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.     

Degradation of synthetic lignin by the protoplasts of Phanerochaete chrysosporium in the presence of lignin peroxidase or manganese peroxidase

Lignin was mineralized in the experiments in which ¹⁴C-lignin was incubated with lignin peroxidase or manganese peroxidase in a tartrate buffer in the presence of cycloheximide-treated protoplasts obtained from the ligninolytic mycelia of Phanerochaete chrysosporium. The rate of lignin mineralization was dependent on the lignin peroxidase or manganese peroxidase concentration in the medium. In the experiments in which lignin was incubated with lignin peroxidase or manganese peroxidase, lignin was repolymerized irrespective of the presence of protoplasts mineralizing lignin, suggesting that an active degradation of lignin and repolymerization took place. Taking into account that lignin peroxidase and manganese peroxidase were the only extracellular enzymes in the experiments in which lignin was mineralized by the protoplasts, it is postulated that lignin peroxidase and/or manganese peroxidase can degrade lignin into small fragments which can then be further absorbed by the fungal cells and subsequently degraded to CO₂.     

Luminol chemiluminescence reaction catalyzed by a microbial peroxidase

A peroxidase produced by microorganisms belonging to the genera Arthromyces and Coprinus was found to be a potent catalyst for the chemiluminescent oxidation of luminol, the luminescence produced per unit of microbial peroxidase protein being well over 100 times as strong as that produced by horseradish peroxidase. No large difference in Km value for H2O2 in the presence of luminol was found between Arthromyces ramosus peroxidase and horseradish peroxidase (7.0 and 15.5 microM, respectively), but Vmax of the Arthromyces peroxidase was 500 times greater than that of the horseradish peroxidase. It was also found that the Arthromyces peroxidase surpasses, beyond expectation, the horseradish peroxidase in the initial velocity of the chemiluminescence reaction with the stopped-flow method. The Arthromyces peroxidase was used for the glucose and cholesterol assays, which were notably more sensitive than the corresponding assays involving the horseradish peroxidase.     

Electron paramagnetic resonance spectroscopy of thyroid peroxidase

Thyroid peroxidase was isolated from porcine thyroids by two methods. Limited trypsin proteolysis was employed to obtain a cleaved enzyme, and affinity chromatography was used to isolate intact thyroid peroxidase. Enzyme isolated by both methods was used in the examination of the heme site of native thyroid peroxidase and its complexes by EPR spectroscopy. Intact thyroid peroxidase showed a homogeneous high-spin EPR signal with axial symmetry, in contrast to the rhombic EPR signal of native lactoperoxidase. Reaction of cyanide or azide ion with native thyroid peroxidase resulted in the loss of the axial EPR signal within several hours. The EPR spectroscopy of the nitrosyl adduct of ferrous thyroid peroxidase exhibited a three-line hyperfine splitting pattern and indicated that the heme-ligand structure of thyroid peroxidase is significantly different from that of lactoperoxidase.     

An inserted loop region of stromal ascorbate peroxidase is involved in its hydrogen peroxide-mediated inactivation

Ascorbate peroxidase isoforms localized in the stroma and thylakoid of higher plant chloroplasts are rapidly inactivated by hydrogen peroxide if the second substrate, ascorbate, is depleted. However, cytosolic and microbody-localized isoforms from higher plants as well as ascorbate peroxidase B, an ascorbate peroxidase of a red alga Galdieria partita, are relatively tolerant. We constructed various chimeric ascorbate peroxidases in which regions of ascorbate peroxidase B, from sites internal to the C-terminal end, were exchanged with corresponding regions of the stromal ascorbate peroxidase of spinach. Analysis of these showed that a region between residues 245 and 287 was involved in the inactivation by hydrogen peroxide. A 16-residue amino acid sequence (249-264) found in this region of the stromal ascorbate peroxidase was not found in other ascorbate peroxidase isoforms. A chimeric ascorbate peroxidase B with this sequence inserted was inactivated by hydrogen peroxide within a few minutes. The sequence forms a loop that binds noncovalently to heme in cytosolic ascorbate peroxidase of pea but does not bind to it in stromal ascorbate peroxidase of tobacco, and binds to cations in both ascorbate peroxidases. The higher susceptibility of the stromal ascorbate peroxidase may be due to a distorted interaction of the loop with the cation and/or the heme.     

