23 results on '"Burner U"'
Search Results
2. The two-Cys peroxiredoxin Bas1: Insight in a new family of plant peroxidases
- Author
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Baier, Margarete, Dietz, Karl-Josef, Obinger, C., Burner, U., Ebermann, R., Penel, C., and Greppin, H.
- Published
- 1996
3. Heavy metal induced changes in peroxidase activity in leaves, roots and cell suspension cultures of Hordeum vulgare L
- Author
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Blinda, A., Abou-Mandour, A., Azarkovich, M., Brune, A., Dietz, Karl-Josef, Obinger, C., Burner, U., Ebermann, R., Penel, C., and Greppin, H.
- Published
- 1996
4. Fabric-mechanical property relationships of trabecular bone allografts are altered by supercritical CO2 treatment and gamma sterilization
- Author
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Schwiedrzik, J.J., primary, Kaudela, K.-H., additional, Burner, U., additional, and Zysset, P.K., additional
- Published
- 2011
- Full Text
- View/download PDF
5. Hypericin - The Facts About a Controversial Agent
- Author
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Kubin, A., primary, Wierrani, F., additional, Burner, U., additional, Alth, G., additional, and Grunberger, W., additional
- Published
- 2005
- Full Text
- View/download PDF
6. The reactivity of myeloperoxidase compound I formed with hypochlorous acid
- Author
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Furtmüller, P.G., primary, Burner, U., additional, Jantschko, W., additional, Regelsberger, G., additional, and Obinger, C., additional
- Published
- 2000
- Full Text
- View/download PDF
7. Oxidation of hydroquinone, 2,3-dimethylhydroquinone and 2,3,5-trimethylhydroquinone by human myeloperoxidase
- Author
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Burner, U., primary, Krapfenbauer, G., additional, Furtmüller, P.G., additional, Regelsberger, G., additional, and Obinger, C., additional
- Published
- 2000
- Full Text
- View/download PDF
8. Transient-state and steady-state kinetics of the oxidation of aliphatic and aromatic thiols by horseradish peroxidase
- Author
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Burner, U., primary and Obinger, C., additional
- Published
- 1997
- Full Text
- View/download PDF
9. Purification and Characterization of a Homodimeric Catalase-Peroxidase from the CyanobacteriumAnacystis nidulans
- Author
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Obinger, C., primary, Regelsberger, G., additional, Strasser, G., additional, Burner, U., additional, and Peschek, G.A., additional
- Published
- 1997
- Full Text
- View/download PDF
10. Transient and steady-state kinetics of the oxidation of substituted benzoic acid hydrazides by myeloperoxidase.
- Author
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Burner, U, Obinger, C, Paumann, M, Furtmüller, P G, and Kettle, A J
- Abstract
Myeloperoxidase is the most abundant protein in neutrophils and catalyzes the production of hypochlorous acid. This potent oxidant plays a central role in microbial killing and inflammatory tissue damage. 4-Aminobenzoic acid hydrazide (ABAH) is a mechanism-based inhibitor of myeloperoxidase that is oxidized to radical intermediates that cause enzyme inactivation. We have investigated the mechanism by which benzoic acid hydrazides (BAH) are oxidized by myeloperoxidase, and we have determined the features that enable them to inactivate the enzyme. BAHs readily reduced compound I of myeloperoxidase. The rate constants for these reactions ranged from 1 to 3 x 10(6) M-1 s-1 (15 degrees C, pH 7.0) and were relatively insensitive to the substituents on the aromatic ring. Rate constants for reduction of compound II varied between 6.5 x 10(5) M-1 s-1 for ABAH and 1.3 x 10(3) M-1 s-1 for 4-nitrobenzoic acid hydrazide (15 degrees C, pH 7.0). Reduction of both compound I and compound II by BAHs adhered to the Hammett rule, and there were significant correlations with Brown-Okamoto substituent constants. This indicates that the rates of these reactions were simply determined by the ease of oxidation of the substrates and that the incipient free radical carried a positive charge. ABAH was oxidized by myeloperoxidase without added hydrogen peroxide because it underwent auto-oxidation. Although BAHs generally reacted rapidly with compound II, they should be poor peroxidase substrates because the free radicals formed during peroxidation converted myeloperoxidase to compound III. We found that the reduction of ferric myeloperoxidase by BAH radicals was strongly influenced by Hansch's hydrophobicity constants. BAHs containing more hydrophilic substituents were more effective at converting the enzyme to compound III. This implies that BAH radicals must hydrogen bond to residues in the distal heme pocket before they can reduce the ferric enzyme. Inactivation of myeloperoxidase by BAHs was related to how readily they were oxidized, but there was no correlation with their rate constants for reduction of compounds I or II. We propose that BAHs destroy the heme prosthetic groups of the enzyme by reducing a ferrous myeloperoxidase-hydrogen peroxide complex.
