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The extracellular peroxidase from Streptomyces albidoflavus BSII#1 was purified to near homogeneity using sequential steps of acid and acetone precipitation, followed by ultrafiltration. The purified peroxidase was characterised and tested for the ability to catalyse coupling reactions between selected phenolic monomer pairs. A 46-fold purification of the peroxidase was achieved, and it was shown to be a 46 kDa haem peroxidase. Unlike other actinobacteria-derived peroxidases, it was only inhibited (27 % inhibition) by relatively high concentrations of sodium azide (5 mM) and was capable of oxidising eleven (2,4-dichlorophenol, 2,6-dimethoxyphenol, 4-tert-butylcatechol, ABTS, caffeic acid, catechol, guaiacol, l-DOPA, o-aminophenol, phenol, pyrogallol) of the seventeen substrates tested. The peroxidase remained stable at temperatures of up to 80 °C for 60 min and retained >50 % activity after 24 h between pH 5.0-9.0, but was most sensitive to incubation with hydrogen peroxide (H 2 O 2 ; 0.01 mM), l-cysteine (0.02 mM) and ascorbate (0.05 mM) for one hour. It was significantly inhibited by all organic solvents tested (p ≤ 0.05). The K m and V max values of the partially purified peroxidase with the substrate 2,4-DCP were 0.95 mM and 0.12 mmol min -1 , respectively. The dyes reactive blue 4, reactive black 5, and Azure B, were all decolourised to a certain extent: approximately 30 % decolourisation was observed after 24 h (1 μM dye). The peroxidase successfully catalysed coupling reactions between several phenolic monomer pairs including catechin-caffeic acid, catechin-catechol, catechin-guaiacol and guaiacol-syringaldazine under the non-optimised conditions used in this study. Genome sequencing confirmed the identity of strain BSII#1 as a S. albidoflavus strain. In addition, the genome sequence revealed the presence of one peroxidase gene that includes the twin arginine translocation signal sequence of extracellular proteins. Functional studies confirmed that the peroxidase produced by S. albidoflavus BSII#1 is part of the dye-decolourising peroxidase (DyP-type) family., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Alkene cleavage is a possibility to generate aldehydes with olfactory properties for the fragrance and flavor industry. A dye-decolorizing peroxidase (DyP) of the basidiomycete Pleurotus sapidus (PsaPOX) cleaved the aryl alkene trans -anethole. The PsaPOX was semi-purified from the mycelium via FPLC, and the corresponding gene was identified. The amino acid sequence as well as the predicted tertiary structure showed typical characteristics of DyPs as well as a non-canonical Mn 2+ -oxidation site on its surface. The gene was expressed in Komagataella pfaffii GS115 yielding activities up to 142 U/L using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) as substrate. PsaPOX exhibited optima at pH 3.5 and 40 °C and showed highest peroxidase activity in the presence of 100 µM H 2 O 2 and 25 mM Mn 2+ . PsaPOX lacked the typical activity of DyPs towards anthraquinone dyes, but oxidized Mn 2+ to Mn 3+ . In addition, bleaching of β -carotene and annatto was observed. Biotransformation experiments verified the alkene cleavage activity towards the aryl alkenes ( E )-methyl isoeugenol, α -methylstyrene, and trans -anethole, which was increased almost twofold in the presence of Mn 2+ . The resultant aldehydes are olfactants used in the fragrance and flavor industry. PsaPOX is the first described DyP with alkene cleavage activity towards aryl alkenes and showed potential as biocatalyst for flavor production.

Industrial synthetic dyes cause health and environmental problems. This work describes the isolation of 84 bacterial strains from the midgut of the Lasius niger ant and the evaluation of their potential application in dye bioremediation. Strains were identified and classified as judged by rRNA 16S. The most abundant isolates were found to belong to Actinobacteria (49%) and Firmicutes (47.2%). We analyzed the content in laccase, azoreductase and peroxidase activities and their ability to degrade three known dyes (azo, thiazine and anthraquinone) with different chemical structures. Strain Ln26 (identified as Brevibacterium permense) strongly decolorized the three dyes tested at different conditions. Strain Ln78 (Streptomyces ambofaciens) exhibited a high level of activity in the presence of Toluidine Blue (TB). It was determined that 8.5 was the optimal pH for these two strains, the optimal temperature conditions ranged between 22 and 37 °C, and acidic pHs and temperatures around 50 °C caused enzyme inactivation. Finally, the genome of the most promising candidate (Ln26, approximately 4.2 Mb in size) was sequenced. Genes coding for two DyP-type peroxidases, one laccase and one azoreductase were identified and account for the ability of this strain to effectively oxidize a variety of dyes with different chemical structures.

Streptococcus pneumoniae is a facultative anaerobic pathogen. Although it maintains fermentative metabolism, during aerobic growth pneumococci produce high levels of H 2 O 2 , which can have adverse effects on cell viability and DNA, and influence pneumococcal interaction with its host. The pneumococcus is unusual in its dealing with toxic reactive oxygen species (ROS) in that it neither has catalase nor the global regulators of peroxide stress resistance. Previously, we identified pneumococcal thiol peroxidase (TpxD) as the key enzyme for enzymatic removal of H 2 O 2 , and showed that TpxD synthesis is up-regulated upon exposure to H 2 O 2 . This study aimed to reveal the mechanism controlling TpxD expression under H 2 O 2 stress. We hypothesize that H 2 O 2 activates a transcription factor which in turn up-regulates tpxD expression. Microarray analysis revealed a pneumococcal global transcriptional response to H 2 O 2 . Mutation of tpxD abolished H 2 O 2 -mediated response to high H 2 O 2 levels, signifying the need for an active TpxD under oxidative stress conditions. Bioinformatic tools, applied to search for a transcription factor modulating tpxD expression, pointed toward CodY as a potential candidate. Indeed, a putative 15-bp consensus CodY binding site was found in the proximal region of tpxD- coding sequence. Binding of CodY to this site was confirmed by EMSA, and genetic engineering techniques demonstrated that this site is essential for TpxD up-regulation under H 2 O 2 stress. Furthermore, tpxD expression was reduced in a Δ codY mutant. These data indicate that CodY is an activator of tpxD expression, triggering its up-regulation under H 2 O 2 stress. In addition we show that H 2 O 2 specifically oxidizes the 2 CodY cysteines. This oxidation may trigger a conformational change in CodY, resulting in enhanced binding to DNA. A schematic model illustrating the contribution of TpxD and CodY to pneumococcal global transcriptional response to H 2 O 2 is proposed.

DyP-type peroxidases are a heme peroxidase family with unique properties whose members are widely distributed from prokaryotes to eukaryotes. DyP-type peroxidases are subdivided into class P, I and V based on structure-based sequence alignment. Class V enzymes possess degradation activities for anthraquinone dyes, and include extra sequences compared with class P and I. Class V enzymes are mainly found in fungi, with only two such proteins, AnaPX and DyP2, reported in bacteria. Here, we heterologously expressed, purified and biochemically characterized SaDyP2 protein, predicted to belong to class V. SaDyP2 was purified as a ∼50 kDa enzyme containing a heme cofactor and was found to oxidize the typical peroxidase substrates, ABTS and DMP. SaDyP2 was generally thermostable and exhibited a lower optimal pH, a feature typical of DyP-type peroxidases. It also degraded anthraquinone dyes, a specific substrate of DyP-type peroxidases, although the k cat for SaDyP2 was lower than that for other class V enzymes. The K m value of SaDyP2 for anthraquinone dye was similar to that of other enzymes of this class. Homology modeling revealed that the structure of SaDyP2 best fit that of class V enzymes., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Unlabelled: Although Mn(2+) is the most abundant substrate of versatile peroxidases (VPs), repression of Pleurotus ostreatus vp1 expression occurred in Mn(2+)-sufficient medium. This seems to be a biological contradiction. The aim of this study was to explore the mechanism of direct oxidation by VP1 under Mn(2+)-deficient conditions, as it was found to be the predominant enzyme during fungal growth in the presence of synthetic and natural substrates. The native VP1 was purified and characterized using three substrates, Mn(2+), Orange II (OII), and Reactive Black 5 (RB5), each oxidized by a different active site in the enzyme. While the pH optimum for Mn(2+) oxidation is 5, the optimum pH for direct oxidation of both dyes was found to be 3. Indeed, effective in vivo decolorization occurred in media without addition of Mn(2+) only under acidic conditions. We have determined that Mn(2+) inhibits in vitro the direct oxidation of both OII and RB5 while RB5 stabilizes both Mn(2+) and OII oxidation. Furthermore, OII was found to inhibit the oxidation of both Mn(2+) and RB5. In addition, we could demonstrate that VP1 can cleave OII in two different modes. Under Mn(2+)-mediated oxidation conditions, VP1 was able to cleave the azo bond only in asymmetric mode, while under the optimum conditions for direct oxidation (absence of Mn(2+) at pH 3) both symmetric and asymmetric cleavages occurred. We concluded that the oxidation mechanism of aromatic compounds by VP1 is controlled by Mn(2+) and pH levels both in the growth medium and in the reaction mixture., Importance: VP1 is a member of the ligninolytic heme peroxidase gene family of the white rot fungus Pleurotus ostreatus and plays a fundamental role in biodegradation. This enzyme exhibits a versatile nature, as it can oxidize different substrates under altered environmental conditions. VPs are highly interesting enzymes due to the fact that they contain unique active sites that are responsible for direct oxidation of various aromatic compounds, including lignin, in addition to the well-known Mn(2+) binding active site. This study demonstrates the limits of versatility of P. ostreatus VP1, which harbors multiple active sites, exhibiting a broad range of enzymatic activities, but they perform differently under distinct conditions. The versatility of P. ostreatus and its enzymes is an advantageous factor in the fungal ability to adapt to changing environments. This trait expands the possibilities for the potential utilization of P. ostreatus and other white rot fungi., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