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.     

Stable high level expression of human thyroid peroxidase in cultured Chinese hamster ovary cells

An expression plasmid containing both human thyroid peroxidase and mouse dihydrofolate reductase cDNAs was transfected into chinese hamster ovary cells. The stably transformed cells constitutively expressed immunoreactive thyroid peroxidase on the cell surface. These cells were further used to establish a subline producing a large amount of thyroid peroxidase by selecting clones resistant to methotrexate. The molecular weight of the expressed thyroid peroxidase was the same as purified human thyroid peroxidase. This expressed protein had peroxidase activity when determined by guaiacol oxidation. Furthermore, the expressed thyroid peroxidase was immunoreactive to sera of patients with autoimmune thyroid disease in which autoantibodies to thyroid peroxidase appeared.     

Mechanisms of electron transfer from sulfite to horseradish peroxidase-hydroperoxide compounds.

Using a rapid-scan spectrophotometer equipped with a stopped-flow apparatus, reactions of sulfite with compounds I and II of two horseradish peroxidase isoenzymes A and C were investigated. The direct two-electron reduction of peroxidase compound I by sulfite occurred at acidic pH but the mechanism gradually changed to the two-step reduction with the intermediate formation of compound II as the pH increased. The pH at which the one- and two-electron changes occurred at the same speed was 4.5 for peroxidase A and 7.7 for peroxidase C. A new peroxidase intermediate was found in the reaction between peroxidase compound II and sulfite. The sulfite compound showed a characteristic absorption band at 850 nm and the optical spectrum was similar to that of isoporphyrins but was quite different from that of sulfhemoproteins. The rate (k) of conversion from the sulfite-compound II complex to the sulfite compound was proportional to the concentration of H+ and the log k vs. pH plot for peroxidase A moved to the acidic side by 1.1 pH unit from that for peroxidase C.     

Characterization of the hydroperoxide-reducing activity of human plasma

A peroxidase was identified in human plasma using a novel peroxidase assay. In this assay both the substrate 5-phenyl-4-pentenyl hydroperoxide (PPHP) and its reduction product, 5-phenyl-4-pentenyl alcohol (PPA) are quantitated by HPLC. Substrate specificity studies indicated that the peroxidase requires glutathione as reducing substrate. No reduction was detected using the classical heme peroxidase reducing substrates, phenol and hydroquinone. Peroxidase activity was not due to glutathione transferases. Failure to saturate the peroxidase activity with reduced glutathione and inhibition by Cd+2 indicated that it is probably selenium dependent. The enzyme appears to be different from erythrocyte glutathione peroxidase based on kinetic and immunological experiments. The apparent Km values for PPHP are 25 microM for erythrocyte peroxidase and 54 microM for plasma peroxidase at 0.5 mM reduced glutathione. Anti-peroxidase prepared against bovine erythrocyte glutathione peroxidase partially inhibited human erythrocyte peroxidase but did not inhibit human plasma peroxidase.     

