Thermal destruction and stability of peroxidase in acid foods.
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The regio- and stereospecificity of bimolecular phenoxy radical coupling reactions, of especial importance in lignin and lignan biosynthesis, are clearly controlled in some manner in vivo; yet in vitro coupling by oxidases, such as laccases, only produce racemic products. In other words, laccases, peroxidases, and comparable oxidases are unable to control regio- or stereospecificity by themselves and thus some other agent must exist. A 78-kilodalton protein has been isolated that, in the presence of an oxidase or one electron oxidant, effects stereoselective bimolecular phenoxy radical coupling in vitro. Itself lacking a catalytically active (oxidative) center, its mechanism of action is presumed to involve capture of E-coniferyl alcohol-derived free-radical intermediates, with consequent stereoselective coupling to give (+)-pinoresinol.
The objective of this study was to establish a hepatic lipase (HL) assay method that can be applied to automatic clinical analyzers. Seventy-four hyperlipidemic subjects (men/women 45/29) were recruited. Lipase activity was assayed measuring the increase in absorbance at 546 nm due to quinonediimine dye production. Reaction mixture R-1 contained 50 mM Tris-HCl (pH 9.5), 0.5 mM glycerol-1,2-dioleate, 0.4% (unless otherwise noted) polyoxyethylene-nonylphenylether, 3 mM ATP, 3 mM MgCl(2), 1.5 mM CaCl(2), monoacylglycerol-specific lipase, glycerol kinase, glycerol-3-phosphate oxidase, 0.075% N,N-bis-(4-sulfobutyl)-3-methylaniline-2 Na, peroxidase, ascorbic acid oxidase. Reaction mixture R-2 contained 50 mM Tris-HCl (pH9.5), 0.15% 4-aminoantypirine. Automated assay for activity was performed with a Model 7080 Hitachi analyzer. In the lipase assay, 160 microl of R-1 was incubated at 37 degrees C with 3 microl of samples for 5 min, and 80 microl of R-2 was added. Within-run coefficient of variations was 0.9-1.0%. Calibration curve of lipase activity was linear (r = 0.999) between 0 and 320 U/l. Analytical recoveries of purified HL added to plasma were 96.6-99.8%. HL activity in postheparin plasma measured in this method had a closer correlation with HL mass by a sandwich ELISA (r = 0.888, P < 0.0001) than those in the conventional method using [(14)C-]triolein (r = 0.730, P < 0.0001). This assay method for HL activity can be applied to an automatic clinical analyzer.
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Perception of microbial signal molecules is part of the strategy evolved by plants to survive attacks by potential pathogens. To gain a more complete understanding of the early signaling events involved in these responses, we used fungal components of Fusarium under in vitro condition and checked the rise in signal molecule, salicylic acid (SA), and marker enzymes in defense reactions against the pathogen. SA level increased by 21 folds in elicitor treated plantlets as compared to that of control plantlets and there was marked increase in phenylalanine ammonia-lyase(PAL), peroxidase(POX), polyphenol oxidase(PPO) along with higher total phenolic content. Present results indicated that use of fungal components had successfully induced systemic resistance in in vitro cultured banana plantlets.
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Lung cancer is a common pathology with high mortality due to late diagnosis. Glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST), catalase (CAT), xanthine oxidase (XO), Cu-Zn superoxide dismutase (Cu-Zn SOD) activities, total glutathione (TGSH), nitric oxide (NO*), and malondialdehyde (MDA) levels were investigated in erythrocytes of patients with non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC), and healthy control group. We aimed to investigate serum GSH, GSH-dependent enzymes activities (GSH-Px and GST), XO, CAT, Cu-Zn SOD activity, and NO*, and MDA levels in patients with NSCLC and with SCLC and correlate with the cancer stage. Erythrocyte MDA, NO*, TGSH levels and erythrocyte SOD, CAT and XO activities were significantly higher in patients with NSCLC and SCLC than in controls. Slightly increased erythrocyte GSH-Px and GST activities were not significantly different from the controls. Erythrocyte MDA level positively correlated with erythrocyte NO* levels in patients with early stage (I+II) in NSCLC groups. Erythrocyte MDA level positively correlated with erythrocyte XO activity in patients with advanced stage (III+IV) in NSCLC groups. However, no other correlation could be found among the parameters in healthy controls and patients with NSCLC and with SCLC. Results obtained in this study indicate significant changes in antioxidant defence system in NSCLC and SCLC patients, which may lead to enhanced action of oxygen radical, resulting in lipid peroxidation.