This study is one of the few investigations which analyze albumen prints, perhaps the most important photographic heritage of the late 19(th) and early 20(th) centuries. The chemical composition of photographic samples was assessed using Fourier-transform infrared spectroscopy and X-ray fluorescence. These two non-invasive techniques revealed the complex nature of albumen prints, which are composed of a mixture of proteins, cellulose and salts. Microbial sampling was performed using cellulose nitrate membranes which also permitted the trapped microflora to be observed with a scanning electron microscope. Microbial analysis was performed using the combination of culture-dependent (cultivation in different media, including one 3% NaCl) and culture-independent (bacterial and fungal cloning and sequencing) approaches. The isolated microorganisms were screened for their lipolytic, proteolytic, cellulolytic, catalase and peroxidase activities. The combination of the culture-dependent and -independent techniques together with enzymatic assays revealed a substantial microbial diversity with several deteriogen microorganisms from the genera Bacillus, Kocuria, Streptomyces and Geobacillus and the fungal strains Acrostalagmus luteoalbus, Bjerkandera adusta, Pleurotus pulmonarius and Trichothecium roseum.

Streptococcus thermophilus is a facultative anaerobic bacterium that has the ability to grow and survive in aerobic environments, but the mechanism for this remains unclear. In this study, the efeB gene, encoding a dye-decolorizing peroxidase, was identified in the genome of Streptococcus thermophilus CGMCC 7.179, and purified EfeB was able to decolorize reactive blue 5. Strikingly, genes encoding two components (TatA and TatC) of the twin-arginine translocation (TAT) system were also found in the same operon with the efeB gene. Knocking out efeB or tatC resulted in decreased growth of the strain under aerobic conditions, and complementation of the efeB-deficient strains with the efeB gene enhanced the biomass of the hosts only in the presence of the tatC gene. Moreover, it was proved for both S. thermophilus CGMCC 7.179 and Escherichia coli DE3 that EfeB could be translocated by the TAT system of S. thermophilus. In addition, the transcriptional levels of efeB and tatC increased when the strain was cultured under aerobic conditions. Overall, these results provide the first evidence that EfeB plays a role in protecting cells of S. thermophilus from oxidative stress, with the assistance of the TAT system., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

We report a novel method to synthesize organic-inorganic nanoflowers for crude soybean peroxidase (SBP) purification. A hierarchical flower-like spherical structure with hundreds of nanopetals was self-assembled by using crude SBP as the organic component and Cu3(PO4)2·3H2O as the inorganic component. The structure of the hybrid nanoflowers was confirmed by Fourier-transform infrared spectroscopy, X-ray diffraction, and energy-dispersive X-ray spectroscopy, and the enzymatic activity of SBP embedded in the hybrid nanoflowers was evaluated using guaiacol as substrate. Compared with free crude SBP in solution, SBP embedded in hybrid nanoflowers exhibited enhanced enzymatic activity (∼446%). The hybrid nanoflowers also exhibited excellent reusability and reproducibility during cycle analysis. These results demonstrate that synthesis of hybrid nanoflowers is an effective enzyme purification strategy., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Soybean peroxidase (SBP)-catalyzed removal of phenol from wastewater has been demonstrated as a feasible wastewater treatment strategy and a non-ionic surfactant, Triton X-100, has the potential for increasing the enzyme economy of the process. Systematic studies on the enzyme-surfactant system have been lacking as well as demonstration of its applicability to industrial wastewater. This paper addresses those two gaps, the latter based on real wastewater from alkyd resin manufacture. The minimum effective Triton X-100 concentrations for crude SBP-catalyzed phenol conversion (≥95%) over 1-10 mM showed a linear trend. To illustrate translation of such lab results to real-world samples, this data were used to optimize crude SBP needed for phenol conversion over that concentration range. Triton X-100 increases enzyme economy by 10- to 13-fold. This treatment protocol was directly applied to tote-scale (700-1000 L) treatment of alkyd resin wastewater, with phenol ranging from 7 to 28 mM and total organic carbon content of >40 g/L, using a crude SBP extract derived from dry soybean hulls by simple aqueous elution. This extract can be used to remove phenol from a complex industrial wastewater and the process is markedly more efficient in the presence of Triton X-100. The water is thus rendered amenable to conventional biological treatment whilst the hulls could still be used in feed, thus adding further value to the crop., (Copyright © 2013 Elsevier Inc. All rights reserved.)

An acidic peroxidase was extracted from garlic (Allium sativum) and was partially purified threefold by ammonium sulphate precipitation, dialysis, and gel filtration chromatography using sephadex G-200. The specific activity of the enzyme increased from 4.89 U/mg after ammonium sulphate precipitation to 25.26 U/mg after gel filtration chromatography. The optimum temperature and pH of the enzyme were 50°C and 5.0, respectively. The Km and V max for H2O2 and o-dianisidine were 0.026 mM and 0.8 U/min, and 25 mM and 0.75 U/min, respectively. Peroxidase from garlic was effective in decolourizing Vat Yellow 2, Vat Orange 11, and Vat Black 27 better than Vat Green 9 dye. For all the parameters monitored, the decolourization was more effective at a pH range, temperature, H2O2 concentration, and enzyme concentration of 4.5-5.0, 50°C, 0.6 mM, and 0.20 U/mL, respectively. The observed properties of the enzyme together with its low cost of extraction (from local sources) show the potential of this enzyme for practical application in industrial wastewater treatment especially with hydrogen peroxide. These Vat dyes also exhibited potentials of acting as peroxidase inhibitors at alkaline pH range.

As a production platform for recombinant proteins, plant leaf tissue has many advantages, but commercialization of this technology has been hindered by high recovery and purification costs. Vacuum infiltration-centrifugation (VI-C) is a technique to obtain extracellularly-targeted products from the apoplast wash fluid (AWF). Because of its selective recovery of secreted proteins without homogenizing the whole tissue, VI-C can potentially reduce downstream production costs. Lab scale experiments were conducted to quantitatively evaluate the VI-C method and compared to homogenization techniques in terms of product purity, concentration, and other desirable characteristics. From agroinfiltrated Nicotiana benthamiana leaves, up to 81% of a truncated version of E1 endoglucanase from Acidothermus cellulolyticus was recovered with VI-C versus homogenate extraction, and average purity and concentration increases of 4.2-fold and 3.1-fold, respectively, were observed. Formulas were developed to predict recovery yields of secreted protein obtained by performing multiple rounds of VI-C on the same leaf tissue. From this, it was determined that three rounds of VI-C recovered 97% of the total active recombinant protein accessible to the VI-C procedure. The results suggest that AWF recovery is an efficient process that could reduce downstream processing steps and costs for plant-made recombinant proteins.

