiation induces cell death by initiating the selective peroxidation of cardiolipin in mitochondria by the peroxidase activity of its complex with cytochrome c leading to release of haemoprotein into the cytosol and commitment to the apoptotic program. Here we design and synthesize mitochondria-targeted triphenylphosphonium-conjugated imidazole-substituted oleic and stearic acids that blocked peroxidase activity of cytochrome c/cardiolipin complex by specifically binding to its haem-iron. We show that both compounds inhibit pro-apoptotic oxidative events, suppress cyt c release, prevent cell death, and protect mice against lethal doses of irradiation. Significant radioprotective/radiomitigative effects of imidazole-substituted oleic acid are observed after pretreatment of mice from 1 h before through 24 h after the irradiation. PMID:21988913

  9. In vitro depolymerization of lignin by manganese peroxidase of Phanerochaete chrysosporium

    SciTech Connect

    Wariishi, H.; Valli, K.; Gold, M.H. )

    1991-04-15

    Homogeneous manganese peroxidase catalyzed the in vitro partial depolymerization of four different {sup 14}C-labeled synthetic lignin preparations. Gel permeation profiles demonstrated significant depolymerization of {sup 14}C-sidechain-labeled syringyl lignin, a {sup 14}C-sidechain-labeled syringyl-guaiacyl copolymer (angiosperm lignin), and depolymerization of {sup 14}C-sidechain- and {sup 14}C-ring-labeled guaiacyl lignins (gymnosperm lignin). 3,5-Dimethoxy-1,4-benzo-quinone, 3,5-dimethoxy-1,4-hydroquinone, and syringylaldehyde were identified as degradation products of the syringyl and syringyl-guaiacyl lignins. These results suggest that manganese peroxidase plays a significant role in the depolymerization of lignin by Phanerochaete chrysosporium.

  10. Peroxidase-mediated biodegradation of carbon nanotubes in vitro and in vivo.

    PubMed

    Kotchey, Gregg P; Zhao, Yong; Kagan, Valerian E; Star, Alexander

    2013-12-01

    As a result of their unique electronic, optical, and mechanical properties, carbon nanotubes (CNTs) have been implemented in therapeutic and imaging applications. In an idealized situation, CNTs would be disposed of after they transport their theranostic payloads. Biodegradation represents an attractive pathway for the elimination of CNT carriers post-delivery and may be integral in catalyzing the release of the cargo from the delivery vehicle. Accordingly, recent research efforts have focused on peroxidase-driven biodegradation of CNTs. In this review, we not only summarize recent efforts to biodegrade CNTs in the test tube, in vitro, and in vivo, but also attempt to explore the fundamental parameters underlying degradation. Encouraged by the in vivo results obtained to date, we envision a future, where carbon-based nano-containers, which are specifically designed to target organs/cells, deliver their cargo, and biodegrade via peroxidase-driven mechanism, will represent an attractive therapeutic delivery option in nanomedicine. PMID:23856412

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

    PubMed

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

    2000-06-13

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

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

    PubMed

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

    2000-01-01

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

  13. 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

  14. Peroxidase-Catalyzed Oxidative Coupling of Phenols in the Presence of Geosorbents

    SciTech Connect

    Huang, Qingguo; Weber, Walter J., Jr.

    2003-03-26

    This study focuses on elucidation of the reaction behaviors of peroxidase-mediated phenol coupling in the presence of soil/sediment materials. Our goal is a mechanistic understanding of the influences of geosorbent materials on enzymatic coupling reactions in general and the development of methods for predicting such influences. Extensive experimental investigations of coupling reactions were performed under strategically selected conditions in systems containing model geosorbents having different properties and chemical characteristics. The geosorbents tested were found to influence peroxidase-mediated phenol coupling through one or both of two principal mechanisms; i.e., (1) mitigation of enzyme inactivation and/or (2) participation in cross-coupling reactions. Such influences were found to correlate with the chemical characteristics of the sorbent materials and to be simulated well by a modeling approach designed in this paper. The results of the study have important implications for potential engineering implementation and enhancement of enzymatic coupling reactions in soil/subsurface remediation practice.

  15. Determining inhibition effects of some aromatic compounds on peroxidase enzyme purified from white and red cabbage

    NASA Astrophysics Data System (ADS)

    Öztekin, Aykut; Almaz, Züleyha; Özdemir, Hasan

    2016-04-01

    Peroxidases (E.C.1.11.1.7) catalyze the one electron oxidation of wide range of substrates. They are used in synthesis reaction, removal of peroxide from industrial wastes, clinical biochemistry and immunoassays. In this study, the white cabbage (Brassica Oleracea var. capitata f. alba) and red cabbage (Brassica oleracea L. var. capitata f. rubra) peroxidase enzymes were purified for investigation of inhibitory effect of some aromatic compounds on these enzymes. IC50 values and Ki constants were calculated for the molecules of 6-Amino nicotinic hydrazide, 6-Amino-5-bromo nicotinic hydrazide, 2-Amino-5-hydroxy benzohydrazide, 4-Amino-3-hydroxy benzohydrazide on purified enzymes and inhibition type of these molecules were determined. (This research was supported by Ataturk University. Project Number: BAP-2015/98).

  16. Emerging pollutants and plants--Metabolic activation of diclofenac by peroxidases.

    PubMed

    Huber, Christian; Preis, Martina; Harvey, Patricia J; Grosse, Sylvia; Letzel, Thomas; Schröder, Peter

    2016-03-01

    Human pharmaceuticals and their residues are constantly detected in our waterbodies, due to poor elimination rates, even in the most advanced waste water treatment plants. Their impact on the environment and human health still remains unclear. When phytoremediation is applied to aid water treatment, plants may transform and degrade xenobiotic contaminants through phase I and phase II metabolism to more water soluble and less toxic intermediates. In this context, peroxidases play a major role in activating compounds during phase I via oxidation. In the present work, the ability of a plant peroxidase to oxidize the human painkiller diclofenac was confirmed using stopped flow spectroscopy in combination with LC-MS analysis. Analysis of an orange colored product revealed the structure of the highly reactive Diclofenac-2,5-Iminoquinone, which may be the precursor of several biological conjugates and breakdown products in planta. PMID:26741549

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

    PubMed

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

    2015-09-01

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

  18. Glutathione peroxidase's reaction intermediate selenenic acid is stabilized by the protein microenvironment.

    PubMed

    Li, Fei; Liu, Jun; Rozovsky, Sharon

    2014-11-01

    Selenenic acids are highly reactive intermediates of selenoproteins' enzymatic reactions. Knowledge of how the protein environment protects and stabilizes them is fundamental not only to descriptions of selenoproteins' reactivity but also potentially for proteomics and therapeutics. However, selenenic acids are considered particularly short-lived and are not yet identified in wild-type selenoproteins. Here, we report trapping the selenenic acid in glutathione peroxidase, an antioxidant enzyme that efficiently eliminates hydroperoxides. It has long been thought that selenium-containing glutathione peroxidases form a selenenic acid intermediate. However, this putative species has eluded detection. Here, we report its identification. The selenenic acid in bovine glutathione peroxidase 1 was chemically trapped using dimedone, an alkylating agent specific to sulfenic and selenenic acids. The alkylation of the catalytic selenocysteine was verified by electrospray ionization mass spectrometry. In the presence of glutathione, the selenocysteine was not alkylated because the selenenic acid condenses faster with glutathione than the alkylation reaction. In the absence of thiols, the selenenic acid was surprisingly long-lived with 95% of the protein still able to react with dimedone 10 min after hydrogen peroxide was removed, indicating that the protein environment stabilizes the selenenic acid by shielding it from reactive groups in the protein. After 30 min, the selenocysteine was no longer modified but became accessible once the protein was exposed to reducing agents. This suggests that the selenenic acid reacted with a protein's amide or amine to form a selenylamide bond. Such a modification may play a role in protecting glutathione peroxidase׳' reactivity. PMID:25124921

  19. Bioinformatic and functional characterization of the basic peroxidase 72 from Arabidopsis thaliana involved in lignin biosynthesis.

    PubMed

    Herrero, Joaquín; Fernández-Pérez, Francisco; Yebra, Tatiana; Novo-Uzal, Esther; Pomar, Federico; Pedreño, Ma Ángeles; Cuello, Juan; Guéra, Alfredo; Esteban-Carrasco, Alberto; Zapata, José Miguel

    2013-06-01

    Lignins result from the oxidative polymerization of three hydroxycinnamyl (p-coumaryl, coniferyl, and sinapyl) alcohols in a reaction mediated by peroxidases. The most important of these is the cationic peroxidase from Zinnia elegans (ZePrx), an enzyme considered to be responsible for the last step of lignification in this plant. Bibliographical evidence indicates that the arabidopsis peroxidase 72 (AtPrx72), which is homolog to ZePrx, could have an important role in lignification. For this reason, we performed a bioinformatic, histochemical, photosynthetic, and phenotypical and lignin composition analysis of an arabidopsis knock-out mutant of AtPrx72 with the aim of characterizing the effects that occurred due to the absence of expression of this peroxidase from the aspects of plant physiology such as vascular development, lignification, and photosynthesis. In silico analyses indicated a high homology between AtPrx72 and ZePrx, cell wall localization and probably optimal levels of translation of AtPrx72. The histochemical study revealed a low content in syringyl units and a decrease in the amount of lignin in the atprx72 mutant plants compared to WT. The atprx72 mutant plants grew more slowly than WT plants, with both smaller rosette and principal stem, and with fewer branches and siliques than the WT plants. Lastly, chlorophyll a fluorescence revealed a significant decrease in ΦPSII and q L in atprx72 mutant plants that could be related to changes in carbon partitioning and/or utilization of redox equivalents in arabidopsis metabolism. The results suggest an important role of AtPrx72 in lignin biosynthesis. In addition, knock-out plants were able to respond and adapt to an insufficiency of lignification. PMID:23508663

  20. Gut Microbiota Conversion of Dietary Ellagic Acid into Bioactive Phytoceutical Urolithin A Inhibits Heme Peroxidases

    PubMed Central

    Saha, Piu; Yeoh, Beng San; Singh, Rajbir; Chandrasekar, Bhargavi; Vemula, Praveen Kumar; Haribabu, Bodduluri; Vijay-Kumar, Matam; Jala, Venkatakrishna R.

    2016-01-01

    Numerous studies signify that diets rich in phytochemicals offer many beneficial functions specifically during pathologic conditions, yet their effects are often not uniform due to inter-individual variation. The host indigenous gut microbiota and their modifications of dietary phytochemicals have emerged as factors that greatly influence the efficacy of phytoceutical-based intervention. Here, we investigated the biological activities of one such active microbial metabolite, Urolithin A (UA or 3,8-dihydroxybenzo[c]chromen-6-one), which is derived from the ellagic acid (EA). Our study demonstrates that UA potently inhibits heme peroxidases i.e. myeloperoxidase (MPO) and lactoperoxidase (LPO) when compared to the parent compound EA. In addition, chrome azurol S (CAS) assay suggests that EA, but not UA, is capable of binding to Fe3+, due to its catechol-like structure, although its modest heme peroxidase inhibitory activity is abrogated upon Fe3+-binding. Interestingly, UA-mediated MPO and LPO inhibition can be prevented by innate immune protein human NGAL or its murine ortholog lipocalin 2 (Lcn2), implying the complex nature of host innate immunity-microbiota interactions. Spectral analysis indicates that UA inhibits heme peroxidase-catalyzed reaction by reverting the peroxidase back to its inactive native state. In support of these in vitro results, UA significantly reduced phorbol myristate acetate (PMA)-induced superoxide generation in neutrophils, however, EA failed to block the superoxide generation. Treatment with UA significantly reduced PMA-induced mouse ear edema and MPO activity compared to EA treated mice. Collectively, our results demonstrate that microbiota-mediated conversion of EA to UA is advantageous to both host and microbiota i.e. UA-mediated inhibition of pro-oxidant enzymes reduce tissue inflammation, mitigate non-specific killing of gut bacteria, and abrogate iron-binding property of EA, thus providing a competitive edge to the microbiota in

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

    PubMed

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

    2012-01-15

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

  2. Improving the Oxidative Stability of a High Redox Potential Fungal Peroxidase by Rational Design

    PubMed Central

    Sáez-Jiménez, Verónica; Acebes, Sandra; Guallar, Victor; Martínez, Angel T.; Ruiz-Dueñas, Francisco J.

    2015-01-01

    Ligninolytic peroxidases are enzymes of biotechnological interest due to their ability to oxidize high redox potential aromatic compounds, including the recalcitrant lignin polymer. However, different obstacles prevent their use in industrial and environmental applications, including low stability towards their natural oxidizing-substrate H2O2. In this work, versatile peroxidase was taken as a model ligninolytic peroxidase, its oxidative inactivation by H2O2 was studied and different strategies were evaluated with the aim of improving H2O2 stability. Oxidation of the methionine residues was produced during enzyme inactivation by H2O2 excess. Substitution of these residues, located near the heme cofactor and the catalytic tryptophan, rendered a variant with a 7.8-fold decreased oxidative inactivation rate. A second strategy consisted in mutating two residues (Thr45 and Ile103) near the catalytic distal histidine with the aim of modifying the reactivity of the enzyme with H2O2. The T45A/I103T variant showed a 2.9-fold slower reaction rate with H2O2 and 2.8-fold enhanced oxidative stability. Finally, both strategies were combined in the T45A/I103T/M152F/M262F/M265L variant, whose stability in the presence of H2O2 was improved 11.7-fold. This variant showed an increased half-life, over 30 min compared with 3.4 min of the native enzyme, under an excess of 2000 equivalents of H2O2. Interestingly, the stability improvement achieved was related with slower formation, subsequent stabilization and slower bleaching of the enzyme Compound III, a peroxidase intermediate that is not part of the catalytic cycle and leads to the inactivation of the enzyme. PMID:25923713

  3. Characterization of structure and activity of garlic peroxidase (POX(1B)).

    PubMed

    El Ichi, Sarra; Miodek, Anna; Sauriat-Dorizon, Hélène; Mahy, Jean-Pierre; Henry, Céline; Marzouki, Mohamed Nejib; Korri-Youssoufi, Hafsa

    2011-01-01

    Structural characterization and study of the activity of new POX(1B) protein from garlic which has a high peroxidase activity and can be used as a biosensor for the detection of hydrogen peroxide and phenolic compounds were performed and compared with the findings for other heme peroxidases. The structure-function relationship was investigated by analysis of the spectroscopic properties and correlated to the structure determined by a new generation of high-performance hybrid mass spectrometers. The reactivity of the enzyme was analyzed by studies of the redox activity toward various ligands and the reactivity with various substrates. We demonstrated that, in the case of garlic peroxidase, the heme group is pentacoordinated, and has an histidine as a proximal ligand. POX(1B) exhibited a high affinity for hydrogen peroxide as well as various reducing cosubstrates. In addition, high enzyme specificity was demonstrated. The k(cat) and K(M) values were 411 and 400 mM(-1) s(-1) for 3,3',5,5'-tetramethylbenzidine and 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), respectively. Furthermore, the reduction of nitro compounds in the presence of POX(1B) was demonstrated by iron(II) nitrosoalkane complex assay. In addition, POX(1B) showed a great potential for application for drug metabolism since its ability to react with 1-nitrohexane in the presence of sodium dithionite was demonstrated by the appearance of a characteristic Soret band at 411 nm. The high catalytic efficiency obtained in the case of the new garlic peroxidase (POX(1B)) is suitable for the monitoring of different analytes and biocatalysis. PMID:21042820

  4. Direct electrochemical detection of kanamycin based on peroxidase-like activity of gold nanoparticles.

    PubMed

    Wang, Chunshuai; Liu, Chang; Luo, Jibao; Tian, Yaping; Zhou, Nandi

    2016-09-14

    An enzyme-free, ultrasensitive electrochemical detection of kanamycin residue was achieved based on mimetic peroxidase activity of gold nanoparticles (AuNPs) and target-induced replacement of the aptamer. AuNPs which were synthesized using tyrosine as a reducing and capping agent, exhibited mimetic peroxidase activity. In the presence of kanamycin-specific aptamer, however, the single-stranded DNA (ssDNA) adsorbed on the surface of AuNPs via the interaction between the bases of ssDNA and AuNPs, and therefore blocked the catalytic site of AuNPs, and inhibited their peroxidase activity. While in the presence of target kanamycin, it bound with the adsorbed aptamer on AuNPs with high affinity, exposed the surface of AuNPs and recovered the peroxidase activity. Then AuNPs catalyzed the reaction between H2O2 and reduced thionine to produce oxidized thionine. The latter exhibited a distinct reduction peak on gold electrode in differential pulse voltammetry (DPV), and could be utilized to quantify the concentration of kanamycin. Under the optimized conditions, the proposed electrochemical assay showed an extremely high sensitivity towards kanamycin, with a linear relationship between the peak current and the concentration of kanamycin in the range of 0.1-60 nM, and a detection limit of 0.06 nM. Moreover, the established approach was successfully applied in the detection of kanamycin in honey samples. Therefore, the proposed electrochemical assay has great potential in the fields of food quality control and environmental monitoring. PMID:27566341

  5. Gut Microbiota Conversion of Dietary Ellagic Acid into Bioactive Phytoceutical Urolithin A Inhibits Heme Peroxidases.

    PubMed

    Saha, Piu; Yeoh, Beng San; Singh, Rajbir; Chandrasekar, Bhargavi; Vemula, Praveen Kumar; Haribabu, Bodduluri; Vijay-Kumar, Matam; Jala, Venkatakrishna R

    2016-01-01

    Numerous studies signify that diets rich in phytochemicals offer many beneficial functions specifically during pathologic conditions, yet their effects are often not uniform due to inter-individual variation. The host indigenous gut microbiota and their modifications of dietary phytochemicals have emerged as factors that greatly influence the efficacy of phytoceutical-based intervention. Here, we investigated the biological activities of one such active microbial metabolite, Urolithin A (UA or 3,8-dihydroxybenzo[c]chromen-6-one), which is derived from the ellagic acid (EA). Our study demonstrates that UA potently inhibits heme peroxidases i.e. myeloperoxidase (MPO) and lactoperoxidase (LPO) when compared to the parent compound EA. In addition, chrome azurol S (CAS) assay suggests that EA, but not UA, is capable of binding to Fe3+, due to its catechol-like structure, although its modest heme peroxidase inhibitory activity is abrogated upon Fe3+-binding. Interestingly, UA-mediated MPO and LPO inhibition can be prevented by innate immune protein human NGAL or its murine ortholog lipocalin 2 (Lcn2), implying the complex nature of host innate immunity-microbiota interactions. Spectral analysis indicates that UA inhibits heme peroxidase-catalyzed reaction by reverting the peroxidase back to its inactive native state. In support of these in vitro results, UA significantly reduced phorbol myristate acetate (PMA)-induced superoxide generation in neutrophils, however, EA failed to block the superoxide generation. Treatment with UA significantly reduced PMA-induced mouse ear edema and MPO activity compared to EA treated mice. Collectively, our results demonstrate that microbiota-mediated conversion of EA to UA is advantageous to both host and microbiota i.e. UA-mediated inhibition of pro-oxidant enzymes reduce tissue inflammation, mitigate non-specific killing of gut bacteria, and abrogate iron-binding property of EA, thus providing a competitive edge to the microbiota in

  6. Improving the oxidative stability of a high redox potential fungal peroxidase by rational design.

    PubMed

    Sáez-Jiménez, Verónica; Acebes, Sandra; Guallar, Victor; Martínez, Angel T; Ruiz-Dueñas, Francisco J

    2015-01-01

    Ligninolytic peroxidases are enzymes of biotechnological interest due to their ability to oxidize high redox potential aromatic compounds, including the recalcitrant lignin polymer. However, different obstacles prevent their use in industrial and environmental applications, including low stability towards their natural oxidizing-substrate H2O2. In this work, versatile peroxidase was taken as a model ligninolytic peroxidase, its oxidative inactivation by H2O2 was studied and different strategies were evaluated with the aim of improving H2O2 stability. Oxidation of the methionine residues was produced during enzyme inactivation by H2O2 excess. Substitution of these residues, located near the heme cofactor and the catalytic tryptophan, rendered a variant with a 7.8-fold decreased oxidative inactivation rate. A second strategy consisted in mutating two residues (Thr45 and Ile103) near the catalytic distal histidine with the aim of modifying the reactivity of the enzyme with H2O2. The T45A/I103T variant showed a 2.9-fold slower reaction rate with H2O2 and 2.8-fold enhanced oxidative stability. Finally, both strategies were combined in the T45A/I103T/M152F/M262F/M265L variant, whose stability in the presence of H2O2 was improved 11.7-fold. This variant showed an increased half-life, over 30 min compared with 3.4 min of the native enzyme, under an excess of 2000 equivalents of H2O2. Interestingly, the stability improvement achieved was related with slower formation, subsequent stabilization and slower bleaching of the enzyme Compound III, a peroxidase intermediate that is not part of the catalytic cycle and leads to the inactivation of the enzyme. PMID:25923713

  7. Interference by acetaminophen in the glucose oxidase-peroxidase method for blood glucose determination.

    PubMed

    Kaufmann-Raab, I; Jonen, H G; Jähnchen, E; Kahl, G F; Groth, U

    1976-10-01

    Acetaminophen, p-aminophenol, and oxyphenbutazone interfere with the glucose oxidase/peroxidase method for glucose. Structurally related compounds that lack a free phenolic hydroxyl group (acetanilide, aniline, and phenylbutazone) do not interfere. During the analytical procedure acetaminophen is consumed. One mole of acetaminophen leads to an apparent loss of four moles of glucose. The hexokinase/glucose-6-phosphate dehydrogenase method (Boehringer Hexokinase method) is not affected by these substances. PMID:975521

  8. Serum Malondialdehyde Concentration and Glutathione Peroxidase Activity in a Longitudinal Study of Gestational Diabetes

    PubMed Central

    Miranda, María; Muriach, María; Romero, Francisco J.; Villar, Vincent M.