- Published
- 1999
11. The reactivity of myeloperoxides compound I formed with hypochlorous acid
- Author
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Paul G. Furtmüller, Burner, U., Jantschko, W., Regelsberger, G., and Obinger, C.
12. Fabric-mechanical property relationships of trabecular bone allografts are altered by supercritical CO2 treatment and gamma sterilization
- Author
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Schwiedrzik, J.J., Kaudela, K.-H., Burner, U., and Zysset, P.K.
- Subjects
- *
BONE mechanics , *BONE cells , *BONE grafting , *SUPERCRITICAL fluids , *STERILIZATION (Disinfection) , *ORTHOPEDIC surgery , *CARBON dioxide - Abstract
Abstract: Tissue grafts are implanted in orthopedic surgery every day. In order to minimize infection risk, bone allografts are often delipidated with supercritical CO2 and sterilized prior to implantation. This treatment may, however, impair the mechanical behavior of the bone graft tissue. The goal of this study was to determine clinically relevant mechanical properties of treated/sterilized human trabecular bone grafts, e.g. the apparent modulus, strength, and the ability to absorb energy during compaction. They were compared with results of identical experiments performed previously on untreated/fresh frozen human trabecular bone from the same anatomical site (Charlebois, 2008). We tested the hypothesis that the morphology–mechanical property relationships of treated cancellous allografts are similar to those of fresh untreated bone. The morphology of the allografts was determined by μCT. Subsequently, cylindrical samples were tested in unconfined and confined compression. To account for various morphologies, the experimental data was fitted to phenomenological mechanical models for elasticity, strength, and dissipated energy density based on bone volume fraction (BV/TV) and the fabric tensor determined by MIL. The treatment/sterilization process does not appear to influence bone graft stiffness. However, strength and energy dissipation of the bone grafts were found to be significantly reduced by 36% to 47% and 66% to 81%, respectively, for a broad range of volume fraction (0.14< BV/TV <0.39) and degree of anisotropy (1.24< DA <2.18). Since the latter properties are strongly dominated by BV/TV, the clinical consequences of this reduction can be compensated by using grafts with lower porosity. The data of this study suggests that an increase of 5–10% in BV/TV is sufficient to compensate for the reduced post-yield mechanical properties of treated/sterilized bone in monotonic compression. In applications where graft stiffness needs to be matched and strength is not a concern, treated allograft with the same BV/TV as an appropriate fresh bone graft may be used. [Copyright &y& Elsevier]
- Published
- 2011
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13. Plant peroxidases : biochemistry and physiology
- Author
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Martinez, Claude, Geiger, Jean-Paul, Bresson, Estelle, Daniel, Jean-François, Dai, G.H., Andray, A., Nicole, Michel, Obinger, C. (ed.), Burner, U. (ed.), Ebermann, R. (ed.), Penel, C. (ed.), and Greppin, H. (ed.)