Myeloperoxidase is a neutrophil enzyme that promotes oxidative stress in numerous inflammatory pathologies. It uses hydrogen peroxide to catalyze the production of strong oxidants including chlorine bleach and free radicals. A physiological defense against the inappropriate action of this enzyme has yet to be identified. We found that myeloperoxidase oxidized 75% of the ascorbate in plasma from ceruloplasmin knock-out mice, but there was no significant loss in plasma from wild type animals. When myeloperoxidase was added to human plasma it became bound to other proteins and was reversibly inhibited. Ceruloplasmin was the predominant protein associated with myeloperoxidase. When the purified proteins were mixed, they became strongly but reversibly associated. Ceruloplasmin was a potent inhibitor of purified myeloperoxidase, inhibiting production of hypochlorous acid by 50% at 25 nm. Ceruloplasmin rapidly reduced Compound I, the Fe(V) redox intermediate of myeloperoxidase, to Compound II, which has Fe(IV) in its heme prosthetic groups. It also prevented the fast reduction of Compound II by tyrosine. In the presence of chloride and hydrogen peroxide, ceruloplasmin converted myeloperoxidase to Compound II and slowed its conversion back to the ferric enzyme. Collectively, our results indicate that ceruloplasmin inhibits myeloperoxidase by reducing Compound I and then trapping the enzyme as inactive Compound II. We propose that ceruloplasmin should provide a protective shield against inadvertent oxidant production by myeloperoxidase during inflammation.

Objective: Increasing evidences have suggested the pathogenic role of anti-neutrophil cytoplasmic antibodies (ANCA) directing myeloperoxidase (MPO) in ANCA-associated vasculitis (AAV). The current study aimed to analyze the association between the linear epitopes of MPO-ANCA and clinicopathological features of patients with AAV., Methods: Six recombinant linear fragments, covering the whole length amino acid sequence of a single chain of MPO, were produced from E.coli. Sera from 77 patients with AAV were collected at presentation. 13 out of the 77 patients had co-existence of serum anti-GBM antibodies. Ten patients also had sequential sera during follow up. The epitope specificities were detected by enzyme-linked immunosorbent assay using the recombinant fragments as solid phase ligands., Results: Sera from 45 of the 77 (58.4%) patients with AAV showed a positive reaction to one or more linear fragments of the MPO chain. The Birmingham Vasculitis Activity Scores and the sera creatinine were significantly higher in patients with positive binding to the light chain fragment than that in patients without the binding. The epitopes recognized by MPO-ANCA from patients with co-existence of serum anti-GBM antibodies were mainly located in the N-terminus of the heavy chain. In 5 out of the 6 patients, whose sera in relapse recognize linear fragments, the reactivity to linear fragments in relapse was similar to that of initial onset., Conclusion: The epitope specificities of MPO-ANCA were associated with disease activity and some clinicopathological features in patients with ANCA-associated vasculitis.

NO and H2O2 are important biological messengers in plants. They are formed during xylem differentiation in Zinnia elegans and apparently play important roles during the xylogenesis. To ascertain the responsiveness of the Z. elegans peroxidase (ZePrx) to these endogenous signals, the effects of NO and H2O2 on ZePrx were studied. The results showed that ZePrx is up-regulated by NO and H2O2, as confirmed by RT-qPCR, and that its promoter contains multiple copies of all the putative cis-elements (ACGT box, OCS box, OPAQ box, L1BX, MYCL box and W box) known to confer regulation by NO and H2O2. Like other OCS elements, the OCS element of ZePrx contains the sequence TACG that is recognized by OBF5, a highly conserved bZIP transcription factor, and the 10 bp sequence, ACAaTTTTGG, which is recognized by OBP1, a Dof domain protein that binds down-stream the OCS element. Furthermore, the ZePrx OCS element is flanked by two CCAAT-like boxes, and encloses one auxin-responsive ARFAT element and two GA3-responsive Pyr boxes. Results also showed that ZePrx may be described as the first protein to be up-regulated by NO and H2O2, whose mRNA contains several short-longevity conferring elements, such as a downstream (DST) sequence analogous to the DSTs contained in the highly unstable SAUR transcripts. The presence of these regulatory elements strongly suggests that ZePrx is finely regulated, as one may expect from an enzyme that catalyzes the last irreversible step of the formation of lignins, the major irreversible sink for the photosynthetically fixed CO2.

The Pseudomonas aeruginosa PAO1 thiol peroxidase homolog (Tpx) belongs to a family of enzymes implicated in the removal of toxic peroxides. We have shown the expression of tpx to be highly inducible with redox cycling/superoxide generators and diamide and weakly inducible with organic hydroperoxides and hydrogen peroxide (H(2)O(2)). The PAO1 tpx pattern is unlike the patterns for other peroxide-scavenging genes in P. aeruginosa. Analysis of the tpx promoter reveals the presence of a putative IscR binding site located near the promoter. The tpx expression profiles in PAO1 and the iscR mutant, together with results from gel mobility shift assays showing that purified IscR specifically binds the tpx promoter, support the role of IscR as a transcriptional repressor of tpx that also regulates the oxidant-inducible expression of the gene. Recombinant Tpx has been purified and biochemically characterized. The enzyme catalyzes thioredoxin-dependent peroxidation and can utilize organic hydroperoxides and H(2)O(2) as substrates. The Δtpx mutant demonstrates differential sensitivity to H(2)O(2) only at moderate concentrations (0.5 mM) and not at high (20 mM) concentrations, suggesting a novel protective role of tpx against H(2)O(2) in P. aeruginosa. Altogether, P. aeruginosa tpx is a novel member of the IscR regulon and plays a primary role in protecting the bacteria from submillimolar concentrations of H(2)O(2).

Cell wall isolated from pea roots was used to separate and characterize two fractions possessing class III peroxidase activity: (i) ionically bound proteins and (ii) covalently bound proteins. Modified SDS-PAGE separated peroxidase isoforms by their apparent molecular weights: four bands of 56, 46, 44, and 41kDa were found in the ionically bound fraction (iPOD) and one band (70kDa) was resolved after treatment of the cell wall with cellulase and pectinase (cPOD). Isoelectric focusing (IEF) patterns for iPODs and cPODs were significantly different: five iPODs with highly cationic pI (9.5-9.2) were detected, whereas the nine cPODs were anionic with pI values between pH 3.7 and 5. iPODs and cPODs showed rather specific substrate affinity and different sensitivity to inhibitors, heat, and deglycosylation treatments. Peroxidase and oxidase activities and their IEF patterns for both fractions were determined in different zones along the root and in roots of different ages. New iPODs with pI 9.34 and 9.5 were induced with root growth, while the activity of cPODs was more related to the formation of the cell wall in non-elongating tissue. Treatment with auxin that inhibits root growth led to suppression of iPOD and induction of cPOD. A similar effect was obtained with the widely used elicitor, chitosan, which also induced cPODs with pI 5.3 and 5.7, which may be specifically related to pathogen defence. The differences reported here between biochemical properties of cPOD and iPOD and their differential induction during development and under specific treatments implicate that they are involved in specific and different physiological processes.

Pentraxin 3 (PTX3), a long pentraxin subfamily member in the pentraxin family, plays an important role in innate immunity as a soluble pattern recognition receptor. Plasma PTX3 is elevated in sepsis (~200 ng/ml) and correlates with mortality. The roles of PTX3 in sepsis, however, are not well understood. To investigate the ligands of PTX3 in sepsis, we performed a targeted proteomic study of circulating PTX3 complexes using magnetic bead-based immunopurification and shotgun proteomics for label-free relative quantitation via spectral counting. From septic patient fluids, we successfully identified 104 candidate proteins, including the known PTX3-interacting proteins involved in complement activation, pathogen opsonization, inflammation regulation, and extracellular matrix deposition. Notably, the proteomic profile additionally showed that PTX3 formed a complex with some of the components of neutrophil extracellular traps. Subsequent biochemical analyses revealed a direct interaction of bactericidal proteins azurocidin 1 (AZU1) and myeloperoxidase with PTX3. AZU1 exhibited high affinity binding (K(D) = 22 ± 7.6 nm) to full-length PTX3 in a calcium ion-dependent manner and bound specifically to an oligomer of the PTX3 N-terminal domain. Immunohistochemistry with a specific monoclonal antibody generated against AZU1 revealed a partial co-localization of AZU1 with PTX3 in neutrophil extracellular traps. The association of circulating PTX3 with components of the neutrophil extracellular traps in sepsis suggests a role for PTX3 in host defense and as a potential diagnostic target.

