Inhibition Of Peroxidase Activity Research Articles

Nitroarachidonic Acid, a Novel Peroxidase Inhibitor of Prostaglandin Endoperoxide H Synthases 1 and 2

Prostaglandin endoperoxide H synthase (PGHS) catalyzes the oxidation of arachidonate to prostaglandin H2. We have previously synthesized and chemically characterized nitroarachidonic acid (AANO2), a novel anti-inflammatory signaling mediator. Herein, the interaction of AANO2 with PGHS was analyzed. AANO2 inhibited oxygenase activity of PGHS-1 but not PGHS-2. AANO2 exhibited time- and concentration-dependent inhibition of peroxidase activity in both PGHS-1 and -2. The plot of kobs versus AANO2 concentrations showed a hyperbolic function with kinact = 0.045 s−1 and Ki*app = 0.019 μm for PGHS-1 and kinact = 0.057 s−1 and Ki*app = 0.020 μm for PGHS-2. Kinetic analysis suggests that inactivation of PGHS by AANO2 involves two sequential steps: an initial reversible binding event (described by Ki) followed by a practically irreversible event (Ki*app) leading to an inactivated enzyme. Inactivation was associated with irreversible disruption of heme binding to the protein. The inhibitory effects of AANO2 were selective because other nitro-fatty acids tested, such as nitrooleic acid and nitrolinoleic acid, were unable to inhibit enzyme activity. In activated human platelets, AANO2 significantly decreased PGHS-1-dependent thromboxane B2 formation in parallel with a decrease in platelet aggregation, thus confirming the biological relevance of this novel inhibitory pathway.

Purification and characterization of two extracellular peroxidases from Streptomyces sp. strain AM2, a decolorizing actinomycetes responsible for the biodegradation of natural humic acids

Two extracellular humic acids peroxidases called HaP1 and HaP2 were isolated from the Streptomyces sp. strain AM2 and, based on MALDI-TOF MS analysis. The purified enzymes were determined as monomers with molecular masses of 40,351.11 and 25,175.19 Da, respectively. The N-terminal amino acid sequences of HaP1 and HaP2 were identified, and their optimum pH values were determined as 6 and 7.5, respectively. Standard 2,4-dichlorophenol (2,4-DCP) assays showed that both enzymes had maximal activity at 55 °C. HaP2 was stable at 55 °C for more than 24 h and had a half-life of 90 min at 65 °C. Although the catalytic properties of HaP1 and HaP2 were nearly identical, their stabilities and Reinheitzahl (RZ) values were substantially different. Both peroxidases were found to be heme proteins that catalyzed the oxidation of a wide range of substrates in the presence of hydrogen peroxide (H 2O 2), with HaP2 exhibiting a broader range of substrate specificity. The characterization of peroxidase activity revealed activity against humic acids, guiacol, 2,4-DCP, l-3,4-dihydroxyphenylalanine, and 2,4,5-trichlorophenol as well as other chlorophenols in the presence of H 2O 2. However, the inhibition of peroxidase activity by the addition of potassium cyanide and sodium azide also indicated the presence of heme components in the tertiary structure of these enzymes.

Acetaminophen and its regioisomer, 3-acetamidophenol interfered with the growth ofEscherichia coli O157:H7

Escherichia coli O157:H7 (O157) is an enterohemorrhagic pathogenic subtype, causing diarrhea and Hemolytic-uremic syndrome (HUS). Treatment for HUS patients might be accompanied with antibiotics and antispasmodics with analgesics for abdominal pain. Antiperistaltic effect of antispasmodics, however, may increase the risk of infection of O157 through the intestine. If analgesics also have antibacteiral effect, the prevention and cure of O157 could get benefits. Akhter, T.et al. (2010) reported the inhibitory effect of ibuprofen (IBF) on the bacterial isolates from infected urinary tract. Here, we also found that acetaminophen (4-AAP) and its regioisomer, 3-acetamidophenol (3-AAP), constrained the growth of O157. Inhibitory effect was compared with IBF, since they were both COX inhibitors for their inhibitory mechanism. The experiment results showed that O157 growth was reduced in the presence of 4-AAP, 3-AAP, and IBF. Among them, the inhibitory effect of 4-AAP was less than 3-AAP and IBF in 1.8 fold at 10 mM concentrations. In addition, 4-AAP and 3-AAP inhibited the activity of endogenous peroxidase ofE. coli, as well as, horseradish peroxidase (HRP), implying its possible inhibitory mechanism. However, IBF did not inhibit peroxidase activity, suggesting the different mechanism to suppress the growth of O157 by 4-AAP and 3-AAP.

