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PubMed1979

Purification of peroxidase-conjugated antibody for enzyme immunoassay by affinity chromatography on concanavalin A.

Preparation of peroxidase-conjugated antibody contaminated with very small amounts of unconjugated antibody for highly sensitive enzyme immunoassays is described. The purification process is dependent upon the differential affinity of antibody and peroxidase-conjugated antibody for concanavalin A (Con A). Application of peroxidase and radioactive labelled IgG alone or as a mixture to Con A-Sepharose columns showed that 62-83% of IgG and 0% of peroxidase could be eluted under starting conditions, while 70-83% of peroxidase and about 6% of IgG could be eluted with appropriate buffer. The contamination of the conjugate with unconjugated antibody is dependent on the ratio of conjugated to unconjugated antibody in the mixture to be purified. Using this procedure on IgG conjugated with peroxidase with a ratio of unconjugated to conjugated IgG of about 2 : 1 a preparation was obtained where the ratio was changed to 0.1 : 1.

Antibodies↗
PubMed1992

Production of Phanerochaete chrysosporium lignin peroxidase.

Liginin peroxidase (ligninase) of the white rot fungus Phanerochaete chrysosporium Burdsall was discovered in 1982 as a secondary metabolite. Today multiple isoenzymes are known, which are often collectively called as lignin peroxidase. Lignin peroxidase has been characterized as a veratryl alcohol oxidizing enzyme, but it is a relatively unspecific enzyme catalyzing a variety of reactions with hydrogen peroxide as the electron acceptor. P. chrysosporium ligninases are heme glycoproteins. At least a number of isoenzymes are also phosphorylated. Two of the major isoenzymes have been crystallized. Until recently lignin peroxidase could only be produced in low yields in very small scale stationary cultures owing to shear sensitivity. Most strains produce the enzyme only after grown under nitrogen or carbon limitation, although strains producing lignin peroxidase under nutrient sufficiency have also been isolated. Activities over 2000 U dm(-3) (as determined at 30 degrees to 37 degrees C) have been reported in small scale Erlenmeyer cultures with the strain INA-12 grown on glycerol in the presence of soybean phospholipids under nitrogen sufficiency. In about 8 dm(3) liquid volume pilot scale higher than 100 U dm(-3) (as determined at 23 degrees C) have been obtained under agitation with immobilized P. chrysosporium strains ATCC 24725 or TKK 20512. Good results have been obtained for example with nylon web, polyurethane foam, sintered glass or silicon tubing as the carrier. The immobilized biocatalyst systems have also made large scale repeated batch and semicontinuous production possible. With nylon web as the carrier, lignin peroxidase production has recently been scaled up to 800 dm(3) liquid volume semicontinuous industrial production process.

Journal Article↗
PubMed1990

Glutathione peroxidase activities during selenium depletion of adult female rats and during selenium repletion of their offspring.

The main purpose of the present investigation was to produce young rats with severe selenium deficiency, but with no clinical signs of this deficiency, and to examine their liver and red blood cell (RBC) glutathione peroxidase activities during selenium repletion. To achieve this goal, female breeders were fed a selenium-deficient diet beginning 2 weeks before mating. The liver glutathione peroxidase activity of the dams was significantly lower than the activity of comparable nonpregnant females after 5 and 10 weeks of selenium depletion. This difference arose exclusively during the period of pregnancy. In contrast, the RBC glutathione peroxidase activity was significantly increased during this period. Only traces of liver enzyme activity were found in the offspring, and the RBC enzyme activity was only 2% of that of the selenium-repleted controls. Body weight was retarded in the male offspring. However, no severe signs of clinical selenium deficiency were observed. The glutathione peroxidase activity in the liver and RBCs of the offspring was determined after 0, 2, 4, 7, 14, and approximately 40 days of selenium repletion. The liver enzyme activity increased faster in females than in males, while the opposite was found for the RBCs. After 14 days of selenium repletion, the glutathione peroxidase activity of the liver was essentially restored, and the RBC enzyme activity was about half that of the control values. This type of rat may prove useful in studies in which young selenium-deficient rats are preferable, as well as in studies of selenium functions that might not be directly related to the role of selenium in glutathione peroxidase.

