Heme Peroxidases Research Articles

The role of Ala134 in controlling substrate binding and reactivity in ascorbate peroxidase

Ascorbate peroxidase (APX) is a class I heme peroxidase. It has two sites for binding of substrates. One is close to the γ-heme edge and is used for oxidation of ascorbate; the other is at the δ-heme edge and is used for binding of aromatic substrates [Gumiero et al., (2010) Arch. Biochem. Biophys. 500, 13–20]. In this work, we have examined the structural factors that control binding at the δ-heme edge by replacement of Ala134 in APX with a proline residue that is more commonly found in other class II and III peroxidases. Kinetic data indicate that replacement of Ala134 by proline has only a small effect on the catalytic mechanism, or the oxidation of ascorbate or guaiacol. Chemical modification with phenylhydrazine indicates that heme accessibility close to the δ-heme edge is only minorly affected by the substitution. We conclude that the A134P mutation alone is not enough to substantially affect the reactivity of APX towards aromatic substrates bound at the δ-heme edge. The data are relevant to the recent application of APX (APEX) in cellular imaging.

Lactoperoxidase, an Antimicrobial Milk Protein, as a Potential Activator of Carcinogenic Heterocyclic Amines in Breast Cancer.

Lactoperoxidase (LPO) is an antimicrobial protein secreted from mammary, salivary and other mucosal glands. It is an important member of heme peroxidase enzymes and the primary peroxidase enzyme present in breast tissues. In addition to the antimicrobial properties, LPO has been shown to be associated with breast cancer etiology. Heterocyclic amines, an important class of environmental and dietary carcinogens, have been increasingly associated with breast cancer etiology. Heterocyclic amines undergo activation in breast tissue as a result of oxidation by LPO. The current study includes three important heterocyclic amines, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methy-6-phenylimidazo[4,5-b]-pyridine (PhIP), that have carcinogenic activity. The structural binding characterization of IQ, MeIQx and PhIP with LPO was done using in silico approaches. Their binding pattern and interactions with LPO amino acid residues were analyzed. The three compounds bound in the distal heme cavity of LPO without replacing the important water molecule required for oxidation of substrate compounds. PhIP displayed lesser binding affinity for LPO in comparison to IQ and MeIQx. The binding mode of heterocyclic amines in distal heme cavity of LPO resembled to that of substrate binding pattern. The three heterocyclic amines are suggested to act as LPO substrate. The undisturbed water molecule present in distal heme cavity of the LPO is expected to facilitate the oxidation and activation of the three heterocyclic amines. These activated compounds may potentially bind with DNA in breast tissues forming DNA adducts and may subsequently lead to breast cancer initiation.

Karmoisin and cardinal ortholog genes participate in the ommochrome synthesis of Nilaparvata lugens (Hemiptera: Delphacidae).

Ommochrome is the major source for eye coloration of all insect species so far examined. Phenoxazinone synthetase (PHS) has always been regarded as the terminal step enzyme for ommochrome formation, which is encoded by cardinal or karmoisin genes. Our previous study indicated that the karmoisin ortholog gene (Nl-karmoisin) product in the brown planthopper (BPH) was a monocarboxylate transporter, while not a PHS. Here, based on full-length complementary DNA, the cardinal ortholog gene in BPH (Nl-cardinal) product was predicted to be a haem peroxidase rather than a PHS. We suggest for the first time that neither karmoisin nor cardinal encodes the PHS, but whether PHS participates in BPH eye pigmentation needs further research. Nymphal RNA interference (RNAi) experiments showed that knockdown Nl-cardinal transcript led the BPH ocelli and compound eye to color change from brown to red, while knockdown Nl-karmoisin only made the ocelli present the red phenotype. Notably, not only the Nl-cardinal transcript, dscd injection (Nl-cardinal targeting double-stranded DNA (dsRNA)) also significantly reduced the Nl-karmoisin transcript by 33.7%, while dska (Nl-karmoisin targeting dsRNA) injection did not significantly change the Nl-cardinal transcript. Considering the above RNAi and quantitative real-time polymerase chain reaction results, we propose that Nl-cardinal plays a more important role in ommochrome synthesis than Nl-karmoisin, and it may be an upstream gene of Nl-karmoisin. The present study suggested that both karmoisin and cardinal ortholog genes play a role in ommochrome synthesis in a hemimetabolous insect.