Inhibition of peroxidase activity and scavenging of reactive oxygen species by astilbin isolated from Dimorphandra mollis (Fabaceae, Caesalpinioideae).

Astilbin (5,7,3',4'-tetrahydroxy-2,3-dihydroflavonol-3-ß-o-rhamnoside), a flavonoid with a large range of biological activities, was isolated from Dimorphandra mollis, a shrub common to the Brazilian Cerrado. The purpose of this study is to verify the effects of astilbin on myeloperoxidase (MPO) and horseradish peroxidase (HRP), and its antioxidant activity against hypochlorous acid (HOCl) and total antioxidant activity (TAC) by the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS•+). Astilbin inhibited MPO and HRP activities in a concentration-dependent relationship and effectively scavenged HOCl. The TAC by ABTS•+ of astilbin (IC50 ~ 20 mM) was higher than that of uric acid, which was used as a positive control. These data demonstrate that astilbin is a potent antioxidant and that it inhibits MPO and HRP activities efficiently.

Inhibition of peroxidase activity and scavenging of reactive oxygen species by astilbin isolated from Dimorphandra mollis (Fabaceae, Caesalpinioideae)

Astilbin (5,7,3',4'-tetrahydroxy-2,3-dihydroflavonol-3-ß-o-rhamnoside), a flavonoid with a large range of biological activities, was isolated from Dimorphandra mollis, a shrub common to the Brazilian Cerrado. The purpose of this study is to verify the effects of astilbin on myeloperoxidase (MPO) and horseradish peroxidase (HRP), and its antioxidant activity against hypochlorous acid (HOCl) and total antioxidant activity (TAC) by the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS•+). Astilbin inhibited MPO and HRP activities in a concentration-dependent relationship and effectively scavenged HOCl. The TAC by ABTS•+ of astilbin (IC50 ~ 20 mM) was higher than that of uric acid, which was used as a positive control. These data demonstrate that astilbin is a potent antioxidant and that it inhibits MPO and HRP activities efficiently.

Production and partial characterization of extracellular peroxidase produced byStreptomyces sp. F6616 isolated in Turkey

Streptomyces sp. F6616 was found to produce higher levels of extracellular peroxidase activity (0.535 U/mL) without any inducers than other actinobacteria which are previously reported. Maximum specific peroxidase activity (6.21 U/mg of protein) was obtained after 72 h of incubation at 30°C in a minimal salt medium (pH 8.0) containing (in wt/v) 0.6% yeast extract and 0.8% ball-milled wheat straw corresponding to a C:N ratio of 4.6:1. Characterization of the peroxidase revealed that the optimal temperature for the enzyme activity, using the standard 2,4-dichlorophenol (2,4-DCP) assay was 50°C, when the enzyme reaction was performed at pH 8.0. A study of the effect of temperature on the stability of peroxidase over time, showed that the enzyme was stable at 50°C, with a half-life of 145 min, while at higher temperature the stability and activity was reduced such that at 60°C the half-life of the enzyme was 30 min. The optimum pH for the activity of the enzyme occurred between pH 9.0 and 10.0. The apparentK m andV max values for the peroxidase preparations were determined to be 1.52 mmol/L and 1.84 U/mg protein, respectively using 2,4-DCP as a substrate. Characterization of the peroxidase activity revealed activity against 2,4-DCP, L-3,4-dihydroxyphenylalanine (L-DOPA), 2,4,5-trichlorophenol and other chlorophenols in the presence of hydrogen peroxide. However, inhibition of peroxidase activity with the addition of potassium cyanide and sodium azide, suggested the presence of heme component in the tertiary structure of the enzyme.