Journal Article↗
PubMed2001

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

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.

Journal Article↗
PubMed2003

Susceptibility of Fusobacterium nucleatum to killing by peroxidase-iodide-hydrogen peroxide combination in buffer solution and in human whole saliva.

Some Gram-negative anaerobic bacteria have been associated with the infection of tooth supporting tissues, i.e. periodontitis. Of these bacteria, Fusobacterium nucleatum is sensitive to lactoperoxidase/myeloperoxidase-iodide-hydrogen peroxide system in vitro, but salivary concentrations of thiocyanate abolishes the bactericidality. These bacteria are located in periodontal pockets, on oral mucosa and in saliva. Although F. nucleatum most probably does not belong to the group of main periodontal pathogens, it sustains its proportion in the periodontal flora when gingivitis progresses to periodontitis. In this study, the sensitivity of F. nucleatum to different horseradish peroxidase-iodide-hydrogen peroxide combinations was tested both in buffer and in sterilized human whole saliva. Horseradish peroxidase was chosen because it does not bind thiocyanate at pH > or = 6. After 1h incubation at 37 degrees C, the cell viability was estimated by plate count and with flow cytometer using LIVE/DEAD BacLight kit (Molecular Probes, USA). In saliva, the horseradish peroxidase (50 microg/mL)-iodide (2.5 mM)-hydrogen peroxide (2.5 mM) combination decreased the amount of viable bacteria to 37% compared to 85% in the control without any of the components when measured with flow cytometer. Replacement of buffer by saliva decreased the bactericidality of the peroxidase system. However, in buffer less iodide and hydrogen peroxide was needed to produce significant decrease in the number of viable bacteria when measured by plate count than with flow cytometer. Our study shows that horseradish peroxidase-iodide-hydrogen peroxide combination is able to kill F. nucleatum cells in saliva. Horseradish peroxidase-iodide-hydrogen peroxide combination may be useful to diminish the degree of re-colonization of periodontitis-associated bacteria after periodontal therapy and to inhibit the transmission of these bacteria via saliva.

Journal Article↗
PubMed2007

Oxyferryl heme and not tyrosyl radical is the likely culprit in prostaglandin H synthase-1 peroxidase inactivation.

Prostaglandin H synthase-1 (PGHS-1) is a bifunctional heme protein catalyzing both a peroxidase reaction, in which peroxides are converted to alcohols, and a cyclooxygenase reaction, in which arachidonic acid is converted into prostaglandin G2. Reaction of PGHS-1 with peroxide forms Intermediate I, which has an oxyferryl heme and a porphyrin radical. An intramolecular electron transfer from Tyr385 to Intermediate I forms Intermediate II, which contains two oxidants: an oxyferryl heme and the Tyr385 radical required for cyclooxygenase catalysis. Self-inactivation of the peroxidase begins with Intermediate II, but it has been unclear which of the two oxidants is involved. The kinetics of tyrosyl radical, oxyferryl heme, and peroxidase inactivation were examined in reactions of PGHS-1 reconstituted with heme or mangano protoporphyrin IX with a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), and ethyl hydrogen peroxide (EtOOH). Tyrosyl radical formation was significantly faster with 15-HPETE than with EtOOH and roughly paralleled oxyferryl heme formation at low peroxide levels. However, the oxyferryl heme intensity decayed much more rapidly than the tyrosyl radical intensity at high peroxide levels. The rates of reactions for PGHS-1 reconstituted with MnPPIX were approximately an order of magnitude slower, and the initial species formed displayed a wide singlet (WS) radical, rather than the wide doublet radical observed with PGHS-1 reconstituted with heme. Inactivation of the peroxidase activity during the reaction of PGHS-1 with EtOOH or 15-HPETE correlated with oxyferryl heme decay, but not with changes in tyrosyl radical intensity or EPR line shape, indicating that the oxyferryl heme, and not the tyrosyl radical, is responsible for the self-destructive peroxidase side reactions. Computer modeling to a minimal mechanism was consistent with oxyferryl heme being the source of peroxidase inactivation.