Phanerochaete chrysosporium produces two classes of extracellular heme proteins, designated lignin peroxidases and manganese peroxidases, that play a key role in lignin degradation. In this study we isolated and characterized a lignin peroxidase-negative mutant (lip mutant) that showed 16% of the ligninolytic activity (14C-labeled synthetic lignin----14CO2) exhibited by the wild type. The lip mutant did not produce detectable levels of lignin peroxidase, whereas the wild type, under identical conditions, produced 96 U of lignin peroxidase per liter. Both the wild type and the mutant produced comparable levels of manganese peroxidase and glucose oxidase, a key H2O2-generating secondary metabolic enzyme in P. chrysosporium. Fast protein liquid chromatographic analysis of the concentrated extracellular fluid of the lip mutant confirmed that it produced only heme proteins with manganese peroxidase activity but no detectable lignin peroxidase activity, whereas both lignin peroxidase and manganese peroxidase activities were produced by the wild type. The lip mutant appears to be a regulatory mutant that is defective in the production of all the lignin peroxidases.
A method for detection of ascorbic acid oxidase, ascorbic acid peroxidase and dehydroascorbic acid reductase is reported. This method allows the qualitative determination of the presence of these enzymes, also in conditions where the commonly used spectrophotometric assays are unreliable.
Enzyme electrodes based on complexing a water-soluble copolymer of acrylamide and vinylimidazole with [Os(dmebpy)2C1]+/2+ (dmebpy = 4,4'-dimethyl-2,2'-bipyridine) and cross-linking with oxidases by water-soluble cross-linkers are described. The potential of the polyacrylamide-based redox polymer is +55 mV (SCE), a typical electron diffusion coefficient (De) in the redox hydrogel that results from its cross-linking is (1.3 +/- 0.1) x 10(-9) cm2/s. The properties of the enzyme electrodes formed when this redox hydrogel "wired" horseradish peroxidase (HRP), lactate oxidase (LOx) or glucose oxidase (GOx) depended on the thickness of the hydrogel film, the chemistry of their cross-linking, and their enzyme content. At the wired HRP electrodes, H2O2 was electrocatalytically reduced to water at 0.0 V (SCE). Lactate and glucose were electrocatalytically oxidized at 0.16 V (SCE). The GOx electrodes, when made with 140 micrograms/cm2 thick polymer films, were selective for glucose in the presence of physiological concentrations of urate and ascorbate.
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A sequential injection analysis (SIA) system is described for the simultaneous determination of L-and D-carnitine using amperometric biosensors as detectors. The SIA system was used, because of its high precision, accuracy and low sample and buffer consumption. The biosensors were designed using physical and chemical immobilization of L-amino acid oxidase and horseradish peroxidase (HRP) for the assay of L- carnitine, and D-amino acid oxidase and HRP for the assay of D-carnitine. The linear concentration ranges are in the pmol/l to nmol/l magnitude order, with very low limits of detection. The biosensors/SIA system was used reliably for on-line process control of the enantiopurity of carnitine with a frequency of 34 samples per hour.
Polyclonal antibodies were raised against isoxazolyl penicillins in rabbits after immunization with a cloxacillin-human serum albumin conjugate. The antisera were tested in direct and indirect competitive enzyme immunoassays (EIAs), using glucose oxidase or horseradish peroxidase conjugates of oxacillin, cloxacillin, or dicloxacillin, respectively, as the labelled antigen. The relative cross-reactivities of each test system with oxacillin, cloxacillin, and dicloxacillin, determined from the amount of antibiotic required for 50% inhibition of labelled antigen binding, were dependent on the antibiotic used as the labelled antigen. Other beta-lactam antibiotics did not cross-react in these test systems. In a direct EIA using a cloxacillin-horseradish peroxidase conjugate, cloxacillin and dicloxacillin in milk were detected at levels of 10 and 30 ng ml-1; the average recoveries at these levels were 102 and 84%, respectively.
Highly sensitive direct visual detection of potyviruses was achieved using a dot blot immunobinding assay (DBIA). The small sample volumes required permit the detection of as little as 0.5 pg virus in purified preparations. The binding of rabbit antibodies could be visualized using goat anti-rabbit IgG (GAR) conjugated to alkaline phosphatase, beta-D-galactosidase, glucose oxidase, or horseradish peroxidase and histochemical substrates. The avidin-biotin system was also useful, but somewhat less sensitive than GAR-enzyme conjugates. Detection of potyviruses in an aphid vector was also attempted, but without success due to endogenous aphid enzymes.