- Subjects
RESISTANCE DE L'HOTE ,ACTIVITE ENZYMATIQUE ,BACTERIOSE ,food and beverages ,PATHOLOGIE VEGETALE ,VARIETE SENSIBLE ,VARIETE RESISTANTE ,COTON - Abstract
Resistant (Reba B50) and susceptible (Acala 44) cotton plants were investigated for intratissular growth of bacterial populations and peroxidase (POx) activity, after infection of cotyledons with races 18 or 20 from #Xanthomonas (#Axonopodis$) campestris$ pv. #malvacearum$. Considerable multiplication of the bacterial population was noticed in the compatible interaction (Acala 44 / Xcm race 18) ; it was much lower during the incompatible interaction when race 18 was infiltrated into cotyledons of Reba B50. An intermediate level of bacterial growth was obtained when Reba B50 was infiltrated with race known to overcome resistance of this line. High increase in POx activity occurred into the infected cotyledons during incompatible interaction, while the increase was much lower when the interactions were compatible. On leaves, a similar and significant difference in enzyme activity was also observed indicating that the "peroxidase response" was systemically induced in entire resistant plants. Five isoperoxidases were evidenced by IEF in both lines, whether they were infected or not. But only two of them accounted for the increase in activity in infected resistant cotyledons. Microscopy revealed that POx activity, detected at the infection sites two hours after infiltration of the resistant line was mainly located in cell walls and the middle lamella bordering intercellular spaces. Our data indicate that bacterial infection of cotton plants enhanced the activity of two of the preexistent isoperoxidases in resistant plants and suggest that stimulation of POx activity is associated with resistance mechanisms. (Résumé d'auteur)
- Published
- 1996
14. Ascorbate Levels and Ozone Decay in Cell Walls of Barley and Wheat Seedlings
- Author
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Kollist, H., Moldau, H., Mortensen, L., Rasmussen, S. K., Bolt-Jørgensen, L., Obinger, C., Burner, U., Ebermann, R., Penel, C., and Greppin, H.
- Published
- 1996
15. Fluorescence diagnosis of bladder cancer with new water soluble hypericin bound to polyvinylpyrrolidone: PVP-hypericin.
- Author
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Kubin A, Meissner P, Wierrani F, Burner U, Bodenteich A, Pytel A, and Schmeller N
- Subjects
- Aged, Anthracenes, Biopsy, Female, Humans, Male, Neoplasm Staging, Perylene chemistry, Sensitivity and Specificity, Solubility, Fluorescent Dyes chemistry, Perylene analogs & derivatives, Povidone chemistry, Urinary Bladder Neoplasms diagnosis, Water chemistry
- Abstract
Although conventional white light endoscopy (WLE) is currently the gold standard for diagnosing bladder tumors, rates of false negative results and residual tumors after transurethral resection are relatively high. The goal of the present clinical study is to investigate whether using new water soluble hypericin (PVP-hypericin) as a fluorescent dye improves bladder cancer detection and diagnosis. Following instillation of PVP-hypericin (total amount of 0.25 mg hypericin bound to 25 mg polyvinylpoyrrolidone [PVP], reconstituted in 50 mL phys. sodium chloride solution), WLE and fluorescence cystoscopy (photodynamic diagnosis; PDD) were performed on patients with suspected primary or recurrent bladder malignancies (n = 57). Incubation time was 1-2 h and biopsies (n = 163) were taken from fluorescing regions and/or from regions which were suspicious under WLE. Histological investigations of the biopsies provided the final proof of malignancy (or the counterevidence). Results indicated that overall sensitivity with PVP-hypericin and PDD is significantly higher (95%) than with WLE (85%). The sensitivity of PDD in the diagnosis of carcinoma in situ (n = 12) was 100% compared with 33% for WLE. In the diagnosis of dysplasia, the sensitivity of PDD was 85% compared with 31% for WLE. PDD has a positive predictive value (PPV) of 0.75% and a negative predictive value (NPV) of 0.86%, in comparison to WLE PPV = 0.66% NPV = 0.58%. Biopsies were not taken from healthy tissues, thus specificity was markedly lower in our study (53%) than that reported in other studies (98-100%). As a conclusion, PDD using PVP-hypericin is superior to WLE in terms of sensitivity in the diagnosis of malignancies of the bladder. Results suggest that PVP-hypericin is a promising formulation for various diagnostic and therapeutic applications.