Animals↗
PubMed2002

In situ characterization of a NO-sensitive peroxidase in the lignifying xylem of Zinnia elegans.

The lignifying xylem from Zinnia elegans stems gives an intense reaction with 3,3',5,5'-tetramethylbenzidine (TMB), a reagent previously reported to be specific for peroxidase/H2O2. However, the staining of lignifying xylem cells with TMB is apparently the result of two independent mechanisms: one, the catalase-sensitive (H2O2-dependent) peroxidase-mediated oxidation of TMB, and the other, the catalase-insensitive oxidation of TMB, probably mediated by xylem oxidases which are specific from lignifying tissues. The catalase-insensitive oxidation of TMB by the Z. elegans xylem was sensitive to sodium nitroprusside (SNP), a nitric oxide (NO)-releasing compound that, when used at 5.0 mM, is capable of sustaining NO concentrations of 6.1 &mgr;M in the aqueous phase. This effect of SNP was totally reversed by 150 &mgr;M 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide (PTIO), an efficient NO scavenger in biological systems, so the above-mentioned effect must be ascribed to NO, and not to other nitrogen oxides. This response of the catalase-insensitive TMB-oxidase activity of the lignifying Z. elegans xylem was similar to that shown by a basic peroxidase isolated from the intercellular washing fluid, which showed TMB-oxidase activity, and which was also inhibited by 5 mM SNP, the effect of SNP also being reversed by 150 &mgr;M PTIO. These results suggest that peroxidase was the enzyme responsible for the NO-sensitive catalase-insensitive TMB-oxidase activity of the lignifying Z. elegans xylem. Further support for this statement was obtained from competitive inhibitor-dissected histochemistry, which showed that this stain responded to peroxidase-selective competitive inhibitors, such as ferulic acid and ferrocyanide, in a similar way to the Z. elegans basic peroxidase. From these results, we conclude that this NO-sensitive catalase-insensitive oxidation of TMB is apparently performed by the Z. elegans basic peroxidase, and that the regulation of this enzyme by NO may constitute an intrinsically programmed event during the differentiation and death of the xylem.

Journal Article↗
PubMed1993

Abolition of an Inducible Highly Anionic Peroxidase Activity in Transgenic Tomato.

Locally induced expression of a highly anionic peroxidase has previously been correlated temporally and spatially with suberization of tissues responding to pathogen assault, wounding, or exogenously applied abscisic acid or fungal elicitors. DNA sequences corresponding to the 5[prime] regions of two tomato (Lycopersicon esculentum) genes encoding homologous anionic peroxidases were fused, inserted into a pTi-based plasmid designed to express a composite antisense transcript, and introduced into tomato via Agrobacterium-mediated transformation. RNA gel-blot analyses showed high expression of the antisense transcript in most transgenic plants and no detectable induction of native anionic peroxidase transcripts in wounded or abscisic acid or pathogen-treated tissues. Plants and fruits expressing the antisense transcript appeared normal in all respects. Electrophoretic analysis of anionic proteins from selected transgenic plants showed no detectable anionic peroxidase protein or activity. Depolymerization of polymeric material from the wound periderm of transgenic tomato fruits and analysis of the aliphatic products by gas-liquid chromatography/mass spectrometry showed that the content and composition of C16/C18 [omega]-hydroxy and dicarboxylic acids, characteristic of suberin, were not affected by the absence of the anionic peroxidase. Autofluorescence generated from cell wall phenolics at the wound lesion was also not affected by the absence of the highly anionic peroxidase.

Journal Article↗
PubMed1968

Peroxidases from the extreme dwarf tomato plant. Identification, isolation, and partial purification.

