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PubMedDate not supplied

4-Phenylylboronic acid: a new type of enhancer for the horseradish peroxidase catalysed chemiluminescent oxidation of luminol.

4-Phenylylboronic acid enhances the light emission from the horseradish peroxidase catalysed oxidation of luminol by hydrogen peroxide. Optimization studies showed that the greatest enhancement was obtained using micromolar concentrations of the new enhancer. The largest degree of enhancement was found with the basic isoenzyme of horseradish peroxidase (Type VIA), and lesser degrees of enhancement were obtained with Type VII and Type IX horseradish peroxidase. The enhancer was also effective in the peroxidase catalysed oxidation of isoluminol by peroxide.

Boronic Acids↗
PubMed2003

Microfluidic peroxidase biochip for polyphenol synthesis.

An enzyme-containing microfluidic biochip has been developed for the oxidative polymerization of phenols. The biochip consists of a simple T-junction with two feed reservoirs 20 mm apart and a microreaction channel 30 mm long. The channel is 15 microm deep and 200 microm wide at the center, giving a reaction volume of 90 nL. The biochip was fabricated using conventional photolithographic methods on a glass substrate etched using a HF-based solution. Fluid transport was enabled using electroosmotic flow. Soybean peroxidase was used as the phenol oxidizing catalyst, and in the presence of p-cresol and H(2)O(2), essentially complete conversion of the H(2)O(2) (the limiting substrate) occurred in the microchannel at a flow rate of ca. 290 nL/min. Thus, peroxidase was found to be intrinsically active even upon dramatic scale-down as achieved in microfluidic reactors. These results were extended to a series of phenols, thereby demonstrating that the microfluidic peroxidase reactor may have application in high-throughput screening of phenolic polymerization reactions for use in phenolic resin synthesis. Finally, rapid growth of poly(p-cresol) on the walls of the microreaction channel could be performed in the presence of higher H(2)O(2) concentrations. This finding suggests that solution-phase peroxidase catalysis can be used in the controlled deposition of polymers on the walls of microreactors.

Bioreactors↗
PubMed2005

The effects of ligand exchange and mobility on the peroxidase activity of a bacterial cytochrome c upon unfolding.

The effect on the heme environment upon unfolding Paracoccus versutus ferricytochrome c-550 and two site-directed variants, K99E and H118Q, has been assessed through a combination of peroxidase activity increase and one-dimensional NMR spectroscopy. At pH 4.5, the data are consistent with a low- to high-spin heme transition, with the K99E mutation resulting in a protein with increased peroxidase activity in the absence of or at low concentrations of denaturant. Furthermore, the mobility of the polypeptide chain at pH 4.5 for the wild-type protein has been monitored in the absence and presence of denaturant through heteronuclear NMR experiments. The results are discussed in terms of local stability differences between bacterial and mitochondrial cytochromes c that are inferred from peroxidase activity assays. At pH 7.0, a mixture of misligated heme states arising from protein-based ligands assigned to lysine and histidine is detected. At low denaturant concentrations, these partially unfolded misligated heme forms inhibit the peroxidase activity. Data from the K99E mutation at pH 7.0 indicate that K99 is not involved in heme misligation, whereas histidine coordination is proven by the data from the H118Q variant.

Amino Acid Substitution↗
PubMed1975

Localization of masticatory motoneurons in the cat and rat by means of retrograde axonal transport of horseradish peroxidase.

Topographical localization of monotoneurons supplying the masticatory muscles was investigated in the cat and rat, utilizing retrograde axonal transport of horseradish peroxidase. Following injection of horseradish peroxidase in each masticatory muscle, motoneurons labelled with peroxidase were seen to be aggregated into a cluster within the motor nucleus of the trigeminal nerve. Such clusters of peroxidase-motoneurons innervating each masticatory muscle were demarcated more sharply in kittens than in adult animals. The pattern of the nuclear representation of the masticatory muscles was found to be essentially the same in the cat and rat; it could be summarized as follows: The motor nucleus of the trigeminal nerve could be divided cytoarchitectonically into the dorsolateral and ventromedial divisions; the former was seen in almost the whole rostrocaudal extent of the nucleus, while the latter was localized at the levels of caudal two thirds of the nucleus. In the dorsolateral division, the temporal muscle was represented dorsally and dorsomedially, the masseter muscle ventrolaterally, and the pterygoid muscles ventromedially. In the ventromedial division, the anterior digastric muscle was represented dorsomedially, and the mylohyoid muscle ventrolaterally. It was also confirmed that the motoneurons supplying the posterior digastric muscle were localized in the accessory facial nucleus.

Animals↗
PubMed1980

Gracilo-Diencephalic relay cells: a quantitative study in the cat using retrograde transport of horseradish peroxidase.

