Prostaglandin H synthase and hydroperoxides: peroxidase reaction and inactivation kinetics

The reaction kinetics of the peroxidase activity of prostaglandin H synthase have been examined with 15-hydroperoxyeicosatetraenoic acid and hydrogen peroxide as substrates and tetramethylphenylenediamine as cosubstrate. The apparent Km and Vmax values for both hydroperoxides were found to increase linearly with the cosubstrate concentration. The overall reaction kinetics could be interpreted in terms of an initial reaction of the synthase with hydroperoxide to form an intermediate equivalent to horseradish peroxidase Compound I, followed by reduction of this intermediate by cosubstrate to regenerate the resting enzyme. The rate constants estimated for the generation of synthase Compound I were 7.1 X 10(7) M-1 s-1 with the lipid hydroperoxide and 9.1 X 10(4) M-1 s-1 with hydrogen peroxide. The rate constants estimated for the rate-determining step in the regeneration of resting enzyme by cosubstrate were 9.2 X 10(6) M-1 s-1 in the case of the reaction with lipid hydroperoxide and 3.5 X 10(6) M-1 s-1 in the case of reaction with hydrogen peroxide. The intrinsic affinities of the synthase peroxidase for substrate (Ks) were estimated to be on the order of 10(-8) M for lipid hydroperoxide and 10(-5) M for hydrogen peroxide. These affinities are quite similar to the reported affinities of the synthase for these hydroperoxides as activators of the cyclooxygenase. The peroxidase activity was found to be progressively inactivated during the peroxidase reaction. The rate of inactivation of the peroxidase was increased by increases in hydroperoxide level, and decreased by increases in peroxidase cosubstrate. The inactivation of the peroxidase appeared to occur by a hydroperoxide-dependent process, originating from synthase Compound I or Compound II.     

Controlled layer-by-layer immobilization of horseradish peroxidase.

Horseradish peroxidase (HRP) was biotinylated with biotinamidocaproate N-hydroxysuccinimide ester (BcapNHS) in a controlled manner to obtain biotinylated horseradish peroxidase (Bcap-HRP) with two biotin moieties per enzyme molecule. Avidin-mediated immobilization of HRP was achieved by first coupling avidin on carboxy-derivatized polystyrene beads using a carbodiimide, followed by the attachment of the disubstituted biotinylated horseradish peroxidase from one of the two biotin moieties through the avidin-biotin interaction (controlled immobilization). Another layer of avidin can be attached to the second biotin on Bcap-HRP, which can serve as a protein linker with additional Bcap-HRP, leading to a layer-by-layer protein assembly of the enzyme. Horseradish peroxidase was also immobilized directly on carboxy-derivatized polystyrene beads by carbodiimide chemistry (conventional method). The reaction kinetics of the native horseradish peroxidase, immobilized horseradish peroxidase (conventional method), controlled immobilized biotinylated horseradish peroxidase on avidin-coated beads, and biotinylated horseradish peroxidase crosslinked to avidin-coated polystyrene beads were all compared. It was observed that in solution the biotinylated horseradish peroxidase retained 81% of the unconjugated enzyme's activity. Also, in solution, horseradish peroxidase and Bcap-HRP were inhibited by high concentrations of the substrate hydrogen peroxide. The controlled immobilized horseradish peroxidase could tolerate much higher concentrations of hydrogen peroxide and, thus, it demonstrates reduced substrate inhibition. Because of this, the activity of controlled immobilized horseradish peroxidase was higher than the activity of Bcap-HRP in solution. It is shown that a layer-by-layer assembly of the immobilized enzyme yields HRP of higher activity per unit surface area of the immobilization support compared to conventionally immobilized enzyme.     

Differential identification of mouse granulocyte (CFU-g) and macrophage (CFU-m) precursors in plasma clots

Because benzidine and its derivatives have possible carcinogenic activity, a safe method is needed to demonstrate endogenous peroxidase activity. Colonies derived from mouse bone marrow cells in plasma clot culture were classified as granulocyte (CFU-g) or macrophage (CFU-m) precursors by peroxidase and naphthol AS acetate (NASA) esterase staining, respectively. Endogenous peroxidase activity was measured using benzidine or p-phenylenediazine-pyrocatechol (PPD-PC). The effectiveness of peroxidase staining with both reagents was evaluated under several conditions, and the enzyme property was confirmed by inactivation with a variety of inhibitors. The level of peroxidase activity did not differ significantly between PPD-PC and benzidine. Colony number and number of cultured cells were strongly correlated (P greater than 0.983). We conclude that PPD-PC safely demonstrates peroxidase activity in cultured cells and is as accurate, reliable, and efficient as benzidine.     