    2016-01-01

    Aims The main goal of this study was to evaluate the presence of oxidative damage and to quantify its level in gestational diabetes. Methods Thirty-six healthy women and thirty-six women with gestational diabetes were studied in the three trimesters of pregnancy regarding their levels of oxidative stress markers. These women were diagnosed with diabetes in the second trimester of pregnancy. Blood glucose levels after 100g glucose tolerance test were higher than 190, 165 or 145 mg/dl, 1, 2 or 3 hours after glucose intake. Results The group of women with gestational diabetes had higher serum malondialdehyde levels, with significant differences between groups in the first and second trimester. The mean values of serum glutathione peroxidase activity in the diabetic women were significantly lower in the first trimester. In the group of women with gestational diabetes there was a negative linear correlation between serum malondialdehyde concentration and glutathione peroxidase activity in the second and third trimester. Conclusions In this observational and longitudinal study in pregnant women, the alterations attributable to oxidative stress were present before the biochemical detection of the HbA1c increase. Usual recommendations once GD is detected (adequate metabolic control, as well as any other normally proposed to these patients) lowered the concentration of malondialdehyde at the end of pregnancy to the same levels of the healthy controls. Serum glutathione peroxidase activity in women with gestational diabetes increased during the gestational period. PMID:27228087

  9. Colorimetric detection of Shewanella oneidensis based on immunomagnetic capture and bacterial intrinsic peroxidase activity

    NASA Astrophysics Data System (ADS)

    Wen, Junlin; Zhou, Shungui; Chen, Junhua

    2014-06-01

    Rapid detection and enumeration of target microorganisms is considered as a powerful tool for monitoring bioremediation process that typically involves cleaning up polluted environments with functional microbes. A novel colorimetric assay is presented based on immunomagnetic capture and bacterial intrinsic peroxidase activity for rapidly detecting Shewanella oneidensis, an important model organism for environmental bioremediation because of its remarkably diverse respiratory abilities. Analyte bacteria captured on the immunomagnetic beads provided a bacterial out-membrane peroxidase-amplified colorimetric readout of the immunorecognition event by oxidizing 3, 3', 5, 5'-tetramethylbenzidine (TMB) in the present of hydrogen peroxide. The high-efficiency of immunomagnetic capture and signal amplification of peroxidase activity offers an excellent detection performance with a wide dynamic range between 5.0 × 103 and 5.0 × 106 CFU/mL toward target cells. Furthermore, this method was demonstrated to be feasible in detecting S. oneidensis cells spiked in environmental samples. The proposed colorimetric assay shows promising environmental applications for rapid detection of target microorganisms.

  10. Immobilization of peroxidase enzyme onto the porous silicon structure for enhancing its activity and stability

    NASA Astrophysics Data System (ADS)

    Sahare, Padmavati; Ayala, Marcela; Vazquez-Duhalt, Rafael; Agrawal, Vivechana

    2014-08-01

    In this work, a commercial peroxidase was immobilized onto porous silicon (PS) support functionalized with 3-aminopropyldiethoxysilane (APDES) and the performance of the obtained catalytic microreactor was studied. The immobilization steps were monitored and the activity of the immobilized enzyme in the PS pores was spectrophotometrically determined. The enzyme immobilization in porous silicon has demonstrated its potential as highly efficient enzymatic reactor. The effect of a polar organic solvent (acetonitrile) and the temperature (up to 50°C) on the activity and stability of the biocatalytic microreactor were studied. After 2-h incubation in organic solvent, the microreactor retained 80% of its initial activity in contrast to the system with free soluble peroxidase that lost 95% of its activity in the same period of time. Peroxidase immobilized into the spaces of the porous silicon support would be perspective for applications in treatments for environmental security such as removal of leached dye in textile industry or in treatment of different industrial effluents. The system can be also applied in the field of biomedicine.

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

    PubMed

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

    2013-01-31

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

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

    PubMed

    Liu, Mei; Li, Baoxin; Cui, Xiang

    2013-10-15

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

  13. Characterization of a purified decolorizing detergent-stable peroxidase from Streptomyces griseosporeus SN9.

    PubMed

    Rekik, Hatem; Nadia, Zaraî Jaouadi; Bejar, Wacim; Kourdali, Sidali; Belhoul, Mouna; Hmidi, Maher; Benkiar, Amina; Badis, Abdelmalek; Sallem, Naim; Bejar, Samir; Jaouadi, Bassem

    2015-02-01

    A novel extracellular lignin peroxidase (called LiP-SN) was produced and purified from a newly isolated Streptomyces griseosporeus strain SN9. The findings revealed that the pure enzyme was a monomeric protein with an estimated molecular mass of 43 kDa and a Reinheitzahl value of 1.63. The 19 N-terminal residue sequence of LiP-SN showed high homology with those of Streptomyces peroxidases. Its optimum pH and temperature were pH 8.5 and 65 °C, respectively. The enzyme was inhibited by sodium azide and potassium cyanide, suggesting the presence of heme components in its tertiary structure. Its catalytic efficiency was higher than that of the peroxidase from Streptomyces albidoflavus strain TN644. Interestingly, LiP-SN showed marked dye-decolorization efficiency and stability toward denaturing, oxidizing, and bleaching agents, and compatibility with EcoVax and Dipex as laundry detergents for 48 h at 40 °C. These properties make LiP-SN a potential candidate for future applications in distaining synthetic dyes and detergent formulations. PMID:25478960

  14. katGI and katGII encode two different catalases-peroxidases in Mycobacterium fortuitum.

    PubMed

    Menéndez, M C; Ainsa, J A; Martín, C; García, M J

    1997-11-01

    It has been suggested that catalase-peroxidase plays an important role in several aspects of mycobacterial metabolism and is a virulence factor in the main pathogenic mycobacteria. In this investigation, we studied genes encoding for this protein in the fast-growing opportunistic pathogen Mycobacterium fortuitum. Nucleotide sequences of two different catalase-peroxidase genes (katGI and katGII) of M. fortuitum are described. They show only 64% homology at the nucleotide level and 55% identity at the amino acid level, and they are more similar to catalases-peroxidases from different bacteria, including mycobacteria, than to each other. Both proteins were found to be expressed in actively growing M. fortuitum, and both could also be expressed when transformed into Escherichia coli and M. aurum. We detected the presence of a copy of IS6100 in the neighboring region of a katG gene in the M. fortuitum strain in which this element was identified (strain FC1). The influence of each katG gene on isoniazid (isonicotinic acid hydrazide; INH) susceptibility of mycobacteria was checked by using the INH-sensitive M. aurum as the host. Resistance to INH was induced when katGI was transformed into INH-sensitive M. aurum, suggesting that this enzyme contributes to the natural resistance of M. fortuitum to the drug. This is the first report showing two different genes encoding same enzyme activity which are actively expressed within the same mycobacterial strain. PMID:9371430

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

    PubMed

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

    1997-12-12

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

  16. Electrochemical Evidence for Multiple Peroxidatic Heme States of the Diheme Cytochrome c Peroxidase of Pseudomonas aeruginosa†

    PubMed Central

    Becker, Clinton F.; Watmough, Nicholas J.; Elliott, Sean J.

    2009-01-01

    The enzyme cytochrome c peroxidase from Pseudomonas aeruginosa and its catalytic mechanism was investigated using protein film voltammetry. Monolayers of the diheme bacterial enzyme were immobilized on both pyrolytic graphite edge and alkanethiol modified Au electrodes. The redox couple associated with the low potential heme could be detected on both electrode surfaces at a reduction potential of −234 mV vs SHE. The midpoint potential displays a distinct pH dependence at acidic pH values, indicative of proton-coupled electron transfer. The non-turnover signal of the LP heme can be transformed into sigmoidal waves upon the addition of substrate. The midpoint potential of the turnover signals were used to calculate Michaelis-Menten kinetics with a Km = 25 μM. Catalysis was inhibited with addition of cyanide (Ki = 50 μM). These kinetic parameters are in good agreement with previously reported solution-based studies, indicated that the activity of the enzyme is unaffected by the immobilization on the electrode surface. The reduction potential of the catalytic wave clearly shows that rate-limiting species during electrocatalysis differs from those previously reported for peroxidases, indicating that PFV may be used in the future to distinguish the requirement for reductive activation in bacterial cytochrome c peroxidases. PMID:19072039

  17. [Characterization of peroxidase-catalyzed oxidation of chromogenic substrates by tetrazole and its 5-substituted derivatives].

    PubMed

    Karaseva, E I; Gaponik, P N; Metelitsa, D I

    2005-01-01

    Peroxidase-catalyzed oxidation of 2,2-azino-di(3-ethyl-benzthiazolydine-6-sulfonic acid) (ABTS) and 3,3',5,5'-tetramethylbenzidine (TMB) is activated by tetrazole and its 5-substituted derivatives--5-amino-(AmT), 5-methyl- (MeT), 5-phenyl- (PhT), and 5-CF3- (CF3-T) tetrazoles. In phosphate-citrate or phosphate buffer (pH 6.4 or 7.2; 20 degrees C), the activating effect of tetrazoles on TMB and ABTS oxidation decreased in the series AmT > MeT > T > PhT > CF3-T and T > AmT > MeT > PhT, respectively. The (coefficient) degree of activation (alpha), expressed in M(-1), determined for both substrates and all activators, depended on substrate type, buffer nature, and pH (it increased as pH increased from 6.4 to 7.2). For TMB oxidation, good correlation between lgalpha and the Hammet constants sigma(meta) for m-substituents in the benzene series NH2, CH3, C6H5, and CF3 was found. It is suggested that AmT, MeT, and T can be used as activators of peroxidase-catalyzed oxidation of TMB and ABTS, as well as in designing peroxidase-based biosensors. PMID:15859456

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

    PubMed

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

    1992-08-01

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

  19. Colorimetric detection of Shewanella oneidensis based on immunomagnetic capture and bacterial intrinsic peroxidase activity

    PubMed Central

    Wen, Junlin; Zhou, Shungui; Chen, Junhua

    2014-01-01

    Rapid detection and enumeration of target microorganisms is considered as a powerful tool for monitoring bioremediation process that typically involves cleaning up polluted environments with functional microbes. A novel colorimetric assay is presented based on immunomagnetic capture and bacterial intrinsic peroxidase activity for rapidly detecting Shewanella oneidensis, an important model organism for environmental bioremediation because of its remarkably diverse respiratory abilities. Analyte bacteria captured on the immunomagnetic beads provided a bacterial out-membrane peroxidase-amplified colorimetric readout of the immunorecognition event by oxidizing 3, 3′, 5, 5′-tetramethylbenzidine (TMB) in the present of hydrogen peroxide. The high-efficiency of immunomagnetic capture and signal amplification of peroxidase activity offers an excellent detection performance with a wide dynamic range between 5.0 × 103 and 5.0 × 106 CFU/mL toward target cells. Furthermore, this method was demonstrated to be feasible in detecting S. oneidensis cells spiked in environmental samples. The proposed colorimetric assay shows promising environmental applications for rapid detection of target microorganisms. PMID:24898751

  20. Quinone-mediated microbial synthesis of reduced graphene oxide with peroxidase-like activity.

    PubMed

    Liu, Guangfei; Zhang, Xin; Zhou, Jiti; Wang, Aijie; Wang, Jing; Jin, Ruofei; Lv, Hong

    2013-12-01

    The effects of different quinones on graphene oxide (GO) reduction by Shewanella oneidensis MR-1 and the peroxidase activity of the resultant reduced graphene oxide (QRGO) were studied. The presence of 100 μM anthraquinone-2-sulfonate (AQS), anthraquinone-2,6-disulfonate and 5-hydroxy-1,4-naphthoquinone could lead to 1.6-2.8-fold increase in GO reduction rate, whereas anthraquinone-2-carboxylate slowed down the reduction. The stimulating effects of AQS increased with the increase of its concentration (10-100 μM). The mediated effects were proved by direct GO reduction by microbially reduced AQS. The mediated reduction of GO to QRGO was characterized by UV-vis, XRD, FTIR, Raman spectra, XPS, TEM and AFM, respectively. The as-prepared QRGO possessed peroxidase-like activity, which could catalyze the oxidation of 3,3'5,5'-tetramethylbenzidine by H2O2, and followed Michealis-Menten kinetics. A colorimetric sensor for quantitative determination of glucose based on the peroxidase activity of QRGO was developed over a range of 1-120 μM with a detection limit of 1 μM. PMID:24140856

  1. Ookinete-induced midgut peroxidases detonate the time bomb in anopheline mosquitoes.

    PubMed

    Kumar, Sanjeev; Barillas-Mury, Carolina

    2005-07-01

    Previous analysis of the temporal-spatial relationship between ookinete migration and the cellular localization of genes mediating midgut immune defense responses suggested that, in order to survive, parasites must complete invasion before toxic chemicals ("a bomb") are generated by the invaded cell. Recent studies indicate that ookinete invasion induces tyrosine nitration as a two-step reaction, in which NOS induction is followed by a localized increase in peroxidase activity. Peroxidases utilize nitrite and hydrogen peroxide as substrates, and detonate the time bomb by generating reactive nitrogen intermediates, such as nitrogen dioxide, which mediate nitration. There is evidence that peroxidases also mediate antimicrobial responses to bacteria, fungi and parasites in a broad range of biological systems including humans and plants. Defense reactions that generate toxic chemicals are also potentially harmful to the host mounting the response and often results in apoptosis. The two-step nitration pathway is probably an ancient response, as it has also been described in vertebrate leukocytes and probably evolved as a mechanism to circumscribe the toxic products generated during defense responses involving protein nitration. PMID:15894189

  2. Vascular peroxidase 1: a novel enzyme in promoting oxidative stress in cardiovascular system.

    PubMed

    Ma, Qi-Lin; Zhang, Guo-Gang; Peng, Jun

    2013-07-01

    Vascular peroxidase 1 (VPO1) is a recently identified novel family member of peroxidases in cardiovascular system. As an enzyme that is downstream of NADPH oxidases (NOX), VPO1 functions to utilize NOX - derived hydrogen peroxide (H2O2) to produce hypochlorous acid (HOCl), a strong oxidant which is believed to greatly promote oxidative stress. Under multiple conditions, NOX is activated concomitantly with an increase in superoxide anion (O2(.-)) and H2O2 production. The latter is converted to HOCl by VPO1. In this process (O2(.-) → H2O2 → HOCl), the oxidant reactivities of reactive oxygen species (ROS) are significantly increased and therefore the oxidative stress is dramatically amplified. Several lines of evidence suggest that the NOX/VPO1 pathway - mediated oxidative stress plays an important role in myocardial ischemia-reperfusion injury, endothelial cell apoptosis and/or smooth muscle cell proliferation. In addition, VPO1 can be secreted into the extracellular space to participate in extracellular matrix formation, suggesting that VPO1 may also play a role in cardiovascular remodeling (such as fibrosis). This function is independent of the peroxidase activity of VPO1. PMID:23357484

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

    PubMed

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

    1991-02-01

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

  4. Extracellular haem peroxidases mediate Mn(II) oxidation in a marine Roseobacter bacterium via superoxide production.

    PubMed

    Andeer, Peter F; Learman, Deric R; McIlvin, Matt; Dunn, James A; Hansel, Colleen M

    2015-10-01

    Manganese (Mn) oxides are among the strongest sorbents and oxidants in environmental systems. A number of biotic and abiotic pathways induce the oxidation of Mn(II) to Mn oxides. Here, we use a combination of proteomic analyses and activity assays, to identify the enzyme(s) responsible for extracellular superoxide-mediated Mn oxide formation by a bacterium within the ubiquitous Roseobacter clade. We show that animal haem peroxidases (AHPs) located on the outer membrane and within the secretome are responsible for Mn(II) oxidation. These novel peroxidases have previously been implicated in direct Mn(II) oxidation by phylogenetically diverse bacteria. Yet, we show that in this Roseobacter species, AHPs mediate Mn(II) oxidation not through a direct reaction but by producing superoxide and likely also by degrading hydrogen peroxide. These findings point to a eukaryotic-like oscillatory oxidative-peroxidative enzymatic cycle by these AHPs that leads to Mn oxide formation by this organism. AHP expression appears unaffected by Mn(II), yet the large energetic investment required to produce and secrete these enzymes points to an as yet unknown physiological function. These findings are further evidence that bacterial peroxidases and secreted enzymes, in general, are unappreciated controls on the cycling of metals and reactive oxygen species (ROS), and by extension carbon, in natural systems. PMID:25923595

  5. Turnover capacity of Coprinus cinereus peroxidase for phenol and monosubstituted phenols

    SciTech Connect

    Aitken, M.D.; Heck, P.E.

    1998-05-01

    Coprinus cinereus peroxidase (CIP) and other peroxidases are susceptible to mechanism-based inactivation during the oxidation of phenolic substrates. The turnover capacity of CIP was quantified for phenol and 11 monosubstituted phenols under conditions in which enzyme inactivation by mechanisms involving hydrogen peroxide alone were minimized. Turnover capacities varied by nearly 2 orders of magnitude, depending on the substituent. On a mass basis, the enzyme consumption corresponding to the lowest turnover capacities is considerable and may influence the economic feasibility of proposed industrial applications of peroxidases. Within a range of substituent electronegativity values, molar turnover capacities correlated well (r{sup 2} = 0.89) with substituent effects quantified by radical {sigma} values and semiquantitatively with homolytic O-H bond dissociation energies of the phenolic substrates, suggesting that phenoxyl radical intermediates are probably involved in the suicide inactivation of CIP. The correlation range in each case did not include phenols with highly electron-withdrawing (nitro and cyano) substituents because they are not oxidized by CIP, nor phenols with highly electron-donating (hydroxy and amino) substituents because they led to virtually complete inactivation of the enzyme with minimal substrate removal.

  6. Immobilization of peroxidase enzyme onto the porous silicon structure for enhancing its activity and stability

    PubMed Central

    2014-01-01

    In this work, a commercial peroxidase was immobilized onto porous silicon (PS) support functionalized with 3-aminopropyldiethoxysilane (APDES) and the performance of the obtained catalytic microreactor was studied. The immobilization steps were monitored and the activity of the immobilized enzyme in the PS pores was spectrophotometrically determined. The enzyme immobilization in porous silicon has demonstrated its potential as highly efficient enzymatic reactor. The effect of a polar organic solvent (acetonitrile) and the temperature (up to 50°C) on the activity and stability of the biocatalytic microreactor were studied. After 2-h incubation in organic solvent, the microreactor retained 80% of its initial activity in contrast to the system with free soluble peroxidase that lost 95% of its activity in the same period of time. Peroxidase immobilized into the spaces of the porous silicon support would be perspective for applications in treatments for environmental security such as removal of leached dye in textile industry or in treatment of different industrial effluents. The system can be also applied in the field of biomedicine. PMID:25221454

  7. Cationic Peptide Conjugation Enhances the Activity of Peroxidase-Mimicking DNAzymes.

    PubMed

    Xiao, Lu; Zhou, Zhaojuan; Feng, Mengli; Tong, Aijun; Xiang, Yu

    2016-03-16

    Peroxidase-mimicking DNAzymes containing G-quadruplex structures are widely applied in chemistry as catalysts and signal amplification for biosensing. Enhancing the catalytic activity of these DNAzymes can therefore improve the performance of many catalysts and biosensors using them. In this work, we synthesized cationic peptide conjugates of peroxidase-mimicking DNAzymes, which were found to exhibit both enhanced peroxidase and oxidase activities up to 4-fold and 3-fold compared with the original DNAzymes, respectively. Further investigation suggested that the enhanced activity was ascribed to the stabilization of parallel DNA G-quadruplex structures and hemin binding by the cationic peptide covalently attached to the DNAzyme. Such a mechanism of activity enhancement was successfully utilized for biosensing applications with improved sensitivity and broadened target range. Hydrogen peroxide (H2O2) detection in K(+)-free solutions by the DNAzyme-peptide conjugate showed 2-fold sensitivity enhancement over the unmodified DNAzyme under the same condition, and the activity switch by target-induced cleavage of the DNAzyme-peptide conjugate was also used for the detection of caspase 3 protease with enzymatic amplification in homogeneous solutions. PMID:26751843

  8. Improved detection and quantification of the (immuno) peroxidase product using reflection contrast microscopy.

    PubMed

    Cornelese-ten Velde, I; Wiegant, J; Tanke, H J; Ploem, J S

    1989-01-01

    Reflection contrast microscopy (RCM) is a sensitive tool to detect minor amounts of precipitated diaminobenzidine (DABox) in immunoperoxidase stained specimens. One of the main issues in immunocytochemistry is the ongoing need for more sensitive and quantitative techniques. Therefore we applied RCM, using a new simple model system, to methods previously described for increased sensitivity in immunocytochemistry with bright field microscopy. Addition of imidazole was found the most sensitive method and addition of Nickel and Cobalt ions gave the most enhanced colour intensity. Variation of the enzyme reaction parameters yielded a continuous increase in reflection with time. This was then discussed in view of other model studies of peroxidase kinetics. A quantitative relationship between the amount of peroxidase and the reflection of DABox was observed, indicating that quantitative immunoperoxidase studies with RCM are feasible. In situ hybridization (ISH) was then used as a useful biological model for RCM to test the optimal conditions for DAB staining found in the model system (high concentrations of DAB and peroxidase and 2 h incubation time). There was no background staining in the model system, also after prolonged incubation time. The ISH experiments showed that the contrast (ratio) between specific signal and chromosome background did not increase in time, whereas only the use of high avPO concentrations yielded the highest contrast. PMID:2475474

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

    PubMed

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

    2014-08-01

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

  10. Purification, crystallization and preliminary X-ray analysis of glutathione peroxidase Gpx3 from Saccharomyces cerevisiae

    SciTech Connect

    Yang, Zhu; Zhou, Cong-Zhao

    2006-06-01

    The glutathione peroxidase Gpx3 from the yeast S. cerevisiae has been overexpressed, purified, crystallized and diffracted to 2.6 Å resolution. Gpx3 is a monomer in solution which is different from its counterparts in mammals. The glutathione peroxidase Gpx3 from the yeast Saccharomyces cerevisiae has been overexpressed, purified and crystallized. Both gel-filtration and dynamic light-scattering (DLS) results indicate that Gpx3 is a monomer in solution at a concentration of about 2 mg ml{sup −1}, whereas glutathione peroxidases are normally tetrameric or dimeric. X-ray diffraction data from a single crystal of Gpx3 have been collected to 2.6 Å resolution. The crystals are triclinic and belong to space group P1, with unit-cell parameters a = 38.187, b = 43.372, c = 56.870 Å, α = 71.405, β = 73.376, γ = 89.633°. There are two Gpx3 monomers in a crystallographic asymmetric unit. Preliminary analyses show that the yeast Gpx3 is quite different from those of mammals.

  11. Gpx1 is a stationary phase-specific thioredoxin peroxidase in fission yeast

    SciTech Connect

    Lee, Si-Young; Song, Ji-Yoon; Kwon, Eun-Soo; Roe, Jung-Hye

    2008-02-29

    The genome sequence of Schizosaccharomyces pombe reveals only one gene for a putative glutathione peroxidase (gpx1{sup +}). The Gpx1 protein has a peroxidase activity but preferred thioredoxin to glutathione as an electron donor when examined in vitro and in vivo, and therefore is a thioredoxin peroxidase. Besides H{sub 2}O{sub 2}, it can reduce alkyl and phospholipid hydroperoxides. Expression of the gpx1 gene was elevated at the stationary phase, and we found that it supported long-term survival of S. pombe. The mutant also exhibited some defect in the activity of aconitase, an oxidation-labile Fe-S enzyme in mitochondria. Activity of sulfite reductase, a labile Fe-S enzyme in the cytosol, was also dramatically lowered in the mutant in the stationary phase. The Gpx1 protein, without any obvious targeting sequence, was localized in mitochondria as well as in the cytosol. Therefore, Gpx1 must serve to ensure optimal mitochondrial function and cytosolic environment, especially in the stationary phase.