Fungal Hybrid B heme peroxidases \u2013 unique fusions of a heme peroxidase domain with a carbohydrate-binding domain

Heme peroxidases, essential peroxide converting oxidoreductases are divided into four independently evolved superfamilies. Within the largest one – the peroxidase-catalase superfamily - two hybrid lineages were described recently. Whereas Hybrid A heme peroxidases represent intermediate enzymes between ascorbate peroxidases and cytochrome c peroxidases, Hybrid B heme peroxidases are unique fusion proteins comprised of a conserved N-terminal heme peroxidase domain and a C-terminal domain of various sugar binding motifs. So far these peculiar peroxidases are only found in the kingdom of Fungi. Here we present a phylogenetic reconstruction of the whole superfamily with focus on Hybrid B peroxidases. We analyse the domain assembly and putative structure and function of the newly discovered oligosaccharide binding domains. Two distinct carbohydrate binding modules (CBM21 and CBM34) are shown to occur in phytopathogenic ascomycetous orthologs of Hybrid B heme peroxidases only. Based on multiple sequence alignment and homology modeling the structure-function relationships are discussed with respect to physiological function. A concerted action of peroxide cleavage with specific cell-wall carbohydrate binding can support phytopathogens survival within the plant host.

Mechanistic Insights into Dye-Decolorizing Peroxidase Revealed by Solvent Isotope and Viscosity Effects.

Dye-decolorizing peroxidases (DyPs) are a family of H2O2-dependent heme peroxidases, which have shown potential applications in lignin degradation and valorization. However, the DyP kinetic mechanism remains underexplored. Using structural biology and solvent isotope (sKIE) and viscosity effects, many mechanistic characteristics have been uncovered for the B-class ElDyP from Enterobacter lignolyticus. Its structure revealed that a water molecule acts as the sixth axial ligand with two channels at diameters of ~3.0 and 8.0 Å leading to the heme center. A conformational change of ERS* to ERS, which have identical spectral characteristics, was proposed as the final step in DyPs' bisubstrate Ping-Pong mechanism. This step is also the rate-determining step in ABTS oxidation. The normal KIE of wild-type ElDyP with D2O2 at pH 3.5 suggested that cmpd 0 deprotonation by the distal aspartate is rate-limiting in the formation of cmpd I, which is more reactive under acidic pH than under neutral or alkaline pH. The viscosity effects and other biochemical methods implied that the reducing substrate binds with cmpd I instead of the free enzyme. The significant inverse sKIEs of kcat/KM and kERS* suggested that the aquo release in DyPs is mechanistically important and may explain the enzyme's adoption of two-electron reduction for cmpd I. The distal aspartate is catalytically more important than the distal arginine and plays key roles in determining DyPs' acidic pH optimum. The kinetic mechanism of D143H-ElDyP was also briefly studied. The results obtained will pave the way for future protein engineering to improve DyPs' lignolytic activity.

Fungal Unspecific Peroxygenases Oxidize the Majority of Organic EPA Priority Pollutants.