Inhibition of horseradish peroxidase by N-ethylamide of o-sulfobenzoylacetic acid

The carboxylic groups of horseradish peroxidase were modified by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate by the Koshland method. The catalytic properties of the native and modified peroxidase were studied in the presence of N-ethylamide of o-sulfobenzoylacetic acid (EASBA) at pH 5.0-7.5. In the oxidation of o-dianisidine, EASBA is a competitive inhibitor of the carbidiimide-modified peroxidase, and it increases both K(m) and Vm in the case of the native enzyme. These data show that at least one of the carboxylic groups modified with carbodiimide is located at the area of the peroxidase active site.     

Extracellular accumulation of high specific-activity peroxidase by cell suspension cultures of cowpea

Cell suspension cultures of cowpea (Vigna sp.) were able to produce extracellular peroxidase. Different growth regulator concentrations induced different peroxidase activity in callus. The crude extracellular medium after four weeks of culture showed higher (6 times) specific peroxidase activity and higher thermo stability than commercial horse-radish peroxidase. The commercial production of peroxidase enzyme from cowpea by utilizing plant cell cultures is discussed.     

The role of enzymes produced by white-rot fungus Irpex lacteus in the decolorization of the textile industry effluent

The textile industry wastewater has been decolorized efficiently by the white rot fungus, Irpex lacteus, without adding any chemicals. The degree of the decolorization of the dye effluent by shaking or stationary cultures is 59 and 93%, respectively, on the 8th day. The higher level of manganese-dependent peroxidase (MnP) and non-specific peroxidase (NsP) was detected in stationary cultures than in the cultures shaken. Laccase activities were equivalent in both cultures and its level was not affected significantly by the culture duration. Neither lignin peroxidase (LiP) nor Remazol Brilliant Blue R oxidase (RBBR ox) was detected in both cultures. The absorbance of the dye effluent was significantly decreased by the stationary culture filtrate of 7 days in the absence of Mn (II) and veratryl alcohol. In the stationary culture filtrate, three or more additional peroxidase bands were detected by the zymogram analysis.     

Oxidation of luminol with peroxidase from royal palm leaves

We optimized the conditions for luminol oxidation by hydrogen peroxide in the presence of peroxidase (EC 1.11.1.7) from royal palm leaves (Roystonea regia). The pH range (8.3-8.6) corresponding to maximum chemiluminescence was similar for palm tree peroxidase and horseradish peroxidase. Variations in the concentration of the Tris buffer were accompanied by changes in chemiluminescence. Note that maximum chemiluminescence was observed in the 30 mM solution. The detection limit of the enzyme assay during luminol oxidation by hydrogen peroxide was 1 pM. The specific feature of palm tree peroxidase was the generation of a long-term chemiluminescent signal. In combination with the data on the high stability of palm tree peroxidase, our results indicate that this enzyme is promising for its use in analytical studies.     

Characterization of 12-L-hydroperoxyeicosa-5,8,10,14-tetraenoic acid peroxidase in platelets by monoclonal antibody against glutathione peroxidase

In the present investigation, 12-L-hydroxyeicosa-5,8,14-tetraenoic acid (12-HPETE) peroxidase in the platelet 12-lipoxygenase pathway was characterized by using a monoclonal antibody to erythrocyte glutathione peroxidase. Pure glutathione peroxidase was used for the immunization of mice. Monoclonal antibody directed against the erythrocyte glutathione peroxidase was obtained from hybridomas, following fusion of mouse NS-1 myeloma cells with spleen cells from a mouse immunized with the enzyme. The subclass of monoclonal antibody was immunoglobulin M with kappa-light chain. Enzyme activity assays using cumene hydroperoxide and [1-14C]12-HPETE as substrates were employed. The monoclonal antibody reacted with glutathione peroxidase in the cumene hydroperoxide assay. In order to see whether platelet 12-HPETE peroxidase reacts with the monoclonal antibody, platelet cytosol and glutathione peroxidase were incubated with the monoclonal antibody and the antibody was precipitated by goat anti-mouse immunoglobulin M. The activities of platelet 12-HPETE peroxidase and glutathione peroxidase remaining were then assayed by using [1-14C]12-HPETE as substrate. The ability of glutathione peroxidase to transform 12-HPETE to 12-HETE was removed by the monoclonal antibody; however, the activity of platelet cytosol was not removed by the antibody. The results indicated that the antigenic specificity of 12-HPETE peroxidase in the platelet 12-lipoxygenase pathway is different from that of erythrocyte glutathione peroxidase.     