  12. Purification, crystallization and preliminary X-ray diffraction analysis of royal palm tree (Roystonea regia) peroxidase

    SciTech Connect

    Watanabe, Leandra; Nascimento, Alessandro S.; Zamorano, Laura S.; Shnyrov, Valery L.; Polikarpov, Igor

    2007-09-01

    The purification, crystallization, X-ray diffraction data acquisition and molecular-replacement results of royal palm tree (R. regia) peroxidase are described. Royal palm tree peroxidase (RPTP), which was isolated from Roystonea regia leaves, has an unusually high stability that makes it a promising candidate for diverse applications in industry and analytical chemistry [Caramyshev et al. (2005 ▶), Biomacromolecules, 6, 1360–1366]. Here, the purification and crystallization of this plant peroxidase and its X-ray diffraction data collection are described. RPTP crystals were obtained by the hanging-drop vapour-diffusion method and diffraction data were collected to a resolution of 2.8 Å. The crystals belong to the trigonal space group P3{sub 1}21, with unit-cell parameters a = b = 116.83, c = 92.24 Å, and contain one protein molecule per asymmetric unit. The V{sub M} value and solvent content are 4.07 Å{sup 3} Da{sup −1} and 69.8%, respectively.

  13. Identification and characterization of a selenium-dependent glutathione peroxidase in Setaria cervi

    SciTech Connect

    Singh, Anchal; Rathaur, Sushma . E-mail: sushmarathaur@yahoo.com

    2005-06-17

    Setaria cervi a bovine filarial parasite secretes selenium glutathione peroxidase during in vitro cultivation. A significant amount of enzyme activity was detected in the somatic extract of different developmental stages of the parasite. Among different stages, microfilariae showed a higher level of selenium glutathione peroxidase activity followed by males then females. However, when the activity was compared in excretory secretory products of these stages males showed higher activity than microfilariae and female worms. The enzyme was purified from female somatic extract using a combination of glutathione agarose and gel filtration chromatography, which migrated as a single band of molecular mass {approx}20 kDa. Selenium content of purified enzyme was estimated by atomic absorption spectroscopy and found to be 3.5 ng selenium/{mu}g of protein. Further, inhibition of enzyme activity by potassium cyanide suggested the presence of selenium at the active site of enzyme. This is the first report of identification of selenium glutathione peroxidase from any filarial parasite.

  14. A thermostable humic acid peroxidase from Streptomyces sp. strain AH4: purification and biochemical characterization.

    PubMed

    Fodil, Djamila; Jaouadi, Bassem; Badis, Abdelmalek; Nadia, Zaraî Jaouadi; Ferradji, Fatma Zohra; Bejar, Samir; Boutoumi, Houcine

    2012-05-01

    An extracellular thermostable humic acid peroxidase (HaP3) was isolated from a Streptomyces sp. strain AH4. MALDI-TOF MS analysis showed that the purified enzyme was a monomer with a molecular mass of 60,215.18Da. The 26N-terminal residues of HaP3 displayed high homology with Streptomyces peroxidases. Optimal peroxidase activity was obtained at pH 5 and 80°C. HaP3 was stable at pH and temperature ranges of 4-8 and 60-90°C for 72 and 4h, respectively. HaP3 catalyzed the oxidation of 2,4-dichlorophenol, commercial humic acid, guiacol, and 2,6-dichlorophenol (50mM); L-3,4-dihydroxyphenylalanine (40 mM); 4-chlorophenol, 2,4,5-trichlorophenol, and 2,4,6-trichlorophenol (30 mM) in the presence of hydrogen peroxide. Sodium azide and potassium cyanide inhibited HaP3, which indicated the presence of heme components. These properties make HaP3 a potential strong candidate for future application in the elimination of natural humic acids in drinking water. PMID:22342039

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

    PubMed

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

    2007-08-25

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

  16. The peroxidase activity of mitochondrial superoxide dismutase (MnSOD/SOD2)

    PubMed Central

    Ansenberger-Fricano, Kristine; Ganini, Douglas da Silva; Mao, Mao; Chatterjee, Saurabh; Dallas, Shannon; Mason, Ronald P.; Stadler, Krisztian; Santos, Janine H.; Bonini, Marcelo G.

    2014-01-01

    Manganese superoxide dismutase (MnSOD) is an integral mitochondrial protein known as a first line antioxidant defense against superoxide radical anions produced as by-products of the electron transport chain. Recent studies have shaped the idea that by regulating the mitochondrial redox status and H2O2 outflow, MnSOD acts as a fundamental regulator of cellular proliferation, metabolism and apoptosis thereby assuming roles that extend far beyond its proposed antioxidant functions. Accordingly, allelic variations of MnSOD that have been shown to augment levels of MnSOD in mitochondria result in a 10-fold increase in prostate cancer risk. In addition, epidemiologic studies indicate that reduced glutathione peroxidase (GPx) activity along with increases in H2O2 further increase cancer risk in the face MnSOD overexpression. These facts led us to hypothesize that, like the Cu, Zn-counterpart, MnSOD may work as a peroxidase, utilizing H2O2 to promote mitochondrial damage, a known cancer risk factor. Here we report that MnSOD indeed possesses peroxidase activity that manifests in mitochondria when the enzyme is overexpressed. PMID:22982047

  17. Novel tungsten carbide nanorods: an intrinsic peroxidase mimetic with high activity and stability in aqueous and organic solvents.

    PubMed

    Li, Nan; Yan, Ya; Xia, Bao-Yu; Wang, Jing-Yuan; Wang, Xin

    2014-04-15

    Tungsten carbide nanorods (WC NRs) are demonstrated for the first time to possess intrinsic peroxidase-like activity towards typical peroxidase substrates, such as 3, 3', 5, 5'-tetramethylbenzidine (TMB) and ο-phenylenediamine (OPD) in the presence of hydrogen peroxide (H2O2). The reactions catalyzed by these nanorods follow the Michaelis-Menten kinetics. The excellent catalytic performance of WC NRs could be attributed to their intrinsic catalytic activity to efficiently accelerate the electron-transfer process and facilitate the decomposition of H2O2 to generate more numbers of reactive oxygen species (ROS). Based upon the strong peroxidase-like activity of these WC NRs, a colorimetric sensor for H2O2 is designed, which provides good response towards H2O2 concentration over a range of 2×10(-7)-8×10(-5) M with a detection limit of 60 nM. Moreover, the peroxidase-like activities of WC NRs with TMB as the substrate are investigated in both protic and aprotic organic media, showing different colorimetric reactions from that performed in aqueous solutions. In comparison with the natural horse radish peroxidase, WC NR exhibits excellent robustness of catalytic activity and considerable reusability, thus making it a promising mimic of peroxidase catalysts. PMID:24325981

  18. Cell Wall Free Space of Cucumis Hypocotyls Contains NAD and a Blue Light-Regulated Peroxidase Activity.

    PubMed

    Shinkle, J R; Swoap, S J; Simon, P; Jones, R L

    1992-04-01

    Solutions were obtained from the cell wall free space of red light-grown cucumber (Cucumis sativus L.) hypocotyl sections by a low-speed centrifugation technique. The centrifugate contained NAD and peroxidase but no detectable cytoplasmic contamination, as indicated by the absence of the activity of glucose-6-phosphate dehydrogenase from the cell wall solution. Peroxidase activity centrifuged from the cell wall of red light-grown cucumber hypocotyl section could be resolved into at least three cathodic isoforms and two anodic isoforms by isoelectric focusing. Treatment of red light-grown cucumber seedlings with a 10-minute pulse of high-intensity blue light increased the level of cell wall peroxidase by about 60% and caused a qualitative change in the anodic isoforms of this enzyme. The increase in peroxidase activity was detectable within 25 minutes after the start of the blue light pulse, was maximal at 35 minutes, and declined to control levels by 45 minutes of irradiation. The inhibitory effect of blue light on hypocotyl elongation was more rapid than the effect of blue light on total wall peroxidase activity, leading to the conclusion that growth and peroxidase activity are not causally related. PMID:16668797

  19. Enhancement of Peroxidase Release from Non-Malignant and Malignant Cells through Low-Dose Irradiation with Different Radiation Quality.

    PubMed

    Abdelrazzak, Abdelrazek B; Pottgießer, Stefanie J; Hill, Mark A; O'Neill, Peter; Bauer, Georg

    2016-02-01

    The release of peroxidase by nontransformed or transformed fibroblasts or epithelial cells (effector cells) triggers apoptosis induction selectively in transformed fibroblasts or transformed epithelial cells (target cells) through intercellular apoptosis-inducing signaling. The release of peroxidase can be induced either by treatment with transforming growth factor beta 1 or by low doses of alpha particles, gamma rays or ultrasoft X rays. In addiation, data indicates that radiation quality does not determine the overall efficiency of peroxidase release and the effects among a wide range of radiation doses are indistinguishable. These findings suggested that peroxidase release might be being triggered through intercellular bystander signaling. We show here that maximal peroxidase release does indeed occur after coculture of a small number of irradiated cells with an excess of unirradiated cells and demonstrate an enhanced effector function of nontransformed cells after the addition of a small number of irradiated cells. These data strongly indicate that peroxidase release is indeed triggered through bystander signaling mechanisms in mammalian cells. PMID:26849404

  20. Cell Wall Free Space of Cucumis Hypocotyls Contains NAD and a Blue Light-Regulated Peroxidase Activity 1

    PubMed Central

    Shinkle, James R.; Swoap, Steven J.; Simon, Patrice; Jones, Russell L.

    1992-01-01

    Solutions were obtained from the cell wall free space of red light-grown cucumber (Cucumis sativus L.) hypocotyl sections by a low-speed centrifugation technique. The centrifugate contained NAD and peroxidase but no detectable cytoplasmic contamination, as indicated by the absence of the activity of glucose-6-phosphate dehydrogenase from the cell wall solution. Peroxidase activity centrifuged from the cell wall of red light-grown cucumber hypocotyl section could be resolved into at least three cathodic isoforms and two anodic isoforms by isoelectric focusing. Treatment of red light-grown cucumber seedlings with a 10-minute pulse of high-intensity blue light increased the level of cell wall peroxidase by about 60% and caused a qualitative change in the anodic isoforms of this enzyme. The increase in peroxidase activity was detectable within 25 minutes after the start of the blue light pulse, was maximal at 35 minutes, and declined to control levels by 45 minutes of irradiation. The inhibitory effect of blue light on hypocotyl elongation was more rapid than the effect of blue light on total wall peroxidase activity, leading to the conclusion that growth and peroxidase activity are not causally related. ImagesFigure 2 PMID:16668797

  1. Phytochrome induces changes in the immunodetectable level of a wall peroxidase that precede growth changes in maize seedlings

    NASA Technical Reports Server (NTRS)

    Kim, S. H.; Shinkle, J. R.; Roux, S. J.

    1989-01-01

    The regulatory pigment phytochrome induces rapid and opposite growth changes in different regions of etiolated maize seedlings: it stimulates the elongation rate of coleoptiles and inhibits that of mesocotyls. As measured by a quantitative immunoassay, phytochrome also promotes rapid and opposite changes in the extractable content of a Mr 98,000 anionic isoperoxidase in the cell walls of these same organs: it induces a decrease of this peroxidase in coleoptiles and an increase in mesocotyls. The peroxidase changes precede the growth changes. As measured by video stereomicroscopy or a position transducer, red light (R), which photoactivates phytochrome, stimulates coleoptile elongation with a lag of about 15-20 min and suppresses mesocotyl growth with a lag of 45-50 min. R also induces a 50% reduction in the extractable level of the anionic peroxidase in coleoptile walls in less than 10 min and a 40% increase in the level of this peroxidase in mesocotyl walls within 30 min. Ascorbic acid, an inhibitor of peroxidase activity, blocks the effects of R on mesocotyl section growth. These results are relevant to hypotheses that postulate that certain wall peroxidases can participate in light-induced changes in growth rate by their effects on wall extensibility.

  2. Spectroscopic and Kinetic Characterization of Peroxidase-Like π-Cation Radical Pinch-Porphyrin-Iron(III) Reaction Intermediate Models of Peroxidase Enzymes.

    PubMed

    Hernández Anzaldo, Samuel; Arroyo Abad, Uriel; León García, Armando; Ramírez Rosales, Daniel; Zamorano Ulloa, Rafael; Reyes Ortega, Yasmi

    2016-01-01

    The spectroscopic and kinetic characterization of two intermediates from the H₂O₂ oxidation of three dimethyl ester [(proto), (meso), (deuteroporphyrinato) (picdien)]Fe(III) complexes ([FePPPic], [FeMPPic] and [FeDPPic], respectively) pinch-porphyrin peroxidase enzyme models, with s = 5/2 and 3/2 Fe(III) quantum mixed spin (qms) ground states is described herein. The kinetic study by UV/Vis at λmax = 465 nm showed two different types of kinetics during the oxidation process in the guaiacol test for peroxidases (1-3 + guaiacol + H₂O₂ → oxidation guaiacol products). The first intermediate was observed during the first 24 s of the reaction. When the reaction conditions were changed to higher concentration of pinch-porphyrins and hydrogen peroxide only one type of kinetics was observed. Next, the reaction was performed only between pinch-porphyrins-Fe(III) and H₂O₂, resulting in only two types of kinetics that were developed during the first 0-4 s. After this time a self-oxidation process was observed. Our hypotheses state that the formation of the π-cation radicals, reaction intermediates of the pinch-porphyrin-Fe(III) family with the ligand picdien [N,N'-bis-pyridin-2-ylmethyl-propane-1,3-diamine], occurred with unique kinetics that are different from the overall process and was involved in the oxidation pathway. UV-Vis, ¹H-NMR and ESR spectra confirmed the formation of such intermediates. The results in this paper highlight the link between different spectroscopic techniques that positively depict the kinetic traits of artificial compounds with enzyme-like activity. PMID:27355940

  3. Different peroxidase activities and expression of abiotic stress-related peroxidases in apical root segments of wheat genotypes with different drought stress tolerance under osmotic stress.

    PubMed

    Csiszár, Jolán; Gallé, Agnes; Horváth, Edit; Dancsó, Piroska; Gombos, Magdolna; Váry, Zsolt; Erdei, László; Györgyey, János; Tari, Irma

    2012-03-01

    One-week-old seedlings of Triticum aestivum L. cv. Plainsman V, a drought tolerant; and Cappelle Desprez, a drought sensitive wheat cultivar were subjected gradually to osmotic stress using polyethylene glycol (PEG 6000) reaching 400 mOsm on the 11th day. Compared to controls cv. Plainsman V maintained the root growth and relative water content of root tissues, while these parameters were decreased in the drought sensitive cv. Cappelle Desprez under PEG-mediated osmotic stress. Simultaneously, H(2)O(2) content in 1-cm-long apical segment of roots comprising the proliferation and elongation zone, showed a transient increase in cv. Plainsman V and a permanent raise in cv. Cappelle Desprez. Measurements of the transcript levels of selected class III peroxidase (TaPrx) coding sequences revealed significant differences between the two cultivars on the 9th day, two days after applying 100 mOsm PEG. The abundance of TaPrx04 transcript was enhanced transitionally in the root apex of cv. Plainsman V but decreased in cv. Cappelle Desprez under osmotic stress while the expression of TaPrx01, TaPrx03, TaPrx19, TaPrx68, TaPrx107 and TaPrx109-C decreased to different extents in both cultivars. After a transient decrease, activities of soluble peroxidase fractions of crude protein extracts rose in both cultivars on day 11, but the activities of cell wall-bound fractions increased only in cv. Cappelle Desprez under osmotic stress. Parallel with high H(2)O(2) content of the tissues, certain isoenzymes of covalently bound fraction in cv. Cappelle Desprez showed increased activity suggesting that they may limit the extension of root cell walls in this cultivar. PMID:22305075

  4. 18O studies of the peroxidase-catalyzed oxidation of N-methylcarbazole. Mechanisms of carbinolamine and carboxaldehyde formation.

    PubMed

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

    1986-12-01

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

  5. Changes in the activity of ascorbate peroxidase under anaerobiosis in cocoyam (Colocasia esculenta).

    PubMed

    Chibueze, Nwose

    2014-01-01

    This study was conducted to determine the activity of ascorbate peroxidase in the cormels of cocoyam (Colocasia esculenta var. antiquorum) immediately after harvest and in storage under anaerobiosis for one and three weeks, respectively. During stress condition in plants, hydrogen peroxide is released and mechanisms to detoxify it must be maintained. The cocoyam tubers that were neither damaged nor affected by disease were harvested from a local farm in Ugbogui, Ovia North Local Government Area in Edo State, Nigeria. The selected cocoyam tubers were peeled manually, washed with ice cold water and cut into pieces. The root tissues (50 g) were homogenised with 100 mL of ice cold 0.05 M phosphate buffer. The extract obtained was clarified by centrifugation for 15 min at 8000 g at 4 degrees C. Ascorbate-peroxidising activity was assayed using the initial rate of decrease in ascorbate concentration as measured by its absorbance at 290 nm using Milton Roy Spectron 21D. Results showed the weight of the cormels decreased all through during storage. Immediately after harvest the activity of ascorbate peroxidase was 15.49 unit mL(-1) with a significant increase (p < 0.05) after one week to 73.05 U mL(-1). Thereafter there was a significant decrease in activity of the enzyme after three weeks of storage to 33.33 U mL(-1). This increase in activity of ascorbate peroxidase after three weeks of storage may be related to increase in response to various biotic stresses. Therefore, manipulation of the capacity of cocoyam to tolerate anaerobiosis is a function of its ability to modulate the antioxidant enzymes' armory in case of need. PMID:24783794

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

    PubMed

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

    2016-06-01

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

  7. Vascular defense responses in rice: peroxidase accumulation in xylem parenchyma cells and xylem wall thickening

    NASA Technical Reports Server (NTRS)

    Hilaire, E.; Young, S. A.; Willard, L. H.; McGee, J. D.; Sweat, T.; Chittoor, J. M.; Guikema, J. A.; Leach, J. E.

    2001-01-01

    The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae is a vascular pathogen that elicits a defensive response through interaction with metabolically active rice cells. In leaves of 12-day-old rice seedlings, the exposed pit membrane separating the xylem lumen from the associated parenchyma cells allows contact with bacterial cells. During resistant responses, the xylem secondary walls thicken within 48 h and the pit diameter decreases, effectively reducing the area of pit membrane exposed for access by bacteria. In susceptible interactions and mock-inoculated controls, the xylem walls do not thicken within 48 h. Xylem secondary wall thickening is developmental and, in untreated 65-day-old rice plants, the size of the pit also is reduced. Activity and accumulation of a secreted cationic peroxidase, PO-C1, were previously shown to increase in xylem vessel walls and lumen. Peptide-specific antibodies and immunogold-labeling were used to demonstrate that PO-C1 is produced in the xylem parenchyma and secreted to the xylem lumen and walls. The timing of the accumulation is consistent with vessel secondary wall thickening. The PO-C1 gene is distinct but shares a high level of similarity with previously cloned pathogen-induced peroxidases in rice. PO-C1 gene expression was induced as early as 12 h during resistant interactions and peaked between 18 and 24 h after inoculation. Expression during susceptible interactions was lower than that observed in resistant interactions and was undetectable after infiltration with water, after mechanical wounding, or in mature leaves. These data are consistent with a role for vessel secondary wall thickening and peroxidase PO-C1 accumulation in the defense response in rice to X. oryzae pv. oryzae.

  8. Characterization and expression of the Schistosoma japonicum thioredoxin peroxidase-2 gene.

    PubMed

    Hong, Yang; Han, Yanhui; Fu, Zhiqiang; Han, Hongxiao; Qiu, Chunhui; Zhang, Min; Yang, Jianmei; Shi, Yaojun; Li, Xiangrui; Lin, Jiaojiao

    2013-02-01

    We analyzed proteins that were differentially expressed by 10-day-old schistosomula from 3 different hosts and determined that a functional thioredoxin peroxidase-2 gene has an important antioxidant role in Schistosoma japonicum , which we investigated further. A full-length cDNA encoding the S. japonicum thioredoxin peroxidase-2 (SjTPx-2) had an open reading frame of 681 bp that encoded 226 amino acids with a signal peptide of 24 amino acids. A cDNA encoding SjTPx-2 without the signal peptide sequence was isolated from 42-day-old schistosome cDNAs. Real-time quantitative RT-PCR analysis revealed that SjTPx-2 was upregulated in 7- and 13-day-old schistosomes, while the expression level in females was around 2-fold higher than that in male worms at 42 days. SjTPx was subcloned into pET28a(+) and expressed as both inclusion bodies and supernatant in Escherichia coli BL21 (DE3) cells. Western blotting showed that the recombinant SjTPx-2 (rSjTPx-2) was immunogenic. The purified recombinant protein could form disulfide-bonded dimers and it had peroxidase activity in vitro. An immunoprotection experiment in BALB/c mice showed that vaccination with recombinant SjTPx-2 could induce 31.2% and 34.0% reductions in the numbers of worms and eggs in the liver, respectively. This study suggests that SjTPx-2 may be an important antioxidative enzyme in scavenging ROS, and it may be a potential vaccine candidate or new drug target for schistosomiasis. PMID:22924918

  9. Pleurotus ostreatus manganese‐dependent peroxidase silencing impairs decolourization of Orange II

    PubMed Central

    Salame, Tomer M.; Yarden, Oded; Hadar, Yitzhak

    2010-01-01

    Summary Decolourization of azo dyes by Pleurotus ostreatus, a white‐rot fungus capable of lignin depolymerization and mineralization, is related to the ligninolytic activity of enzymes produced by this fungus. The capacity of P. ostreatus to decolourize the azo dye Orange II (OII) was dependent and positively co‐linear to Mn2+ concentration in the medium, and thus attributed to Mn2+‐dependent peroxidase (MnP) activity. Based on the ongoing P. ostreatus genome deciphering project we identified at least nine genes encoding for MnP gene family members (mnp1–9), of which only four (mnp1–4) were previously known. Relative real‐time PCR quantification analysis confirmed that all the nine genes are transcribed, and that Mn2+ amendment results in a drastic increase in the transcript levels of the predominantly expressed MnP genes (mnp3 and mnp9), while decreasing versatile peroxidase gene transcription (mnp4). A reverse genetics strategy based on silencing the P. ostreatus mnp3 gene by RNAi was implemented. Knock‐down of mnp3 resulted in the reduction of fungal OII decolourization capacity, which was co‐linear with marked silencing of the Mn2+‐dependent peroxidase genes mnp3 and mnp9. This is the first direct genetic proof of an association between MnP gene expression levels and azo dye decolourization capacity in P. ostreatus, which may have significant implication on understanding the mechanisms governing lignin biodegradation. Moreover, this study has proven the applicability of RNAi as a tool for gene function studies in Pleurotus research. PMID:21255310

  10. Evidence for radical formation at Tyr-353 in Mycobacterium tuberculosis catalase-peroxidase (KatG).

    PubMed

    Zhao, Xiangbo; Girotto, Stefania; Yu, Shengwei; Magliozzo, Richard S

    2004-02-27

    Mycobacterium tuberculosis KatG is a heme-containing catalase-peroxidase responsible for activation, through its peroxidase cycle, of the front line antituberculosis antibiotic isoniazid (isonicotinic acid hydrazide). Formation of Compound I (oxyferryl heme-porphyrin pi-cation radical), the classical peroxidase intermediate generated when the resting enzyme turns over with alkyl peroxides, is rapidly followed by production of a protein-centered tyrosyl radical in this enzyme. In our efforts to identify the residue at which this radical is formed, nitric oxide was used as a radical scavenging reagent. Quenching of the tyrosyl radical generated in the presence of NO was shown using electron paramagnetic resonance spectroscopy, and formation of nitrotyrosine was confirmed by proteolytic digestion followed by high performance liquid chromatography analysis of the NO-treated enzyme. These results are consistent with formation of nitrosyltyrosine by addition of NO to tyrosyl radical and oxidation of this intermediate to nitrotyrosine. Two predominant nitrotyrosine-containing peptides were identified that were purified and sequenced by Edman degradation. Both peptides were derived from the same M. tuberculosis KatG sequence spanning residues 346-356 with the amino acid sequence SPAGAWQYTAK, and both peptides contained nitrotyrosine at residue 353. Some modification of Trp-351 most probably into nitrosotryptophan was also found in one of the two peptides. Control experiments using denatured KatG or carried out in the absence of peroxide did not produce nitrotyrosine. In the mutant enzyme KatG(Y353F), which was constructed using site-directed mutagenesis, a tyrosyl radical was also formed upon turnover with peroxide but in poor yield compared with wild-type KatG. Residue Tyr-353 is unique to M. tuberculosis KatG and may play a special role in the function of this enzyme. PMID:14665627

  11. Overproduction of lignin peroxidase by Phanerochaete chrysosporium (BKM-F-1767) under nonlimiting nutrient conditions.