Hydroxyurea and p-aminophenol are the suicide inhibitors of ascorbate peroxidase

Guaiacol peroxidase from spinach catalyzes the oxidation of p-aminophenol to produce the aminophenoxy radical as the primary product which is converted further into a stable oxidation product with an absorption peak at 470 nm. The p-aminophenol radicals oxidize ascorbate (AsA) to produce monodehydroascorbate radicals. Kinetic analysis indicates that p-aminophenol radicals also oxidize monodehydroascorbate to dehydroascorbate. Incubation of AsA peroxidase from tea leaves and hydrogen peroxide with p-aminophenol, p-cresol, hydroxyurea, or hydroxylamine results in the inactivation of the enzyme. No inactivation of the enzyme was found upon incubation of the enzyme with these compounds either in the absence of hydrogen peroxide or with the stable oxidized products of these compounds. The enzyme was protected from inactivation by the inclusion of AsA in the incubation mixture. The radicals of p-aminophenol and hydroxyurea were produced by AsA peroxidase as detected by their ESR signals. These signals disappeared upon the addition of AsA, and the signal characteristic of monodehydroascorbate was found. Thus, AsA peroxidase is inactivated by the radicals of p-aminophenol, p-cresol, hydroxyurea, and hydroxylamine which are produced by the peroxidase reaction, and it is protected from inactivation by AsA via the scavenging of the radicals. Thus, these compounds are the suicide inhibitors for AsA peroxidase. Isozyme II of AsA peroxidase, which is localized in chloroplasts, is more sensitive to these compounds than isozyme I. In contrast to AsA peroxidase, guaiacol peroxidase was not affected by these various compounds, even though each was oxidized by it and the corresponding radicals were produced.     

Enzymatic enhancement of the catalytic rate of sulfhydryl oxidase

The rate of oxidation of glutathione by solubilized sulfhydryl oxidase was significantly enhanced in the presence of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7). This enhancement was proportional to the amount of active peroxidase in the assay, but could not be attributed solely to the oxidation of glutathione catalyzed by the peroxidase. A change in the Soret region of the horseradish peroxidase spectrum was observed when both glutathione and peroxidase were present. Moreover, addition of glutathione to a sulfhydryl oxidase/horseradish peroxidase mixture resulted in a rapid shift of the absorbance maximum from 403 nm to 417 nm. This shift indicates the oxidation of horseradish peroxidase. Spectra for three isozyme preparations of horseradish peroxidase, two acidic and one basic, all underwent this red-shift in the presence of sulfhydryl oxidase and glutathione. Cysteine and N-acetylcysteine could replace glutathione. Addition of catalase had no effect on the oxidation of peroxidase, indicating that the peroxide involved in the reaction was not derived from that released into the bulk solution by sulfhydryl oxidase-catalyzed thiol oxidation. Further evidence for a direct transfer of the hydrogen peroxide moiety was obtained by addition of glutaraldehyde to a sulfhydryl oxidase/horseradish peroxidase/N-acetylcysteine mixture. Size exclusion chromatography revealed the formation of a high-molecular-weight species with peroxidase activity, which was completely resolved from native horseradish peroxidase. Formation of this species was absolutely dependent on the presence of both the cysteine-containing substrate and sulfhydryl oxidase. The observed enhancement of sulfhydryl oxidase catalytic activity by the addition of horseradish peroxidase supports a bi uni ping-pong mechanism proposed previously for sulfhydryl oxidase.     

COLORIMETRIC INVESTIGATION OF THE UPTAKE OF AN INTRAVENOUSLY INJECTED PROTEIN (HORSERADISH PEROXIDASE) BY RAT KIDNEY AND EFFECTS OF COMPETITION BY EGG WHITE

After intravenous injection of horseradish peroxidase into rats, the foreign protein appeared in the kidney first in the small phagosomes and its concentration there decreased quickly; it then was concentrated and "stored" for several days in the large phagosomes. After injection of 10 mg of peroxidase per 100 gm of body weight, the concentration of peroxidase in blood and urine decreased exponentially during the first 6 hours; small amounts of peroxidase were excreted in the urine for several days. When 0.05 to 1.0 mg of peroxidase per 100 gm were administered, most of the peroxidase was taken up by the liver and little by the kidney, and a portion was excreted in the urine even at the lowest dose. At doses above 1.5 mg per 100 gm, the liver cells were saturated, and large reabsorption droplets appeared in the tubule cells of the kidney. With further dosage increase, the concentration of peroxidase in the phagosomes of the kidney increased rapidly until saturation was reached at doses of 13 mg per 100 gm. After intraperitoneal injection of egg white 18 hours prior to the administration of peroxidase, the concentration of peroxidase in all kidney fractions was only 10 to 25 per cent of the values for the untreated animals, the disappearance of peroxidase from the blood was delayed, and 81 percent more peroxidase was excreted in the urine. The treatment with egg white had no effect on the uptake of peroxidase by the liver. The ability of kidney tissue to degrade and adsorb peroxidase in vitro was tested.     