Rubrerythrin (Rbr) is a non-heme iron protein composed of two distinctive domains and functions as a peroxidase in anaerobic organisms. A novel Rbr-like protein, ferriperoxin (Fpx), was identified in Hydrogenobacter thermophilus and was found not to possess the rubredoxin-like domain that is present in typical Rbrs. Although this protein is widely distributed among aerobic organisms, its function remains unknown. In this study, Fpx exhibited ferredoxin:NADPH oxidoreductase (FNR)-dependent peroxidase activity and reduced both hydrogen peroxide (H(2)O(2)) and organic hydroperoxide in the presence of NADPH and FNR as electron donors. The calculated K(m) and V(max) values of Fpx for organic hydroperoxides were comparable to that for H(2)O(2), demonstrating a multiple reactivity of Fpx towards hydroperoxides. An fpx gene disruptant was unable to grow under aerobic conditions, whereas its growth profiles were comparable to those of the wild-type strain under anaerobic and microaerobic conditions, clearly indicating the indispensability of Fpx as an antioxidant of H. thermophilus in aerobic environments. Structural analysis suggested that domain-swapping occurs in Fpx, and this domain-swapped structure is well conserved among thermophiles, implying the importance of structural stability of domain-swapped conformation for thermal environments. In addition, Fpx was located on a deep branch of the phylogenetic tree of Rbr and Rbr-like proteins. This finding, taken together with the wide distribution of Fpx among Bacteria and Archaea, suggests that Fpx is an ancestral type of Rbr homolog that functions as an essential antioxidant and may be part of an ancestral peroxide-detoxification system.

A peroxidase (PD-cP; 0.47 mg/100 g leaves) was purified from autumn leaves of Phytolacca dioica L. and characterized. PD-cP was obtained by acid precipitation followed by gel-filtration and cation exchange chromatography. Amino acid composition and N-terminal sequence of PD-cP up to residue 15 were similar to that of Spinacia oleracea (N-terminal pairwise comparison showing four amino acid differences). PD-cP showed a molecular mass of approx. 36 kDa by SDS-PAGE, pH and temperature optima at 3.0 and 50.0°C, respectively and seasonal variation. The Michaelis-Menten constant (K(M)) for H(2)O(2) was 5.27 mM, and the velocity maximum (V(max)) 1.31 nmol min(-1), while the enzyme turnover was 0.148 s(-1). Finally, the presence of Ca(2+) and Mg(2+) enhanced the PD-cP activity, with Mg(2+) 1.4-fold more effective than Ca(2+)

Surfactant proteins A (SP-A) and D (SP-D), both members of the collectin family, play a well established role in apoptotic cell recognition and clearance. Recent in vitro data show that SP-A and SP-D interact with apoptotic neutrophils in a distinct manner. SP-A and SP-D bind in a Ca(2+)-dependent manner to viable and early apoptotic neutrophils whereas the much greater interaction with late apoptotic neutrophils is Ca(2+)-independent. Cell surface molecules on the apoptotic target cells responsible for these interactions had not been identified and this study was done to find candidate target molecules. Myeloperoxidase (MPO), a specific intracellular defense molecule of neutrophils that becomes exposed on the outside of the cell upon apoptosis, was identified by affinity purification, mass-spectrometry and western blotting as a novel binding molecule for SP-A and SP-D. To confirm its role in recognition, it was shown that purified immobilised MPO binds SP-A and SP-D, and that MPO is surface-exposed on late apoptotic neutrophils. SP-A and SP-D inhibit binding of an anti-MPO monoclonal Ab to late apoptotic cells. Fluorescence microscopy confirmed that anti-MPO mAb and SP-A/SP-D colocalise on late apoptotic neutrophils. Desmoplakin was identified as a further potential ligand for SP-A, and neutrophil defensin as a target for both proteins.

Atherosclerosis is linked to inflammation. HDL protects against atherosclerotic cardiovascular disease, mainly by mediating cholesterol efflux and reverse cholesterol transport (RCT). The present study aimed to test the impact of acute inflammation as well as selected acute phase proteins on RCT with a macrophage-to-feces in vivo RCT assay using intraperitoneal administration of [(3)H]cholesterol-labeled macrophage foam cells. In patients with acute sepsis, cholesterol efflux toward plasma and HDL were significantly decreased (P < 0.001). In mice, acute inflammation (75 microg/mouse lipopolysaccharide) decreased [(3)H]cholesterol appearance in plasma (P < 0.05) and tracer excretion into feces both within bile acids (-84%) and neutral sterols (-79%, each P < 0.001). In the absence of systemic inflammation, overexpression of serum amyloid A (SAA, adenovirus) reduced overall RCT (P < 0.05), whereas secretory phospholipase A(2) (sPLA(2), transgenic mice) had no effect. Myeloperoxidase injection reduced tracer appearance in plasma (P < 0.05) as well as RCT (-36%, P < 0.05). Hepatic expression of bile acid synthesis genes (P < 0.01) and transporters mediating biliary sterol excretion (P < 0.01) was decreased by inflammation. In conclusion, our data demonstrate that acute inflammation impairs cholesterol efflux in patients and macrophage-to-feces RCT in vivo in mice. Myeloperoxidase and SAA contribute to a certain extent to reduced RCT during inflammation but not sPLA(2). However, reduced bile acid formation and decreased biliary sterol excretion might represent major contributing factors to decreased RCT in inflammation.

A new type of manganese-oxidizing enzyme has been identified in two alphaproteobacteria, "Aurantimonas manganoxydans" strain SI85-9A1 and Erythrobacter sp. strain SD-21. These proteins were identified by tandem mass spectrometry of manganese-oxidizing bands visualized by native polyacrylamide gel electrophoresis in-gel activity assays and fast protein liquid chromatography-purified proteins. Proteins of both alphaproteobacteria contain animal heme peroxidase and hemolysin-type calcium binding domains, with the 350-kDa active Mn-oxidizing protein of A. manganoxydans containing stainable heme. The addition of both Ca(2+) ions and H(2)O(2) to the enriched protein from Aurantimonas increased manganese oxidation activity 5.9-fold, and the highest activity recorded was 700 microM min(-1) mg(-1). Mn(II) is oxidized to Mn(IV) via an Mn(III) intermediate, which is consistent with known manganese peroxidase activity in fungi. The Mn-oxidizing protein in Erythrobacter sp. strain SD-21 is 225 kDa and contains only one peroxidase domain with strong homology to the first 2,000 amino acids of the peroxidase protein from A. manganoxydans. The heme peroxidase has tentatively been named MopA (manganese-oxidizing peroxidase) and sheds new light on the molecular mechanism of Mn oxidation in prokaryotes.

Recombinant Aspergillus oryzae expressing a dye-decolorizing peroxidase gene (dyp) was cultivated for repeated-batch production of recombinant dye-decolorizing peroxidase (rDyP) using maltose as a carbon source. High-level rDyP activity in limitation of carbon and nitrogen sources was maintained stably for 26 cycles of repeated 1-d batches of A. oryzae pellets without any additional pH control. Cultures maintained at 4 degrees C for 20 d resumed rDyP production following a single day of incubation. One liter filtrated crude rDyP containing 4600 U rDyP decolorized 5.07 g RBBR at the apparent decolorization rate of 17.7 mg l(-1) min(-1).

An original method called SIEFED (specific immunological extraction followed by enzymatic detection) was developed for the specific detection of the activity of equine myeloperoxidase (MPO). The method consists of the extraction of MPO from aqueous solutions by immobilized anti-MPO antibodies followed by washing (to eliminate proteins and interfering molecules) and measurement of MPO activity using a detection system containing a fluorogenic substrate, hydrogen peroxide, and nitrite as reaction enhancer. The SIEFED technique was applied to study active MPO in horse biological fluids and the effects of 2 polyphenolic molecules, curcumin and resveratrol, on MPO activity. The detection limit of the SIEFED was 0.23 mU/ml. The SIEFED exhibited good precision with intra-assay and interassay coefficients of variation below 10% and 20%, respectively, for MPO activities ranging from 0.25 to 6.4 mU/ml. The activity of MPO was generally higher than 1 mU/ml in the fluids collected from horses with inflammatory diseases. Curcumin and resveratrol exerted a dose-dependent inhibition on MPO activity and, as they were removed before the enzymatic detection of MPO, the results suggest a direct drug-nzyme interaction or an enzyme structure modification by the drug. The SIEFED is a new tool that would be useful for specific detection of active MPO in complex media and for selection of MPO activity modulators.