Utilizing starch-gel electrophoresis, 12 discrete peroxidases were identified in purified extracts of the dwarf tomato shoot (Lycopersicon esculentum). Nine of the peroxidases moved toward the anode while 3 moved toward the cathode. In addition, a continuous peroxidase smear was separated from the above 12 peroxidases. This smear moved toward the anode and accounted for 50% of the total peroxidase activity in the crude extract.Four of the major peroxidases were isolated and partially purified, using acetone and ammonium sulfate precipitations, preparative starch-gel electrophoresis and chromatography on DEAE and CM-cellulose.

Journal Article↗
PubMed1971

The role of peroxidase isozymes in resistance to wheat stem rust disease.

In common with other disease situations, rust-resistant wheat leaves show a large increase in peroxidase activity during infection. Peroxidase isozymes from healthy or infected lines of wheat (Triticum aestivum L.) near isogenic for resistance and susceptibility to race 56 of Puccinia graminis tritici were separated by gel electrophoresis and the activity of each was estimated by photometric scanning. In order to ensure that the activity of isozymes observed on gels reflected the changes found in peroxidase enzymes assayed spectrophotometrically in extracts, a study was made of extraction procedures, substrates, and reaction conditions for both types of enzyme measurements. Of the 14 isozymes detected in both healthy and infected leaves, increases in only 1 (isozyme 9) were associated consistently with the development of resistant disease reaction at 20 C. Additional evidence was obtained to show that this isozyme can account for the increased peroxidase activity observed in extracts from resistant plants. When plants with high induced peroxidase activity due to resistance at 20 C were treated with ethylene or transferred to 25 C, they reverted to complete susceptibility. However, the disease-induced activity of isozyme 9 did not fall. The data suggest that, in this case, the association of peroxidase with resistance was a consequence of, not a determinant in, resistance.

Journal Article↗
PubMed1973

Peroxidase Activity in the Abscission Zone of Bean Leaves during Abscission.

Peroxidase activity and localization in the abscission zone of bean leaves were studied histochemically and by gel electrophoresis. Deblading of bean leaves resulted in an increase in peroxidase activity in the abscission zone 2 to 4 days after deblading with highest activity just prior to separation. In debladed plants, the cell division in six to eight layers of cells preceded separation. An ethylene treatment (8 microliters per liter) induced separation of debladed petioles in approximately 24 hours and of intact plants in 36 to 48 hours. Ethylene treatment produced similar results in both debladed and intact plants. In ethylene-treated plants, whether debladed or not, enzyme localization was restricted to only two to three layers of cells with no cell division apparent prior to separation. Infrequent cell divisions were observed after treatment with 2-chloroethylphosphonic acid (1000 micrograms per liter) (Ethephon); however, other changes were similar to those observed with ethylene. Deblading and ethylene treatment resulted in changes in the six peroxidase isozymes observed in the abscission zone. Only four were observed in samples collected 2 centimeters below the abscission zone. Peroxidase bands IV and V increased significantly in debladed and ethylene-treated plants and peroxidase VI decreased only in debladed plants. The changes in peroxidase activity were invariably observed prior to separation in all treatments.

Journal Article↗
PubMed1976

Soluble and cell wall peroxidases in reed canarygrass in relation to disease resistance and localized lignin formation.

The relationship of peroxidases to an inducible disease-resistance mechanism involving lignification of leaf epidermal cell walls was studied. Reed canarygrass (Phalaris arundinacea L.) leaf discs were inoculated with Helminthosporium avenae Eidam and floated on water. In inoculated discs, the activity of soluble, ionic wall-bound and covalent wall-bound peroxidases was about twice the level of activity in noninoculated discs. The increase was attributable to increases in activity of three cathodic isoperoxidases and to the appearance of a new cathodic isoperoxidase. Peroxidase activity in cryostat microtome sections of inoculated discs was histochemically localized in the wall near the site of attempted penetration. When inoculated discs were floated on solutions of cycloheximide (25 mug/ml), increases in peroxidase activity were inhibited, and the fungus penetrated the tissue. The inhibition of peroxidase activity was related to inhibition of cathodic isoperoxidase activity. Anodic isoperoxidase activity did not show changes in response to inoculation or cycloheximide treatment.It was suggested that the resistance mechanism in P. arundinacea involves an induction of cathodic isoperoxidases in challenged tissue. These peroxidases may function in the biosynthesis of lignin at the site of attempted penetration.