- Published
- 2008
- Full Text
- View/download PDF
16. How to make hypericin water-soluble.
- Author
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Kubin A, Loew HG, Burner U, Jessner G, Kolbabek H, and Wierrani F
- Subjects
- Anthracenes, Carcinoma, Transitional Cell pathology, Chemical Phenomena, Chemistry, Physical, Drug Delivery Systems, Flow Cytometry, Humans, Hydrogen Bonding, K562 Cells, Leukocytes chemistry, Leukocytes metabolism, Perylene chemistry, Pharmaceutic Aids chemistry, Photochemistry, Povidone chemistry, Solubility, Spectrometry, Fluorescence, Water, Antidepressive Agents chemistry, Perylene analogs & derivatives
- Abstract
Unlabelled: Hypericin, isolated from Hypericum perforatum, is an effective photodynamic substance as demonstrated by various studies. Practical forms of applications of hypericin solutions for systemic use and introduction into body cavities are, however, lacking. We developed an aqueous solution of hypericin non-covalently bound to polyvinylpyrrolidone (PVP). PVP is a poly-N-vinylamide of various degrees of polymerization and forms of intermolecular crosslinks suitable for diagnostic and therapeutic applications. We used PVP (molecular weights of PVP between 10 kD and 40 kD) as a complex forming agent to prepare hypericin for photodynamic therapy and diagnostics. In pure water, hypericin forms aggregates which are non-soluble and non-fluorescent. The hypericin-PVP complex binds more than 1000 mg of hypericin in presence of 100 g PVP or less and is soluble in 1 liter of pure water. Aqueous complex solutions of hypericin-PVP display a characteristic absorption spectrum and fluorescence emission band around 600 nm wavelength. Varying concentrations of hypericin do not cause a blue- or red-shift in the absorption maximum at 595 nm. Excitation at 200 nm to 500 nm leads to emission at 590 nm; a property conducive to diagnostic investigations both in vitro and in vivo. Furthermore, hypericin-PVP exhibits high photostability in the presence of oxygen and broad band light which ensures reproducible photodynamic therapy and diagnosis., Conclusion: Hypericin forms liquid molecular chromophore complexes in water when bound to PVP thus allowing investigations in biological media.
- Published
- 2008
17. Spectral and kinetic studies on the formation of eosinophil peroxidase compound I and its reaction with halides and thiocyanate.
- Author
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Furtmüller PG, Burner U, Regelsberger G, and Obinger C
- Subjects
- Eosinophil Peroxidase, Humans, Oxidation-Reduction, Peroxidases chemistry, Substrate Specificity, Thiocyanates metabolism, Eosinophils enzymology, Peroxidases metabolism
- Abstract
Compound I of peroxidases takes part in both the peroxidation and the halogenation reaction. This study for the first time presents transient kinetic measurements of the formation of compound I of human eosinophil peroxidase (EPO) and its reaction with halides and thiocyanate, using the sequential-mixing stopped-flow technique. Addition of 1 equiv of hydrogen peroxide to native EPO leads to complete formation of compound I. At pH 7 and 15 degrees C, the apparent second-order rate constant is (4.3 +/- 0.4) x 10(7) M(-1) s(-1). The rate for compound I formation by hypochlorous acid is (5.6 +/- 0.7) x 10(7) M(-1) s(-1). EPO compound I is unstable and decays to a stable intermediate with a compound II-like spectrum. At pH 7, the two-electron reduction of compound I to the native enzyme by thiocyanate has a second-order rate constant of (1.0 +/- 0. 5) x 10(8) M(-1) s(-1). Iodide [(9.3 +/- 0.7) x 10(7) M(-1) s(-1)] is shown to be a better electron donor than bromide [(1.9 +/- 0.1) x 10(7) M(-1) s(-1)], whereas chloride oxidation by EPO compound I is extremely slow [(3.1 +/- 0.3) x 10(3) M(-1) s(-1)]. The pH dependence studies suggest that a protonated form of compound I is more competent in oxidizing the anions. The results are discussed in comparison with those of the homologous peroxidases myeloperoxidase and lactoperoxidase and with respect to the role of EPO in host defense and tissue injury.