    PubMed Central

    Dosoretz, C G; Rothschild, N; Hadar, Y

    1993-01-01

    The ligninolytic enzymes synthesized by Phanerochaete chrysosporium BKM-F-1767 immobilized on polyurethane foam were characterized under limiting, sufficient, and excess nutrient conditions. The fungus was grown in a nonimmersed liquid culture system under conditions close to those occurring in nature, with nitrogen concentrations ranging from 2.4 to 60 mM. This nonimmersed liquid culture system consisted of fungal mycelium immobilized on porous pieces of polyurethane foam saturated with liquid medium and highly exposed to gaseous oxygen. Lignin peroxidase (LIP) activity decreased to almost undetectable levels as the initial NH4+ levels were increased over the range from 2.4 to 14 mM and then increased with additional increases in initial NH4+ concentration. At 45 mM NH4+, LIP was overproduced, reaching levels of 800 U/liter. In addition, almost simultaneous secretion of LIP and secretion of manganese-dependent lignin peroxidase were observed on the third day of incubation. Manganese-dependent lignin peroxidase activity was maximal under nitrogen limitation conditions (2.4 mM NH4+) and then decreased to 40 to 50% of the maximal level in the presence of sufficient or excess initial NH4+ concentrations. Overproduction of LIP in the presence of a sufficient nitrogen level (24 mM NH4+) and excess nitrogen levels (45 to 60 mM NH4+) seemed to occur as a response to carbon starvation after rapid glucose depletion. The NH4+ in the extracellular fluid reappeared as soon as glucose was depleted, and an almost complete loss of CO2 was observed, suggesting that an alternative energy source was generated by self-proteolysis of cell proteins.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8328807

  12. Expression of a Phanerochaete chrysosporium manganese peroxidase gene in the yeast Pichia pastoris.

    PubMed

    Gu, Lina; Lajoie, Curtis; Kelly, Christine

    2003-01-01

    A gene encoding manganese peroxidase (mnp1) from Phanerochaete chrysosporium was cloned downstream of a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter in the methylotrophic yeast Pichia pastoris. Three different expression vectors were constructed: pZBMNP contains the native P. chrysosporium fungal secretion signal, palphaAMNP contains an alpha-factor secretion signal derived from Saccharomyces cerevisiae, and pZBIMNP has no secretion signal and was used for intracellular expression. Both the native fungal secretion signal sequence and alpha-factor secretion signal sequence directed the secretion of active recombinant manganese peroxidase (rMnP) from P. pastoris transformants. The majority of the rMnP produced by P. pastoris exhibited a molecular mass (55-100 kDa) considerably larger than that of the wild-type manganese peroxidase (wtMnP, 46 kDa). Deletion of the native fungal secretion signal yielded a molecular mass of 39 kDa for intracellular rMnP in P. pastoris. Treatment of the secreted rMnP with endoglycosidase H (Endo H) resulted in a considerable decrease in the mass of rMnP, indicating N-linked hyperglycosylation. Partially purified rMnP showed kinetic characteristics similar to those of wtMnP. Both enzymes also had similar pH stability profiles. Addition of exogenous Mn(II), Ca(II), and Fe(III) conferred additional thermal stability to both enzymes. However, rMnP was slightly less thermostable than wtMnP, which demonstrated an extended half-life at 55 degrees C. PMID:14524699

  13. A peroxidase biomimetic system based on Fe3O4 nanoparticles in non-enzymatic sensors.

    PubMed

    Guivar, Juan A Ramos; Fernandes, Edson G R; Zucolotto, Valtencir

    2015-08-15

    Magnetic nanoparticles have been applied in many areas of nanomedicine. Sensing platforms based on this type of nanoparticles have received attention due to the relative low cost and biocompatibility. Biosensor is the most widely investigated type of analytical device. This type of sensor combines the physicochemical transduction with the incorporation of biological sensing components. Among the biological components, enzymes are the most commonly used as sensitive elements. However, natural enzymes may exhibit serious disadvantages such as lack of stability and loss of catalytic activity after immobilization. The study of enzymatic biomimetic systems are of great interest. This study reports the development of a new sensor composed of Fe3O4@CTAB and poly(sodium 4-styrenesulfonate) (PSS) films assembled via Layer-by-Layer (LbL) technique and used as peroxidase mimetic systems. Magnetic nanoparticles (MNps) were synthesized using thermal decomposition method and further dispersed to aqueous medium by ligand modification reaction using cetyltrimethylammonium bromide (CTAB). The amperometric detection limit of H2O2 was found to be ca. 103 µmol L(-1). By chronoamperometry, the peroxidase biomimetic sensor exhibited a linear response for H2O2 in the range from 100 µmol L(-1) to 1.8 mmol L(-1) (R(2) = 0.994) with sensitivity of 16 nA mol(-1) L. The apparent Michaelis-Menten constant was 5.3 mmol L(-1), comparable with some biosensors based on peroxidase enzyme. Moreover, the sensor presented a reproducibility of ca. 7.7% (n = 4) and their response (response time: 90 s) is not significantly affected in the presence of some interferents including K(+), Na(+), Cl(-), Mg(2+), Ca(2+), and Uric Acid. PMID:25966419

  14. Molecular characterization of fruit-specific class III peroxidase genes in tomato (Solanum lycopersicum).

    PubMed

    Wang, Chii-Jeng; Chan, Yuan-Li; Shien, Chin Hui; Yeh, Kai-Wun

    2015-04-01

    In this study, expression of four peroxidase genes, LePrx09, LePrx17, LePrx35 and LePrxA, was identified in immature tomato fruits, and the function in the regulation of fruit growth was characterized. Analysis of amino acid sequences revealed that these genes code for class III peroxidases, containing B, D and F conserved domains, which bind heme groups, and a buried salt bridge motif. LePrx35 and LePrxA were identified as novel peroxidase genes in Solanum lycopersicum (L.). The temporal expression patterns at various fruit growth stages revealed that LePrx35 and LePrxA were expressed only in immature green (IMG) fruits, whereas LePrx17 and LePrx09 were expressed in both immature and mature green fruits. Tissue-specific expression profiles indicated that only LePrx09 was expressed in the mesocarp but not the inner tissue of immature fruits. The effects of hormone treatments and stresses on the four genes were examined; only the expression levels of LePrx17 and LePrx09 were altered. Transcription of LePrx17 was up-regulated by jasmonic acid (JA) and pathogen infection and expression of LePrx09 was induced by ethephon, salicylic acid (SA) and JA, in particular, as well as wounding, pathogen infection and H2O2 stress. Tomato plants over-expressing LePrx09 displayed enhanced resistance to H2O2 stress, suggesting that LePrx09 may participate in the H2O2 signaling pathway to regulate fruit growth and disease resistance in tomato fruits. PMID:25703772

  15. Functionally Distinct Bacterial Cytochrome c Peroxidases Proceed through a Common (Electro)catalytic Intermediate.

    PubMed

    Frato, Katherine E; Walsh, Kelly A; Elliott, Sean J

    2016-01-12

    The diheme cytochrome c peroxidase from Shewanella oneidensis (So CcP) requires a single electron reduction to convert the oxidized, as-isolated enzyme to an active conformation. We employ protein film voltammetry to investigate the mechanism of hydrogen peroxide turnover by So CcP. When the enzyme is poised in the active state by incubation with sodium l-ascorbate, the graphite electrode specifically captures a highly active state that turns over peroxide in a high potential regime. This is the first example of an on-pathway catalytic intermediate observed for a bacterial diheme cytochrome c peroxidase that requires reductive activation, consistent with the observed voltammetric response from the diheme cytochrome c peroxidase from Nitrosomonas europaea (Ne), which is constitutively active and does not require the same one electron activation. Mutational analysis at the active site of So CcP confirms that the rate-limiting step involves a proton-coupled single electron reduction of a high valent iron species centered on the low-potential heme, consistent with the same mutation in Ne CcP. The pH dependence of catalysis for wild-type So CcP suggests that reduction shifts the pK(a)'s of at least two amino acids. Mutation of His81 in "loop 1", a surface exposed loop thought to shift conformation during the reductive activation process, eliminated one of the pH dependent features, confirming that the loop 1 shifts, changing the environment of His81 during the rate-limiting step. The observed catalytic intermediate has the same electron stoichiometry and similar pH dependence to that previously reported for Ne CcP, which is constitutively active and therefore hypothesized to follow a different catalytic mechanism. The prominent similarities between the rate-limiting steps of differing mechanistic classes of bCcPs suggest unexpected similarities in the intermediates formed. PMID:26575087

  16. Crystallographic, kinetic, and spectroscopic study of the first ligninolytic peroxidase presenting a catalytic tyrosine.

    PubMed

    Miki, Yuta; Calviño, Fabiola R; Pogni, Rebecca; Giansanti, Stefania; Ruiz-Dueñas, Francisco J; Martínez, María Jesús; Basosi, Riccardo; Romero, Antonio; Martínez, Angel T

    2011-04-29

    Trametes cervina lignin peroxidase (LiP) is a unique enzyme lacking the catalytic tryptophan strictly conserved in all other LiPs and versatile peroxidases (more than 30 sequences available). Recombinant T. cervina LiP and site-directed variants were investigated by crystallographic, kinetic, and spectroscopic techniques. The crystal structure shows three substrate oxidation site candidates involving His-170, Asp-146, and Tyr-181. Steady-state kinetics for oxidation of veratryl alcohol (the typical LiP substrate) by variants at the above three residues reveals a crucial role of Tyr-181 in LiP activity. Moreover, assays with ferrocytochrome c show that its ability to oxidize large molecules (a requisite property for oxidation of the lignin polymer) originates in Tyr-181. This residue is also involved in the oxidation of 1,4-dimethoxybenzene, a reaction initiated by the one-electron abstraction with formation of substrate cation radical, as described for the well known Phanerochaete chrysosporium LiP. Detailed spectroscopic and kinetic investigations, including low temperature EPR, show that the porphyrin radical in the two-electron activated T. cervina LiP is unstable and rapidly receives one electron from Tyr-181, forming a catalytic protein radical, which is identified as an H-bonded neutral tyrosyl radical. The crystal structure reveals a partially exposed location of Tyr-181, compatible with its catalytic role, and several neighbor residues probably contributing to catalysis: (i) by enabling substrate recognition by aromatic interactions; (ii) by acting as proton acceptor/donor from Tyr-181 or H-bonding the radical form; and (iii) by providing the acidic environment that would facilitate oxidation. This is the first structure-function study of the only ligninolytic peroxidase described to date that has a catalytic tyrosine. PMID:21367853

  17. Airway Peroxidases Catalyze Nitration of the β2-Agonist Salbutamol and Decrease Its Pharmacological Activity

    PubMed Central

    Sallans, Larry; Macha, Stephen; Brown, Kari; McGraw, Dennis W.; Kovacic, Melinda Butsch; Britigan, Bradley E.

    2011-01-01

    β2-Agonists are the most effective bronchodilators for the rapid relief of asthma symptoms, but for unclear reasons, their effectiveness may be decreased during severe exacerbations. Because peroxidase activity and nitrogen oxides are increased in the asthmatic airway, we examined whether salbutamol, a clinically important β2-agonist, is subject to potentially inactivating nitration. When salbutamol was exposed to myeloperoxidase, eosinophil peroxidase or lactoperoxidase in the presence of hydrogen peroxide (H2O2) and nitrite (NO2−), both absorption spectroscopy and mass spectrometry indicated formation of a new metabolite with features expected for the nitrated drug. The new metabolites showed an absorption maximum at 410 nm and pKa of 6.6 of the phenolic hydroxyl group. In addition to nitrosalbutamol (m/z 285.14), a salbutamol-derived nitrophenol, formed by elimination of the formaldehyde group, was detected (m/z 255.13) by mass spectrometry. It is noteworthy that the latter metabolite was detected in exhaled breath condensates of asthma patients receiving salbutamol but not in unexposed control subjects, indicating the potential for β2-agonist nitration to occur in the inflamed airway in vivo. Salbutamol nitration was inhibited in vitro by ascorbate, thiocyanate, and the pharmacological agents methimazole and dapsone. The efficacy of inhibition depended on the nitrating system, with the lactoperoxidase/H2O2/NO2− being the most affected. Functionally, nitrated salbutamol showed decreased affinity for β2-adrenergic receptors and impaired cAMP synthesis in airway smooth muscle cells compared with the native drug. These results suggest that under inflammatory conditions associated with asthma, phenolic β2-agonists may be subject to peroxidase-catalyzed nitration that could potentially diminish their therapeutic efficacy. PMID:20974700

  18. Thiol Peroxidase Is an Important Component of Streptococcus pneumoniae in Oxygenated Environments

    PubMed Central

    Hajaj, Barak; Yesilkaya, Hasan; Benisty, Rachel; David, Maayan; Andrew, Peter W.

    2012-01-01

    Streptococcus pneumoniae is an aerotolerant Gram-positive bacterium that causes an array of diseases, including pneumonia, otitis media, and meningitis. During aerobic growth, S. pneumoniae produces high levels of H2O2. Since S. pneumoniae lacks catalase, the question of how it controls H2O2 levels is of critical importance. The psa locus encodes an ABC Mn2+-permease complex (psaBCA) and a putative thiol peroxidase, tpxD. This study shows that tpxD encodes a functional thiol peroxidase involved in the adjustment of H2O2 homeostasis in the cell. Kinetic experiments showed that recombinant TpxD removed H2O2 efficiently. However, in vivo experiments revealed that TpxD detoxifies only a fraction of the H2O2 generated by the pneumococcus. Mass spectrometry analysis demonstrated that TpxD Cys58 undergoes selective oxidation in vivo, under conditions where H2O2 is formed, confirming the thiol peroxidase activity. Levels of TpxD expression and synthesis in vitro were significantly increased in cells grown under aerobic versus anaerobic conditions. The challenge with D39 and TIGR4 with H2O2 resulted in tpxD upregulation, while psaBCA expression was oppositely affected. However, the challenge of ΔtpxD mutants with H2O2 did not affect psaBCA, implying that TpxD is involved in the regulation of the psa operon, in addition to its scavenging activity. Virulence studies demonstrated a notable difference in the survival time of mice infected intranasally with D39 compared to that of mice infected intranasally with D39ΔtpxD. However, when bacteria were administered directly into the blood, this difference disappeared. The findings of this study suggest that TpxD constitutes a component of the organism's fundamental strategy to fine-tune cellular processes in response to H2O2. PMID:23027531

  19. Characterization of the W321F mutant of Mycobacterium tuberculosis catalase-peroxidase KatG.

    PubMed

    Yu, Shengwei; Chouchane, Salem; Magliozzo, Richard S

    2002-01-01

    A single amino acid mutation (W321F) in Mycobacterium tuberculosis catalase-peroxidase (KatG) was constructed by site-directed mutagenesis. The purified mutant enzyme was characterized using optical and electron paramagnetic resonance spectroscopy, and optical stopped-flow spectrophotometry. Reaction of KatG(W321F) with 3-chloroperoxybenzoic acid, peroxyacetic acid, or t-butylhydroperoxide showed formation of an unstable intermediate assigned as Compound I (oxyferryl iron:porphyrin pi-cation radical) by similarity to wild-type KatG, although second-order rate constants were significantly lower in the mutant for each peroxide tested. No evidence for Compound II was detected during the spontaneous or substrate-accelerated decay of Compound I. The binding of isoniazid, a first-line anti-tuberculosis pro-drug activated by catalase-peroxidase, was noncooperative and threefold weaker in KatG(W321F) compared with wild-type enzyme. An EPR signal assigned to a protein-based radical tentatively assigned as tyrosyl radical in wild-type KatG, was also observed in the mutant upon reaction of the resting enzyme with alkyl peroxide. These results show that mutation of residue W321 in KatG does not lead to a major alteration in the identity of intermediates formed in the catalytic cycle of the enzyme in the time regimes examined here, and show that this residue is not the site of stabilization of a radical as might be expected based on homology to yeast cytochrome c peroxidase. Furthermore, W321 is indicated to be important in KatG for substrate binding and subunit interactions within the dimer, providing insights into the origin of isoniazid resistance in clinically isolated KatG mutants. PMID:11742122

  20. Construction of a Fusion Enzyme Exhibiting Superoxide Dismutase and Peroxidase Activity.

    PubMed

    Sharapov, M G; Novoselov, V I; Ravin, V K

    2016-04-01

    A chimeric gene construct encoding human peroxiredoxin 6 and Mn-superoxide dismutase from Escherichia coli was developed. Conditions for expression of the fusion protein in E. coli cell were optimized. Fusing of the enzymes into a single polypeptide chain with peroxiredoxin 6 at the N-terminus (PSH) did not affect their activities. On the contrary, the chimeric protein with reverse order of enzymes (SPH) was not obtained in a water-soluble active form. The active chimeric protein (PSH) exhibiting both peroxidase and superoxide dismutase activities was prepared and its physicochemical properties were characterized. PMID:27293100

  1. 3,3',5,5'-Tetramethylbenzidine Oxidation on Paper Devices for Horseradish Peroxidase-based Assays.

    PubMed

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

    2016-01-01

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

  2. Efficient expression of a Phanerochaete chrysosporium manganese peroxidase gene in Aspergillus oryzae

    SciTech Connect

    Stewart, P.; Whitwam, R.E.; Tien, Ming

    1996-03-01

    A manganese peroxidase (mnp1) from Phanerochaete chrysosporium was efficiently expressed in Aspergillus oryzae. Expression was achieved by fusing the mature cDNA of mnp1 with the A. oryzae Taka amylase promoter and secretion signal. The 3{prime} untranslated region of the glucoamylase gene of Asperigillus awamori provided the terminator. The recombinant protein (rMnP) was secreted in an active form, permitting rapid detection and purification. Physical and kinetic properties of rMnP were similar to those of the native protein. The A. oryzae expression system is well suited for both mechanistic and site-directed mutagenesis studies. 34 refs., 7 figs., 1 tab.

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

    PubMed

    Brück, Thomas B; Brück, Dieter W

    2011-02-01

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

  4. Peroxidase-like oxidative activity of a manganese-coordinated histidyl bolaamphiphile self-assembly

    NASA Astrophysics Data System (ADS)

    Kim, Min-Chul; Lee, Sang-Yup

    2015-10-01

    A peroxidase-like catalyst was constructed through the self-assembly of histidyl bolaamphiphiles coordinated to Mn2+ ions. The prepared catalyst exhibited oxidation activity for the organic substrate o-phenylenediamine (OPD) in the presence of hydrogen peroxide (H2O2). The histidyl bolaamphiphiles of bis(N-alpha-amido-histidine)-1,7-heptane dicarboxylates self-assembled to make spherical structures in an aqueous solution. Subsequent association of Mn2+ ions with the histidyl imidazoles in the self-assembly produced catalytic active sites. The optimal Mn2+ ion concentration was determined and coordination of the Mn2+ ion with multiple histidine imidazoles was investigated using spectroscopy analysis. The activation energy of the produced catalysts was 55.0 kJ mol-1, which was comparable to other peroxidase-mimetic catalysts. A detailed kinetics study revealed that the prepared catalyst followed a ping-pong mechanism and that the turnover reaction was promoted by increasing the substrate concentration. Finally, application of the prepared catalyst for glucose detection was demonstrated through cascade enzyme catalysis. This study demonstrated a facile way to prepare an enzyme-mimetic catalyst through the self-assembly of an amphiphilic molecule containing amino acid segments.A peroxidase-like catalyst was constructed through the self-assembly of histidyl bolaamphiphiles coordinated to Mn2+ ions. The prepared catalyst exhibited oxidation activity for the organic substrate o-phenylenediamine (OPD) in the presence of hydrogen peroxide (H2O2). The histidyl bolaamphiphiles of bis(N-alpha-amido-histidine)-1,7-heptane dicarboxylates self-assembled to make spherical structures in an aqueous solution. Subsequent association of Mn2+ ions with the histidyl imidazoles in the self-assembly produced catalytic active sites. The optimal Mn2+ ion concentration was determined and coordination of the Mn2+ ion with multiple histidine imidazoles was investigated using spectroscopy

  5. Polymerization of cardanol using soybean peroxidase and its potential application as anti-biofilm coating material.

    PubMed

    Kim, Yong Hwan; An, Eun Suk; Song, Bong Keun; Kim, Dong Shik; Chelikani, Rahul

    2003-09-01

    Soybean peroxidase (20 mg) catalyzed the oxidative polymerization of cardanol in 2-propanol/phospate buffer solution (25 ml, 1:1 v/v) and yielded 62% polycardanol over 6 h. Cobalt naphthenate (0.5% w/w) catalyzed the crosslinking of polycardanol and the final hardness of crosslinked polycardanol film exceeded 9 H scale as pencil scratch hardness, which shows a high potential as a commercial coating material. In addition, it showed an excellent anti-biofouling activity to Pseudomonas fluorescens compared to other polymeric materials such as polypropylene. PMID:14571976

  6. Apoplastic Peroxidases are Required for Salicylic Acid-Mediated Defense Against Pseudomonas syringae

    PubMed Central

    Mammarella, Nicole D.; Cheng, Zhenyu; Fu, Zheng Qing; Daudi, Arsalan; Bolwell, G. Paul; Dong, Xinnian; Ausubel, Frederick M.