Identification of subcellular compartments involved in biosynthetic processing of cathepsin D.

We have assigned the biosynthetic processing steps of cathepsin D to intracellular compartments which are involved in its transport to lysosomes in HepG2 cells. Cathepsin D was synthesized as a 51-kDa proenzyme. After formation of 51-55-kDa intermediates due to processing of N-linked oligosaccharides, procathepsin D was proteolytically processed to an intermediate 44-kDa and the mature 31-kDa enzyme. The intersection of the biosynthetic pathway of cathepsin D with the endocytic pathway was labeled with horseradish peroxidase and monitored biochemically by 3,3'-diaminobenzidine cytochemistry. Horseradish peroxidase was used either as a fluid-phase marker to label the entire endocytic pathway or conjugated to transferrin (Tf) to label endosomes only. Directly after biosynthesis cathepsin D was accessible neither to horseradish peroxidase nor Tf-horseradish peroxidase. Newly synthesized 51-55-kDa species of cathepsin D present in the trans-Golgi reticulum were accessible to both horseradish peroxidase and Tf-horseradish peroxidase. The accessibility of trans-Golgi reticulum to both endocytosed horseradish peroxidase and Tf-horseradish peroxidase was monitored by colocalization with a secretory protein, alpha 1anti-trypsin. The proteolytic processing of 51-55-kDa to 44-kDa cathepsin D occurred in compartments which were fully accessible to fluid-phase horseradish peroxidase. Tf-horseradish peroxidase had access to only 20% of 44-kDa cathepsin D while it had no access to 31-kDa cathepsin D. In contrast, the 31-kDa species was completely accessible to fluid-phase horseradish peroxidase. We conclude that proteolytic processing of 51-55-kDa to 44-kDa cathepsin D occurs in endosomes, whereas the processing of 44-31-kDa cathepsin D takes place in lysosomes.     

beta-Adrenergic receptors stimulated peroxidase secretion from rat lacrimal gland

Incubation of rat extraorbital lacrimal gland slices with the beta-agonist isoproterenol caused peroxidase secretion but no K+ release. The peroxidase secretion was inhibited by propranolol. Addition of dibutyryl cyclic AMP or adenosine 3'5'-cyclic phosphorothioate to lacrimal slices produced peroxidase secretion at a higher rate than that obtained with optimal concentration of isoproterenol. Methyl isobutylxanthine is also a strong stimulator of peroxidase secretion. Peroxidase activity was determined by a modified sensitive guaiacol method. Membrane fraction of lacrimal cells was shown to contain an isoproterenol-stimulated adenylate cyclase activity. It is therefore suggested that there is a beta-adrenergic receptor in the rat lacrimal gland and that its stimulation causes activation of an adenylate cyclase which leads to peroxidase secretion.     

Rapid isolation of plant peroxidase. Purification of peroxidase a from Petunia

A rapid isolation procedure was developed for purification of peroxidase a from Petunia hybrida . Rapid isolation was possible since about 15% of the extracellular protein from stem tissue obtained by vacuum infiltration followed by centrifugation of the tissue represents peroxidase. Purification of peroxidase a from intercellular fluid was achieved by two acetone precipitation steps followed by DEAE-cellulose chromatography.
Three different forms of peroxidase were eluted from DEAE-cellulose at different NaCl concentrations. Isoelectric focusing showed, however, a pI of 3.8 for all three forms of peroxidase a . Only part of the peroxidase a enzymes bound to Concanavalin A indicating heterogeneity in the carbohydrate part. Homology of peroxidase a to the peroxidase G₁ group from tobacco is discussed.     