- Published
- 2000
- Full Text
- View/download PDF
18. Two-electron reduction and one-electron oxidation of organic hydroperoxides by human myeloperoxidase.
- Author
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Furtmüller PG, Burner U, Jantschko W, Regelsberger G, and Obinger C
- Subjects
- Electrons, Humans, Hydrogen Peroxide chemistry, Hydrogen-Ion Concentration, Kinetics, Oxidation-Reduction, Peroxidase chemistry, Spectrum Analysis, Hydrogen Peroxide metabolism, Peroxidase metabolism
- Abstract
The reaction of native myeloperoxidase (MPO) and its redox intermediate compound I with hydrogen peroxide, ethyl hydroperoxide, peroxyacetic acid, t-butyl hydroperoxide, 3-chloroperoxybenzoic acid and cumene hydroperoxide was studied by multi-mixing stopped-flow techniques. Hydroperoxides are decomposed by MPO by two mechanisms. Firstly, the hydroperoxide undergoes a two-electron reduction to its corresponding alcohol and heme iron is oxidized to compound I. At pH 7 and 15 degrees C, the rate constant of the reaction between 3-chloroperoxybenzoic acid and ferric MPO was similar to that with hydrogen peroxide (1.8x10(7) M(-1) s(-1) and 1.4x10(7) M(-1) s(-1), respectively). With the exception of t-butyl hydroperoxide, the rates of compound I formation varied between 5.2x10(5) M(-1) s(-1) and 2.7x10(6) M(-1) s(-1). Secondly, compound I can abstract hydrogen from these peroxides, producing peroxyl radicals and compound II. Compound I reduction is shown to be more than two orders of magnitude slower than compound I formation. Again, with 3-chloroperoxybenzoic acid this reaction is most effective (6. 6x10(4) M(-1) s(-1) at pH 7 and 15 degrees C). Both reactions are controlled by the same ionizable group (average pK(a) of about 4.0) which has to be in its conjugated base form for reaction.
- Published
- 2000
- Full Text
- View/download PDF
19. Mechanism of reaction of myeloperoxidase with nitrite.
- Author
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Burner U, Furtmuller PG, Kettle AJ, Koppenol WH, and Obinger C
- Subjects
- Humans, Hydrogen Peroxide metabolism, Hydrogen-Ion Concentration, Kinetics, Nitric Oxide metabolism, Oxidation-Reduction, Spectrophotometry, Neutrophils enzymology, Nitrites chemistry, Peroxidase chemistry
- Abstract
Myeloperoxidase (MPO) is a major neutrophil protein and may be involved in the nitration of tyrosine residues observed in a wide range of inflammatory diseases that involve neutrophils and macrophage activation. In order to clarify if nitrite could be a physiological substrate of myeloperoxidase, we investigated the reactions of the ferric enzyme and its redox intermediates, compound I and compound II, with nitrite under pre-steady state conditions by using sequential mixing stopped-flow analysis in the pH range 4-8. At 15 degrees C the rate of formation of the low spin MPO-nitrite complex is (2.5 +/- 0.2) x 10(4) m(-1) s(-1) at pH 7 and (2.2 +/- 0.7) x 10(6) m(-1) s(-1) at pH 5. The dissociation constant of nitrite bound to the native enzyme is 2.3 +/- 0.1 mm at pH 7 and 31.3 +/- 0.5 micrometer at pH 5. Nitrite is oxidized by two one-electron steps in the MPO peroxidase cycle. The second-order rate constant of reduction of compound I to compound II at 15 degrees C is (2.0 +/- 0.2) x 10(6) m(-1) s(-1) at pH 7 and (1.1 +/- 0.2) x 10(7) m(-1) s(-1) at pH 5. The rate constant of reduction of compound II to the ferric native enzyme at 15 degrees C is (5.5 +/- 0.1) x 10(2) m(-1) s(-1) at pH 7 and (8.9 +/- 1.6) x 10(4) m(-1) s(-1) at pH 5. pH dependence studies suggest that both complex formation between the ferric enzyme and nitrite and nitrite oxidation by compounds I and II are controlled by a residue with a pK(a) of (4.3 +/- 0.3). Protonation of this group (which is most likely the distal histidine) is necessary for optimum nitrite binding and oxidation.
- Published
- 2000
- Full Text
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20. Dynamics of NF kappa B and Ikappa Balpha studied with green fluorescent protein (GFP) fusion proteins. Investigation of GFP-p65 binding to DNa by fluorescence resonance energy transfer.