    2014-01-01

    Reactive oxygen species (ROS) generated by NADPH oxidases or apoplastic peroxidases play an important role in the plant defense response. Diminished expression of at least two Arabidopsis thaliana peroxidase encoding genes, PRX33 (At3g49110) and PRX34 (At3g49120), as a consequence of anti-sense expression of a heterologous French bean peroxidase gene (asFBP1.1), were previously shown to result in reduced levels of ROS following pathogen attack, enhanced susceptibility to a variety of bacterial and fungal pathogens, and reduced levels of callose production and defense-related gene expression in response to the microbe associated molecular pattern (MAMP) molecules flg22 and elf26. These data demonstrated that the peroxidase-dependent oxidative burst plays an important role in the elicitation of pattern-triggered immunity (PTI). Further work reported in this paper, however, shows that asFBP1.1 antisense plants are not impaired in all PTI-associated responses. For example, some but not all flg22-elicited genes are induced to lower levels by flg22 in asFPB1.1, and callose deposition in asFPB1.1 is similar to wild-type following infiltration with a Pseudomonas syringae hrcC mutant or with non-host P. syringae pathovars. Moreover, asFPB1.1 plants did not exhibit any apparent defect in their ability to mount a hypersensitive response (HR). On the other hand, salicylic acid (SA)-mediated activation of PR1 was dramatically impaired in asFPB1.1 plants. In addition, P. syringae-elicited expression of many genes known to be SA-dependent was significantly reduced in asFBP1.1 plants. Consistent with this latter result, in asFBP1.1 plants the key regulator of SA-mediated responses, NPR1, showed both dramatically decreased total protein abundance and a failure to monomerize, which is required for its translocation into the nucleus. PMID:25096754

  7. Glutamate dehydrogenase 1 signals through antioxidant glutathione peroxidase 1 to regulate redox homeostasis and tumor growth

    PubMed Central

    Jin, Lingtao; Li, Dan; Alesi, Gina N.; Fan, Jun; Kang, Hee-Bum; Lu, Zhou; Boggon, Titus J.; Jin, Peng; Yi, Hong; Wright, Elizabeth R.; Duong, Duc; Seyfried, Nicholas T.; Egnatchik, Robert; DeBerardinis, Ralph J.; Magliocca, Kelly R.; He, Chuan; Arellano, Martha L.; Khoury, Hanna J.; Shin, Dong M.; Khuri, Fadlo R.; Kang, Sumin

    2015-01-01

    SUMMARY How mitochondrial glutaminolysis contributes to redox homeostasis in cancer cells remains unclear. Here we report that the mitochondrial enzyme glutamate dehydrogenase 1 (GDH1) is commonly upregulated in human cancers. GDH1 is important for redox homeostasis in cancer cells by controlling the intracellular levels of its product alpha-ketoglutarate (α-KG) and subsequent metabolite fumarate. Mechanistically, fumarate binds to and activates a ROS scavenging enzyme glutathione peroxidase 1 (GPx1). Targeting GDH1 by shRNA or a small molecule inhibitor R162 resulted in imbalanced redox homeostasis, leading to attenuated cancer cell proliferation and tumor growth. PMID:25670081

  8. Abnormal fucosylation of ileal mucus in cystic fibrosis: I. A histochemical study using peroxidase labelled lectins.

    PubMed Central

    Thiru, S; Devereux, G; King, A

    1990-01-01

    Peroxidase conjugated lectins were used to analyse the glycoproteins of small intestinal mucins in normal infants and those with cystic fibrosis to ascertain whether there are any detectable histochemical differences in saccharide composition. A significant decrease in Lotus tetragonolobus (LTG) binding fucose was shown in normal small intestinal mucin starting around 36 weeks' gestation with total absence of staining at term and beyond. In contrast, the age matched patients with cystic fibrosis showed persistent and intense LTG binding of fucose. These results provide the first clear histochemical evidence that cystic fibrosis mucin is abnormal and confirm the findings of previous biochemical studies. Images PMID:2266173

  9. Unusually high thermal stability and peroxidase activity of cytochrome c in ionic liquid colloidal formulation.

    PubMed

    Bharmoria, Pankaj; Kumar, Arvind

    2016-01-11

    Ionic liquid (IL) surfactant choline dioctylsulfosuccinate, [Cho][AOT], formed polydispersed vesicular structures in the IL, ethylmethylimidazolium ethylsulfate, [C2mim][C2OSO3]. Cytochrome c dissolved in such a colloidal medium has shown very high peroxidase activity (∼2 times to that in neat IL and ∼4 times to that in an aqueous buffer). Significantly, the enzyme retained both structural stability and functional activity in IL colloidal solutions up to 180 °C, demonstrating the suitability of the system as a high temperature bio-catalytic reactor. PMID:26529242

  10. Chemical enhancement of footwear impressions in blood on fabric - part 2: peroxidase reagents.

    PubMed

    Farrugia, Kevin J; Savage, Kathleen A; Bandey, Helen; Ciuksza, Tomasz; Nic Daéid, Niamh

    2011-09-01

    This study investigates the optimisation of peroxidase based enhancement techniques for footwear impressions made in blood on various fabric surfaces. Four different haem reagents: leuco crystal violet (LCV), leuco malachite green (LMG), fluorescein and luminol were used to enhance the blood contaminated impressions. The enhancement techniques in this study were used successfully to enhance the impressions in blood on light coloured surfaces, however, only fluorescent and/or chemiluminescent techniques allowed visualisation on dark coloured fabrics, denim and leather. Luminol was the only technique to enhance footwear impressions made in blood on all the fabrics investigated in this study. PMID:21889107

  11. Epithelial Nitration by a Peroxidase/NOX5 System Mediates Mosquito Antiplasmodial Immunity

    PubMed Central

    de Almeida Oliveira, Giselle; Lieberman, Joshua; Barillas-Mury, Carolina

    2012-01-01

    Plasmodium ookinetes traverse midgut epithelial cells before they encounter the complement system in the mosquito hemolymph. We identified a heme peroxidase (HPX2) and NADPH oxidase 5 (NOX5) as critical mediators of midgut epithelial nitration and antiplasmodial immunity that enhance nitric oxide toxicity in Anopheles gambiae. We show that the two immune mechanisms that target ookinetes—epithelial nitration and thioester-containing protein 1 (TEP1)-mediated lysis—work sequentially and propose that epithelial nitration works as an opsonization-like system that promotes activation of the mosquito complement cascade. PMID:22282475

  12. Epithelial nitration by a peroxidase/NOX5 system mediates mosquito antiplasmodial immunity.

    PubMed

    Oliveira, Giselle de Almeida; Lieberman, Joshua; Barillas-Mury, Carolina

    2012-02-17

    Plasmodium ookinetes traverse midgut epithelial cells before they encounter the complement system in the mosquito hemolymph. We identified a heme peroxidase (HPX2) and NADPH oxidase 5 (NOX5) as critical mediators of midgut epithelial nitration and antiplasmodial immunity that enhance nitric oxide toxicity in Anopheles gambiae. We show that the two immune mechanisms that target ookinetes-epithelial nitration and thioester-containing protein 1 (TEP1)-mediated lysis-work sequentially, and we propose that epithelial nitration works as an opsonization-like system that promotes activation of the mosquito complement cascade. PMID:22282475

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

    PubMed

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

    1980-02-01

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

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

    PubMed

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

    2016-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  17. Characterization and application of a novel class II thermophilic peroxidase from Myceliophthora thermophila in biosynthesis of polycatechol.

    PubMed

    Zerva, Anastasia; Christakopoulos, Paul; Topakas, Evangelos

    2015-01-01

    A peroxidase from the thermophilic fungus Myceliophthora thermophila that belongs to ascomycete Class II based on PeroxiBase classification was functionally expressed in methylotrophic yeast Pichia pastoris. The putative peroxidase from the genomic DNA was successfully cloned in P. pastoris X-33 under the transcriptional control of the alcohol oxidase (AOX1) promoter. The heterologous production was greatly enhanced by the addition of hemin with a titer of 0.41 U mL(-1) peroxidase activity at the second day of incubation. The recombinant enzyme was purified to homogeneity (50 kDa) and characterized using a series of phenolic substrates that indicated similar characteristics with those of generic peroxidases. In addition, the enzyme was found thermostable, retaining its activity for temperatures up to 60 °C after eight hours incubation. Moreover, the enzyme displayed remarkable H2O2 stability, retaining more than 80% of its initial activity after 24h incubation in 5000-fold molar excess of H2O2. The ability of the peroxidase to polymerize catechol at high superoxide concentrations, together with its high thermostability and substrate specificity, indicate a potential commercial significance of the enzyme. PMID:26047916

  18. Purification, Characterization, and Gene Cloning of Ceriporiopsis sp. Strain MD-1 Peroxidases That Decolorize Human Hair Melanin▿

    PubMed Central

    Nagasaki, Kenji; Kumazawa, Masaro; Murakami, Shuichiro; Takenaka, Shinji; Koike, Kenzo; Aoki, Kenji

    2008-01-01

    Ceriporiopsis sp. strain MD-1, isolated from forest soil, produced several extracellular enzymes that decolorized human hair melanin. Among them, three enzymes (E1, E2-1, and E2-2) were purified to homogeneity and characterized. The enzymes required hydrogen peroxide in their enzyme reactions and, typical of other fungal peroxidases, oxidized various phenol compounds such as guaiacol, but not 3,4-dimethoxybenzyl alcohol. The spectra of the three enzymes showed an absorption maximum at 406 nm, indicating that they were heme proteins. However, the A406/A280 values of the enzymes were below 0.4, which was lower than those of other peroxidases. E2-1 and E2-2 were similar to each other in their molecular and catalytic properties, and they possibly represent products of posttranslational modifications and/or allelic variants of the same gene, mdcA. The corresponding cDNA was cloned and sequenced; the deduced amino acid sequence showed high identities to the manganese peroxidases from other microorganisms. The specific activities and Km values of E2-1 and E2-2 for synthetic and human hair melanins were much higher than those of Phanerochaete chrysosporium manganese peroxidase and lignin peroxidase. PMID:18586974

  19. Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I

    SciTech Connect

    Chinchilla, Diana Kilheeney, Heather Vitello, Lidia B. Erman, James E.

    2014-01-03

    Highlights: •Cytochrome c peroxidase (CcP) binds acrylonitrile in a pH-independent fashion. •The spectrum of the CcP/acrylonitrile complex is that of a 6c–ls ferric heme. •The acrylonitrile/CcP complex has a K{sub D} value of 1.1 ± 0.2 M. •CcP compound I oxidizes acrylonitrile with a maximum turnover rate of 0.61 min{sup −1}. -- Abstract: Ferric heme proteins bind weakly basic ligands and the binding affinity is often pH dependent due to protonation of the ligand as well as the protein. In an effort to find a small, neutral ligand without significant acid/base properties to probe ligand binding reactions in ferric heme proteins we were led to consider the organonitriles. Although organonitriles are known to bind to transition metals, we have been unable to find any prior studies of nitrile binding to heme proteins. In this communication we report on the equilibrium and kinetic properties of acrylonitrile binding to cytochrome c peroxidase (CcP) as well as the oxidation of acrylonitrile by CcP compound I. Acrylonitrile binding to CcP is independent of pH between pH 4 and 8. The association and dissociation rate constants are 0.32 ± 0.16 M{sup −1} s{sup −1} and 0.34 ± 0.15 s{sup −1}, respectively, and the independently measured equilibrium dissociation constant for the complex is 1.1 ± 0.2 M. We have demonstrated for the first time that acrylonitrile can bind to a ferric heme protein. The binding mechanism appears to be a simple, one-step association of the ligand with the heme iron. We have also demonstrated that CcP can catalyze the oxidation of acrylonitrile, most likely to 2-cyanoethylene oxide in a “peroxygenase”-type reaction, with rates that are similar to rat liver microsomal cytochrome P450-catalyzed oxidation of acrylonitrile in the monooxygenase reaction. CcP compound I oxidizes acrylonitrile with a maximum turnover number of 0.61 min{sup −1} at pH 6.0.

  20. Direct measurements of intramolecular electron transfer rates between cytochrome c and cytochrome c peroxidase: effects of exothermicity and primary sequence on rate

    SciTech Connect

    Cheung, E.; Taylor, K.; Kornblatt, J.A.; English, A.M.; McLendon, G.; Miller, J.R.

    1986-03-01

    Rapid mixing of ferrocytochrome c peroxidase (cyt c peroxidase(II)) and ferricytochrome c (cyt c(III)) results in the reduction of cyt c(III) by cyt c peroxidase(II). In 10 mM phosphate, pH 7.0, the rate of decay of cyt c peroxidase(II) and the rate of accumulation of cyt c(II) give equal first-order rate constants. Equivalent results are obtained by pulse radiolysis using isopropanol radical as the reducing agent. This rate is independent of the initial cyt c(III):cyt c peroxidase(II) ratios. These results are consistent with unimolecular electron transfer occurring within a cyt c(III)-cyt c peroxidase(II) complex. When cyt c is replaced by porphyrin cyt c (iron-free cyt c), a complex still forms with cyt c peroxidase. On radiolysis intracomplex electron transfer occurs from the porphyrin cyt c anion radical to cyt c peroxidase(III). This large rate increase suggest that the barrier for intracomplex electron transfer is large. Finally, the authors have briefly investigated how the cyt c peroxidase(II) ..-->.. cyt c(III) rate depends on the primary structure of cyt c(III). They find the reactivity order to be as follows: yeast > horse > tuna.

  1. Regulation of the activity of Korean radish cationic peroxidase promoter during dedifferentiation and differentiation.

    PubMed

    Kim, Soung Soo; Choi, Suh-Yeon; Park, Jin-Hyoun; Lee, Dong Ju

    2004-12-01

    Studies of the regulation of the activity of the Korean radish cationic peroxidase (KRCP) promoter during dedifferentiation and redifferentiation are reported here. Histochemical staining with 5-bromo-4-chloro-indolyl glucuronide (X-gluc) showed that only dedifferentiated marginal cells of leaf discs of the transgenic plants, but not of the interior region, were stained blue, as leaf discs were incubated on dedifferentiation-inducing medium from 5 days after callus induction (DACI). The levels of cationic peroxidase activity and of KRCP transcripts in Korean radish seedlings (Raphanus sativus L. F1 Handsome Fall) were also upregulated by a low ratio of cytokinin to auxin, but not by high concentrations of cytokinin. To identify important cis-regulatory regions controlling callus-specific expression, a series of 5' promoter deletions was carried out with KRCP::GUS gene fusion systems. The data suggest that at least two positively regulatory regions are involved in the KRCP::GUS expression during dedifferentiation induced by a low ratio of cytokinin to auxin: one from -471 to -242 and another from -241 to +196. GUS expression, however, was quickly decreased to a basal level during regeneration of root and shoot. Thus, the downstream region between +197 and +698 seems to be enough to suppress GUS expression of all constructs during regeneration. We further show that the 142-bp fragment (-471 to -328) has at least one cis-element to bind to the nuclear proteins from Korean radish seedlings induced by dedifferentiation. PMID:15596095

  2. S-Nitrosylation Positively Regulates Ascorbate Peroxidase Activity during Plant Stress Responses1

    PubMed Central

    Yang, Huanjie; Mu, Jinye; Chen, Lichao; Feng, Jian; Hu, Jiliang; Li, Lei; Zhou, Jian-Min; Zuo, Jianru

    2015-01-01

    Nitric oxide (NO) and reactive oxygen species (ROS) are two classes of key signaling molecules involved in various developmental processes and stress responses in plants. The burst of NO and ROS triggered by various stimuli activates downstream signaling pathways to cope with abiotic and biotic stresses. Emerging evidence suggests that the interplay of NO and ROS plays a critical role in regulating stress responses. However, the underpinning molecular mechanism remains poorly understood. Here, we show that NO positively regulates the activity of the Arabidopsis (Arabidopsis thaliana) cytosolic ascorbate peroxidase1 (APX1). We found that S-nitrosylation of APX1 at cysteine (Cys)-32 enhances its enzymatic activity of scavenging hydrogen peroxide, leading to the increased resistance to oxidative stress, whereas a substitution mutation at Cys-32 causes the reduction of ascorbate peroxidase activity and abolishes its responsiveness to the NO-enhanced enzymatic activity. Moreover, S-nitrosylation of APX1 at Cys-32 also plays an important role in regulating immune responses. These findings illustrate a unique mechanism by which NO regulates hydrogen peroxide homeostasis in plants, thereby establishing a molecular link between NO and ROS signaling pathways. PMID:25667317

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

    PubMed

    Shrivastav, Tulsidas G

    2004-01-01

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

  4. Coloidal gold, ferritin and peroxidase as markers for electron microscopic double labeling lectin techniques.

    PubMed

    Roth, J; Binder, M

    1978-03-01

    Three markers, colloidal gold, ferritin and peroxidase, were checked for usefulness in double labeling of lectin-binding sites. The amount of various lectins for the stabilization of good sols of a different particle size was evaluated. Several lectin-gold complexes were prepared for electron microscopic labeling purposes, and the optimal amount of various lectins needed for stabilization of gold solutions of a different particle size was determined. The following combinations were investigated for their usefulness in labeling two different lectin-binding sites: lectin-gold and lectin-gold (different particle size), lectin-gold and lectin-ferritin, as well as lectin-ferritin and lectin-peroxidase. Of these combinations the latter did not give satisfactory results for double labeling. In all single and double labeling techniques with the above mentioned markers the quantitative evaluation of the number of lectin-binding sites is not feasible, but these techniques will be of considerable value for the investigation of the dynamics of different lectin-binding sites on the cell surface. PMID:632554

  5. Peroxidase-like activity of mesoporous silica encapsulated Pt nanoparticle and its application in colorimetric immunoassay.

    PubMed

    Wang, Zhifei; Yang, Xia; Yang, Jingjing; Jiang, Yanyun; He, Nongyue

    2015-03-01

    Nanomaterial-based artificial enzymes have received great attention in recent year due to their potential application in immunoassay techniques. However, such potential is usually limited by poor dispersion stability or low catalytic activity induced by the capping agent essentially required in the synthesis. In an attempt to address these challenges, here, we studied the novel Pt nanoparticles (NPs) based peroxidase-like mimic by encapsulating Pt NP in mesoporous silica (Pt@mSiO2 NPs). Compared with other nanomaterial-based artificial enzymes, the obtained Pt@mSiO2 NPs not only exhibit high peroxidase-like activity but also have good dispersion stability in buffer saline solution when grafted with spacer PEG. Results show that when the thickness of silica shell is about 9 nm the resulting Pt@mSiO2 NPs exhibit the catalytic activity similar to that of Pt NPs, which is approximately 26 times higher than that of Fe3O4 NPs (in terms of Kcat for H2O2). Due to the protection of silica shell, the subsequent surface modification with antibody has little effect on their catalytic activity. The analytical performance of this system in detecting hCG shows that after 5 min incubation the limit of detection can reach 10 ng mL(-1) and dynamic linear working range is 5-200 ng mL(-1). Our findings pave the way for design and development of novel artificial enzyme labeling. PMID:25682428

  6. Spatiotemporal Secretion of PEROXIDASE36 Is Required for Seed Coat Mucilage Extrusion in Arabidopsis[W

    PubMed Central

    Kunieda, Tadashi; Shimada, Tomoo; Kondo, Maki; Nishimura, Mikio; Nishitani, Kazuhiko; Hara-Nishimura, Ikuko

    2013-01-01

    The epidermal cells of the Arabidopsis thaliana seed coat, which correspond to the second layer of the outer integument (oi2), contain large quantities of a pectic polysaccharide called mucilage within the apoplastic space beneath the outer periclinal cell wall. Immediately after seed imbibition, the mucilage is extruded and completely envelops the seed in a gel-like capsule. We found that a class III peroxidase family protein, PEROXIDASE36 (PER36), functions as a mucilage extrusion factor. Expression of PER36 occurred only in oi2 cells for a few days around the torpedo stage. A PER36–green fluorescent protein fusion was secreted into the outer cell wall in a polarized manner. per36 mutants were defective in mucilage extrusion after seed imbibition due to the failure of outer cell wall rupture, although the mutants exhibited normal monosaccharide composition of the mucilage. This abnormal phenotype of per36 was rescued by pectin solubilization, which promoted cell wall loosening. These results suggest that PER36 regulates the degradation of the outer cell wall. Taken together, this work indicates that polarized secretion of PER36 in a developmental stage-dependent manner plays a role in cell wall modification of oi2 cells. PMID:23572548

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

    PubMed

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

    2015-03-15

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

  8. Peroxidase-like activity of apoferritin paired gold clusters for glucose detection.

    PubMed

    Jiang, Xin; Sun, Cuiji; Guo, Yi; Nie, Guangjun; Xu, Li

    2015-02-15

    The discovery and application of noble metal nanoclusters have received considerable attention. In this paper, we reported that apoferritin paired gold clusters (Au-Ft) could efficiently catalyze oxidation of 3.3',5.5'-tetramethylbenzidine (TMB) by H2O2 to produce a blue color reaction. Compared with natural enzyme, Au-Ft exhibited higher activity near acidic pH and could be used over a wide range of temperatures. Apoferritin nanocage enhanced the reaction activity of substrate TMB by H2O2. The reaction catalyzed by Au-Ft was found to follow a typical Michaelis-Menten kinetics. The kinetic parameters exhibited a lower K(m) value (0.097 mM) and a higher K(cat) value (5.8 × 10(4) s(-1)) for TMB than that of horse radish peroxidase (HRP). Base on these findings, Au-Ft, acting as a peroxidase mimetic, performed enzymatic spectrophotometric analysis of glucose. This system exhibited acceptable reproducibility and high selectivity in biosening, suggesting that it could have promising applications in the future. PMID:25218100

  9. Fructans, ascorbate peroxidase, and hydrogen peroxide in ryegrass exposed to ozone under contrasting meteorological conditions.