Effects of decreased glutathione peroxidase activity on the pentose phosphate cycle in mouse lung

The effects of reducing glutathione peroxidase activity in the lung by changing dietary selenium intake has been investigated. In animals that were exposed to room air, selenium effects were confined to glutathione peroxidase activity, whereas under conditions of oxidant stress (ozone) the decrease in glutathione peroxidase activity prevented the stimulation of the pentose phosphate cycle (assayed by measuring glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities) which has been reported to increase in response to oxidant stress. The suppression of glutathione peroxidase activity was found to depend on dietary selenium concentration. The physiological significance of this observation may be related to the process of injury and repair in the lung.     

The effect of manganese on the production of Phanerochaete flavido-alba ligninolytic peroxidases in nitrogen limited cultures

Manganese supplementation of culture medium affected Phanerochaete flavido-alba FPL 106507 growth, glucose consumption and extracellular protein accumulation. Both the titre and time of detection of lignin peroxidase (LiP) were affected by manganese concentration in the medium, whereas with manganese peroxidase (MnP) only the titre was affected. In high Mn(II) containing cultures highest manganese peroxidase levels and a decrease in extracellular veratryl alcohol accumulation were observed. After FPLC a number of haemprotein peaks showing manganese peroxidase activity were detected in Mn(II) supplemented cultures. On the contrary, only haemprotein peaks of lignin peroxidase were detected in culture medium not supplemented with Mn(II).     

Effect of Organic Acids on Reactions Catalyzed by Manganese Peroxidase from Phanerochaete chrysosporium

The effect of several organic acids on the oxidation of Mn(II) catalyzed by manganese peroxidase was studied. Reactivities of manganese peroxidase and chemically prepared Mn(III) organic acid complexes towards phenolic compounds were compared. If lactate appears to be the best complexant for manganese peroxidase activity, chemically prepared Mn(III)—lactate complex is a less effective oxidant towards phenolic compounds than other Mn(III)—complexes. Our results agree with the hypothesis that certain organic acids are involved in the catalytic cycle of manganese peroxidase. Malonate and lactate seem to be the most attractive complexants for practical applications of manganese peroxidase and were used in enzymatic treatment of hardwood kraft pulp. Bleaching of kraft pulp was studied and after alkaline extraction, a significant decrease of kappa number was measured. The bleaching was enhanced in lactate buffer.     

Studies on mammalian intestinal peroxidase.

A peroxidase, purified from rat small intestine to apparent homogeneity as judged by polyacrylamide-gel electrophoresis, exhibited an absorbance ratio (A412/A280) of 0.783. Its Mr (44000 +/- 1000) and spectral properties were similar to those of the pig intestinal enzyme. The velocity constant for the reaction between rat intestinal peroxidase and hydrogen peroxide was found to be 1.8 x 10(7) M-1 . s-1. Benzhydroxamic acid inhibited the peroxidative oxidation of guaiacol by intestinal peroxidase from both species but the concentration required to cause half-inhibition of the enzyme from the rat was higher by one order of magnitude than for the pig enzyme. The amino acid composition of highly-purified pig intestinal peroxidase showed a relative abundance of basic amino acids (lysine and arginine) and was similar to that of lactoperoxidase, but not that of myeloperoxidase. The initial ten amino acid residues of this enzyme (the first reported partial sequence for a mammalian peroxidase) were also determined.     