Unspecific peroxygenases (UPOs) are secreted fungal enzymes with promiscuity for oxygen transfer and oxidation reactions. Functionally, they represent hybrids of P450 monooxygenases and heme peroxidases; phylogenetically they belong to the family of heme-thiolate peroxidases. Two UPOs from the basidiomycetous fungi Agrocybe aegerita (AaeUPO) and Marasmius rotula (MroUPO) converted 35 out of 40 compounds listed as EPA priority pollutants, including chlorinated benzenes and their derivatives, halogenated biphenyl ethers, nitroaromatic compounds, polycyclic aromatic hydrocarbons (PAHs) and phthalic acid derivatives. These oxygenations and oxidations resulted in diverse products and—if at all—were limited for three reasons: (i) steric hindrance caused by multiple substitutions or bulkiness of the compound as such (e.g., hexachlorobenzene or large PAHs), (ii) strong inactivation of aromatic rings (e.g., nitrobenzene), and (iii) low water solubility (e.g., complex arenes). The general outcome of our study is that UPOs can be considered as extracellular counterparts of intracellular monooxygenases, both with respect to catalyzed reactions and catalytic versatility. Therefore, they should be taken into consideration as a relevant biocatalytic detoxification and biodegradation tool used by fungi when confronted with toxins, xenobiotics and pollutants in their natural environments.

Role of Hypohalous Acids in Basement Membrane Homeostasis.

Basement membranes (BMs) are sheet-like structures of specialized extracellular matrix that underlie nearly all tissue cell layers including epithelial, endothelial, and muscle cells. BMs not only provide structural support but are also critical for the development, maintenance, and repair of organs. Animal heme peroxidases generate highly reactive hypohalous acids extracellularly and, therefore, target BMs for oxidative modification. Given the importance of BMs in tissue structure and function, hypohalous acid-mediated oxidative modifications of BM proteins represent a key mechanism in normal development and pathogenesis of disease. Recent Advances: Peroxidasin (PXDN), a BM-associated animal heme peroxidase, generates hypobromous acid (HOBr) to form sulfilimine cross-links within the collagen IV network of BM. These cross-links stabilize BM and are critical for animal tissue development. These findings highlight a paradoxical anabolic role for HOBr, which typically damages protein structure leading to dysfunction. The molecular mechanism whereby PXDN uses HOBr as a reactive intermediate to cross-link collagen IV, yet avoid collateral damage to nearby BM proteins, remains unclear. The exact identification and functional impact of specific hypohalous acid-mediated modifications of BM proteins need to be addressed to connect these modifications to tissue development and pathogenesis of disease. As seen with the sulfilimine cross-link of collagen IV, hypohalous acid oxidative events may be beneficial in select situations rather than uniformly deleterious. Antioxid. Redox Signal. 27, 839-854.

Design of anti-thyroid drugs: Binding studies and structure determination of the complex of lactoperoxidase with 2-mercaptoimidazole at 2.30 Å resolution.

Lactoperoxidase (LPO) belongs to mammalian heme peroxidase superfamily, which also includes myeloperoxidase (MPO), eosinophil peroxidase (EPO), and thyroid peroxidase (TPO). LPO catalyzes the oxidation of a number of substrates including thiocyanate while TPO catalyzes the biosynthesis of thyroid hormones. LPO is also been shown to catalyze the biosynthesis of thyroid hormones indicating similar functional and structural properties. The binding studies showed that 2-mercaptoimidazole (MZY) bound to LPO with a dissociation constant of 0.63 µM. The inhibition studies showed that the value of IC50 was 17 µM. The crystal structure of the complex of LPO with MZY showed that MZY bound to LPO in the substrate-binding site on the distal heme side. MZY was oriented in the substrate-binding site in such a way that the sulfur atom is at a distance of 2.58 Å from the heme iron. Previously, a similar compound, 3-amino-1,2,4-triazole (amitrole) was also shown to bind to LPO in the substrate-binding site on the distal heme side. The amino nitrogen atom of amitrole occupied the same position as that of sulfur atom in the present structure indicating a similar mode of binding. Recently, the structure of the complex of LPO with a potent antithyroid drug, 1-methylimidazole-2-thiol (methimazole, MMZ) was also determined. It showed that MMZ bound to LPO in the substrate-binding site on the distal heme side with 2 orientations. The position of methyl group was same in the 2 orientations while the positions of sulfur atom differed indicating a higher preference for a methyl group.