    PubMed

    Pasqualetti, C B; Sandrin, C Z; Pedroso, A N V; Domingos, M; Figueiredo-Ribeiro, R C L

    2015-03-01

    Ozone (O3) is the most abundant tropospheric oxidant as well as an important component of photochemical pollution. Once inside the plant, ozone can produce reactive oxygen species that change the antioxidative pool and the carbohydrate metabolism. The current study aimed to analyze whether the contents and the composition of the fructan, the ascorbate peroxidase activity, and the H2O2 accumulation were changed in Lolium multiflorum ssp. italicum cv. Lema plants as response to short-term exposure to ozone and/or to different meteorological conditions, in two contrasting seasons (winter and summer). Results showed that higher solar radiation tends to decrease fructose content and, along with temperature, increases the ascorbate peroxidase (APX) activity. Such activity and levels of fructans practically did not vary during the time the experiment was being done, but APX daylight variation was modified by the ozone. Thus, the higher levels of this pollutant decreased the APX activity and increased fructose content, as well as changed the size of the fructan chains. Hydrogen peroxide (H2O2) accumulation was higher in plants that were fumigated with ozone when compared to the control, and it decreased throughout the day. As a conclusion, fructan contents increased when the APX activity decreased. It suggested that fructans could also help the defense system when there is a reduction on the APX activity in the plant. PMID:25583262

  10. Identifying peroxidases and their oxidants in the early pathology of cystic fibrosis.

    PubMed

    Thomson, Eline; Brennan, Siobhain; Senthilmohan, Revathy; Gangell, Catherine L; Chapman, Anna L; Sly, Peter D; Kettle, Anthony J; Balding, Elizabeth; Berry, Luke J; Carlin, John B; Carzino, Rosemary; de Klerk, Nick; Douglas, Tonia; Foo, Clara; Garratt, Luke W; Hall, Graham L; Harrison, Jo; Kicic, Anthony; Laing, Ingrid A; Logie, Karla M; Massie, John; Mott, Lauren S; Murray, Conor; Parsons, Faith; Pillarisetti, Naveen; Poreddy, Srinivas R; Ranganathan, Sarath C; Robertson, Colin F; Robins-Browne, Roy; Robinson, Philip J; Skoric, Billy; Stick, Stephen M; Sutanto, Erika N; Williamson, Elizabeth

    2010-11-15

    We aimed to determine whether myeloperoxidase (MPO) is the main peroxidase present in the airways of children with cystic fibrosis (CF) and to assess which oxidants it produces and whether they are associated with clinical features of CF. Children with CF (n=54) and without CF (n=16) underwent bronchoscopy and bronchoalveolar lavage (BAL) for assessment of pulmonary infection and inflammation. BAL fluid was analyzed for MPO, halogenated tyrosines as markers of hypohalous acids, thiocyanate, and protein carbonyls. MPO was the only peroxidase detected in BAL samples from children with CF and its concentration was markedly higher than in controls. Levels of 3-chlorotyrosine and 3-bromotyrosine in proteins were higher in the CF group. They correlated with neutrophils and MPO. The concentration of thiocyanate in BAL samples was below 1μM. Protein carbonyl levels correlated with MPO and halogenated tyrosines in patients with CF. Levels of MPO and halogenated tyrosines were higher in children with infections, especially Pseudomonas aeruginosa, and in the presence of respiratory symptoms. They also correlated with the Kanga clinical score. Our findings suggest that MPO produces hypobromous acid as well as hypochlorous acid in the airways of children with CF and that these oxidants are involved in the early pathogenesis of CF. PMID:20647044

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

    PubMed Central

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

    2012-01-01

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

  12. Isonicotinic acid hydrazide conversion to Isonicotinyl-NAD by catalase-peroxidases.

    PubMed

    Wiseman, Ben; Carpena, Xavi; Feliz, Miguel; Donald, Lynda J; Pons, Miquel; Fita, Ignacio; Loewen, Peter C

    2010-08-20

    Activation of the pro-drug isoniazid (INH) as an anti-tubercular drug in Mycobacterium tuberculosis involves its conversion to isonicotinyl-NAD, a reaction that requires the catalase-peroxidase KatG. This report shows that the reaction proceeds in the absence of KatG at a slow rate in a mixture of INH, NAD(+), Mn(2+), and O(2), and that the inclusion of KatG increases the rate by >7 times. Superoxide, generated by either Mn(2+)- or KatG-catalyzed reduction of O(2), is an essential intermediate in the reaction. Elimination of the peroxidatic process by mutation slows the rate of reaction by 60% revealing that the peroxidatic process enhances, but is not essential for isonicotinyl-NAD formation. The isonicotinyl-NAD(*+) radical is identified as a reaction intermediate, and its reduction by superoxide is proposed. Binding sites for INH and its co-substrate, NAD(+), are identified for the first time in crystal complexes of Burkholderia pseudomallei catalase-peroxidase with INH and NAD(+) grown by co-crystallization. The best defined INH binding sites were identified, one in each subunit, on the opposite side of the protein from the entrance to the heme cavity in a funnel-shaped channel. The NAD(+) binding site is approximately 20 A from the entrance to the heme cavity and involves interactions primarily with the AMP portion of the molecule in agreement with the NMR saturation transfer difference results. PMID:20554537

  13. Multidomain Human Peroxidasin 1 Is a Highly Glycosylated and Stable Homotrimeric High Spin Ferric Peroxidase*

    PubMed Central

    Soudi, Monika; Paumann-Page, Martina; Delporte, Cedric; Pirker, Katharina F.; Bellei, Marzia; Edenhofer, Eva; Stadlmayr, Gerhard; Battistuzzi, Gianantonio; Boudjeltia, Karim Zouaoui; Furtmüller, Paul G.; Van Antwerpen, Pierre; Obinger, Christian

    2015-01-01

    Human peroxidasin 1 (hsPxd01) is a multidomain heme peroxidase that uses bromide as a cofactor for the formation of sulfilimine cross-links. The latter confers critical structural reinforcement to collagen IV scaffolds. Here, hsPxd01 and various truncated variants lacking nonenzymatic domains were recombinantly expressed in HEK cell lines. The N-glycosylation site occupancy and disulfide pattern, the oligomeric structure, and unfolding pathway are reported. The homotrimeric iron protein contains a covalently bound ferric high spin heme per subunit with a standard reduction potential of the Fe(III)/Fe(II) couple of −233 ± 5 mV at pH 7.0. Despite sequence homology at the active site and biophysical properties similar to human peroxidases, the catalytic efficiency of bromide oxidation (kcat/KMapp) of full-length hsPxd01 is rather low but increased upon truncation. This is discussed with respect to its structure and proposed biosynthetic function in collagen IV cross-linking. PMID:25713063

  14. Peroxidase is involved in Pepper yellow mosaic virus resistance in Capsicum baccatum var. pendulum.

    PubMed

    Gonçalves, L S A; Rodrigues, R; Diz, M S S; Robaina, R R; do Amaral Júnior, A T; Carvalho, A O; Gomes, V M

    2013-01-01

    Pathogenesis-related proteins (PRs) are among the defense mechanisms of plants that work as an important barrier to the development of pathogens. These proteins are classified into 17 families according to their amino acid sequences, serology, and/or biological or enzyme activity. The present study aimed to identify PRs associated with the pathosystem of Capsicum baccatum var. pendulum: Pepper yellow mosaic virus (PepYMV). Forty-five-day-old plants from accession UENF 1624, previously identified as resistant to PepYMV, were inoculated with the virus. Control and infected leaves were collected for analysis after 24, 48, 72, and 96 h. The inoculated and control plants were grown in cages covered with anti-aphid screens. Proteins were extracted from leaf tissue and the presence of β-1,3-glucanase, chitinase, peroxidase, and lipid transport protein was verified. No difference was observed between the protein pattern of control and infected plants when β-1,3-glucanase, chitinase, and lipid transport protein were compared. However, increased peroxidase expression was observed in infected plants at 48 and 72 h after inoculation, indicating that this PR is involved in the response of resistance to PepYMV in C. baccatum var. pendulum. PMID:23661464

  15. Colorimetric Glucose Assay Based on Magnetic Particles Having Pseudo-peroxidase Activity and Immobilized Glucose Oxidase.

    PubMed

    Martinkova, Pavla; Opatrilova, Radka; Kruzliak, Peter; Styriak, Igor; Pohanka, Miroslav

    2016-05-01

    Magnetic particles (MPs) are currently used as a suitable alternative for peroxidase in the construction of novel biosensors, analytic and diagnostic methods. Their better chemical and thermal stabilities predestine them as appropriate pseudo-enzymatic catalysts. In this point of view, our research was focused on preparation of simply and fast method for immobilization of glucose oxidase onto surface of MPs with peroxidase-like activity. Spectrophotometric method (wavelength 450 nm) optimized for glucose determination using modified MPs has been successfully developed. Concentration curve for optimization of method was assayed, and Michaelis-Menten constant (K m) calculated, maximum reaction rate (V max), limit of detection, and correlation coefficient were determined to be 0.13 mmol/l (2.34 mg/dl), 1.79 pkat, 3.74 µmol/l (0.067 mg/dl), and 0.996, respectively. Interferences of other sugars such as sucrose, sorbitol, deoxyribose, maltose, and fructose were determined as well as effect of substances presenting in plasma (ascorbic acid, reduced glutathione, trolox, and urea). Results in comparison with positive and negative controls showed no interferences of the other sugars and no influence of plasma substances to measuring of glucose. The constructed method showed corresponding results with linear dependence and a correlation coefficient of 0.997. Possibility of repeated use of modified MPs was successfully proved. PMID:27041274

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

    PubMed Central

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

    1991-01-01

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

  17. Crystal Structure of Two Anti-Porphyrin Antibodies with Peroxidase Activity

    PubMed Central

    Maréchal, Jean-Didier; Bahloul, Amel; Sari, Marie-Agnès; Mahy, Jean-Pierre

    2012-01-01

    We report the crystal structures at 2.05 and 2.45 Å resolution of two antibodies, 13G10 and 14H7, directed against an iron(III)-αααβ-carboxyphenylporphyrin, which display some peroxidase activity. Although these two antibodies differ by only one amino acid in their variable λ-light chain and display 86% sequence identity in their variable heavy chain, their complementary determining regions (CDR) CDRH1 and CDRH3 adopt very different conformations. The presence of Met or Leu residues at positions preceding residue H101 in CDRH3 in 13G10 and 14H7, respectively, yields to shallow combining sites pockets with different shapes that are mainly hydrophobic. The hapten and other carboxyphenyl-derivatized iron(III)-porphyrins have been modeled in the active sites of both antibodies using protein ligand docking with the program GOLD. The hapten is maintained in the antibody pockets of 13G10 and 14H7 by a strong network of hydrogen bonds with two or three carboxylates of the carboxyphenyl substituents of the porphyrin, respectively, as well as numerous stacking and van der Waals interactions with the very hydrophobic CDRH3. However, no amino acid residue was found to chelate the iron. Modeling also allows us to rationalize the recognition of alternative porphyrinic cofactors by the 13G10 and 14H7 antibodies and the effect of imidazole binding on the peroxidase activity of the 13G10/porphyrin complexes. PMID:23240001

  18. Polyoxometalates as peroxidase mimetics and their applications in H2O2 and glucose detection.

    PubMed

    Wang, Jingjing; Han, Dongxue; Wang, Xiaohong; Qi, Bin; Zhao, Meisheng

    2012-01-01

    Polyoxometalates (H(3)PW(12)O(40), H(4)SiW(12)O(40) and H(3)PMo(12)O(40)) have been proven to possess intrinsic peroxidase-like activity for the first time, which can catalyze oxidation of the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) by H(2)O(2) to form a blue color in aqueous solution. Among them, H(3)PW(12)O(40) (PW(12)) exhibits higher catalytic activity to TMB than natural enzyme HRP and other two POMs. In addition, H(3)PW(12)O(40)/graphene exhibited higher activity than H(3)PW(12)O(40) in this catalytic oxidation reaction due to the effect of graphene in promoting the electron transfer between the substrate and catalyst. POMs/H(2)O(2)/TMB system provides a simple, accurate approach to colorimetric detection for H(2)O(2) or glucose. The colorimetric method based on POMs showed good response toward H(2)O(2) and glucose detection with a linear range from 1.34×10(-7) to 6.7×10(-5) mol/L and 1×10(-7) to 1×10(-4) mol/L, respectively. The results showed that it is a simple, cheap, more convenient, highly selective, sensitive, and easy handling colorimetric assay. PMID:22560441

  19. Soybean peroxidase-mediated degradation of an azo dye– a detailed mechanistic study

    PubMed Central

    2013-01-01

    Background Peroxidases are emerging as an important class of enzymes that can be used for the efficient degradation of organic pollutants. However, detailed studies identifying the various intermediates produced and the mechanisms involved in the enzyme-mediated pollutant degradation are not widely published. Results In the present study, the enzymatic degradation of an azo dye (Crystal Ponceau 6R, CP6R) was studied using commercially available soybean peroxidase (SBP) enzyme. Several operational parameters affecting the enzymatic degradation of dye were evaluated and optimized, such as initial dye concentration, H2O2 dosage, mediator amount and pH of the solution. Under optimized conditions, 40 ppm dye solution could be completely degraded in under one minute by SBP in the presence of H2O2 and a redox mediator. Dye degradation was also confirmed using HPLC and TOC analyses, which showed that most of the dye was being mineralized to CO2 in the process. Conclusions Detailed analysis of metabolites, based on LC/MS results, showed that the enzyme-based degradation of the CP6R dye proceeded in two different reaction pathways- via symmetric azo bond cleavage as well as asymmetric azo bond breakage in the dye molecule. In addition, various critical transformative and oxidative steps such as deamination, desulfonation, keto-oxidation are explained on an electronic level. Furthermore, LC/MS/MS analyses confirmed that the end products in both pathways were small chain aliphatic carboxylic acids. PMID:24308857

  20. Peroxidase Mechanism of Lipid-dependent Cross-linking of Synuclein with Cytochrome c

    PubMed Central

    Bayır, Hülya; Kapralov, Alexandr A.; Jiang, Janfei; Huang, Zhentai; Tyurina, Yulia Y.; Tyurin, Vladimir A.; Zhao, Qing; Belikova, Natalia A.; Vlasova, Irina I.; Maeda, Akihiro; Zhu, Jianhui; Na, Hye-Mee; Mastroberardino, Pier-Giorgio; Sparvero, Louis J.; Amoscato, Andrew A.; Chu, Charleen T.; Greenamyre, John T.; Kagan, Valerian E.

    2009-01-01

    Damage of presynaptic mitochondria could result in release of proapoptotic factors that threaten the integrity of the entire neuron. We discovered that α-synuclein (Syn) forms a triple complex with anionic lipids (such as cardiolipin) and cytochrome c, which exerts a peroxidase activity. The latter catalyzes covalent hetero-oligomerization of Syn with cytochrome c into high molecular weight aggregates. Syn is a preferred substrate of this reaction and is oxidized more readily than cardiolipin, dopamine, and other phenolic substrates. Co-localization of Syn with cytochrome c was detected in aggregates formed upon proapoptotic stimulation of SH-SY5Y and HeLa cells and in dopaminergic substantia nigra neurons of rotenone-treated rats. Syn-cardiolipin exerted protection against cytochrome c-induced caspase-3 activation in a cell-free system, particularly in the presence of H2O2. Direct delivery of Syn into mouse embryonic cells conferred resistance to proapoptotic caspase-3 activation. Conversely, small interfering RNA depletion of Syn in HeLa cells made them more sensitive to dopamine-induced apoptosis. In human Parkinson disease substantia nigra neurons, two-thirds of co-localized Syn-cytochrome c complexes occurred in Lewy neurites. Taken together, these results indicate that Syn may prevent execution of apoptosis in neurons through covalent hetero-oligomerization of cytochrome c. This immediate protective function of Syn is associated with the formation of the peroxidase complex representing a source of oxidative stress and postponed damage. PMID:19351880

  1. Cytochrome P450 peroxidase/peroxygenase mediated xenobiotic metabolic activation and cytotoxicity in isolated hepatocytes.

    PubMed

    Anari, M R; Khan, S; Liu, Z C; O'Brien, P J

    1995-12-01

    Cytochrome P450 (P450) can utilize organic hydroperoxides and peracids to support hydroxylation and dealkylation of various P450 substrates. However, the biological significance of this P450 peroxygenase/peroxidase activity in the bioactivation of xenobiotics in intact cells has not been demonstrated. We have shown that tert-butyl hydroperoxide (tBHP) markedly enhances 3-20-fold the cytotoxicity of various aromatic hydrocarbons and their phenolic metabolites. The tBHP-enhanced hepatocyte cytotoxicity of 4-nitroanisole (4-NA) and 4-hydroxyanisole (4-HA) was also accompanied by an increase in the hepatocyte O-demethylation of 4-NA and 4-HA up to 7.5- and 21-fold, respectively. Hepatocyte GSH conjugation by 4-HA was also markedly increased by tBHP. An LC/MS analysis of the GSH conjugates identified hydroquinone-GSH and 4-methoxy-catechol:GSH conjugates as the predominant adducts. Pretreatment of hepatocytes with P450 inhibitors, e.g., phenylimidazole, prevented tBHP-enhanced 4-HA metabolism, GSH depletion, and cytotoxicity. In conclusion, hydroperoxides can therefore be used by intact cells to support the bioactivation of xenobiotics through the P450 peroxidase/peroxygenase system. PMID:8605292

  2. Lignin Peroxidase from Streptomyces viridosporus T7A: Enzyme Concentration Using Ultrafiltration

    NASA Astrophysics Data System (ADS)

    Gottschalk, Leda M. F.; Bon, Elba P. S.; Nobrega, Ronaldo

    It is well known that lignin degradation is a key step in the natural process of biomass decay whereby oxidative enzymes such as laccases and high redox potential ligninolytic peroxidases and oxidases play a central role. More recently, the importance of these enzymes has increased because of their prospective industrial use for the degradation of the biomass lignin to increase the accessibility of the cellulose and hemicellulose moieties to be used as renewable material for the production of fuels and chemicals. These biocatalysts also present potential application on environmental biocatalysis for the degradation of xenobiotics and recalcitrant pollutants. However, the cost for these enzymes production, separation, and concentration must be low to permit its industrial use. This work studied the concentration of lignin peroxidase (LiP), produced by Streptomyces viridosporus T7A, by ultrafiltration, in a laboratory-stirred cell, loaded with polysulfone (PS) or cellulose acetate (CA) membranes with molecular weight cutoffs (MWCO) of 10, 20, and 50 KDa. Experiments were carried out at 25 °C and pH 7.0 in accordance to the enzyme stability profile. The best process conditions and enzyme yield were obtained using a PS membrane with 10 KDa MWCO, whereby it was observed a tenfold LiP activity increase, reaching 1,000 U/L and 90% enzyme activity upholding.

  3. Identification of a bacterial di-haem cytochrome c peroxidase from Methylomicrobium album BG8.

    PubMed

    Karlsen, O A; Larsen, Ø; Jensen, H B

    2010-09-01

    The nucleotide sequence of an open reading frame (corB) downstream of the copper-repressible CorA-encoding gene of the methanotrophic bacterium Methylomicrobium album BG8 was obtained by restriction enzyme digestion and inverse PCR. The amino acid sequence deduced from this gene showed significant sequence similarity to the surface-associated di-haem cytochrome c peroxidase (SACCP) previously isolated from Methylococcus capsulatus (Bath), including both c-type haem-binding motifs. Homology analysis placed this protein, phylogenetically, within the subfamily containing the M. capsulatus SACCP of the bacterial di-haem cytochrome c peroxidase (BCCP) family of proteins. Immunospecific recognition confirmed synthesis of the M. album CorB as a protein non-covalently associated with the outer membrane and exposed to the periplasm. corB expression is regulated by the availability of copper ions during growth and the protein is most abundant in M. album when grown at a low copper-to-biomass ratio, indicating an important physiological role of CorB under these growth conditions. corB was co-transcribed with the gene encoding CorA, constituting a copper-responding operon, which appears to be under the control of a sigma(54)-dependent promoter. M. album CorB is the second isolated member of the recently described subfamily of the BCCP family of proteins. So far, these proteins have only been described in methanotrophic bacteria. PMID:20576687

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

    PubMed

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

    2013-06-01

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

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

    PubMed

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

    2012-01-01

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

  6. Prophage-Encoded Peroxidase in 'Candidatus Liberibacter asiaticus' Is a Secreted Effector That Suppresses Plant Defenses.

    PubMed

    Jain, Mukesh; Fleites, Laura A; Gabriel, Dean W

    2015-12-01

    'Candidatus Liberibacter asiaticus' is transmitted by psyllids and causes huanglongbing (HLB), a lethal disease of citrus. Most pathogenic 'Ca. L. asiaticus' strains carry two nearly identical prophages similar to SC1 and SC2 in strain UF506. SC2 was observed to replicate as a moderately high-copy excision plasmid encoding a reactive oxygen species-scavenging peroxidase (SC2_gp095), a predicted lysogenic conversion factor. SC2_gp095 was expressed at significantly higher levels in periwinkle than in citrus and was suppressed in psyllids. SC2_gp095 was cloned in a shuttle vector and transformed into Escherichia coli and Liberibacter crescens, a culturable proxy for 'Ca. L. asiaticus'. Transformed L. crescens cells showed 20 to 25% enhanced resistance to H₂O₂on agar plates, 47% greater enzymatic activity, and enhanced growth in liquid cultures. A nonclassical secretion potential was predicted for SC2_gp095 and secretion from L. crescens was confirmed by enzymatic and Western blot analyses. Transient expression of SC2_gp095 in planta resulted in strong transcriptional downregulation of RbohB, the key gatekeeper of the H₂O₂-mediated defense signaling in plants, helping explain the surprisingly long incubation period (years) before HLB symptoms appear in 'Ca. L. asiaticus'-infected citrus. 'Ca. L. asiaticus' peroxidase is likely a secreted, horizontally acquired effector that suppresses host symptom development, a tactic used by most biotrophic plant pathogens. PMID:26313412

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

    SciTech Connect

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

    1999-05-01

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

  8. 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.

  9. Functional and Structural Characterization of a Thiol Peroxidase from Mycobacterium tuberculosis

    SciTech Connect

    Rho,B.; Hung, L.; Holton, J.; Vigil, D.; Kim, S.; Park, M.; Terwilliger, T.; Pedelacq, j.

    2006-01-01

    A thiol peroxidase (Tpx) from Mycobacterium tuberculosis was functionally analyzed. The enzyme shows NADPH-linked peroxidase activity using a thioredoxin-thioredoxin reductase system as electron donor, and anti-oxidant activity in a thiol-dependent metal-catalyzed oxidation system. It reduces H{sub 2}O{sub 2}, t-butyl hydroperoxide, and cumene hydroperoxide, and is inhibited by sulfhydryl reagents. Mutational studies revealed that the peroxidatic (Cys60) and resolving (Cys93) cysteine residues are critical amino acids for catalytic activity. The X-ray structure determined to a resolution of 1.75 Angstroms shows a thioredoxin fold similar to that of other peroxiredoxin family members. Superposition with structural homologues in oxidized and reduced forms indicates that the M. tuberculosis Tpx is a member of the atypical two-Cys peroxiredoxin family. In addition, the short distance that separates the Ca atoms of Cys60 and Cys93 and the location of these cysteine residues in unstructured regions may indicate that the M. tuberculosis enzyme is oxidized, though the side-chain of Cys60 is poorly visible. It is solely in the reduced Streptococcus pneumoniae Tpx structure that both residues are part of two distinct helical segments. The M. tuberculosis Tpx is dimeric both in solution and in the crystal structure. Amino acid residues from both monomers delineate the active site pocket.