Proteolytic and peroxidatic reactions of commercial horseradish peroxidase with myelin basic protein

Degradation of myelin basic protein during incubations with high concentrations of horseradish peroxidase has been demonstrated [Johnson & Cammer (1977) J. Histochem. Cytochem.25, 329-336]. Possible mechanisms for the interaction of the basic protein with peroxidase were investigated in the present study. Because the peroxidase samples previously observed to degrade basic protein were mixtures of isoenzymes, commercial preparations of the separated isoenzymes were tested, and all three degraded basic protein, but to various extents. Three other basic proteins, P(2) protein from peripheral nerve myelin, lysozyme and cytochrome c, were not degraded by horseradish peroxidase under the same conditions. Inhibitor studies suggested a minor peroxidatic component in the reaction. Therefore the peroxidatic reaction with basic protein was studied by using low concentrations of peroxidase along with H(2)O(2). Horseradish peroxidase plus H(2)O(2) caused the destruction of basic protein, a reaction inhibited by cyanide, azide, ferrocyanide, tyrosine, di-iodotyrosine and catalase. Lactoperoxidase plus H(2)O(2) and myoglobin plus H(2)O(2) were also effective in destroying the myelin basic protein. Low concentrations of horseradish peroxidase plus H(2)O(2) were not active against other basic proteins, but did destroy casein and fibrinogen. Although high concentrations of peroxidase alone degraded basic protein to low-molecular-weight products, suggesting the operation of a proteolytic enzyme contaminant in the absence of H(2)O(2), incubations with catalytic concentrations of peroxidase in the presence of H(2)O(2) converted basic protein into products with high molecular weights. Our data suggest a mechanism for the latter, peroxidatic, reaction where polymers would form by linking the tyrosine side chains in basic-protein molecules. These data show that the myelin basic protein is unusually susceptible to peroxidatic reactions.     

Copper-induced oxidative stress in maize shoots (Zea mays L.): H2O2 accumulation and peroxidases modulation

The effect of copper excess on growth, H2O2 level and peroxidase activities were studied in maize shoots. Ten-day-old seedlings were cultured in nutrient solution that contained Cu2+ ions at various concentrations (50 and 100 microM) for seven days. High concentrations of Cu2+ ions caused significant decrease both in matter production and elongation of maize shoots. In addition, treatment with CuSO4 increased levels of H2O2 and induced changes in several peroxidase activities. Moreover, the disturbance of the physiological parameters was accompanied by the modulation of the peroxidase activities: GPX (Guaiacol peroxidase, EC 1.11.1.7), CAPX (Coniferyl alcohol peroxidase, EC 1.11.1.4) and APX (Ascorbate peroxidase, EC. 1.11.1.11). Furthermore, this modulation becomes highly significant, especially, in the presence of 100 microM of CuSO4.     

Reactive oxygen species and human spermatozoa: analysis of the cellular mechanisms involved in luminol- and lucigenin-dependent chemiluminescence.

We have shown that human spermatozoa generate and release reactive oxygen species that can be detected by chemiluminescence techniques. Analysis of the cellular mechanisms responsible for this activity suggests that the probe, luminol, undergoes an intracellular dioxygenation reaction mediated by hydrogen peroxide and a sperm peroxidase located within the acrosome. Support for this model included the following observations: (1) the luminol-dependent signal could be suppressed with peroxidase inhibitors, phenylhydrazine and sodium azide; (2) this suppression could be reversed by the addition of an azide-insensitive peroxidase, horse radish peroxidase (HRP); (3) inhibition of intracellular superoxide dismutase (SOD) with potassium cyanide (KCN) suppressed the luminol signal; (4) peroxidase activity could be detected in purified populations of human spermatozoa with 3,3',5,5' tetramethylbenzidine (TMB); (5) this peroxidase was active at the pH prevailing within the acrosomal vesicle; and (6) peroxidase activity and luminol-dependent chemiluminescence were minimal in spermatozoa exhibiting a congenital absence of acrosomes. Human spermatozoa could also generate lucigenin-dependent chemiluminescent signals that could neither be suppressed with peroxidase inhibitors nor enhanced by the addition of peroxidase. However, these signals could be enhanced by suppression of intracellular SOD with KCN or inhibited by exogenous SOD, suggesting that lucigenin was responding to superoxide anion released into the extracellular space. The ability of chemiluminescent techniques to detect and discriminate the production of superoxide and hydrogen peroxide by spermatozoa should facilitate the further analysis of reactive oxygen species as mediators of normal and abnormal human sperm function.     