P 008 - Formation of cyanogen iodide by heme peroxidases

The heme-containing proteins lactoperoxidase (LPO) and myeloperoxidase are important enzymes involved in immune defence reactions. They are well known for their ability to oxidise in a hydrogen peroxide-driven reaction thiocyanate, iodide and in case of myeloperoxidase also bromide and chloride to the corresponding hypo(pseudo)halous acids. Here we describe the formation of cyanogen iodide (ICN) as a novel product of these enzymes. The formation of ICN was evidenced by both 13 C-nuclear magnetic resonance spectroscopy by applying 13 C-labeled thiocyanate and by head-space gas-chromatography mass spectrometry. This product is only formed when iodide is used in excess over thiocyanate during these reactions. Furthermore, the formation of bactericidal components by the LPO-hydrogen peroxide-thiocyanate/iodide system was investigated on a luminescent E. coli strain. This approach delivered time-resolved data about the killing of these microorganisms. In the mixed presence of thiocyanate and iodide (with an excess of iodide) a considerably higher killing rate was observed than in the sole application of either ion species. These data correspond well with the direct apllication of ICN. Control experiments of the determination of colony-forming units confirmed the results. Our data offer a novel product pathway that may be important for numerous biotechnological application of LPO.

Crystal structure and functional analysis of Leishmania major pseudoperoxidase.

Leishmania major pseudoperoxidase (LmPP) is a recently discovered heme protein expressed by the human pathogen. Previous in vivo and in vitro studies suggest that LmPP is a crucial element of the pathogen's defense mechanism against the reactive nitrogen species peroxynitrite produced during the host immune response. To shed light on the potential mechanism of peroxynitrite detoxification, we have determined the 1.76-Å X-ray crystal structure of LmPP, revealing a striking degree of homology with heme peroxidases. The most outstanding structural feature is a Cys/His heme coordination, which corroborates previous spectroscopic and mutagenesis studies. We also used a combination of stopped-flow and electron paramagnetic spectroscopies that together suggest that peroxynitrite is not a substrate for LmPP catalysis, leaving the function of LmPP an open question.

Urate hydroperoxide oxidizes human peroxiredoxin 1 and peroxiredoxin 2

Urate hydroperoxide is a product of the oxidation of uric acid by inflammatory heme peroxidases. The formation of urate hydroperoxide might be a key event in vascular inflammation, where there is large amount of uric acid and inflammatory peroxidases. Urate hydroperoxide oxidizes glutathione and sulfur-containing amino acids and is expected to react fast toward reactive thiols from peroxiredoxins (Prxs). The kinetics for the oxidation of the cytosolic 2-Cys Prx1 and Prx2 revealed that urate hydroperoxide oxidizes these enzymes at rates comparable with hydrogen peroxide. The second-order rate constants of these reactions were 4.9 × 105 and 2.3 × 106 m-1 s-1 for Prx1 and Prx2, respectively. Kinetic and simulation data suggest that the oxidation of Prx2 by urate hydroperoxide occurs by a three-step mechanism, where the peroxide reversibly associates with the enzyme; then it oxidizes the peroxidatic cysteine, and finally, the rate-limiting disulfide bond is formed. Of relevance, the disulfide bond formation was much slower in Prx2 (k3 = 0.31 s-1) than Prx1 (k3 = 14.9 s-1). In addition, Prx2 was more sensitive than Prx1 to hyperoxidation caused by both urate hydroperoxide and hydrogen peroxide. Urate hydroperoxide oxidized Prx2 from intact erythrocytes to the same extent as hydrogen peroxide. Therefore, Prx1 and Prx2 are likely targets of urate hydroperoxide in cells. Oxidation of Prxs by urate hydroperoxide might affect cell function and be partially responsible for the pro-oxidant and pro-inflammatory effects of uric acid.