- Author
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Schmid JA, Birbach A, Hofer-Warbinek R, Pengg M, Burner U, Furtmüller PG, Binder BR, and de Martin R
- Subjects
- Animals, Base Sequence, CHO Cells, Cricetinae, DNA metabolism, DNA-Binding Proteins metabolism, Green Fluorescent Proteins, Kinetics, Microscopy, Fluorescence, Protein Binding, Spectrometry, Fluorescence, Transcription Factor RelA, I-kappa B Proteins metabolism, Luminescent Proteins metabolism, NF-kappa B metabolism, Recombinant Fusion Proteins metabolism
- Abstract
We investigated the dynamics of nuclear transcription factor kappaB (NF-kappaB) by using fusion proteins of the p65 subunit with mutants of green fluorescent protein (GFP). GFP-NF-kappaB chimeras were functional both in vitro and in vivo, as demonstrated by electrophoretic mobility shift assays and reporter gene studies. GFP-p65 was regulated by IkappaBalpha similar to wild type p65 and associated with its inhibitor even if both proteins were linked to a GFP protein. This finding was also verified by fluorescence resonance energy transfer (FRET) microscopy and studies showing mutual regulation of the intracellular localization of both GFP chimerae. Incubation of GFP-p65 with fluorescently labeled NF-kappaB-binding oligonucleotides also resulted in FRET. This effect was DNA sequence-specific and exhibited saturation characteristics. Application of stopped-flow fluorometry to measure the kinetics of FRET between GFP-p65 and oligonucleotides revealed a fast increase of acceptor fluorescence with a plateau after about 10 ms. The observed initial binding rate showed a temperature-dependent linear correlation with the oligonucleotide concentration. The association constant calculated according to pre-steady state kinetics was 3 x 10(6) m(-1), although equilibrium binding studies implied significantly higher values. This observation suggests that the binding process involves a rapid association with a rather high off-rate followed by a conformational change resulting in an increase of the association constant.
- Published
- 2000
- Full Text
- View/download PDF
21. Kinetics of oxidation of aliphatic and aromatic thiols by myeloperoxidase compounds I and II.
- Author
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Burner U, Jantschko W, and Obinger C
- Subjects
- Cysteine analogs & derivatives, Cysteine metabolism, Electrons, Glutathione metabolism, Homovanillic Acid pharmacology, Humans, Hydrogen Peroxide pharmacology, Hydrogen-Ion Concentration, Kinetics, Neutrophils enzymology, Oxidants pharmacology, Oxidation-Reduction, Penicillamine metabolism, Pentetic Acid pharmacology, Structure-Activity Relationship, Substrate Specificity, Sulfhydryl Compounds chemistry, Thioctic Acid metabolism, Thiouridine metabolism, Peroxidase metabolism, Sulfhydryl Compounds metabolism
- Abstract
Myeloperoxidase (MPO) is the most abundant protein in neutrophils and plays a central role in microbial killing and inflammatory tissue damage. Because most of the non-steroidal anti-inflammatory drugs and other drugs contain a thiol group, it is necessary to understand how these substrates are oxidized by MPO. We have performed transient kinetic measurements to study the oxidation of 14 aliphatic and aromatic mono- and dithiols by the MPO intermediates, Compound I (k3) and Compound II (k4), using sequential mixing stopped-flow techniques. The one-electron reduction of Compound I by aromatic thiols (e.g. methimidazole, 2-mercaptopurine and 6-mercaptopurine) varied by less than a factor of seven (between 1.39 +/- 0.12 x 10(5) M(-1) s(-1) and 9.16 +/- 1.63 x 10(5) M(-1) s(-1)), whereas reduction by aliphatic thiols was demonstrated to depend on their overall net charge and hydrophobic character and not on the percentage of thiol deprotonation or redox potential. Cysteamine, cysteine methyl ester, cysteine ethyl ester and alpha-lipoic acid showed k3 values comparable to aromatic thiols, whereas a free carboxy group (e.g. cysteine, N-acetylcysteine, glutathione) diminished k3 dramatically. The one-electron reduction of Compound II was far more constrained by the nature of the substrate. Reduction by methimidazole, 2-mercaptopurine and 6-mercaptopurine showed second-order rate constants (k4) of 1.33 +/- 0.08 x 10(5) M(-1) s(-1), 5.25 +/- 0.07 x 10(5) M(-1) s(-1) and 3.03 +/- 0.07 x 10(3) M(-1) s(-1). Even at high concentrations cysteine, penicillamine and glutathione could not reduce Compound II, whereas cysteamine (4.27 +/- 0.05 x 10(3) M(-1) s(-1)), cysteine methyl ester (8.14 +/- 0.08 x 10(3) M(-1) s(-1)), cysteine ethyl ester (3.76 +/- 0.17 x 10(3) M(-1) s(-1)) and alpha-lipoic acid (4.78 +/- 0.07 x 10(4) M(-1) s(-1)) were demonstrated to reduce Compound II and thus could be expected to be oxidized by MPO without co-substrates.
- Published
- 1999
- Full Text
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22. Reaction of myeloperoxidase compound I with chloride, bromide, iodide, and thiocyanate.