Loading the multilayer dextran sulfate/protamine microsized capsules with peroxidase

Stable polyelectrolyte capsules were produced by the layer-by-layer (LbL) assembling of biodegradable polyelectrolytes, dextran sulfate and protamine, on melamine formaldehyde (MF) microcores followed by the cores decomposition at low pH. The mean diameter of the capsules at pH 3-5 was 8.0 +/- 0.2 microm, which is more than that diameter of the templates (5.12 +/- 0.15 microm). With pH growing up to 7-8, the capsules enlarged, swelling up to the diameter 9-10 microm. The microcapsules were loaded with horseradish peroxidase. Seemingly, peroxidase is embedded in the gellike structure in the microcapsule interior formed by MF residues in the complex with polymers used for LbL coating as proved by Raman confocal spectroscopy. The amount of finally incorporated peroxidase increased from 0.2 x 10(8) to 2.2 x 10(8) peroxidase molecules per capsule with pH growing from 5 to 8. The pH shifts causing changes in capsule swelling and the replacement of solutions without pH shifts lead to the protein loss. The encapsulated peroxidase showed a high activity (57%), which remained stable for 12 months.     

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

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.     

Homologous expression of recombinant manganese peroxidase in Phanerochaete chrysosporium.

The promoter region of the glyceraldehyde-3-phosphate dehydrogenase gene (gpd) was used to drive expression of mnp1, the gene encoding Mn peroxidase isozyme 1, in primary metabolic cultures of Phanerochaete chrysosporium. A 1,100-bp fragment of the P. chrysosporium gpd promoter region was fused upstream of the mnp1 gene to construct plasmid pAGM1, which contained the Schizophyllum commune ade5 gene as a selectable marker. pAGM1 was used to transform a P. chrysosporium ade1 auxotroph to prototrophy. Ade+ transformants were screened for peroxidase activity on a solid medium containing high carbon and high nitrogen (2% glucose and 24 mM NH4 tartrate) and o-anisidine as the peroxidase substrate. Several transformants that expressed high peroxidase activities were purified and analyzed further in liquid cultures. Recombinant Mn peroxidase (rMnP) was expressed and secreted by transformant cultures on day 2 under primary metabolic growth conditions (high carbon and high nitrogen), whereas endogenous wild-type mnp genes were not expressed under these conditions. Expression of rMnP was not influenced by the level of Mn in the culture medium, as previously observed for the wild-type Mn peroxidase (wtMnP). The amount of active rMnP expressed and secreted in this system was comparable to the amount of enzyme expressed by the wild-type strain under ligninolytic conditions. rMnP was purified to homogeneity by using DEAE-Sepharose chromatography, Blue Agarose chromatography, and Mono Q column chromatography. The M(r) and absorption spectrum of rMnP were essentially identical to the M(r) and absorption spectrum of wtMnP, indicating that heme insertion, folding, and secretion were normal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Some enzymes present in the walls of mesophyll cells of tobacco leaves.

Cell-wall enzymes were assayed by the difference between enzyme activities in the whole cell and the protoplast. Both peroxidase (85.2%) and acid phosphatase (21.9%) were located in the wall. However, malate dehydrogenase was found only in the protoplast. A study of the time-course of the release of peroxidase and malate dehydrogenase into the incubation medium from cells either treated with cellulase or untreated, also indicated that peroxidase and not malate dehydrogenase was located in the wall. Only two anodic isoenzymes of peroxidase were present in the cell wall. These were more negatively charged than those of horseradish peroxidase.     

Regulation of synthesis of nitrite reductase in pea leaves: in-vivo and in-vitro studies

The cellular location of three peroxidase isoenzymes (PRX) in mature leaf tissue of Petunia and their affinity for Concanavalin A-Sepharose were investigated. The isoenzymes PRXa, PRXb and PRXc were identified by their positions in starch-gel zymograms. The fast-moving anodic and cathodic peroxidase bands, the isoenzymes PRXa and PRXc respectively, were the most active peroxidases in extracellular extracts. The molecular forms of PRXa showed a tissue-specific distribution between midrib and remaining leaf tissue. An intermediate-moving anodic peroxidase band, the isoenzyme PRXb, was the most active peroxidase released after extraction of isolated mesophyll protoplasts. Small amounts of the peroxidase isoenzymes were present in cell-wall-bound fractions. Incubation of a crude protein fraction with Concanavalin A-Sepharose showed that the isoenzyme PRXb bound more firmly to Concanavalin A-Sepharose than the isoenzymes PRXa and PRXc, of which only one molecular form bound partly. The results are discussed with respect to a possible function of one of the peroxidase isoenzymes, and a possible role of oligosaccharide chains in determining the cellular location of plant peroxidases is suggested.Abbreviations Con A Concanavalin A - PRX peroxidase (isoenzyme)