The major cationic peroxidase in sorghum grain (SPC4) , which is ubiquitously present in all sorghum varieties was purified to apparent homogeneity, and found to be a highly basic protein (pI approximately 11). MS analysis showed that SPC4 consists of two glycoforms with molecular masses of 34,227 and 35,629 Da and it contains a type-b heme. Chemical deglycosylation allowed to estimate sugar contents of 3.0% and 6.7% (w/w) in glycoform I and II, respectively, and a mass of the apoprotein of 33,246 Da. High performance anion exchange chromatography allowed to determine the carbohydrate constituents of the polysaccharide chains. The N-terminal sequence of SPC4 is not blocked by pyroglutamate. MS analysis showed that six peptides, including the N-terminal sequence of SPC4 matched with the predicted tryptic peptides of gene indice TC102191 of sorghum chromosome 1, indicating that TC102191 codes for the N-terminal part of the sequence of SPC4, including a signal peptide of 31 amino acids. The N-terminal fragment of SPC4 (213 amino acids) has a high sequence identity with barley BP1 (85%), rice Prx23 (90%), wheat WSP1 (82%) and maize peroxidase (58%), indicative for a common ancestor. SPC4 is activated by calcium ions. Ca2+ binding increased the protein conformational stability by raising the melting temperature (Tm) from 67 to 82 degrees C. SPC4 catalyzed the oxidation of a wide range of aromatic substrates, being catalytically more efficient with hydroxycinnamates than with tyrosine derivatives. In spite of the conserved active sites, SPC4 differs from BP1 in being active with aromatic compounds above pH 5.

The mushroom Agrocybe aegerita secretes a peroxidase (AaP) that catalyzes halogenations and hydroxylations. Phenol was brominated to 2- and 4-bromophenol (ratio 1:4) and chlorinated to a lesser extent to 2-chlorophenol. The purified enzyme was found to oxidize toluene via benzyl alcohol and benzaldehyde into benzoic acid. A second fraction of toluene was hydroxylated to give p-cresol as well as o-cresol and methyl-p-benzoquinone. The UV-Vis absorption spectrum of purified AaP showed high similarity to a resting state cytochrome P450 with the Soret band at 420 nm and additional maxima at 278, 358, 541 and 571 nm; the AaP CO-complex had a distinct absorption maximum at 445 nm that is characteristic for heme-thiolate proteins. AaP regioselectively hydroxylated naphthalene to 1-naphthol and traces of 2-naphthol (ratio 36:1). H2O2 was necessarily required for AaP function and hence the hydroxylations catalyzed by AaP can be designated as peroxygenation and the enzyme as an extracellular peroxygenase.

The major basic peroxidase from Zinnia elegans (ZePrx) suspension cell cultures was purified and cloned, and its properties and organ expression were characterized. The ZePrx was composed of two isoforms with a M(r) (determined by matrix-assisted laser-desorption ionization time of flight) of 34,700 (ZePrx34.70) and a M(r) of 33,440 (ZePrx33.44). Both isoforms showed absorption maxima at 403 (Soret band), 500, and 640 nm, suggesting that both are high-spin ferric secretory class III peroxidases. M(r) differences between them were due to the glycan moieties, and were confirmed from the total similarity of the N-terminal sequences (LSTTFYDTT) and by the 99.9% similarity of the tryptic fragment fingerprints obtained by reverse-phase nano-liquid chromatography. Four full-length cDNAs coding for these peroxidases were cloned. They only differ in the 5'-untranslated region. These differences probably indicate different ways in mRNA transport, stability, and regulation. According to the k(cat) and apparent K(m)(RH) values shown by both peroxidases for the three monolignols, sinapyl alcohol was the best substrate, the endwise polymerization of sinapyl alcohol by both ZePrxs yielding highly polymerized lignins with polymerization degrees > or =87. Western blots using anti-ZePrx34.70 IgGs showed that ZePrx33.44 was expressed in tracheary elements, roots, and hypocotyls, while ZePrx34.70 was only expressed in roots and young hypocotyls. None of the ZePrx isoforms was significantly expressed in either leaves or cotyledons. A neighbor-joining tree constructed for the four full-length cDNAs suggests that the four putative paralogous genes encoding the four cDNAs result from duplication of a previously duplicated ancestral gene, as may be deduced from the conserved nature and conserved position of the introns.

A peroxidase is present in the chorion of Aedes aegypti eggs and catalyzes chorion protein cross-linking during chorion hardening, which is critical for egg survival in the environment. The unique chorion peroxidase (CPO) is a glycoprotein. This study deals with the N-glycosylation site, structures, and profile of CPO-associated oligosaccharides using mass spectrometric techniques and enzymatic digestion. CPO was isolated from chorion by solubilization and several chromatographic methods. Mono-saccharide composition was analyzed by HPLC with fluorescent detection. Our data revealed that carbohydrate (D-mannose, N-acetyl D-glucosamine, D-arabinose, N-acetyl D-galactosamine, and L-fucose) accounted for 2.24% of the CPO molecular weight. A single N-glycosylation site (Asn328-Cys- Thr) was identified by tryptic peptide mapping and de novo sequencing of native and PNGase A-deglycosylated CPO using matrix-assisted laser/desorption/ionization time-of-flight mass spectrometry (MALDI/TOF/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The Asn328 was proven to be a major fully glycosylated site. Potential tryptic glycopeptides and profile were first assessed by MALDI/TOF/MS and then by precursor ion scanning during LC/MS/MS. The structures of N-linked oligosaccharides were elucidated from the MS/MS spectra of glycopeptides and exoglycosidase sequencing of PNGase A-released oligosaccharides. These CPO-associated oligosaccharides had dominant Man3GlcNAc2 and Man3 (Fuc) GlcNAc2 and high mannose-type structures (Man(4-8)GlcNAc2). The truncated structures, Man2GlcNAc2 and Man2 (Fuc) GlcNAc2, were also identified. Comparison of CPO activity and Stokes radius between native and deglycosylated CPO suggests that the N-linked oligosaccharides influence the enzyme activity by stabilizing its folded state.

The nature of the peroxidase isoenzyme complement responsible for cell wall lignification in both Zinnia elegans seedlings and Z. elegans tracheary single-cell cultures have been studied. Results showed that both hypocotyls and stems from lignifying Z. elegans seedlings express a cell wall-located basic peroxidase of pI approximately 10.2, which was purified to homogeneity. Molecular mass determination under non-denaturing conditions showed an M(r) of about 43 000, similar to that of other plant peroxidases. The purified Z. elegans peroxidase showed absorption maxima at 403 (Soret band), and at 496-501 and 632-635 (alpha and beta absorption bands), indicating that this enzyme is a high spin ferric haem protein, belonging to the plant peroxidase superfamily, the prosthetic group being ferric protoporphyrin IX. The N-terminal amino acid sequence of this Z. elegans basic peroxidase was KVAVSPLS (peptide motif in bold), which shows strong homologies with the N-amino acid terminus of other strongly basic plant peroxidases. Isoenzyme and western blot analyses showed that this peroxidase isoenzyme is also expressed in trans-differentiating Z. elegans tracheary single-cell cultures. The results also showed that Z. elegans tracheary single-cell cultures not only express the same peroxidase isoenzyme as the Z. elegans lignifying xylem, but that this peroxidase isoenzyme acts as a marker of tracheary element differentiation in Z. elegans mesophyll single-cell cultures. From these results, it may be concluded that Z. elegans uses a single programme, i.e. an identical peroxidase isoenzyme complement, for lignification of the xylem, regardless of the existence of different ontogenesis pathways from either mesophyll cells (in the case of tracheary elements) or cambial derivatives (in the case of xylem vessels).