Modeling the design and operational mode of a continuous membrane reactor for enzymatic lignin modification

The modification of lignin-derived compounds such as technical lignins, which are highly aromatic and therefore valuable as renewable feedstocks for the biobased product industry, is still a challenging and multidisciplinary task. An enzyme membrane reactor system (EMRS) featuring a continuous stirred-tank reactor and an external ceramic crossflow ultrafiltration membrane is a promising configuration, particularly when combined with ligninolytic heme peroxidases (PODs) as biocatalysts, such as the new versatile peroxidases (VP) described herein. However, time-dependent irreversible enzyme inactivation caused by the co-substrate H2O2 and the fouling of the filtration membrane are limiting factors. To facilitate rational bioprocess development and reactor design, we present an overall modeling concept for a continuous operating mode that addresses both limitations. When including H2O2-related VP inactivation dynamic model analyses showed that two H2O2 molecules were required to convert one molecule of the reducing substrate (here, adlerol). In this context, an (initial) enzyme inactivation rate ki0 caused by factors other than H2O2 (e.g. the used buffer system) was introduced. Because oxygen utilization is characteristic of normal POD actions, the continuous online measurement of dissolved oxygen concentration was useful to monitor enzyme inactivation and therefore an excess of H2O2.

A Novel Role of Vesicles as Templates for the Oxidation and Oligomerization of p‐Aminodiphenylamine by Cytochrome c

The oxidation and subsequent oligo‐ or polymerization of aniline or the N‐C‐para coupled aniline dimer p‐aminodiphenylamine (N‐phenyl‐1,4‐phenylenediamine) with H2O2‐dependent heme peroxidases in aqueous medium at pH = 4.3 is strongly influenced in a positive way by the presence of anionic polymers, micelles or vesicles as soft ‘templates’ (macromolecular or polymolecular additives), to yield products which resemble the emeraldine salt form of polyaniline (PANI‐ES). The positive effect the templates exert on the reaction mainly originates from interactions between the templates and the monomers, reaction intermediates and products, whereby the reaction occurs localized in the vicinity of the templates, suppressing undesired side reactions. As shown in the present work, the templates may even play an additional role, depending on the type of catalyst. Through interactions between the heme protein cytochrome c and anionic vesicles, cytochrome c gains increased peroxidase activity. In this way, the templates not only serve for hosting the oxidation and oligo‐ or polymerization reactions for obtaining PANI‐ES type products, but also simultaneously activate the catalyst in order to trigger the reaction.

Lignin-degrading peroxidases in white-rot fungus Trametes hirsuta 072. Absolute expression quantification of full multigene family.

Ligninolytic heme peroxidases comprise an extensive family of enzymes, which production is characteristic for white-rot Basidiomycota. The majority of fungal heme peroxidases are encoded by multigene families that differentially express closely related proteins. Currently, there were very few attempts to characterize the complete multigene family of heme peroxidases in a single fungus. Here we are focusing on identification and characterization of peroxidase genes, which are transcribed and secreted by basidiomycete Trametes hirsuta 072, an efficient lignin degrader. The T. hirsuta genome contains 18 ligninolytic peroxidase genes encoding 9 putative lignin peroxidases (LiP), 7 putative short manganese peroxidases (MnP) and 2 putative versatile peroxidases (VP). Using ddPCR method we have quantified the absolute expression of the 18 peroxidase genes under different culture conditions and on different growth stages of basidiomycete. It was shown that only two genes (one MnP and one VP) were prevalently expressed as well as secreted into cultural broth under all conditions investigated. However their transcriptome and protein profiles differed in time depending on the effector used. The expression of other peroxidase genes revealed a significant variability, so one can propose the specific roles of these enzymes in fungal development and lifestyle.

Anopheles stephensi Heme Peroxidase HPX15 Suppresses Midgut Immunity to Support Plasmodium Development.

