Partial purification and characterization of a peroxidase activity from human placenta.
ABSTRACT: Peroxidases can metabolize a variety of xenobiotics to reactive intermediates capable of binding to protein or DNA. The potential role of these enzymes in fetotoxicity has not been explored. In this study, the presence of peroxidase activity was observed in human term and pre-term placenta. Human term placental peroxidase activity (HTPP) was partially purified by concanavalin A affinity chromatography from CaCl2 extracts of the particulate fraction. HTPP appears to be a membrane-bound glycoprotein. Arachidonic acid-dependent oxidation of guaiacol was not observed, suggesting that the peroxidase activity was not due to prostaglandin synthase. Moreover, HTPP preparations were devoid of catalase and spectrally dissimilar from human haemoglobin, cytochrome P-450, eosinophil peroxidase and myloperoxidase, suggesting an endogenous origin. An Mr of approx. 119,000 was determined for HTPP by gel filtration. Cathodic slab-PAGE of cetyltrialkylammonium bromide-solubilized HTPP yielded two peroxidase-staining bands.
Project description:Due to changing climate, flooding (waterlogged soils and submergence) becomes a major problem in agriculture and crop production. In the present study, the effect of waterlogging was investigated on peroxidases of maize (Zea mays L.) leaves. The plants showed typical adaptations to flooding stress, i.e., alterations in chlorophyll a/b ratios and increased basal shoot diameter. Seven peroxidase bands could be detected by first dimension modified SDS-PAGE and 10 bands by first dimension high resolution Clear Native Electrophoresis that altered in dependence on plant development and time of waterlogging. Native isoelectric focusing revealed three acidic to neutral and four alkaline guaiacol peroxidases that could be further separated by high resolution Clear Native Electrophorese in the second dimension. One neutral peroxidase (pI 7.0) appeared to be down-regulated within four hours after flooding, whereas alkaline peroxidases (pI 9.2, 8.0 and 7.8) were up-regulated after 28 or 52 h. Second dimensions revealed molecular masses of 133 kDa and 85 kDa for peroxidases at pI 8.0 and 7.8, respectively. Size exclusion chromatography revealed native molecular masses of 30-58 kDa for peroxidases identified as class III peroxidases and ascorbate peroxidases by mass spectrometry. Possible functions of these peroxidases in flooding stress will be discussed.
Project description:Ceriporiopsis sp. strain MD-1, isolated from forest soil, produced several extracellular enzymes that decolorized human hair melanin. Among them, three enzymes (E1, E2-1, and E2-2) were purified to homogeneity and characterized. The enzymes required hydrogen peroxide in their enzyme reactions and, typical of other fungal peroxidases, oxidized various phenol compounds such as guaiacol, but not 3,4-dimethoxybenzyl alcohol. The spectra of the three enzymes showed an absorption maximum at 406 nm, indicating that they were heme proteins. However, the A(406)/A(280) values of the enzymes were below 0.4, which was lower than those of other peroxidases. E2-1 and E2-2 were similar to each other in their molecular and catalytic properties, and they possibly represent products of posttranslational modifications and/or allelic variants of the same gene, mdcA. The corresponding cDNA was cloned and sequenced; the deduced amino acid sequence showed high identities to the manganese peroxidases from other microorganisms. The specific activities and K(m) values of E2-1 and E2-2 for synthetic and human hair melanins were much higher than those of Phanerochaete chrysosporium manganese peroxidase and lignin peroxidase.
Project description:Plant peroxidases are involved in numerous cellular processes in plant development and stress responses. Four plasma membrane-bound peroxidases have been identified and characterized in maize (Zea mays L.) roots. In the present study, maize seedlings were treated with different stresses and signal compounds, and a functional analysis of these membrane-bound class III peroxidases (pmPOX1, pmPOX2a, pmPOX2b, and pmPOX3) was carried out. Total guaiacol peroxidase activities from soluble and microsomal fractions of maize roots were compared and showed weak changes. By contrast, total plasma membrane and washed plasma membrane peroxidase activities, representing peripheral and integral membrane proteins, revealed strong changes after all of the stresses applied. A proteomic approach using 2D-PAGE analysis showed that pmPOX3 was the most abundant class III peroxidase at plasma membranes of control plants, followed by pmPOX2a >pmPOX2b >pmPOX1. The molecular mass (63 kDa) and the isoelectric point (9.5) of the pmPOX2a monomer were identified for the first time. The protein levels of all four enzymes changed in response to multiple stresses. While pmPOX2b was the only membrane peroxidase down-regulated by wounding, all four enzymes were differentially but strongly stimulated by methyl jasmonate, salicylic acid, and elicitors (Fusarium graminearum and Fusarium culmorum extracts, and chitosan) indicating their function in pathogen defence. Oxidative stress applied as H(2)O(2) treatment up-regulated pmPOX2b >pmPOX2a, while pmPOX3 was down-regulated. Treatment with the phosphatase inhibitor chantharidin resulted in distinct responses.