Although several investigations have demonstrated a plasma membrane (PM)-bound peroxidase activity in plants, this study is the first, to our knowledge, to purify and characterize the enzymes responsible. Proteins were extracted from highly enriched and thoroughly washed PMs. Washing and solubilization procedures indicated that the enzymes were tightly bound to the membrane. At least two distinct peroxidase activities could be separated by cation exchange chromatography (pmPOX1 and pmPOX2). Prosthetic groups were identified in fractions with peroxidase activity by absorption spectra, and the corresponding protein bands were identified by heme staining. The activities of the peroxidase enzymes responded different to various substrates and effectors and had different thermal stabilities and pH and temperature optima. Because the enzymes were localized at the PM and were not effected by p-chloromercuribenzoate, they were probably class III peroxidases. Additional size exclusion chromatography of pmPOX1 revealed a single activity peak with a molecular mass of 70 kD for the native enzyme, whereas pmPOX2 had two activity peaks (155 and 40 kD). Further analysis of these fractions by a modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with heme staining confirmed the estimated molecular masses of the size exclusion chromatography.

Myeloperoxidase (MPO), an abundant heme enzyme released by activated phagocytes, catalyzes the formation of a number of reactive species that can modify low-density lipoprotein (LDL) to a form that converts macrophages into lipid-laden or 'foam' cells, the hallmark of atherosclerotic lesions. Since MPO has been shown to bind to a number of different cell types, we investigated binding of MPO to LDL. Using the precipitation reagents phosphotungstate or isopropanol, MPO co-precipitated with LDL, retaining its catalytic activity. The association of MPO with LDL was confirmed using native gel electrophoresis. MPO was also found to co-precipitate with apolipoprotein B-100-containing lipoproteins in whole plasma. No precipitation of MPO was observed in lipoprotein-deficient plasma, and there was a dose-dependent increase in precipitation following addition of LDL to lipoprotein-deficient plasma. Binding of MPO to LDL could potentially enhance site-directed oxidation of the lipoprotein and limit scavenging of reactive oxygen species by antioxidants.

The reinvestigation of the kinetics of myeloperoxidase (MPO) activity with the use of NADPH as a probe has allowed us to determine the effects of H(2)O(2), Cl(-) ion and pH on the MPO-dependent production of HOCl. The chlorination rate of NADPH did not depend on NADPH concentration and was entirely related to the rate of production of HOCl by MPO. The overall oxidation of NADPH occurred similarly in the absence of O(2) and was insensitive to scavengers of the superoxide radical anion. Experiments performed on the direct oxidation of NADPH by MPO in the presence and the absence of H(2)O(2) showed that neither the rate nor the stoichiometry of the reaction could interfere in the NADPH oxidation process involved in the steady-state chlorination cycle. The oxidation of NADPH was characterized by a decrease in the A(339) of the reduced nicotinamide with the concomitant appearance of a new chromophore with absorbance maximum at 274 nm, characterized by isosbestic points at 300 and 238 nm. The reaction product did not possess any enzymic properties with dehydrogenases and led to a metabolite other than NADP(+). Its amount accounted for a stoichiometric conversion of H(2)O(2) into HOCl. Analyses of the NADPH reaction allowed the determination of both kinetic (k(cat) and K(m)) and thermodynamic (K(d)) parameters. When the values of kinetic parameters were compared with previously published ones, the main discrepancy was found with data obtained with the chlorination of monochlorodimedon and a better agreement with diethanolchloramine formation or H(2)O(2) consumption. Variations in the extent of NADPH oxidation with Cl(-) concentration enabled us to determine the dissociation constant for the enzyme-Cl(-) complex. In the course of titration studies, the spectral properties of NADPH reacting with either HOCl or the MPO/H(2)O(2)/Cl(-) system were quantitatively similar in terms of stoichiometry and absorbance coefficient and thus led to identical chlorinated products. However, no spectral modification occurred with NADP(+) and adenine nucleotide analogues under the same conditions. A quantitative comparison of difference spectra obtained with NADPH and NMNH indicated that chlorination occurred on the nicotinamide part of the molecule.

The first temperature-dependent proteins (expressed at 37 degrees C, but not 26 degrees C) to be identified in Yersinia pestis were antigens 3 (fraction 1), 4 (pH 6 antigen), and 5 (hereafter termed KatY). Antigens 3 and 4 are now established virulence factors, whereas little is known about KatY, except that it is encoded chromosomally, produced in abundance, possesses modest catalase activity, and is shared by Yersinia pseudotuberculosis, but not Yersinia enterocolitica. We report here an improved chromatographic method (DEAE-cellulose, calcium hydroxylapatite, and Sephadex G-150) that yields enzymatically active KatY (2,423 U/mg of protein). Corresponding mouse monoclonal antibody 1B70.1 detected plasminogen activator-mediated hydrolysis of KatY, and a polyclonal rabbit antiserum raised against outer membranes of Y. pestis was enriched for anti-KatY. A sequenced approximately 16-kb Y. pestis DNA insert of a positive pLG338 clone indicated that katY encodes an 81.4-kDa protein (pI 6.98) containing a leader sequence of 2.6 kDa; the deduced molecular mass and pI of processed KatY were 78.8 kDa and 6. 43, respectively. A minor truncated variant (predicted molecular mass of 53.6 kDa) was also expressed. KatY is similar (39 to 59% identity) to vegetative bacterial catalase-peroxidases (KatG in Escherichia coli) and is closely related to plasmid-encoded KatP of enterohemorrhagic E. coli O157:H7 (75% identity). katY encoded a putative Ca2+-binding site, and its promoter contained three homologues to the consensus recognition sequence of the pCD-encoded transcriptional activator LcrF. rbsA was located upstream of katY, and cybB, cybC, dmsABC, and araD were mapped downstream. These genes are not linked to katG or katP in E. coli.

Salivary homogenates of the adult female mosquito Anopheles albimanus have been shown previously to contain a vasodilatory activity associated with a catechol oxidase/peroxidase activity. We have now purified the salivary peroxidase using high-performance liquid chromatography. The pure enzyme is able to relax rabbit aortic rings pre-constricted with norepinephrine. The peroxidase has a relative molecular mass of 66 907 as estimated by mass spectrometry. Amino-terminal sequencing allowed us to design oligonucleotide probes for isolation of cDNA clones derived from the salivary gland mRNA from female mosquitoes. The full sequence of the cDNA demonstrated homology between A. albimanus salivary peroxidase and several members of the myeloperoxidase gene family. A close comparison of A. albimanus salivary peroxidase with canine myeloperoxidase, for which the crystal structure is known, showed that all six disulfide bridges were conserved and demonstrated identity for all five residues associated with a Ca2+-binding site. In addition, 16 of 26 residues shown to be in close proximity to the heme moiety in the canine myeloperoxidase were identical. We conclude that the salivary peroxidase of A. albimanus belongs to the myeloperoxidase gene family. Other possible functions for this molecule in blood feeding are discussed.

An acidic peroxidase (EC 1.11.1.7) 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.

Increases of plasma concentrations of neutrophil myeloperoxidase (MPO) can be used as markers of polymorphonuclear leucocytes (PMN) activation in pathological situations (sepsis, acute lung injury, acute inflammation). To develop an assay for measurement of plasma MPO in horses during the above-mentioned infectious and inflammatory conditions, MPO was purified from equine PMN isolated from blood anticoagulated with citrate. PMN were extracted in a saline milieu (0.2 M Na acetate, 1 M NaCl, pH 4.7) to eliminate most of cellular proteins. Pellets were then extracted in the same buffer containing cationic detergent (1% cetyltrimethyl ammonium bromide). The supernatant was further purified by ion exchange chromatography (Hiload S Sepharose HP column 0.5 x 26 cm, equilibrated with 25 mM Na acetate, 0.2 M NaCl, pH 4.7) with a NaCl gradient (until 1 M). Most of the peroxidase activity of MPO (spectrophotometrically measured by the oxidation of orthodianisidine by hydrogen peroxide) was eluted at 0.65 M NaCl. MPO was further purified by gel filtration chromatography (Sephacryl S 200 column 2.6 x 42 cm with 25 mM Na acetate, 0.2 M NaCl, pH 4.7). MPO (specific activity: 74.3 U/mg) was obtained with a yield of 30% from the detergent extraction supernatant. Electrophoresis (non-reducing conditions) showed 3 bands identified, by comparison with human MPO, (i) the mature tetrameric enzyme (150 kDa) with 2 light and 2 heavy subunits, (ii) the precursor form (88 kDa) and (iii) a form of the heavy subunit without the prosthetic heme group (40 kDa). The mature enzyme and its precursor were glycosylated and possessed peroxidase activity. Equine MPO showed strong similarities with human and bovine MPO, with an absorption peak at 430 nm (Soret peak) characteristic of ferrimyeloperoxidase. Enzymatic activity was pH dependent (optimal value at pH 5.5).