Salacia campestris root bark extract: peroxidase inhibition, antioxidant and antiradical profile

Reactive oxygen species (ROS) and free radical species have been implicated in initiating or accompanying many diseases in living organisms; there is thus, a continual need for antioxidants molecules to inactivate ROS/free radicals. Many studies of plants crude extracts have demonstrated free-radical scavenging and antioxidant action. Salacia species have long been used, in several countries, as traditional medicines against certain diseases and for their anti-inflammatory properties. In this study, Salacia campestris Walp (Hippocrateaceae) root bark ethanol extract (ScEtOH) was assessed for its ability to scavenge free radicals and reactive oxygen species; the results were expressed as percentage inhibition of the active species. ScEtOH was efficient against studied species: DPPH radical (obtained inhibition = 30%), ABTS•+ (IC50 = 1.8±0.8 μg/mL), HOCl (IC50 = 1.7 ± 0.1 μg/mL), O2•- (obtained inhibition = 32%), and NO• (obtained inhibition = 18 %). Peroxidase activity inhibition was evaluated through the guaiacol oxidation reaction catalyzed by hemin, HRP and myeloperoxidase (MPO); data showed that ScEtOH at 10 μg/mL led to 54 and 51% of inhibition, respectively, for the hemin and HRP systems. In the MPO system, ScEtOH promoted a 50% inhibition at 8.9 μg/mL, whereas quercetin, a powerful MPO inhibitor, inhibited this system at 1.35 μg/mL.

Peroxiredoxin 2 and Peroxidase Enzymatic Activity of Mammalian Spermatozoa1

Peroxiredoxin 2 (PRDX2) is a highly efficient redox protein that neutralizes hydrogen peroxide, resulting in protection of cells from oxidative damage and in regulation of peroxide-mediated signal transduction events. The oxidized form of PRDX2 is reverted back to the reduced form by the thioredoxin system. In the present study, we investigated the presence of PRDX2 in mouse and boar spermatozoa and in mouse spermatids using proteomic techniques and immunocytochemistry. Sperm and spermatid extracts displayed a 20-kDa PRDX2 band on Western blotting. PRDX2 occurred as a Triton-soluble form in spermatids and as a Triton-insoluble form in mature spermatozoa. Boar seminiferous tubule extracts were immunoprecipitated with PRDX2 antibody and separated by SDS-PAGE. Peptide mass fingerprinting by matrix-assisted laser desorption ionization-time of flight (TOF) and microsequencing by nanospray quadrupole-quadrupole TOF tandem mass spectrometry revealed the presence of PRDX2 ions in the immunoprecipitated band, along with sperm mitochondria-associated cysteine-rich protein, cellular nucleic acid-binding protein, and glutathione peroxidase 4. In mouse spermatocytes and spermatids, diffuse labeling of PRDX2 was observed in the cytoplasm and residual bodies. After spermiation, PRDX2 localization became confined to the mitochondrial sheath of the sperm tail midpiece. Boar spermatozoa displayed similar PRDX2 localization as in mouse spermatozoa. Boar spermatozoa with disrupted acrosomes expressed PRDX2 in the postacrosomal sheath region. Peroxidase enzyme activity of boar sperm extracts was evaluated by estimating the rate of NADPH oxidation in the presence or absence of a glutathione depletor (diethyl maleate) or a glutathione reductase inhibitor (carmustine). Diethyl maleate partially inhibited peroxidase activity, whereas carmustine showed an insignificant effect. These observations suggest that glutathione and glutathione reductase activity contribute only partially to the total peroxidase activity of the sperm extract. While the specific role of PRDX2 in the total peroxidase activity of sperm extract is still an open question, the present study for the first time (to our knowledge) shows the presence of PRDX2 in mammalian spermatozoa. Peroxidase activity in sperm extracts is not due to the glutathione system and therefore possibly involves PRDX2 and other peroxiredoxins.