Journal Article↗
PubMed1979

Pyrophosphatase, Peroxidase and Polyphenoloxidase Activities during Leaf Development and Senescence.

Inorganic pyrophosphatase, peroxidase, and polyphenoloxidase activities were studied as the function of leaf insertion level in eight monocotyledonous and eight dicotyledonous species. Alkaline inorganic pyrophosphatase shows a declining activity toward the end of senescence whereas no regular drift in either peroxidase or polyphenoloxidase activities was noticed during senescence of attached leaves. In the primary leaves of rice, peroxidase and polyphenoloxidase activities were high in the senescent leaves and there exists a correlation between chlorophyll content and peroxidase activity though not with polyphenoloxidase activity. Upon detachment leaves exhibit increasing peroxidase and polyphenoloxidase activities with time. The distribution of the enzyme activities during senescence of attached leaves is suggested to be species-specific, and an increase in peroxidase and polyphenoloxidase activities cannot be taken as an indicator of leaf senescence.

Journal Article↗
PubMed1991

Characterization of peroxidases in lignifying peach fruit endocarp.

Developing peach (Prunus persica L. Batsch ;Redskin') fruit were used to characterize the role of peroxidases in lignification. During development, the endocarp of these drupes becomes lignified while the mesocarp remains parenchymatous. Acidic peroxidase from lignifying endocarp were similar to those of the fleshy mesocarp. The endocarp had a larger amount and number of basic peroxidases than the mesocarp. Cultured peach leaf cells are thought to be lignified because their walls give a positive reaction with phloroglucinol-HCI. These cells also secreted a basic peroxidase. Peroxidases were difficult to extract from endocarp tissue as they lignified. This was also demonstrated by tissue printing on nitrocellulose. Flesh, but not endocarp peroxidase was evident in tissue prints. This suggests that tissue printing may fail to reveal the presence of enzymes which are firmly attached to the cell.

Journal Article↗
PubMed1992

Cell Wall Free Space of Cucumis Hypocotyls Contains NAD and a Blue Light-Regulated Peroxidase Activity.

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.

Journal Article↗
PubMed1989

Detection of lignin peroxidase and xylanase by immunocytochemical labeling in wood decayed by basidiomycetes.

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.

Journal Article↗
PubMed1992

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

The discovery in 1983 of fungal lignin peroxidases able to catalyze the oxidation of nonphenolic aromatic lignin model compounds and release some CO(2) 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.

Journal Article↗
PubMed1991

A novel tetrazolium method for peroxidase histochemistry and immunohistochemistry.

We developed a new method for the histochemical demonstration of peroxidase. This method, which has a novel reaction mechanism, is based on the oxidation of phenol by peroxidase and coupling of this reaction to the reduction of a tetrazolium salt, with the deposition of an insoluble formazan at sites of enzyme activity. This new method was compared with an established diaminobenzidine (DAB) technique for peroxidase histochemistry and immunohistochemistry. Although both methods identified peroxidase activity in myeloid cells of bone marrow biopsy specimens, there was no interference from red cell pseudoperoxidase activity with the phenol-tetrazolium method, in contrast to the diaminobenzidine method. The detection of cytokeratin using an indirect immunoperoxidase technique was compared with both methods for demonstrating peroxidase activity. The phenol-tetrazolium method gave results similar to that obtained with DAB and appeared to be at least as sensitive as DAB in detecting low amounts of antigen. In addition, the production of a formazan as the final reaction product means that the phenol-tetrazolium method is ideally suited for quantitative peroxidase histochemistry. Therefore, the phenol-tetrazolium method represents a useful alternative method to DAB and for certain applications offers significant advantages over DAB.

Bone Marrow↗