We examined the effects of a human milk diet on rats with chemical colitis induced with a 4% acetic acid enema. Colonic myeloperoxidase activity was used as a surrogate marker for neutrophil infiltration. Control rats fed rat chow had little colonic myeloperoxidase activity; geometric mean, 0.27 U/g of tissue. Rats with colitis fed rat chow had significantly increased colonic myeloperoxidase activity (geometric mean, 6.76 U/g, p < 0.01 versus no colitis), as did rats with colitis fed infant formula or Pedialyte (geometric mean, 6.92 and 8.13 U/g, respectively, both p < 0.01 versus no colitis). Animals with colitis fed human milk had significantly lower colonic myeloperoxidase activity (geometric mean, 2.34 U/g) than did animals with colitis fed either chow or infant formula (p < 0.001). Similar effects were seen in rats with colitis fed infant formula supplemented with recombinant human IL-1 receptor antagonist (geometric mean, 1.95 U/g). These data show that orally administered human milk has an antiinflammatory effect on chemically induced colitis in rats, which may be mediated in part by IL-1 receptor antagonist contained in human milk.

The neutrophil enzyme myeloperoxidase uses H2O2 to oxidize chloride, bromide, iodide and thiocyanate to their respective hypohalous acids. Chloride is considered to be the physiological substrate. However, a detailed kinetic study of its substrate preference has not been undertaken. Our aim was to establish whether myeloperoxidase oxidizes thiocyanate in the presence of chloride at physiological concentrations of these substrates. We determined this by measuring the rate of H2O2 loss in reactions catalysed by the enzyme at various concentrations of each substrate. The relative specificity constants for chloride, bromide and thiocyanate were 1:60:730 respectively, indicating that thiocyanate is by far the most favoured substrate for myeloperoxidase. In the presence of 100 mM chloride, myeloperoxidase catalysed the production of hypothiocyanite at concentrations of thiocyanate as low as 25 microM. With 100 microM thiocyanate, about 50% of the H2O2 present was converted into hypothiocyanite, and the rate of hypohalous acid production equalled the sum of the individual rates obtained when each of these anions was present alone. The rate of H2O2 loss catalysed by myeloperoxidase in the presence of 100 mM chloride doubled when 100 microM thiocyanate was added, and was maximal with 1mM thiocyanate. This indicates that at plasma concentrations of thiocyanate and chloride, myeloperoxidase is far from saturated. We conclude that thiocyanate is a major physiological substrate of myeloperoxidase, regardless of where the enzyme acts. As a consequence, more consideration should be given to the oxidation products of thiocyanate and to the role they play in host defence and inflammation.

A distant relative of catalase that is specialized for metabolism of a fatty acid hydroperoxide was identified. This heme peroxidase occurs in coral as part of a fusion protein, the other component of which is a lipoxygenase that forms the hydroperoxide substrate. The end product is an unstable epoxide (an allene oxide) that is a potential precursor of prostaglandin-like molecules. These results extend the known chemistry of catalase-like proteins and reveal a distinct type of enzymatic construct involved in the metabolism of polyunsaturated fatty acids.

A novel, basic (isoelectric point > 10), heme peroxidase isoenzyme (TP; relative molecular weight = 34,660 +/- 10, mean +/- SE) that can account for a significant part of the ascorbate peroxidase activity in tea (Camellia sinensis) leaves has been purified to homogeneity. The ultraviolet/visible absorption spectrum is typical of heme-containing plant peroxidases, with a Soret peak at 406 nm (epsilon = 115 mM-1 cm-1) and an A406/A280 value of 3.4. The enzyme has a high specific activity for ascorbate oxidation (151 mumol min-1 mg-1), with a pH optimum in the range of 4.5 to 5.0. Substrate-specificity studies have revealed significant differences between TP and other class III peroxidases, as well as similarities with class I ascorbate peroxidases. TP, like ascorbate peroxidase, exhibits a preference for ascorbate over guaiacol, whereas other class III isoenzymes are characterized by 2-orders-of-magnitude higher activity for guaiacol than for ascorbate. TP also forms an unstable porphyrin pi cation radical-type compound I, which is converted to compound II within approximately 2 min in the absence of added reductant. Amino acid sequence data show TP to be the first example, to our knowledge, of a class III peroxidase with a high specificity for ascorbate as an electron donor.

The predominant peroxidase (pI 3.5) (E.C. 1.11.1.7) of an Arabidopsis thaliana cell suspension culture was purified and partially sequenced. Oligonucleotides were designed and a specific probe was obtained. A cDNA clone was isolated from an Arabidopsis cell suspension cDNA library and completely sequenced. The cDNA clone comprised 1194 bp and encodes a 30 residue signal peptide and a 305 residue mature protein (Mr 31,966). The sequence of the mature protein is 95% identical to the well-characterized horseradish peroxidase HRP A2 and has therefore been designated ATP A2. Three introns at positions identical to those found in Arabidopsis and horseradish genes encoding cationic peroxidases were identified. RT-PCR analysis revealed root-specific expression.

Many animal-bacteria cooperative associations occur in highly modified host organs that create a unique environment for housing and maintaining the symbionts. It has been assumed that these specialized organs develop through a program of symbiosis-specific or -enhanced gene expression in one or both partners, but a clear example of this process has been lacking. In this study, we provide evidence for the enhanced production of an enzyme in the symbiotic organ of the squid Euprymna scolopes, which harbors a culture of the luminous bacterium Vibrio fischeri. Our data show that this enzyme has a striking biochemical similarity to mammalian myeloperoxidase (MPO; EC 1.11.17), an antimicrobial dianisidine peroxidase that occurs in neutrophils. MPO and the squid peroxidase catalyze the same reaction, have similar apparent subunit molecular masses, and a polyclonal antibody to native human MPO specifically localized a peroxidase-like protein to the bacteria-containing regions of the symbiotic organ. We also provide evidence that a previously described squid cDNA encodes the protein (LO4) that is responsible for the observed dianisidine peroxidase activity. An antibody made against a fragment of LO4 immunoprecipiated dianisidine peroxidase activity from extracts of the symbiotic organ, and reacted against these extracts and human MPO in Western blot analysis. These data suggest that related biochemical mechanisms for the control of bacterial number and growth operate in associations that are as functionally diverse as pathogenesis and mutualism, and as phylogenetically distant as molluscs and mammals.

The first dimeric catalase-peroxidase of eucaryotic origin, an intracellular hydroperoxidase from Penicillium simplicissimum which exhibited both catalase and peroxidase activities, has been isolated. The enzyme has an apparent molecular mass of about 170 kDa and is composed of two identical subunits. The purified protein has a pH optimum for catalase activity at 6.4 and for peroxidase at 5.4. Both activities are inhibited by cyanide and azide whereas 3-amino-1,2,4-triazole has no effect. 3,3'-Diaminobenzidine, 3,3'-dimethoxybenzidine, guaiacol, 2,6-dimethoxyphenol and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) all serve as substrates. The optical spectrum of the purified enzyme shows a Soret band at 407 nm. Reduction by dithionite results in the disappearance of the Soret band and formation of three absorption maxima at 440, 562 and 595 nm. The prosthetic group was identified as a protoheme IX and EPR spectroscopy revealed the presence of a histidine residue as proximal ligand. In addition to the catalase-peroxidase, an atypical catalase which is active over a broad pH range was also partially purified from P. simplicissimum. This catalase is located in the periplasm and contains a chlorin-type heme as prosthetic group.