Mitochondria‐targeted disruptors and inhibitors of cytochrome c/cardiolipin peroxidase complexes: A new strategy in anti‐apoptotic drug discovery

The critical role of mitochondria in programmed cell death leads to the design of mitochondriotropic agents as a strategy in regulating apoptosis. For anticancer therapy, stimulation of proapoptotic mitochondrial events in tumor cells and their suppression in surrounding normal cells represents a promising paradigm for new therapies. Different approaches targeting regulation of components of mitochondrial antioxidant system such as Mn-SOD demonstrated significant antitumor efficiency, particularly in combination therapy. This review is focused on a newly discovered early stage of mitochondria-dependent apoptosis - oxidative lipid signaling involving a mitochondria-specific phospholipid cardiolipin (CL). Cytochrome c (cyt c) acts as a CL-specific peroxidase very early in apoptosis. At this stage, the hostile events are still secluded within the mitochondria and do not reach the cytosolic targets. CL oxidation process is required for the release of pro-apoptotic factors into the cytosol. Manipulation of cyt c interactions with CL, inhibition of peroxidase activity, and prevention of CL peroxidation are prime targets for the discovery of anti-apoptotic drugs acting before the "point-of-no-return" in the fulfillment of the cell death program. Therefore, mitochondria-targeted disruptors and inhibitors of cyt c/CL peroxidase complexes and suppression of CL peroxidation represent new strategies in anti-apoptotic drug discovery.

INHIBITORY EFFECT OF ANTHOCYANIN EXTRACT FROM SEED COAT OF BLACK BEAN ON PERICARP BROWNING AND LIPID PEROXIDATION OF LITCHI FRUIT DURING STORAGE

ABSTRACT Anthocyanins were extracted from seed coats of black beans (Glycine max[L.]) and the inhibitory effects of anthocyanin extract on pericarp browning and lipid peroxidation of litchi fruit were investigated. Litchi fruit were infiltrated for 3 min with 0 (control) or 50 mg/L of anthocyanin extract at a reduced pressure of 53 kPa, then packed in 0.03 mm thick polyethylene bags, and finally stored at 28C for 6 days. Changes in browning index, contents of anthocyanins and total phenol, peroxidase (POD) activity, levels of relative leakage rate and lipid peroxidation, α,α-diphenyl-β-picrylhydrazy (DPPH) radical scavenging activity and reducing power were evaluated. Application of anthocyanin extract from black bean delayed pericarp browning of litchi fruit during storage, which was associated with reduced POD activity and higher contents of anthocyanins and total phenol. Moreover, the anthocyanin extract was found to have a direct inhibition on the POD activity in vitro. Furthermore, application of the anthocyanin extract apparently reduced lipid peroxidation and relatively maintained membrane integrity, which may account for browning inhibition to an extent. Finally, higher DPPH radical scavenging activity and reducing power of the fruits treated with the anthocyanin extract than control fruits possibly benefited in scavenging free radicals and reducing lipid peroxidation. It is, therefore, suggested that inhibited POD activity and reduced lipid peroxidation by the anthocyanin extract from seed coats of black beans were responsible for the inhibition of pericarp browning of litchi fruits. PRACTICAL APPLICATIONS Lipid peroxidation is a major cause of quality deterioration of postharvest fruits and vegetables. Some synthetic antioxidants are beneficial in inhibiting lipid peroxidation. However, considering that synthetic antioxidants, such as butylhydroxyanisole and butylated hydroxytoluene (BHT), have potential toxicity, the use of natural extracts to extend the shelf life of postharvest fruits and vegetables is a tendency. In our previous study, it has been found that anthocyanin extract from litchi pericarp has stronger antioxidant activities or free radical scavenging activities than BHT and ascorbic acid (Duan et al. 2007). In this study, the application of anthocyanin extract from seed coat of black soybean showed reduced lipid peroxidation and pericarp browning. It could be used potentially as a postharvest technology for reducing or replacing the use of other chemicals, but it requires further investigation.