  10. Manganese Peroxidase, Produced by Trametes versicolor during Pulp Bleaching, Demethylates and Delignifies Kraft Pulp

    PubMed Central

    Paice, M. G.; Reid, I. D.; Bourbonnais, R.; Archibald, F. S.; Jurasek, L.

    1993-01-01

    Previous work has shown that Trametes (Coriolus) versicolor bleaches kraft pulp brownstock with the concomitant release of methanol. In this work, the fungus is shown to produce both laccase and manganese peroxidase (MnP) but not lignin peroxidase during pulp bleaching. MnP production was enhanced by the presence of pulp and/or Mn(II) ions. The maximum level of secreted MnP was coincident with the maximum rate of fungal bleaching. Culture filtrates isolated from bleaching cultures produced Mn(II)- and hydrogen peroxide-dependent pulp demethylation and delignification. Laccase and MnP were separated by ion-exchange chromatography. Purified MnP alone produced most of the demethylation and delignification exhibited by the culture filtrates. On the basis of the methanol released and the total and phenolic methoxyl contents of the pulp, it appears that MnP shows a preference for the oxidation of phenolic lignin substructures. The extensive increase in brightness observed in the fungus-treated pulp was not found with MnP alone. Therefore, either the MnP effect must be optimized or other enzymes or compounds from the fungus are also required for brightening. Images PMID:16348850

  11. Soybean ascorbate peroxidase suppresses Bax-induced apoptosis in yeast by inhibiting oxygen radical generation.

    PubMed

    Moon, Haejeong; Baek, Dongwon; Lee, Boyoung; Prasad, D Theertha; Lee, Sang Yeol; Cho, Moo Je; Lim, Chae Oh; Choi, Myung Suk; Bahk, Jeongdong; Kim, Myeong Ok; Hong, Jong Chan; Yun, Dae-Jin

    2002-01-11

    Bax, a mammalian proapoptotic member of the Bcl-2 family, can induce cell death when expressed in yeast or plant cells. To identify plant Bax inhibitors, we cotransformed a soybean cDNA library and the Bax gene into yeast cells and screened for expressed genes that prevented Bax-induced apoptosis. From the Bax-inhibiting genes isolated, ascorbate peroxidase (sAPX) was selected for characterization. The transcription of sAPX in plants was specifically induced by oxidative stress. Moreover, overexpression of sAPX partially suppressed the H(2)O(2)-sensitive phenotype of yeast cytosolic catalase T (Deltactt)- and thermosensitive phenotype of cytochrome c peroxidase (Deltaccp)-deleted mutant cells. Examination of reactive oxygen species (ROS) production using the fluorescence method of dihydrorhodamine 123 oxidation revealed that expression of Bax in yeast cells generated ROS, which was greatly reduced by coexpression with sAPX. Our results collectively suggest that sAPX inhibits the generation of ROS by Bax, which in turn suppresses Bax-induced cell death in yeast. PMID:11779192

  12. Effect of Diffusion on Discoloration of Congo Red by Alginate Entrapped Turnip (Brassica rapa) Peroxidase

    PubMed Central

    Ahmedi, Afaf; Abouseoud, Mahmoud; Abdeltif, Amrane; Annabelle, Couvert

    2015-01-01

    Enzymatic discoloration of the diazo dye, Congo red (CR), by immobilized plant peroxidase from turnip “Brassica rapa” is investigated. Partially purified turnip peroxidase (TP) was immobilized by entrapment in spherical particles of calcium alginate and was assayed for the discoloration of aqueous CR solution. Experimental data revealed that pH, reaction time, temperature, colorant, and H2O2 concentration play a significant role in dye degradation. Maximum CR removal was found at pH 2.0, constant temperature of 40°C in the presence of 10 mM H2O2, and 180 mg/L of CR. More than 94% of CR was removed by alginate immobilized TP after 1 h of incubation in a batch process under optimal conditions. About 74% removal efficiency was retained after four recycles. Diffusional limitations in alginate beads such as effectiveness factor η, Thiele modulus Φ, and effective diffusion coefficients (De) of Congo red were predicted assuming a first-order biodegradation kinetic. Results showed that intraparticle diffusion resistance has a significant effect on the CR biodegradation rate. PMID:25734011

  13. Effect of Diffusion on Discoloration of Congo Red by Alginate Entrapped Turnip (Brassica rapa) Peroxidase.

    PubMed

    Ahmedi, Afaf; Abouseoud, Mahmoud; Abdeltif, Amrane; Annabelle, Couvert

    2015-01-01

    Enzymatic discoloration of the diazo dye, Congo red (CR), by immobilized plant peroxidase from turnip "Brassica rapa" is investigated. Partially purified turnip peroxidase (TP) was immobilized by entrapment in spherical particles of calcium alginate and was assayed for the discoloration of aqueous CR solution. Experimental data revealed that pH, reaction time, temperature, colorant, and H2O2 concentration play a significant role in dye degradation. Maximum CR removal was found at pH 2.0, constant temperature of 40°C in the presence of 10 mM H2O2, and 180 mg/L of CR. More than 94% of CR was removed by alginate immobilized TP after 1 h of incubation in a batch process under optimal conditions. About 74% removal efficiency was retained after four recycles. Diffusional limitations in alginate beads such as effectiveness factor η, Thiele modulus Φ, and effective diffusion coefficients (D e ) of Congo red were predicted assuming a first-order biodegradation kinetic. Results showed that intraparticle diffusion resistance has a significant effect on the CR biodegradation rate. PMID:25734011

  14. Inactivation of glutathione peroxidase activity contributes to UV-induced squamous cell carcinoma formation.

    PubMed

    Walshe, Jennifer; Serewko-Auret, Magdalena M; Teakle, Ngari; Cameron, Sarina; Minto, Kelly; Smith, Louise; Burcham, Philip C; Russell, Terry; Strutton, Geoffrey; Griffin, Anthony; Chu, Fong-Fong; Esworthy, Stephen; Reeve, Vivienne; Saunders, Nicholas A

    2007-05-15

    Cutaneous squamous cell carcinomas (CSCC) are a common malignancy of keratinocytes that arise in sites of the skin exposed to excessive UV radiation. In the present study, we show that human SCC cell lines, preneoplastic solar keratoses (SK), and CSCC are associated with perturbations in glutathione peroxidase (GPX) activity and peroxide levels. Specifically, we found that two of three SKs and four of five CSCCs, in vivo, were associated with decreased GPX activity and all SKs and CSCCs were associated with an elevated peroxide burden. Given the association of decreased GPX activity with CSCC, we examined the basis for the GPX deficiency in the CSCCs. Our data indicated that GPX was inactivated by a post-translational mechanism and that GPX could be inactivated by increases in intracellular peroxide levels. We next tested whether the decreased peroxidase activity coupled with an elevated peroxidative burden might contribute to CSCC formation in vivo. This was tested in Gpx1(-/-) and Gpx2(-/-) mice exposed to solar-simulated UV radiation. These studies showed that Gpx2 deficiency predisposed mice to UV-induced CSCC formation. These results suggest that inactivation of GPX2 in human skin may be an early event in UV-induced SCC formation. PMID:17510403

  15. Mechanism-based suicide inactivation of white Spanish broom (Cytisus multiflorus) peroxidase by excess hydrogen peroxide.

    PubMed

    Galende, Patricia Pérez; Cuadrado, Nazaret Hidalgo; Kostetsky, Eduard Ya; Roig, Manuel G; Kennedy, John F; Shnyrov, Valery L

    2015-11-01

    Suicide inactivation is a common mechanism observed for haem peroxidases, in which the enzyme is inactivated as a result of self-oxidation mediated by intermediate highly oxidizing enzyme forms during the catalytic cycle. The time-dependence and the inactivation mechanism of Cytisus multiflorus peroxidase (CMP) by hydrogen peroxide were studied kinetically with four co-substrates (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), ferulic acid, guaiacol and o-dianisidine). Catalytic activity decreased following the sequence ABTS>guaiacol>ferulic acid>o-dianisidine. Once the intermediate complex (compound III-H2O2) had been formed, competition was established between the catalytic pathway and the suicide inactivation pathway. One mole of CMP afforded around 3790 turnovers of H2O2 for ABTS before its complete inactivation. These results suggest that CMP follows a suicide mechanism, the enzyme not being protected in this case. The mechanism of suicide inactivation is discussed with a view to establishing a broad knowledge base for future rational protein engineering. PMID:26407901

  16. The kinetics of the enzyme-substrate compound of peroxidase. 1943.

    PubMed

    Chance, B

    1999-01-01

    Under the narrow range of experimental conditions, and at a temperature of approximately 25 degrees, the following data were obtained. 1. The equilibrium constant of peroxidase and hydrogen peroxide has a minimum value of 2 x 10(-8). 2. The velocity constant for the formation of peroxidase-H2O2 Complex I is 1.2 x 10(7) liter mole-1 sec.-1, +/- 0.4 x 10(7). 3. The velocity constant for the reversible breakdown of peroxidase-H2O2 Complex I is a negligible factor in the enzyme-substrate kinetics and is calculated to be less than 0.2 sec.-1. 4. The velocity constant, k3, for the enzymatic breakdown of peroxidase-H2O2 Complex I varies from nearly zero to higher than 5 sec.-1, depending upon the acceptor and its concentration. The quotient of k3 and the leucomalachite green concentration is 3.0 x 10(4) liter mole-1 sec.-1. For ascorbic acid this has a value of 1.8 x 10(5) liter mole-1 sec.-1. 5. For a particular acceptor concentration, k3 is determined solely from the enzyme-substrate kinetics and is found to be 4.2 sec.-1. 6. For the same conditions, k3 is determined from a simple relationship derived from mathematical solutions of the Michaelis theory and is found to be 5.2 sec.-1. 7. For the same conditions, k3 is determined from the over-all enzyme action and is found to be 5.1 sec.-1. 8. The Michaelis constant determined from kinetic data alone is found to be 0.44 x 10(-6). 9. The Michaelis constant determined from steady state measurements is found to be 0.41 x 10(-6). 10. The Michaelis constant determined from measurement of the overall enzyme reaction is found to be 0.50 x 10(-6). 11. The kinetics of the enzyme-substrate compound closely agree with mathematical solutions of an extension of the Michaelis theory obtained for experimental values of concentrations and reaction velocity constants. 12. The adequacy of the criteria by which experiment and theory were correlated has been examined critically and the mathematical solutions have been found to be sensitive

  17. Differential Regulation of mnp2, a New Manganese Peroxidase-Encoding Gene from the Ligninolytic Fungus Trametes versicolor PRL 572

    PubMed Central

    Johansson, Tomas; Nyman, Per Olof; Cullen, Daniel

    2002-01-01

    A peroxidase-encoding gene, mnp2, and its corresponding cDNA were characterized from the white-rot basidiomycete Trametes versicolor PRL 572. We used quantitative reverse transcriptase-mediated PCR to identify mnp2 transcripts in nutrient-limited stationary cultures. Although mnp2 lacks upstream metal response elements (MREs), addition of MnSO4 to cultures increased mnp2 transcript levels 250-fold. In contrast, transcript levels of an MRE-containing gene of T. versicolor, mnp1, increased only eightfold under the same conditions. Thus, the manganese peroxidase genes in T. versicolor are differentially regulated, and upstream MREs are not necessarily involved. Our results support the hypothesis that fungal and plant peroxidases arose through an ancient duplication and folding of two structural domains, since we found the mnp1 and mnp2 polypeptides to have internal homology. PMID:11916737

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

    PubMed

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

    2015-09-21

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

  19. Kinetics of Spanish broom peroxidase obeys a Ping-Pong Bi-Bi mechanism with competitive inhibition by substrates.

    PubMed

    Pérez Galende, Patricia; Hidalgo Cuadrado, Nazaret; Kostetsky, Eduard Ya; Roig, Manuel G; Villar, Enrique; Shnyrov, Valery L; Kennedy, John F

    2015-11-01

    In plants, adverse conditions often induce an increase in reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). H2O2 is reduced to water, and thus becomes detoxified by enzymes such as Cytisus multiflorus peroxidase (CMP). Here, the steady-state kinetics of the H2O2-supported oxidation of different organic substrates by CMP was investigated. Analysis of the initial rates vs. H2O2 and reducing substrate concentrations proved to be consistent with a substrate-inhibited Ping-Pong Bi-Bi reaction mechanism. The phenomenological approach expresses the peroxidase Ping-Pong mechanism in the form of the Michaelis-Menten equation and affords an interpretation of the effects in terms of the kinetic parameters [Formula: see text] , [Formula: see text] , kcat, [Formula: see text] , [Formula: see text] and of the microscopic rate constants, k1 and k3, of the shared three-step catalytic cycle of peroxidases. PMID:26416239

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2016-05-18

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

  2. Candida albicans erythroascorbate peroxidase regulates intracellular methylglyoxal and reactive oxygen species independently of D-erythroascorbic acid.

    PubMed

    Kwak, Min-Kyu; Song, Sung-Hyun; Ku, MyungHee; Kang, Sa-Ouk

    2015-07-01

    Candida albicans D-erythroascorbate peroxidase (EAPX1), which can catalyze the oxidation of D-erythroascorbic acid (EASC) to water, was observed to be inducible in EAPX1-deficient and EAPX1-overexpressing cells via activity staining. EAPX1-deficient cells have remarkably increased intracellular reactive oxygen species and methylglyoxal independent of the intracellular EASC content. The increased methylglyoxal caused EAPX1-deficient cells to activate catalase-peroxidase and cytochrome c peroxidase, which led to defects in cell growth, viability, mitochondrial respiration, filamentation and virulence. These findings indicate that EAPX1 mediates cell differentiation and virulence by regulating intracellular methylglyoxal along with oxidative stresses, regardless of endogenous EASC biosynthesis or alternative oxidase expression. PMID:25957768

  3. Comparison of the peroxidase-like activity of unmodified, amino-modified, and citrate-capped gold nanoparticles.

    PubMed

    Wang, Sheng; Chen, Wei; Liu, Ai-Lin; Hong, Lei; Deng, Hao-Hua; Lin, Xin-Hua

    2012-04-10

    The origin of the peroxidase-like activity of gold nanoparticles and the impact of surface modification are studied. Furthermore, some influencing factors, such as fabrication process, redox property of the modifier, and charge property of the substrate, are investigated. Compared to amino-modified or citrate-capped gold nanoparticles, unmodified gold nanoparticles show significantly higher catalytic activity toward peroxidase substrates, that is, the superficial gold atoms are a contributing factor to the observed peroxidase-like activity. The different catalytic activities of amino-modified and citrate-capped gold nanoparticles toward 3,3',5,5'-tetramethylbenzidine (TMB) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) show that the charge characteristics of the nanoparticles and the substrate also play an important role in the catalytic reactions. PMID:22383315

  4. Bicarbonate is required for the peroxidase function of Cu, Zn-superoxide dismutase at physiological pH.

    PubMed

    Sankarapandi, S; Zweier, J L

    1999-01-15

    Cu,Zn-superoxide dismutase (SOD1) acts as a peroxidase in the presence of H2O2 at high pH (pH > 9). The high pH species of H2O2, HO2-, was previously implicated as the reactive species. However, recent EPR studies of the enzyme performed in the physiological pH range 7.4-7.6 with the spin trap 5,5'-dimethyl-1-pyrolline-N-oxide attributed the intense EPR signal of 5, 5'-dimethyl-1-pyrolline-N-oxide-OH obtained from SOD1 and H2O2 to the peroxidase activity of the enzyme. The present study establishes that this intense signal is obtained only in the presence of bicarbonate. To explore the critical role of HCO3-, a comprehensive EPR investigation of the radical production and redox state of the active site copper was performed. The results indicate that HCO3- competes with other anions for the anion-binding site of SOD1 (Arg141) but does not bind directly to the copper. Structurally different anions that bind to Arg141 did not stimulate, but rather blocked, peroxidase function, ruling out an effect due to mere anion binding. However, the structurally similar anions HSeO3- and HSO3- mimic HCO3- in stimulating peroxidase function. These data suggest that HCO3- bound to Arg141 anchors the neutral H2O2 molecule at the active site copper, enabling its redox cleavage. Thus, SOD1 acquires peroxidase activity at physiological pH only in the presence of HCO3- or structurally similar anions. Alterations in pH that shift the HCO3-/CO2 equilibrium as occur in disease processes such as ischemia, sepsis, or shock would modulate the peroxidase function of SOD1. PMID:9880490

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

    PubMed

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

    2014-04-01

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

  6. Changes in chalazal cell walls and in the peroxidase enzymes of the crease region during grain development in barley.

    PubMed

    Cochrane, M P; Paterson, L; Gould, E

    2000-03-01

    In an investigation of the role of peroxidase enzymes in the differentiation of the tissues of the crease region of barley, plants of winter barley cv. Halcyon were grown from anthesis onwards in controlled conditions at a constant temperature of 16 degrees C. Four ears were harvested at 2-d intervals from 6 d after anthesis (daa) until 50 daa. Grains from mid-ear were used for (i) fresh and dry weight determinations, (ii) extraction of crease tissue for the determination of peroxidase activity and for the separation of isozymes of peroxidase by isoelectric focusing (IEF) and (iii) detection of lignin and suberin in the tissues of the crease using autofluorescence and cytochemistry. Peroxidase activity was located histochemically in the crease tissue of cv. Chariot. Scanning electron microscopy studies were carried out on developing grains of cv. Blenheim. Maximum grain water content was achieved at 14 daa. Lignin and suberin were detected in the walls of the chalazal cells from 18 daa onwards. No changes in the staining of chalazal cell walls were detected at the end of grain filling (32 daa), but loss of autofluorescence and staining were observed at 42 daa, just prior to the final, rapid phase of grain dehydration. Peroxidase activity per fresh weight of crease tissue was high at 6 daa and low at 22 daa. It was also low between 32 and 40 daa, but it rose again from 42 daa onwards. IEF demonstrated that both anionic and cationic isozymes of peroxidase were present in crease tissue, the pattern of bands showing some marked changes during the course of grain development. PMID:10938807

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

    PubMed

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

    2014-07-01

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

  8. Characterization of a Pinus pinaster cDNA encoding an auxin up-regulated putative peroxidase in roots.

    PubMed

    Charvet-Candela, V; Hitchin, S; Reddy, M S; Cournoyer, B; Marmeisse, R; Gay, G

    2002-03-01

    As part of a study to identify host plant genes regulated by fungal auxin during ectomycorrhiza formation, we differentially screened a cDNA library constructed from roots of auxin-treated Pinus pinaster (Ait.) Sol. seedlings. We identified three cDNAs up-regulated by auxin. Sequence analysis of one of these cDNAs, PpPrx75, revealed the presence of an open reading frame of 216 amino acids with the characteristic consensus sequences of plant peroxidases. The deduced amino acid sequence showed homology with Arabidopsis thaliana (L.) Heynh., Arachis hypogaea L. and Stylosanthes humilis HBK cationic peroxidases. Amino acid sequence identities in the conserved domains of plant peroxidases ranged from 60 to 100%. In PpPrx75, there are five cysteine residues and one histidine residue that are found at conserved positions among other peroxidases. A potential glycosylation site (NTS) is present in the deduced sequence. Phylogenetic analysis showed that PpPrx75 is closely related to two A. thaliana peroxidases. The PpPrx75 cDNA was induced by active auxins, ethylene, abscisic acid and quercetin, a flavonoid possibly involved in plant-microorganism interactions. Transcript accumulation was detected within 3 h following root induction by auxin, and the amount of mRNA increased over the following 24 h. The protein synthesis inhibitor cycloheximide did not inhibit indole-3-acetic acid-induced transcript accumulation, suggesting that PpPrx75 induction is a primary (direct) response to auxin. This cDNA can be used to study expression of an auxin-regulated peroxidase during ectomycorrhiza formation. PMID:11874719

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

    PubMed

    McDonald, Andrew G; Tipton, Keith F

    2010-12-16

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

  10. Enhanced chemiluminescence of the luminol-hydrogen peroxide system by colloidal cupric oxide nanoparticles as peroxidase mimic.

    PubMed

    Chen, Wei; Hong, Lei; Liu, Ai-Lin; Liu, Jian-Qing; Lin, Xin-Hua; Xia, Xing-Hua

    2012-09-15

    As a peroxidase mimic, cupric oxide nanoparticles were found to enhance the chemiluminescence (CL) of luminol-H(2)O(2) system up to 400 folds. The CL spectra and radical scavengers were conducted to investigate the possible CL enhancement mechanism. It was suggested that the enhanced CL could be attributed to the peroxidase-like activity of CuO nanoparticles, which effectively catalyzed the decomposition of hydrogen peroxide into hydroxyl radicals. The effects of the reactant concentrations and some organic compounds were also investigated. The proposed method could be used as a sensitive detection tool for hydrogen peroxide and glucose. PMID:22967606

  11. Evidence for a significant contribution by peroxidase-mediated O2 uptake to root respiration of Brachypodium pinnatum.

    PubMed

    van der Werf, A; Raaimakers, D; Poot, P; Lambers, H

    1991-02-01

    This study describes the O2 uptake characteristics of intact roots of Brachypodium pinnatum. In the presence of 25 mM salicylhydroxamic acid (SHAM), concentrations of KCN below 3.5 νM had no effect on the rate of root respiration, whereas in the absence of 25 mM SHAM a significant inhibition of approx. 18% was observed. This indicates that an O2-consuming reaction, not associated with the cytochrome pathway, the alternative pathway or the "residual component", operates in the absence of any inhibitors in roots of B. pinnatum. We demonstrate here that this fourth O2-consuming reaction is mediated by a peroxidase. A peroxidase which catalyzed O2 reduction in the presence of NADH was readily washed from the roots of B. pinnatum. This peroxidase was stimulated by 5 mM SHAM, whereas ascorbic acid, catalase, catechol, gentisic acid, low concentrations potassium cyanide (3.5 μM), sodium azide, sodium sulfide, superoxide dismutase and high concentrations SHAM (25 mM) inhibited this reaction. Except for high concentrations of SHAM and concentrations of KCN higher than approx. 3.5 μM, these effectors could not be used to inhibit the peroxidase-mediated O2 uptake in intact roots of B. pinnatum. Concentrations of SHAM below 10 mM stimulated O2 uptake up to 15% of the control rate, depending on concentration, whereas 25 mM SHAM inhibited O2 uptake by 35%. The stimulation at low concentrations resulted from a SHAM-stimulated peroxidase activity, whereas 25 mM SHAM completely inhibited both the peroxidase-mediated O2 uptake and the activity of the alternative pathway. A method is presented for determining the relative contributions of each of the four O2-consuming reactions, i.e. the cytochrome pathway, the alternative pathway, the "residual component" and the peroxidase-mediated O2 uptake. The peroxidase-mediated O2 uptake contributed 21% to the total rate of oxygen uptake in roots of B. pinnatum, the cytochrome pathway contributed 41%, the alternative pathway 14% and the

  12. Oxidation-reduction potentials and ionization states of extracellular peroxidases from the lignin-degrading fungus Phanerochaete chrysosporium.