Myeloperoxidase and eosinophil peroxidase catalyzed the oxidation of bromide ion by hydrogen peroxide (H2O2) and produced a brominating agent that reacted with amine compounds to form bromamines, which are long-lived oxidants containing covalent nitrogen-bromine bonds. Results were consistent with oxidation of bromide to an equilibrium mixture of hypobromous acid (HOBr) and hypobromite ion (OBr-). Up to 1 mol of bromamine was produced per mole of H2O2, indicating that bromamine formation prevented the reduction of HOBr/OBr- by H2O2 and the loss of oxidizing and brominating activity. Bromamines differed from HOBr/OBr- in that bromamines reacted slowly with H2O2, were not reduced by dimethyl sulfoxide, and had absorption spectra similar to those of chloramines, but shifted 36 nm toward higher wavelengths. Mono- and di-bromo derivatives (RNHBr and RNHBr2) of the beta-amino acid taurine were relatively stable with half-lives of 70 and 16 h at pH 7, 37 degrees C. The mono-bromamine was obtained with a 200-fold excess of amine over the amount of HOBr/OBr- and the di-bromamine at a 2:1 ratio of HOBr/OBr- to the amine. In the presence of physiologic levels of both bromide (0.1 mM) and chloride (0.1 M), myeloperoxidase and eosinophil peroxidase produced mixtures of bromamines and chloramines containing 6 +/- 4% and 88 +/- 4% bromamine. In contrast, only the mono-chloramine derivative (RNHCl) was formed when a mixture of hypochlorous acid (HOCl) and hypochlorite ion (OCl-) was added to solutions containing bromide and excess amine. The rapid formation of the chloramine prevented the oxidation of bromide by HOCl/OCl-, and the chloramine did not react with bromide within 1 h at 37 degrees C. The results indicate that when enzyme-catalyzed bromide or chloride oxidation took place in the presence of an amine compound at 10 mM or higher, bromamines were not produced in secondary reactions such as the oxidation of bromide by HOCl/OCl- and the exchange of bromide with chlorine atoms of chloramines. Therefore, the amount of bromamine produced by myeloperoxidase or eosinophil peroxidase was equal to the amount of bromide oxidized by the enzyme. Bromide was preferred over chloride as the substrate for both enzymes.

Peroxidasin is a novel protein combining peroxidase and extracellular matrix motifs. Hemocytes differentiate early from head mesoderm, make peroxidasin and later phagocytose apoptotic cells. As hemocytes spread throughout the embryo, they synthesize extracellular matrix and peroxidasin, incorporating it into completed basement membranes. Cultured cells secrete peroxidasin; it occurs in larvae and adults. Each 1512 residue chain of the three-armed, disulfide-linked homotrimer combines a peroxidase domain with six leucine-rich regions, four Ig loops, a thrombospondin/procollagen homology and an amphipathic alpha-helix. The peroxidase domain is homologous with human myeloperoxidase and eosinophil peroxidase. This heme protein catalyzes H2O2-driven radioiodinations, oxidations and formation of dityrosine. We propose that peroxidasin functions uniquely in extracellular matrix consolidation, phagocytosis and defense.

Rapidly progressive glomerulonephritis with or without other signs of systemic vasculitis is often accompanied by antibodies to myeloperoxidase. Such antibodies normally produce a perinuclear pattern on ethanol-fixed neutrophils (perinuclear anti-neutrophil cytoplasm antibodies (P-ANCA)) at indirect immunofluorescence. We report here sera from three patients that are anti-myeloperoxidase-positive in ELISA that instead produce a cytoplasmic pattern (classical anti-neutrophil cytoplasmic antibodies (C-ANCA)), a pattern normally seen in conjunction with antibodies to proteinase 3. These sera did not react with proteinase 3. For two of the sera the specificity of the anti-myeloperoxidase reaction was confirmed with inhibition-ELISA experiments and with immunoblotting. A mouse anti-myeloperoxidase MoAb that produces a cytoplasmic pattern is also described. Competition ELISA experiments show that this antibody and anti-myeloperoxidase sera with cytoplasmic pattern recognize epitopes that are separate from epitopes recognized by another perinuclear pattern producing anti-myeloperoxidase MoAb. 'Cytoplasmic pattern' epitopes as well as 'perinuclear pattern' epitopes can be found on all three major myeloperoxidase isoforms, after separation by ion exchange chromatography. Affinity chromatography, using the cytoplasmic pattern producing anti-myeloperoxidase monoclonal antibody, shows that the epitope recognized by this MoAb is present on all myeloperoxidase molecules. This epitope is not confined to any special subpopulation. These findings indicate that all myeloperoxidase do not relocate after ethanol fixation, and that C-ANCA and P-ANCA epitopes exist simultaneously on the same myeloperoxidase molecule. We propose that the two immunofluorescence patterns arise due to different availabilities of the epitopes in the microenvironment where myeloperoxidase is present.

Myeloperoxidase, in the presence of noncytotoxic concentrations of H2O2, was used to induce cytotoxicity to the lung epithelial cell line, AKD. When the cationic aminoglycosides, tobramycin and gentamicin were added to the cells in the presence of myeloperoxidase and H2O2, cytotoxicity was completely inhibited. In addition, tobramycin prevented cytotoxicity induced by cystic fibrosis sputum and H2O2. Protection against myeloperoxidase and H2O2 was also observed with the thioether-containing antibiotics, ticarcillin and ceftazidime, but at higher concentrations than with the aminoglycosides. Analysis of spectral properties, dimethylsulfoxide-mediated reduction, and ethyl acetate/NaCl partitioning, demonstrated that aminoglycosides converted HOCl to hydrophilic noncytotoxic chloramines, but were unable to prevent the oxidation of sulfhydryls and methionine by HOCl. In contrast, ticarcillin and ceftazidime were highly effective inhibitors of HOCl-mediated sulfhydryl and methionine oxidation. These results suggest that aminoglycosides protect lung epithelial cells against myeloperoxidase-dependent oxidant injury by binding to anionic cell surfaces and converting HOCl to hydrophilic noncytotoxic chloramines, whereas penicillins and cephalosporins are potent HOCl scavengers capable of protecting critical extracellular molecules against oxidation.

Myeloperoxidase (MPO) is a functionally important component of the normal human neutrophil host defense system. This enzyme possesses a dimeric structure composed of two heavy subunit (55-63 kDa)/light subunit (10-15 kDa) protomers, each of which is associated with a heme-like prosthetic group. In addition, MPO species of approximately 38 and 22 kDa have been reported by many different investigators, but their nature and mode of origin are not understood. In the present study, we demonstrate that when MPO is heated under nonreducing, denaturing conditions, these two species are produced via a novel autolytic cleavage of the heavy subunit. The 38-kDa species was isolated by fast-protein liquid chromatography and identified by sequencing as the carboxyl-terminal portion of the heavy subunit, and the cleavage was shown to occur exclusively between Met409 and Pro410. In order to further characterize this unusual cleavage reaction, the 22-kDa species was digested with endoproteinase Asp-N, and the peptide corresponding to its carboxyl terminus was isolated and analyzed by sequencing and mass spectrometry. These data indicated that during cleavage of the heavy subunit, Met409 was converted to homoserine lactone. Thus, the cleavage appeared to formally resemble the cyanogen bromide-dependent cleavage of Met-X peptide bonds. Recent x-ray crystallographic data for canine MPO have indicated that Met409 is in close proximity to the heme-like prosthetic group of MPO. Our studies suggest that interaction of Met409 with this group leads to the formation of a methionyl sulfonium derivative which undergoes intramolecular rearrangement with subsequent peptide cleavage under nonreducing conditions. This derivative may be, at least in part, responsible for the unusual spectral characteristics and enzymatic properties of the enzyme. The primary structure of the 22-kDa MPO species is also reported, and direct evidence is provided for asparagine-linked oligosaccharide moieties at two of the three predicted glycosylation sites.

Human neutrophilic myeloperoxidase (MPO) is involved in the defence mechanism of the body against micro-organisms. The enzyme catalyses the generation of the strong oxidant hypochlorous acid (HOCl) from hydrogen peroxide and chloride ions. In normal neutrophils MPO is present in the dimeric form (140 kDa). The disulphide-linked protomers each consist of a heavy subunit and a light one. Reductive alkylation converts the dimeric enzyme into two promoters, 'hemi-myeloperoxidase'. We studied the initial activities of human dimeric MPO and hemi-MPO at the physiological pH of 7.2 and found no significant differences in chlorinating activity. These results indicate that, at least at neutral pH, the protomers of MPO function independently. The absorption spectra of MPO compounds II and III, both inactive forms concerning HOCl generation, and the rate constants of their formation were the same for dimeric MPO and hemi-MPO, but hemi-MPO required a slightly larger excess of H2O2 for complete conversion. Hemi-MPO was less stable at a high temperature (80 degrees C) as compared to the dimeric enzyme. Furthermore, the resistance of the chlorinating activity of hemi-MPO against its oxidative product hypochlorous acid was somewhat lower (IC50 = 32 microM HOCl) compared to dimeric MPO (IC50 = 50 microM HOCl). The higher stability of dimeric MPO in the presence of its oxidative product compared to that of monomeric MPO might be the reason for the occurrence of MPO as a dimer.