Kinetics of inhibition of peroxidase activity of myeloperoxidase by quercetin

AbstractThe inhibition of myeloperoxidase (MPO), isolated from human neutrophils, by quercetin was investigated by following peroxidase activity of the enzyme using o‐dianisidine as the substrate. The inhibition parameters (IC50) were obtained by graphical analysis of the inhibition curves. A reaction mechanism, which involved the enzyme inhibition by quercetin and H2O2 in excess, was proposed. The rate and equilibrium constants for the proposed reaction path were calculated from experimental data. Kinetic analysis in noninhibiting H2O2 concentration range in the absence and the presence of quercetin revealed that the reaction mechanism underwent Michaelis–Menten kinetics. K and V values indicated that quercetin was a mixed inhibitor of MPO activity. The initial reaction rates were recalculated using the obtained results. Calculated curves fitted the experimental results within the range of experimental error. © 2008 Wiley Periodicals, Inc. Int J Chem Kinet 40: 384–394, 2008

The kinetic properties producing the perfunctory pH profiles of catalase-peroxidases

Many structure–function relationship studies performed on the catalase-peroxidase enzymes are based on limited kinetic data. To provide a more substantive understanding of catalase-peroxidase function, we undertook a more exhaustive evaluation of catalase-peroxidase catalysis as a function of pH. Kinetic parameters across a broad pH range for the catalase and peroxidase activities of E. coli catalase peroxidase (KatG) were obtained, including the separate analysis of the oxidizing and reducing substrates of the peroxidase catalytic cycle. This investigation identified ABTS-dependent inhibition of peroxidase activity, particularly at low pH, unveiling that previously reported pH optima are clearly skewed. We show that turnover and efficiency of peroxidase activity increases with decreasing pH until the protein unfolds. The data also suggest that the catalase pH optimum is more complex than it is often assumed to be. The apparent optimum is in fact the intersection of the optimum for binding (7.00) and the optimum for activity (5.75). We also report the apparent pKas for binding and catalysis of catalase activity as well as approximate values for certain peroxidatic and catalatic steps.

Activation of NO donors in mitochondria: Peroxidase metabolism of (2-hydroxyamino-vinyl)-triphenyl-phosphonium by cytochrome c releases NO and protects cells against apoptosis

In mitochondrial apoptosis, the formation of cytochrome c–cardiolipin complex ([CL-cyt c]) with peroxidase properties is an early event in the cascade of reactions that leads to cell death. Herein, we report the synthesis of a new prodrug, (2-hydroxyamino-vinyl)-triphenyl-phosphonium (HVTP), which compartmentalizes exclusively into mitochondria, undergoes a [CL-cyt c]-catalyzed bioactivation to nitric oxide (NO), inhibits peroxidase activity, and protects cells from apoptosis.

Sunitinib Induces Hypothyroidism in Advanced Cancer Patients and May Inhibit Thyroid Peroxidase Activity

Sunitinib is a novel tyrosine kinase inhibitor with antitumor and antiangiogenic effects. An observed higher than expected rate of hypothyroidism in sunitinib-treated patients prompted assessment of the incidence of hypothyroidism. Patients taking sunitinib had their thyroid function tests (TFTs) assessed via chart review. To explore potential effects on the thyroid, we examined the antiperoxidase activity of sunitinib by in vitro testing its effect on guaiacol oxidation and protein iodination by lactoperoxidase. Of the 89 patients who took sunitinib, 49 patients were excluded from analysis for several reasons. Of the remaining 40 patients, 21 (53%, 24% of the original 89) developed elevated thyrotropin (TSH) after a median of 5 months (range 1-36 months). Median TSH was 21.4 mU/L (range 4.6-174 mU/L). In vitro, sunitinib had antiperoxidase activity that was about one-fourth the potency of propylthiouracil. Of the 40 patients who had TFTs assessed after starting sunitinib, 53% developed elevated TSH. We recommend that all patients treated with sunitinib be monitored for hypothyroidism. The mechanism of the antithyroid effect appears to be inhibition of peroxidase activity. Further research is needed to confirm the mechanism by which sunitinib induces hypothyroidism.