    PubMed

    Millis, C D; Cai, D Y; Stankovich, M T; Tien, M

    1989-10-17

    The oxidation-reduction potentials of lignin peroxidase isozymes H1, H2, H8, and H10 as well as the Mn-dependent peroxidase isozymes H3 and H4 are reported. The potentiometric titrations involving the ferrous and ferric states of the enzyme had Nernst plots indicating single-electron transfer. The Em7 values of lignin peroxidase isozymes H1, H2, H8, and H10 are -142, -135, -137, and -127 mV versus standard hydrogen electrode, respectively. The Em7 values for the Mn-dependent peroxidase isozymes H3 and H4 are -88 and -93 mV versus standard hydrogen electrode, respectively. The midpoint potential of H1, H8, and H4 remained unchanged in the presence of their respective substrates, veratryl alcohol and Mn(II). The midpoint potential between the ferric and ferrous forms of isozymes H1 and H4 exhibited a pH-dependent change between pH 3.5 and pH 6.5. These results indicate that the reductive half-reaction of the enzymes is the following: ferric peroxidase + le- + H+----ferrous peroxidase. Above pH 6.5, the effect of pH on the midpoint potential is diminished and indicates that an ionization with an apparent pKa equal to approximately 6.6-6.7 occurs in the reduced form of the enzymes. A heme-linked ionization group in the ferrous form of the enzymes was confirmed by studying the effect of pH on the absorption spectra of isozymes H1 and H4. These spectrophotometric pH titration experiments confirmed the electrochemical results indicating pKa values of 6.59 and 6.69 for reduced isozymes H1 and H4, respectively. These results indicate the presence of a heme-linked ionization of an amino acid in the reduced form of the lignin peroxidase isozymes similar to that of other plant peroxidases. PMID:2605198

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

    PubMed

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

    2015-05-01

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

  14. Ferromagnetic nanoparticles with peroxidase-like activity enhance the cleavage of biological macromolecules for biofilm elimination

    NASA Astrophysics Data System (ADS)

    GaoCurrent Address: University Of Pennsylvania, School Of Dental Medicine, Philadelphia, Pa 19104, Usa. E.-Mail: Gaoliz@Dental. Upenn. Edu, Lizeng; Giglio, Krista M.; Nelson, Jacquelyn L.; Sondermann, Holger; Travis, Alexander J.

    2014-02-01

    Hydrogen peroxide (H2O2) is a ``green chemical'' that has various cleaning and disinfectant uses, including as an anti-bacterial agent for hygienic and medical treatments. However, its efficacy is limited against biofilm-producing bacteria, because of poor penetration into the protective, organic matrix. Here we show new applications for ferromagnetic nanoparticles (Fe3O4, MNPs) with peroxidase-like activity in potentiating the efficacy of H2O2 in biofilm degradation and prevention. Our data show that MNPs enhanced oxidative cleavage of biofilm components (model nucleic acids, proteins, and oligosaccharides) in the presence of H2O2. When challenged with live, biofilm-producing bacteria, the MNP-H2O2 system efficiently broke down the existing biofilm and prevented new biofilms from forming, killing both planktonic bacteria and those within the biofilm. By enhancing oxidative cleavage of various substrates, the MNP-H2O2 system provides a novel strategy for biofilm elimination, and other applications utilizing oxidative breakdown.Hydrogen peroxide (H2O2) is a ``green chemical'' that has various cleaning and disinfectant uses, including as an anti-bacterial agent for hygienic and medical treatments. However, its efficacy is limited against biofilm-producing bacteria, because of poor penetration into the protective, organic matrix. Here we show new applications for ferromagnetic nanoparticles (Fe3O4, MNPs) with peroxidase-like activity in potentiating the efficacy of H2O2 in biofilm degradation and prevention. Our data show that MNPs enhanced oxidative cleavage of biofilm components (model nucleic acids, proteins, and oligosaccharides) in the presence of H2O2. When challenged with live, biofilm-producing bacteria, the MNP-H2O2 system efficiently broke down the existing biofilm and prevented new biofilms from forming, killing both planktonic bacteria and those within the biofilm. By enhancing oxidative cleavage of various substrates, the MNP-H2O2 system provides a novel

  15. Peroxidase Activity and Involvement in the Oxidative Stress Response of Roseobacter denitrificans Truncated Hemoglobin

    PubMed Central

    Wang, Yaya; Barbeau, Xavier; Bilimoria, Astha; Lagüe, Patrick; Couture, Manon; Tang, Joseph Kuo-Hsiang

    2015-01-01

    Roseobacter denitrificans is a member of the widespread marine Roseobacter genus. We report the first characterization of a truncated hemoglobin from R. denitrificans (Rd. trHb) that was purified in the heme-bound form from heterologous expression of the protein in Escherichia coli. Rd. trHb exhibits predominantly alpha-helical secondary structure and absorbs light at 412, 538 and 572 nm. The phylogenetic classification suggests that Rd. trHb falls into group II trHbs, whereas sequence alignments indicate that it shares certain important heme pocket residues with group I trHbs in addition to those of group II trHbs. The resonance Raman spectra indicate that the isolated Rd. trHb contains a ferric heme that is mostly 6-coordinate low-spin and that the heme of the ferrous form displays a mixture of 5- and 6-coordinate states. Two Fe-His stretching modes were detected, notably one at 248 cm-1, which has been reported in peroxidases and some flavohemoglobins that contain an Fe-His-Asp (or Glu) catalytic triad, but was never reported before in a trHb. We show that Rd. trHb exhibits a significant peroxidase activity with a (kcat/Km) value three orders of magnitude higher than that of bovine Hb and only one order lower than that of horseradish peroxidase. This enzymatic activity is pH-dependent with a pKa value ~6.8. Homology modeling suggests that residues known to be important for interactions with heme-bound ligands in group II trHbs from Mycobacterium tuberculosis and Bacillus subtilis are pointing toward to heme in Rd. trHb. Genomic organization and gene expression profiles imply possible functions for detoxification of reactive oxygen and nitrogen species in vivo. Altogether, Rd. trHb exhibits some distinctive features and appears equipped to help the bacterium to cope with reactive oxygen/nitrogen species and/or to operate redox biochemistry. PMID:25658318

  16. Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I.

    PubMed

    Chinchilla, Diana; Kilheeney, Heather; Vitello, Lidia B; Erman, James E

    2014-01-01

    Ferric heme proteins bind weakly basic ligands and the binding affinity is often pH dependent due to protonation of the ligand as well as the protein. In an effort to find a small, neutral ligand without significant acid/base properties to probe ligand binding reactions in ferric heme proteins we were led to consider the organonitriles. Although organonitriles are known to bind to transition metals, we have been unable to find any prior studies of nitrile binding to heme proteins. In this communication we report on the equilibrium and kinetic properties of acrylonitrile binding to cytochrome c peroxidase (CcP) as well as the oxidation of acrylonitrile by CcP compound I. Acrylonitrile binding to CcP is independent of pH between pH 4 and 8. The association and dissociation rate constants are 0.32±0.16 M(-1) s(-1) and 0.34±0.15 s(-1), respectively, and the independently measured equilibrium dissociation constant for the complex is 1.1±0.2 M. We have demonstrated for the first time that acrylonitrile can bind to a ferric heme protein. The binding mechanism appears to be a simple, one-step association of the ligand with the heme iron. We have also demonstrated that CcP can catalyze the oxidation of acrylonitrile, most likely to 2-cyanoethylene oxide in a "peroxygenase"-type reaction, with rates that are similar to rat liver microsomal cytochrome P450-catalyzed oxidation of acrylonitrile in the monooxygenase reaction. CcP compound I oxidizes acrylonitrile with a maximum turnover number of 0.61 min(-1) at pH 6.0. PMID:24291498

  17. Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I

    PubMed Central

    Chinchilla, Diana; Kilheeney, Heather; Vitello, Lidia B.; Erman, James E.

    2013-01-01

    Ferric heme proteins bind weakly basic ligands and the binding affinity is often pH dependent due to protonation of the ligand as well as the protein. In an effort to find a small, neutral ligand without significant acid/base properties to probe ligand binding reactions in ferric heme proteins we were led to consider the organonitriles. Although organonitriles are known to bind to transition metals, we have been unable to find any prior studies of nitrile binding to heme proteins. In this communication we report on the equilibrium and kinetic properties of acrylonitrile binding to cytochrome c peroxidase (CcP) as well as the oxidation of acrylonitrile by CcP compound I. Acrylonitrile binding to CcP is independent of pH between pH 4 and 8. The association and dissociation rate constants are 0.32 ± 0.16 M−1s−1 and 0.34 ± 0.15 s−1, respectively, and the independently measured equilibrium dissociation constant for the complex is 1.1 ± 0.2 M. We have demonstrated for the first time that acrylonitrile can bind to a ferric heme protein. The binding mechanism appears to be a simple, one-step association of the ligand with the heme iron. We have also demonstrated that CcP can catalyze the oxidation of acrylonitrile, most likely to 2-cyanoethylene oxide in a “peroxygenase”-type reaction, with rates that are similar to rat liver microsomal cytochrome P450-catalyzed oxidation of acrylonitrile in the monooxygenase reaction. CcP compound I oxidizes acrylonitrile with a maximum turnover number of 0.61 min−1 at pH 6.0. PMID:24291498

  18. Peroxidase-positive Auer bodies in plasma cells in multiple myeloma: a case report

    PubMed Central

    Zhu, Lin; An, Li; Zhang, Xiao-Yan; Ren, Xue-Rui; Song, Jing-Wen

    2015-01-01

    Reports of clinical cases with Auer bodies in the plasma cells in multiple myeloma (MM) are rare; however, most of those reported contain peroxidase (POX)-negative Auer bodies rather than the POX-positive Auer bodies observed in myeloid progenitors, indicating differences in their chemical properties. Furthermore, the cases with POX-positive Auer bodies similar to those observed in myeloid cells are extremely rare in non-myeloid cells. Here, we report the clinical features, laboratory investigations, diagnosis and treatment of a case of MM with POX-positive Auer bodies in plasma cells and review related the literature to advance the prognostic evaluation, diagnosis and treatment of similar cases. PMID:26823884

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

    PubMed

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

    2014-01-15

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

  20. 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

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

    NASA Astrophysics Data System (ADS)

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

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

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

    PubMed Central

    Huang, Jitao; Huang, Wei; Wang, Titi

    2012-01-01

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

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

    PubMed

    Saa, Laura; Pavlov, Valeri

    2012-11-19

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

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

    PubMed

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

    2016-10-01

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

  5. Immunohistochemistry of fumonisin in poultry using avidin-biotin-peroxidase system.

    PubMed

    Buim, M R; Bracarense, A P; Guimarães, I G; Kawamura, O; Ueno, Y; Hirooka, E Y

    1999-01-01

    Using monoclonal anti-fumonisin B1 antibody (anti-FB1) and avidin-biotin-peroxidase system, liver and kidneys of broiler chicks were evaluated for the detection and distribution of fumonisins (FBs). One hundred and fifty micrograms of FB1 or culture extract of Fusarium moniliforme str. 113F containing 150 microg of FB1 and 4 microg of FB2 were administered into the vitelline sac of 1-day old, specific pathogen-free chicks. The animals were killed 24 h after injection, and renal and hepatic tissues submitted for immunohistochemical analysis. FBs were detected in the epithelial cells of convoluted distal and proximal tubules of the kidneys, as well as in the cytoplasm of hepatocytes. This novel immunohistochemical method developed is expected to be an efficient way for monitoring the target of the FB toxins in tissues. PMID:11122519

  6. Inflammatory bowel disease (IBD) locus 12: is glutathione peroxidase-1 (GPX1) the relevant gene?

    PubMed

    Häuser, F; Rossmann, H; Laubert-Reh, D; Wild, P S; Zeller, T; Müller, C; Neuwirth, S; Blankenberg, S; Lackner, K J

    2015-12-01

    Genome-wide association studies have identified and repeatedly confirmed the association of rs3197999 in MST1 with inflammatory bowel disease (IBD). However, the underlying pathophysiology remains unclear. rs3197999 is a non-synonymous single-nucleotide polymorphism which modifies the function of macrophage stimulating protein-1 (MST1). We show by haplotyping that rs3197999 is in linkage disequilibrium with rs1050450 in GPX1, with almost complete cosegregation of the minor alleles. As shown by immunoassay, rs3197999 influences the MST-1 level in serum. But also rs1050450 causes an amino acid exchange in glutathione peroxidase 1 (GPx-1) and reduced activity of this antioxidant enzyme. The association of GPx deficiency and IBD in mice was already shown. We propose that GPx-1 is a better candidate than MST1 for the pathophysiologic link between IBD locus 12 and IBD. PMID:26355565

  7. Influence of media nutrients on synthesis of lignin peroxidase from Aspergillus sp.

    PubMed

    Ahammed, Shamla; Prema, Parukuttyamma

    2002-01-01

    The effect of carbon and nitrogen sources, lignocellulosic substrates, and metal ions on lignin peroxidase (LiP) activity of Aspergillus sp., which was isolated from a mangrove area, was studied. Glucose (1%) was found to be the best carbon source. Among the various lignocellulosic substrates used, coir pith at 3% concentration increased LiP activity twofold on the second day of incubation. Peptone and KNO3 completely inhibited the enzyme synthesis while (NH4)2SO4 at 12.5 mM produced maximum activity. Since seawater contained all the requisite metal ions, any added ions had a negative effect on activity. Cu2+ had the most inhibiting effect while K+ the least. When all the optimized conditions were provided, in nitrogen- and carbon-sufficient medium, a maximum LiP activity of 345 U/mL was obtained on the second day of incubation. PMID:12396134

  8. Ascorbate peroxidase-related (APx-R) is not a duplicable gene

    PubMed Central

    Dunand, Christophe; Mathé, Catherine; Lazzarotto, Fernanda; Margis, Rogério; Margis-Pinheiro, Marcia

    2011-01-01

    Phylogenetic, genomic and functional analyses have allowed the identification of a new class of putative heme peroxidases, so called APx-R (APx-Related). These new class, mainly present in the green lineage (including green algae and land plants), can also be detected in other unicellular chloroplastic organisms. Except for recent polyploid organisms, only single-copy of APx-R gene was detected in each genome, suggesting that the majority of the APx-R extra-copies were lost after chromosomal or segmental duplications. In a similar way, most APx-R co-expressed genes in Arabidopsis genome do not have conserved extra-copies after chromosomal duplications and are predicted to be localized in organelles, as are the APx-R. The member of this gene network can be considered as unique gene, well conserved through the evolution due to a strong negative selection pressure and a low evolution rate. PMID:22231200

  9. An immunohistological study of feline glomerulonephritis using the peroxidase-antiperoxidase method.

    PubMed

    Arthur, J E; Lucke, V M; Newby, T J; Bourne, F J

    1984-07-01

    Twenty-two cases of feline glomerulonephritis were investigated for the presence of immune complexes within the glomerulus using the peroxidase-antiperoxidase (PAP) method. This method was used with formalin-fixed paraffin-wax embedded tissues which were pretreated with trypsin and with frozen sections of kidney tissue. Of a total of 25 kidney specimens examined (two cats had repeated biopsies) the composition of the deposits was 23/25 IgG, 17/25 C3, 11/25 IgM and 2/25 IgA. Serial studies of two cats showed a progression of the disease from initial nephrotic syndrome to chronic renal failure. With the more severe form of the disease there was a tendency for the deposition of complement and more than one class of immunoglobulin within the glomeruli. PMID:6382492

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2014-01-21

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

  12. Detection of glucose based on the peroxidase-like activity of reduced state carbon dots.

    PubMed

    Long, Yijuan; Wang, Xiliang; Shen, Dongjun; Zheng, Huzhi

    2016-10-01

    It was found that reduced state carbon dots (r-CDs) possessed intrinsic peroxidase-like activity, and could catalytically oxidize 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 to produce a color reaction. The effects of temperature, pH, incubating time and the concentration of H2O2 and TMB on catalytic activity of r-CDs were investigated. Finally we calculated the kinetic constant was ca. 0.00729min(-1) and applied r-CDs to glucose sensing by coupling glucose oxidase. As low as 2µM H2O2 could be detected with a linear range from 0.010 to 0.40mM via this method. This study offered a simple, sensitive, and high selectivity method for glucose determination even in serum. PMID:27474288

  13. Polyphenol oxidase and peroxidase expression in four pineapple varieties (Ananas comosus L.) after a chilling injury.

    PubMed

    Raimbault, Astrid-Kim; Marie-Alphonsine, Paul-Alex; Horry, Jean-Pierre; Francois-Haugrin, Madlyn; Romuald, Karell; Soler, Alain

    2011-01-12

    Pineapple internal browning (IB) is a chilling injury that produces enzymatic browning associated with flesh translucency. Pineapple biodiversity allowed the investigation of how polyphenol oxidase (PPO) and peroxidase (POD) activities with their different isoforms are involved in the IB mechanism. Fruits of four varieties that expressed IB symptoms differently, Smooth Cayenne (SCay) and the hybrids MD2, Flhoran 41 (Flh 41), and Flhoran 53 (Flh 53), were stressed by cold. The susceptible varieties showed classical brown spots but different patterns of IB, whereas MD2 and controls showed no IB. Enzymatic activities were measured on fruit protein extracts and PPO and POD isoforms separated on mini-gels (PhastSystem). Only PPO activity was significantly enhanced in the presence of IB. Up to six PPO isoforms were identified in the susceptible varieties. PPO was barely detectable in the nonsusceptible variety MD2 and in controls. The number of PPO isoforms and the total PPO activity after chilling are varietal characteristics. PMID:21133422

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

    PubMed

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

    2015-11-01

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

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

    PubMed

    Didenko, Vladimir V; Baskin, David S

    2006-03-01

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

  16. Staining electrophoretic gels for laccase and peroxidase activity using 1,8-diaminonaphthalene.

    PubMed

    Hoopes, J T; Dean, J F

    2001-06-01

    A new chromogenic substrate for laccases and peroxidases, 1,8-diaminonapthalene, was used to detect phenoloxidase activity in gels after SDS-PAGE. This substrate has several advantages over other widely used phenoloxidase stains in that it is inexpensive, and the oxidized product has both high molar absorptivity and very low solubility. Furthermore, neither the substrate nor the product is known to have toxicity problems of the type associated with many other phenoloxidase stains. The sensitivity of detection using 1,8-diaminonapthalene was comparable to that obtained using the most sensitive stains commonly used for phenoloxidases, e.g., 3,3-diaminobenzidine, and was close to that attainable for protein detection using silver staining. Zymograms developed with 1,8-diaminonapthalene can be used with video densitometry to monitor the specific enzymatic activity of phenoloxidases during enzyme purification. PMID:11373084

  17. Carbamazepine-induced hemolytic and aplastic crises associated with reduced glutathione peroxidase activity of erythrocytes.

    PubMed

    Yamamoto, Masaki; Suzuki, Nobuhiro; Hatakeyama, Naoki; Kubo, Noriaki; Tachi, Nobutada; Kanno, Hitoshi; Fujii, Hisaichi; Tsutsumi, Hiroyuki

    2007-11-01

    Although pure red cell aplasia is a well-known side effect of carbamazepine treatment, intravascular hemolytic anemia is rare. We describe a 5-year-old boy who developed concurrent intravascular hemolytic anemia and erythroblastopenia, probably due to carbamazepine. Carbamazepine treatment was subsequently discontinued, and the patient was treated with red blood cell transfusions, haptoglobin, and methylprednisolone. His hematologic abnormalities were almost fully recovered within 2 weeks. Examination of the patient's and mother's erythrocyte enzyme activities revealed mildly decreased erythrocyte glutathione peroxidase (GSH-Px) activity. We speculate that patients with reduced GSH-Px activity are at a high risk of developing carbamazepine-induced hemolytic crisis and/or aplastic crisis. PMID:18055338

  18. Electron Transfer between Cytochrome C and Cytochome C Peroxidase in Single Crystals

    SciTech Connect

    Kang, Seong A.; Marjavaara, Pieti J.; Crane, Brian R.

    2010-11-10

    Cytochrome c (Cc) and cytochrome c peroxidase (CcP) form an important redox pair for understanding interprotein electron transfer (ET). Measurements of ET rates from photoexcited CcP substituted with Zn porphyrin to either yeast Fe(III)Cc or horse Fe(III)Cc in crystals reveal that the molecular associations found in the respective crystal structures determine solution reactivity. Similar forward rates for yeast isozyme-1 Cc (yCc) and yCc homologue horse Cc (hCc), despite different orientations relative to CcP, suggest small-amplitude conformational gating of ET even in the crystalline state; faster back ET in the yCc compared to the hCc complex agrees with the relative coupling between redox sites predicted by the structures.

  19. Effect of chemical form of selenium on tissue glutathione peroxidase activity in developing rats

    NASA Technical Reports Server (NTRS)

    Lane, Helen W.; Strength, Ralph; Johnson, Janet; White, Marguerite T.

    1991-01-01

    The hypothesis that the stage of development of rats may affect the availability of various forms of selenium for the activity of glutathione peroxidase (GSHPx) in the rat was experimentally investigated. One experiment evaluated the availability of selenium as selenite or selenomethionine for GSPHx activity during three developmental states in rats: fetus and 7-day old and 14-day old nursing pups. In all tissues studied, GSHPx activity was highest in the 14-day-old pups whose dams were in the selenomethionine group. Rat pups given intraperitoneal selenite had higher liver and kidney GSHPx activity than pups given the same amount of selenium as intraperitoneal selenomethionine. In a second experiment, all dams were fed the same basal diet and pups were weaned to diets containing one of two levels of selenium and one of three forms of selenium (selenite, selenomethionine, or selenocystine). The results also supported the hypothesis these dietary forms of selenium are differentially available for GSHPx activity.

  20. Fully-branched hyperbranched polymers with a diselenide core as glutathione peroxidase mimics.

    PubMed

    Fu, Yu; Chen, Junyi; Xu, Huaping; Van Oosterwijck, Chantal; Zhang, Xi; Dehaen, Wim; Smet, Mario

    2012-05-14

    A novel glutathione peroxidase (GPx) mimic has been prepared by incorporation of a selenium-based catalytic unit into the focal point of a fully-branched hyperbranched polymer. First, an AB(2) monomer consisting of isatin and an electron rich aromatic moiety was polycondensed in the presence of 5-nitroisatin as a core reagent, resulting in a polymer with 100% degree of branching. The latter was coupled to the catalytically active moiety, Br(CH(2))(5) SeSe(CH(2))(5) Br, by nucleophilic substitution of the bromides by the residual amide groups of the incorporated nitroisatin core. The obtained polymer has demonstrated prominent GPx activity as desired, which could be attributed to the hydrophobic, densely branched and core-shell structure of the polymer surrounding the catalytic center. PMID:22434542