The heme peroxidase HPX15 is an evolutionary conserved anopheline lineage-specific gene. Previously, we found that this gene is present in the genome of 19 worldwide distributed different species of Anopheles mosquito and its orthologs are absent in other mosquitoes, insects, or human. In addition, 65–99% amino acid identity among these 19 orthologs permitted us to hypothesize that the functional aspects of this gene might be also conserved in different anophelines. In this study, we found that Anopheles stephensi AsHPX15 gene is mainly expressed in the midgut and highly induced after uninfected or Plasmodium berghei-infected blood feeding. RNA interference-mediated silencing of midgut AsHPX15 gene drastically reduced the number of developing P. berghei oocysts. An antiplasmodial gene nitric oxide synthase was induced 13-fold in silenced midguts when compared to the unsilenced controls. Interestingly, the induction of antiplasmodial immunity in AsHPX15-silenced midguts is in absolute agreement with Anopheles gambiae. In A. gambiae, AgHPX15 catalyzes the formation of a dityrosine network at luminal side of the midgut that suppresses the activation of mosquito immunity against the bolus bacteria. Thus, a low-immunity zone created by this mechanism indirectly supports Plasmodium development inside the midgut lumen. These indistinguishable functional behaviors and conserved homology indicates that HPX15 might be a potent target to manipulate the antiplasmodial immunity of the anopheline midgut, and it will open new frontiers in the field of malaria control.

Oxidation Catalysis by Enzymes in Microemulsions

Microemulsions are regarded as “the ultimate enzyme microreactors” for liquid oxidations. Their structure, composed of water nanodroplets dispersed in a non-polar medium, provides several benefits for their use as media for enzymatic transformations. They have the ability to overcome the solubility limitations of hydrophobic substrates, enhance the enzymatic activity (superactivity phenomenon) and stability, while providing an interface for surface-active enzymes. Of particular interest is the use of such systems to study biotransformations catalyzed by oxidative enzymes. Nanodispersed biocatalytic media are perfect hosts for liquid oxidation reactions catalyzed by many enzymes such as heme peroxidases, phenoloxidases, cholesterol oxidase, and dehydrogenases. The system’s composition and structural properties are important for better understanding of nanodispersion-biocatalyst interactions.

Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP)

The Trypanosoma cruzi ascorbate peroxidase is, by sequence analysis, a hybrid type A member of class I heme peroxidases [TcAPx-cytochrome c peroxidase (CcP)], suggesting both ascorbate (Asc) and cytochrome c (Cc) peroxidase activity. Here, we show that the enzyme reacts fast with H2O2 (k = 2.9 × 107 M-1⋅s-1) and catalytically decomposes H2O2 using Cc as the reducing substrate with higher efficiency than Asc (kcat/Km = 2.1 × 105 versus 3.5 × 104 M-1⋅s-1, respectively). Visible-absorption spectra of purified recombinant TcAPx-CcP after H2O2 reaction denote the formation of a compound I-like product, characteristic of the generation of a tryptophanyl radical-cation (Trp233•+). Mutation of Trp233 to phenylalanine (W233F) completely abolishes the Cc-dependent peroxidase activity. In addition to Trp233•+, a Cys222-derived radical was identified by electron paramagnetic resonance spin trapping, immunospin trapping, and MS analysis after equimolar H2O2 addition, supporting an alternative electron transfer (ET) pathway from the heme. Molecular dynamics studies revealed that ET between Trp233 and Cys222 is possible and likely to participate in the catalytic cycle. Recognizing the ability of TcAPx-CcP to use alternative reducing substrates, we searched for its subcellular localization in the infective parasite stages (intracellular amastigotes and extracellular trypomastigotes). TcAPx-CcP was found closely associated with mitochondrial membranes and, most interestingly, with the outer leaflet of the plasma membrane, suggesting a role at the host-parasite interface. TcAPx-CcP overexpressers were significantly more infective to macrophages and cardiomyocytes, as well as in the mouse model of Chagas disease, supporting the involvement of TcAPx-CcP in pathogen virulence as part of the parasite antioxidant armamentarium.

Systemic Stress and Recovery Patterns of Rice Roots in Response to Graphene Oxide Nanosheets.