- Author
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Furtmüller PG, Burner U, and Obinger C
- Subjects
- Bromides chemistry, Chlorides chemistry, Humans, Hydrogen Peroxide chemistry, Hydrogen-Ion Concentration, Iodides chemistry, Neutrophils, Oxidation-Reduction, Peroxidase isolation & purification, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Anions chemistry, Peroxidase chemistry, Thiocyanates chemistry
- Abstract
Myeloperoxidase plays a fundamental role in oxidant production by neutrophils. The enzyme uses hydrogen peroxide to oxidize chloride (Cl-), bromide (Br-), iodide (I-), and the pseudohalide thiocyanate (SCN-) to their respective hypohalous acids. This study for the first time presents transient kinetic measurements of the oxidation of these halides and thiocyanate by the myeloperoxidase intermediate compound I, using the sequential mixing stopped-flow technique. At pH 7 and 15 degrees C, the two-electron reduction of compound I to the native enzyme by Cl- has a second-order rate constant of (2.5 +/- 0.3) x 10(4) M(-1) s(-1), whereas reduction of compound I by SCN- has a second-order rate constant of (9.6 +/- 0.5) x 10(6) M(-1) s(-1). Iodide [(7.2 +/- 0.7) x 10(6) M(-1) s(-1)] is shown to be a better electron donor for compound I than Br- [(1.1 +/- 0.1) x 10(6) M(-1) s(-1)]. The pH dependence studies suggest that compound I reduction by (pseudo-)halides is controlled by a residue with a pKa of about 4.6. The protonation of this group is necessary for optimum (pseudo-)halide anion oxidation. These transient kinetic results are underlined by steady-state spectral and kinetic investigations. SCN- is shown to be most effective in shifting the system myeloperoxidase/hydrogen peroxide from the peroxidatic cycle to the halogenation cycle, whereas iodide is shown to be more effective than bromide which in turn is much more effective than chloride. Decreasing pH increases the rate of this transition. Our results show that thiocyanate is an important substrate of myeloperoxidase in most environments and that hypothiocyanate is likely to contribute to leukocyte antimicrobial activity.
- Published
- 1998
- Full Text
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23. Purification and characterization of a homodimeric catalase-peroxidase from the cyanobacterium Anacystis nidulans.
- Author
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Obinger C, Regelsberger G, Strasser G, Burner U, and Peschek GA
- Subjects
- Amitrole pharmacology, Chromatography, Gel, Chromatography, Ion Exchange, Cyanides pharmacology, Cytosol enzymology, Dimerization, Dithionite, Electrophoresis, Polyacrylamide Gel, Hydrogen-Ion Concentration, Kinetics, Macromolecular Substances, Peroxidases isolation & purification, Substrate Specificity, Bacterial Proteins, Cyanobacteria enzymology, Peroxidases chemistry, Peroxidases metabolism
- Abstract
Cytosolic extracts of the cyanobacterium Anacystis nidulans exhibit both catalase and o-dianisidine peroxidase activity. Native polyacrylamide gel electrophoresis demonstrates one distinct enzyme, which has been purified to essential homogeneity and found to be composed of two identical subunits of equal size (80.5 kDa). The isoelectric point is at pH 4.7. It is a very efficient catalase with a broad pH optimum between 6.5 and 7.5 and a Km for H2O2 of 4.3 mM, a calculated turnover number of 7200 s(-1), and an overall-rate constant of 3.5 x 10(6) M(-1) s(-1). The behaviour of this protoheme-enzyme is typical of the class of prokaryotic catalase-peroxidases, which is sensitive to cyanide (Ki = 27.2 microM) and insensitive to the eukaryotic catalase inhibitor 3-amino-1,2,4-triazole. The enzyme accepts electrons from o-dianisidine, but not from ascorbate, glutathione, and NADH. With hydrogen peroxide in steady-state conditions the enzyme is mainly in the ferric state indicating that Compound I is much faster reduced by H2O2 than it is formed. The native enzyme is in the high-spin state, which is transformed to low-spin upon addition of cyanide. With peroxoacetic acid Compound I is formed at a rate of 5.9 x 10(4) M(-1) s(-1) at pH 7.0 and 25 degrees C with about 50% hypochromicity, a Soret-maximum at 405 nm and isosbestic points at 354 and 427 nm.
- Published
- 1997
- Full Text
- View/download PDF
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