Sequence analysis and homology modeling of peroxidase from Medicago sativa

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.     

Effects of Peroxidase and Hydrogen Peroxide on the Dityrosine Formation and the Mixing Characteristics of Wheat-Flour Dough

The effects of adding hydrogen peroxide and peroxidase to wheat-flour dough on dityrosine formation and mixing characteristics were investigated. Dityrosine in wheat-flour dough was identified by HPLC with a fluorescence detector and by LC/MS/MS. Formation of dityrosine increased with the addition of hydrogen peroxide, and hydrogen peroxide plus peroxidase, to wheat-flour dough, while the addition of peroxidase had no effect on the amount of dityrosine formed. The mixing curve obtained by a doughgraph changed with the addition of hydrogen peroxide, and hydrogen peroxide plus peroxidase; the peak time was significantly delayed and the dough development time was extended. We found that dityrosine cross-links in wheat-flour dough increased with the addition of peroxidase plus hydrogen peroxide. It is thought that these cross-links can lead to polymerization of the proteins in wheat-flour dough.     

Purification and characterization of pea cytosolic ascorbate peroxidase

The cytosolic isoform of ascorbate peroxidase was purified to homogeneity from 14-day-old pea (Pisum sativum L.) shoots. The enzyme is a homodimer with molecular weight of 57,500, composed of two subunits with molecular weight of 29,500. Spectral analysis and inhibitor studies were consistent with the presence of a heme moiety. When compared with ascorbate peroxidase activity derived from ruptured intact chloroplasts, the purified enzyme was found to have a higher stability, a broader pH optimum for activity, and the capacity to utilize alternate electron donors. Unlike classical plant peroxidases, the cytosolic ascorbate peroxidase had a very high preference for ascorbate as an electron donor and was specifically inhibited by p-chloromercurisulfonic acid and hydroxyurea. Antibodies raised against the cytosolic ascorbate peroxidase from pea did not cross-react with either protein extracts obtained from intact pea chloroplasts or horseradish peroxidase. The amino acid sequence of the N-terminal region of the purified enzyme was determined. Little homology was observed among pea cytosolic ascorbate peroxidase, the tea chloroplastic ascorbate peroxidase, and horseradish peroxidase; homology was, however, found with chloroplastic ascorbate peroxidase isolated from spinach leaves.     

Addition of veratryl alcohol oxidase activity to manganese peroxidase by site-directed mutagenesis

Manganese peroxidase and lignin peroxidase are ligninolytic heme-containing enzymes secreted by the white-rot fungus Phanerochaete chrysosporium. Despite structural similarity, these peroxidases oxidize different substrates. Veratryl alcohol is a typical substrate for lignin peroxidase, while manganese peroxidase oxidizes chelated Mn2+. By a single mutation, S168W, we have added veratryl alcohol oxidase activity to recombinant manganese peroxidase expressed in Escherichia coli. The kcat for veratryl alcohol oxidation was 11 s-1, Km for veratryl alcohol approximately 0.49 mM, and Km for hydrogen peroxide approximately 25 microM at pH 2.3. The Km for veratryl alcohol was higher and Km for hydrogen peroxide was lower for this manganese peroxidase mutant compared to two recombinant lignin peroxidase isoenzymes. The mutant retained full manganese peroxidase activity and the kcat was approximately 2.6 x 10(2) s-1 at pH 4.3. Consistent with relative activities with respect to these substrates, Mn2+ strongly inhibited veratryl alcohol oxidation. The single productive mutation in manganese peroxidase suggested that this surface tryptophan residue (W171) in lignin peroxidase is involved in catalysis.