The interactions between nanomaterials and plants have attracted increasing attention. However, the systemic stress and recovery patterns of plants in response to nanomaterials and the connections between the molecular responses and the phenotypes remain unclear. Herein, rice was exposed to graphene oxide (GO) nanosheets at 0.01-1.0 mg/L for 7 days under hydroponic exposure, followed by a 7-day post exposure (GO-free). The significant upregulation (p < 0.05) of phenylalanine metabolism, secondary metabolism, and heme peroxidase reflected the stress and recovery patterns of rice roots exposed to GO. GO triggered 27% and more than 50% decreases in hydraulic conductivity and aquaporin gene expression (PIP1-3 and PIP2-2), respectively. The uptake of GO was mediated by aquaporin inhibition. Nanomaterial biotransformation reflected the potential for rice roots to adapt to GO stress. Oxidative stress, especially the downregulation of class III peroxidase mRNAs, were suppressed by GO. Lateral root inhibition, primary root growth, and cell wall synthesis, as forms of resistance to GO stress, were related to the significant (p < 0.05) downregulation of salicylic acid and lignin biosynthesis, as well as the upregulation of jasmonic acid and laccases. The present study helps elucidate the molecular and phenotypic responses of plants to nanomaterials, which are closely linked to their environmental risk assessment.

Application of HBOCs electrophoretic method to detect a new blood substitute derived from the giant extracellular haemoglobin of lugworm.

Manipulation of blood and blood components is prohibited in sports by the World Anti-Doping Agency (WADA). This includes the use of blood substitutes to increase oxygen transport, like haemoglobin-based oxygen carriers (HBOCs), which are compounds derived from haemoglobin. Despite their medical interest, the first generation of HBOCs had serious adverse effects and was abandoned. However, new studies are now exploiting the properties of marine worm haemoglobins, which circulate as giant extracellular complexes with high oxygen-binding capacities. HEMOXYCarrier® (HC), developed by Hemarina, is one of the most advanced and promising HBOCs, and HC may become a tempting doping tool for athletes in the future. Here, HC detection in plasma/serum was evaluated with the method used to detect the first HBOCs, based on electrophoresis and heme peroxidase properties. An HC-derived product was identified in human plasma up to 72 h after in vitro incubation at 37 °C. HC degradation also induced methemalbumin formation. After injecting HC at the effective dose of 200 mg/kg into mice, the HC-derived product was detected only for a few hours and no accumulation of methemalbumin was observed. Due to this limited detection window in vivo, measuring specific worm globin degradation products by mass spectrometry might be an alternative for future anti-doping analyses. Copyright © 2016 John Wiley & Sons, Ltd.

Molecular characterization and functional analysis of a salmon louse (Lepeophtheirus salmonis, Krøyer 1838) heme peroxidase with a potential role in extracellular matrixes

Heme peroxidases are the most abundant type of peroxidase catalyzing a H2O2-dependent oxidation of a wide variety of substrates. They are involved in numerous processes like the innate immune response, hormone and prostaglandin synthesis and crosslinking of proteins within extracellular matrixes (ECM) as well as molecules within the cuticle and chorion of arthropods and nematodes. In the present study, a Lepeophtheirus salmonis heme peroxidase (LsHPX) 1 was characterized. Amino acids in the active site of heme peroxidases were conserved, and the predicted protein sequence showed the highest similarity to genes annotated as chorion peroxidases and genes suggested to be involved in cuticle hardening or adhesion. LsHPX1 exhibited a dynamic expression during ontogenesis and during the nauplius molting cycle. Transcripts were localized to muscle cells near the muscle-tendon junction, in nerve tissue especially at neuromuscular junctions, subcuticular epithelium, subepithelial cells facing the hemolymph, exocrine glands within the subepithelial tissue and in isolated cells within the testis. Knock-down of LsHPX1 in nauplius larvae decreased the swimming activity of emerging copepodids. Histological analysis of knock-down animals revealed increased spacing between myofibers and changes in subepithelial and exocrine gland tissue. Considering these results, the potential role of LsHPX1 in crosslinking molecules of salmon louse ECMs is discussed.