Project description:Lignins are aromatic heteropolymers that arise from oxidative coupling of lignin precursors, including lignin monomers (p-coumaryl, coniferyl, and sinapyl alcohols), oligomers, and polymers. Whereas plant peroxidases have been shown to catalyze oxidative coupling of monolignols, the oxidation activity of well-studied plant peroxidases, such as horseradish peroxidase C (HRP-C) and AtPrx53, are quite low for sinapyl alcohol. This characteristic difference has led to controversy regarding the oxidation mechanism of sinapyl alcohol and lignin oligomers and polymers by plant peroxidases. The present study explored the oxidation activities of three plant peroxidases, AtPrx2, AtPrx25, and AtPrx71, which have been already shown to be involved in lignification in the Arabidopsis stem. Recombinant proteins of these peroxidases (rAtPrxs) were produced in Escherichia coli as inclusion bodies and successfully refolded to yield their active forms. rAtPrx2, rAtPrx25, and rAtPrx71 were found to oxidize two syringyl compounds (2,6-dimethoxyphenol and syringaldazine), which were employed here as model monolignol compounds, with higher specific activities than HRP-C and rAtPrx53. Interestingly, rAtPrx2 and rAtPrx71 oxidized syringyl compounds more efficiently than guaiacol. Moreover, assays with ferrocytochrome c as a substrate showed that AtPrx2, AtPrx25, and AtPrx71 possessed the ability to oxidize large molecules. This characteristic may originate in a protein radical. These results suggest that the plant peroxidases responsible for lignin polymerization are able to directly oxidize all lignin precursors.
Project description:An acidic peroxidase (EC 220.127.116.11) produced by cell suspension cultures of Cassia didymobotrya (wild senna) was purified from culture medium collected on the 29th day. The enzyme was shown to be a glycoprotein with a pI of 3.5, a molecular mass of approx. 43 kDa by SDS/PAGE and 50 kDa by gel filtration. The N-terminal sequence was very similar to those of other plant peroxidases. The peroxidase was characterized by a high specificity towards coniferyl alcohol and other natural phenolics such as guaiacol and ferulic and caffeic acids. These findings suggest that the enzyme is involved in lignification processes of the cell wall. Moreover, the enzyme was able to catalyse the oxidation of 4,3',4'-trihydroxychalcone and 4, 3',4'-trihydroxy-3-methoxychalcone to the corresponding 3, 3'-biflavanones, as mixtures of racemic and meso forms.
Project description:Wild-type recombinant horseradish peroxidase purified and refolded from Escherichia coli inclusion bodies has been studied in the system of bis(2-ethylhexyl)sulphosuccinate sodium salt (Aerosol OT)-reversed micelles in octane. In contrast with native horseradish peroxidase the wild-type recombinant enzyme forms dimeric structures as judged by sedimentation analysis. Peroxidase substrates affect the equilibrium between monomeric and dimeric enzyme forms. The dependence of the catalytic activity of recombinant peroxidase on the degree of hydration of the surfactant exhibits two maxima with pyrogallol, o-phenylene- diamine, guaiacol and o-dianisidine, with different ratios of activities for the first and second maxima. The differences in activities of monomeric and dimeric forms of the recombinant horseradish peroxidase provide evidence for active-site screening in dimeric forms. This has been used to model a dimeric structure of recombinant horseradish peroxidase with the screened entrance to the active site. In the model structure obtained, three of eight glycosylation sites were screened. This might explain the absence of dimeric structures in native enzyme peroxidase. The system of reversed micelles provides, for the first time, evidence for the formation of dimeric structures by recombinant plant peroxidase with an altered substrate specificity compared with the native enzyme. Thus one can assume that haem-containing peroxidases in general are able to form dimeric structures.
Project description:The selenite-induced glutathione peroxidase in Chlamydomonas reinhardtii has been purified about 323-fold with a 10% yield, as judged by PAGE. The native enzyme had an Mr of 67,000 and was composed of four identical subunits of Mr 17,000. Glutathione was the only electron donor, giving a specific activity of 193.6 mumol/min per mg of protein. L-Ascorbate, NADH, NADPH, pyrogallol, guaiacol and o-dianisidine did not donate electrons to the enzyme. In addition to H2O2, organic hydroperoxides were reduced by the enzyme. The Km values for glutathione and H2O2 were 3.7 mM and 0.24 mM respectively. The enzyme reaction proceeded by a Ping Pong Bi Bi mechanism. Cyanide and azide had no effect on the activity. The enzyme contained approx. 3.5 atoms of selenium per mol of protein. On immunoprecipitation, Chlamydomonas glutathione peroxidase was precipitated and its activity was inhibited about 90% by the antibody raised against bovine erythrocyte glutathione peroxidase. The antibody also cross-reacted with the subunits of Chlamydomonas glutathione peroxidase in Western blotting SDS/PAGE. In terms of enzymic, physico-chemical and immunological properties, the experimental results demonstrate clearly that Chlamydomonas glutathione peroxidase resembles other well-characterized glutathione peroxidases from animal sources that contain selenium.