Overexpression of Cox-2 in Human Osteosarcoma Cells Decreases Proliferation and Increases Apoptosis

Overexpression of cyclooxygenase-2 (COX-2) is generally considered to promote tumorigenesis. To investigate a potential role of COX-2 in osteosarcoma, we overexpressed COX-2 in human osteosarcoma cells. Saos-2 cells deficient in COX-2 expression were retrovirally transduced or stably transfected with murine COX-2 cDNA. Functional expression of COX-2 was confirmed by Northern and Western analyses and prostaglandin production. Overexpression of COX-2 reduced cell numbers by 50% to 70% compared with controls. Decreased proliferation in COX-2-overexpressing cells was associated with cell cycle prolongation in G(2)-M. Apoptosis, measured by both Annexin V binding assay and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining, was increased in cells overexpressing COX-2, and the increase was not reversed by treatment with NS-398, indicating that the effects were not mediated by prostaglandins. Retroviral COX-2 overexpression in two other human osteosarcoma cell lines, U2OS and TE85, also decreased cell viability. However, in the human colon carcinoma HCT-116 cell line, which is deficient in COX-2, retroviral overexpression of COX-2, at similar efficiency as in Saos-2 cells, increased resistance to apoptosis. Reactive oxygen species (ROS), measured by flow cytometry, were increased by COX-2 overexpression in Saos-2 cells but not in HCT-116 cells. Inhibition of peroxidase activity, but not of COX activity, blocked the ROS increase. Antioxidants blocked the increase in ROS and the increase in apoptosis due to COX-2 overexpression in Saos-2 cells. Our results suggest that (a) COX-2 overexpression in osteosarcoma cells may increase resistance to tumorigenesis by increasing ROS to levels that decrease cell viability and (b) the effects of COX-2 overexpression are cell type/tissue dependent.

Cadmium induced changes in cell wall peroxidase isozyme pattern in barley root tips

Changes in peroxidase activity as well as isozyme peroxidase pattern were analyzed in five cell wall (CW) fractions of barley roots exposed to 1mM CdCl<sub>2</sub>for 48 and 72 h. Whereas strong inhibition of peroxidase activity was detected in fraction CW I and weak inhibition in fractions CW II, III and IV, strong activation of peroxidase was observed in fraction V after Cd application. Despite the inhibition of enzyme activity in most CW fractions, induction of several isoperoxidases was discovered after separation on PAGE. In fraction CW I inhibition of one cathodic isoperoxidase and activation of two anodic and two cathodic isoperoxidases was detected. Additional two anodic and one cathodic Cd-induced isozyme appeared in CW II and CW III. Strongly activated anodic isoperoxidase was detected in fraction CW V. Our results demonstrate that monitoring the stress modified peroxidase activity in enzyme extracts using guaiacol as a non-specific peroxidase substrate is not sufficient enough. Since some isozymes can be activated and some inhibited, isozyme pattern analysis is recommended.

Nitric Oxide Inhibits Peroxidase Activity of Cytochrome c· Cardiolipin Complex and Blocks Cardiolipin Oxidation

The increased production of NO during the early stages of apoptosis indicates its potential involvement in the regulation of programmed cell death through yet to be identified mechanisms. Recently, an important role for catalytically competent peroxidase form of pentacoordinate cytochrome c (cyt c) in a complex with a mitochondria-specific phospholipid, cardiolipin (CL), has been demonstrated during execution of the apoptotic program. Because the cyt c.CL complex acts as CL oxygenase and selectively oxidizes CL in apoptotic cells in a reaction dependent on the generation of protein-derived (tyrosyl) radicals, we hypothesized that binding and nitrosylation of cyt c regulates CL oxidation. Here we demonstrate by low temperature electron paramagnetic resonance spectroscopy that CL facilitated interactions of ferro- and ferri-states of cyt c with NO and NO(-), respectively, to yield a mixture of penta- and hexa-coordinate nitrosylated cyt c. In the nitrosylated cyt c.CL complex, NO chemically reacted with H(2)O(2)-activated peroxidase intermediates resulting in their reduction. A dose-dependent quenching of H(2)O(2)-induced protein-derived radicals by NO donors was shown using direct electron paramagnetic resonance measurements as well as immuno-spin trapping with antibodies against protein 5,5-dimethyl-1-pyrroline N-oxide-nitrone adducts. In the presence of NO donors, H(2)O(2)-induced oligomeric forms of cyt c positively stained for 3-nitrotyrosine confirming the reactivity of NO toward tyrosyl radicals of cyt c. Interaction of NO with the cyt c.CL complex inhibited its peroxidase activity with three different substrates: CL, etoposide, and 3,3'-diaminobenzidine. Given the importance of CL oxidation in apoptosis, mass spectrometry analysis was utilized to assess the effects of NO on oxidation of 1,1'2,2'-tertalinoleoyl cardiolipin. NO effectively inhibited 1,1'2,2'-tertalinoleoyl cardiolipin oxidation catalyzed by the peroxidase activity of cyt c. Thus, NO can act as a regulator of peroxidase activity of cyt c.CL complexes.

Early Detection of Bacterium‐induced Basal Resistance in Tobacco Leaves with Diaminobenzidine and Dichlorofluorescein Diacetate

AbstractThe present study demonstrate that in tobacco leaves the diaminobenzidine (DAB) and 2′,7′‐dichlorofluorescein diacetate (DCFH‐DA) staining is a useful indicator of the basal (also known as general or innate) defence‐associated reactions, especially of the early developing form of basal resistance (EBR). DAB and DCFH‐DA, in the presence of H2O2 and peroxidase converts to a brown polymer and fluorescent DCF respectively. In the present study, the hypersensitive response (HR)‐inducing avirulent Pseudomonas syringae pv. syringae 61, its HR‐negative hrp/hrc mutants and even non‐pathogenic bacteria such as P. fluorescens and Escherichia coli caused DAB and DCFH‐DA staining, if the dyes were injected 3–4 h after bacterial inoculation into tobacco leaves. The conditions that enable the staining of plant leaves infiltrated with HR‐negative bacteria were persisted for 1 to several days depending on the physiological state of the plant, and plant activity was required to the development of the staining. The live virulent P. syringae pv. tabaci was able to suppress the development of the staining reaction. Bacteria that induced more intensive staining reaction triggered stronger local resistance response, which was verified by its ability to inhibit the HR by challenging avirulent bacteria and by expression analysis of genes that are activated during the basal defence response. The peroxidase enzyme activity increased in bacterially treated tobacco tissue, and inhibition of peroxidase activity blocked the development of the staining. The results showed that in tobacco leaves the staining reactions were associated with the general recognition and basal defence reaction of tobacco plant and can be used as markers in tobacco leaves for testing the occurrence of this type of defence.

Decolorization kinetics of the azo dye drimaren blue X3LR by laccase

An extracellular Drimaren Blue X3LR decolorizing enzymatic activity was found in the crude filtrate of Funalia trogii grown by solid-state fermentation using wheat bran and soya bean waste. Decolorization of the azo dye Drimaren Blue X3LR by the crude filtrate and partially purified enzyme of Funalia trogii were investigated and compared. In the absence of additional redox mediator, maximum decolorization ratios of 81.33 % and 77.4 % were observed for Drimaren Blue X3LR using crude filtrate and partially purified enzyme respectively. Decolorization yield was found to be higher with crude enzyme preparations. Na2S2O5 inhibited laccase and dye decolorizing enzyme activities but a significant peroxidase activity inhibition was not observed. Since the reaction was catalyzed in the absence of H2O2 as co-substrate, it could be concluded that this enzyme is not a peroxidase but may be a laccase.. The kinetic parameters of decolorization were calculated according to Michaelis constant (Km of 1.700 x 10-5 mol dm-3 and Wmax = 8.02 x10-7 mol dm-3 sec-1).

Immunolocalization of an anionic peroxidase in differentiating poplar xylem

Peroxidases are the major candidate enzymes involved in dehydrogenative polymerization of monolignols. Peroxidases have the signal sequence at their N-terminus and this suggests that they are transported to extracellular spaces or developing cell walls. In this study, we focused on an anionic peroxidase isozyme encoded by prxA3a, which seems to be related to lignification. To investigate the localization of peroxidase in differentiating xylem cells of poplar (Populus sieboldii × Populus grandidentata), anti-PRX3 antibody was raised against the anionic peroxidase. Western blotting and peroxidase activity inhibition assay showed specificity of the antibody. Labeling by anti-PRX3 antibody was localized in vessels and fibers during the secondary wall formation and was observed along the plasma membrane beside the microtubules. The labeling was not seen in the cell wall, where localization of peroxidases was expected during lignification. The peroxidase isozyme, which is suggested to be involved in monolignol polymerization, is localized on the plasma membrane and its localization might be regulated by microtubules.