Project description:This study sought to determine the process conditions for optimum peroxidase production by a B acillus species (Bacillus sp. FALADE-1-KX640922) isolated from Hogsback forest reserve in South Africa and characterize the peroxidase gene in the bacteria. We optimized peroxidase production by manipulating the environmental and nutritional parameters under submerged fermentation. Subsequently, the gene encoding heme-peroxidase was determined through nested polymerase chain reaction and Sanger DNA sequencing. The studied bacteria had maximum peroxidase production at pH 8, 30 °C and 150 rpm. The addition of guaiacol to lignin fermentation medium enhanced peroxidase production by over 100 % in the studied bacteria. However, the other lignin monomers (veratryl alcohol, vanillin, vanillic acid and ferulic acid) repressed the enzyme activity. Modification of the fermentation medium with ammonium sulphate gave the maximum peroxidase yield (8.87 U mL-1). Under the predetermined culture conditions, Bacillus sp. FALADE-1 expressed maximum specific peroxidase activity at 48 h (8.32 U mg-1). Interestingly, a search of the sequenced gene in PeroxiBase showed 100% similarity to Sporotrichum thermophile catalase-peroxidase gene (katG), as well, the deduced protein sequence clustered with bacterial catalase-peroxidases and had a molecular weight of about 11.45 kDa with 7.01 as the estimated isoelectric point. Subsequently, the nucleotide sequence was deposited in the National Center for Biotechnology Information (NCBI) repository with the accession number MF407314. In conclusion, Bacillus sp. FALADE-1 is a promising candidate for improved peroxidase production.
Project description:Alleviation of cadmium-induced root genotoxicity and cytotoxicity by calcium chloride (CaCl2) in faba bean (Vicia faba L. var. minor) seedlings were studied. Faba bean seeds were treated with H2O or 2% CaCl2 for 6 h before germination. Seeds were then exposed to 0 and 50 µM CdCl2 concentrations for 7 days. Genotoxic damaging effects of Cd was examined through the determination of the mitotic index (MI), chromosomal aberrations (CA) and micronucleus (MN) in the meristem cells of faba bean roots. Similarly, effects of Cd stress on metal accumulation, total membrane lipid contents, total fatty acid composition (TFA), lipid peroxidation as indicated by malondialdehyde production, soluble protein and non-protein thiols (NP-SH) contents, hydrogen peroxide production and the activities of superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (GPX) were evaluated after 7 days of Cd stress in the seedling roots. Cd stress resulted in the reduction of MI, in addition to MN formation and CA induction in the roots of non-primed seeds (treated with H2O). Moreover, Cd induced lipid peroxidation, H2O2 overproduction and loss of membrane lipid amount and soluble protein content, and changes in the TFA composition in roots of faba bean seedlings. SOD activity declined, but CAT and GPX activities increased. However, seed pre-treatment with CaCl2 attenuated the genotoxic and cytotoxic effects of Cd on Vicia faba roots. The results showed that CaCl2 induced reduction of Cd accumulation, improved cell membrane stability and increased the antioxidant defence systems, thus reducing and alleviating Cd genotoxicity and oxidative damage.
Project description:Water deficit induces reactive oxygen species (ROS) overproduction, which in turn inhibits plant growth and development. High concentrations of ROS disrupt the osmotic balance in plant cells and alter membrane integrity. Chromosomes carrying structural or regulatory genes must be detected to better understand plant response mechanisms to stress. The aim of our study was to identify Triticum aestivum L. chromosomes involved in early responses to short-term water-deficit stress (1, 3 and 6 h). In the present study, intervarietal substitution lines of drought-tolerant 'Saratovskaya 29' and sensitive 'Janetzkis Probat' wheat cultivars were examined. We studied the biochemical plant response system and conducted an analysis of catalase, ascorbate peroxidase and guaiacol peroxidase activities, levels of lipid peroxidation and changes in relative water content. Our results determined that the first reaction was a significant increase in guaiacol peroxidase (GPX) activity. However, the strongest impact on plant responses was found for catalase (CAT), which caused a significant decrease in lipid peroxidation (LPO) levels. Our findings indicate that chromosomes 5A, 4B, 6B and 7D are associated with early responses to short-term osmotic stress in wheat.