In a quantitative study in the cat, gracilo-diencephalic relay cells were labeled by the use of retrograde axonal transport of horseradish peroxidase injected into the ventroposterolateral nucleus of the thalamus. An initial series comprising 22 animals with survival periods varying between 2 h and 4 days showed maximal labeling in the gracile nucleus after 24-48 h. The earliest appearing peroxidase-positive neurons were found after only 6 h, implying a transport rate in the medial lemniscus of at least 100 mm/day. In a second series of five cats, which were killed 24 h after injection, serial sections from the gracile nucleus were embedded in Epon following peroxidase processing, and cut at 2 micrometer. By stratified random sampling, about 2,000 cells were selected for light microscopic examination. The total number of nerve cells in a single gracile nucleus was calculated to be about 50,000, out of which about 15,000 were retrogradely labeled. In agreement with previous reports, rostrocaudal differences were observed. Thus, less than a third of the neurons in the rostral part of the nucleus were peroxidase-positive, whereas about half of those in the middle and caudal regions were retrogradely labeled. The total number of labeled neurons was, however, about the same in the rostral as in the middle-caudal part of the nucleus. The size of the nerve cell bodies, measured as the cross-sectional area in the nucleolar plane, differed significantly between labeled and unlabeled neurons. The estimated average diameter of the former was 22 micrometer and of the latter 17 micrometer. There was a considerable overlap between the two groups, however, and labeled cells as small s 12 micrometer in diameter were found. Mainly because of the uneven distribution of labeled neurons, cells in the rostral region were, on the average, significantly smaller than those in the middle and caudal regions; these mean diameters were calculated to be 18, 23, and 21 micrometer, respectively. The results of this study support the idea of a heterogeneous organization of the gracile nucleus. However, a much larger proportion of the gracilo-diencephalic relay cells is situated in the rostral part of the gracile nucleus than has previously been thought. The concept that only medium-sized neurons project to the diencephalon also seems to need revision. It is concluded that although gracilo-diencephalic relay cells are, on the average, larger than the nonlabeled neurons, single nerve cells cannot be identified on the basis of size alone. The function of the unlabeled neurons is discussed. Although many of these might relay to various extra-diencephalic sites, it is suggested that a large number are internuncial neurons.

Afferent Pathways↗
PubMed1992

Using coomassie blue to stabilize H2O2-guaiacol stained peroxidases on polyacrylamide gels.

Shimoni and Reuveni [1] have reported a procedure which uses Coomassie Brilliant Blue R-250 staining and destaining to stabilize H2O2-guaiacol stained peroxidases on polyacrylamide gels and at the same time detects nonspecific protein bands. The procedure has the advantage that the otherwise orange and unstable peroxidase bands are visualized as blue bands and it, therefore, facilitates laser densitometry of electrophoretically separated peroxidases. In the modification of the procedure reported here, the Coomassie staining reagent was adjusted and the destaining step was eliminated. The modified procedure has added advantages: it simplifies the procedure, increases the consistency of the results across gels, prevents or reduces the staining of nonspecific proteins, and still gives excellent resolution of stable bands which can be used to quantify peroxidase activity.

Electrophoresis, Polyacrylamide Gel↗
PubMed2007

Mammalian peroxidases activate anticancer drug ellipticine to intermediates forming deoxyguanosine adducts in DNA identical to those found in vivo and generated from 12-hydroxyellipticine and 13-hydroxyellipticine.

Ellipticine is a potent antineoplastic agent, whose mode of action is considered to be based mainly on DNA intercalation, inhibition of topoisomerase II and cytochrome P450-mediated formation of covalent DNA adducts. This is the first report on the molecular mechanism of ellipticine oxidation by peroxidases (human myeloperoxidase, human and ovine cyclooxygenases, bovine lactoperoxidase, horseradish peroxidase) to species forming ellipticine-DNA adducts. Using NMR spectroscopy, the structures of 2 ellipticine metabolites were identified; the major product is the ellipticine dimer, in which the 2 ellipticine skeletons are connected via N(6) of the pyrrole ring of one ellipticine molecule and C9 in the second one. The minor metabolite is ellipticine N(2)-oxide. Using (32)P-postlabeling and [(3)H]-labeled ellipticine, we showed that ellipticine binds covalently to DNA after its activation by peroxidases. The DNA adduct pattern induced by ellipticine consisted of a cluster of up to 4 adducts. The 2 adducts are indistinguishable from the 2 major adducts generated between deoxyguanosine in DNA and either 13-hydroxy- or 12-hydroxyellipticine or in rats treated with ellipticine, or if ellipticine was activated with human hepatic and renal microsomes. The results presented here are the first characterization of the peroxidase-mediated oxidative metabolites of ellipticine and we have proposed species, 2 carbenium ions, ellipticine-13-ylium and ellipticine-12-ylium, as reactive species generating 2 major DNA adducts seen in vivo in rats treated with ellipticine. The study forms the basis to further predict the susceptibility of human cancers to ellipticine.

Animals↗
PubMed1989

The formation of 2-aminofluorene-DNA adducts in vivo: evidence for peroxidase-mediated activation.

Formation of DNA adducts in various tissues of dogs fed a single dose of the carcinogen 2-aminofluorene was investigated. Adduct analysis was performed using a technique that allows measurement of both N-(deoxyguanosin-8-yl)-2-amino-2-aminofluorene-DNA adduct formed by reaction of N-hydroxy-2-aminofluorene with DNA, as well as the polar 2-aminofluorene-DNA adducts formed when 2-aminofluorene is activated by prostaglandin H synthase-peroxidase in vitro. Two male beagle (A and B) dogs were examined and a different DNA adduct profile was observed with each dog. For the dog A, N-(deoxyguanosin-8-yl)-2-aminofluorene was the major adduct found in hepatic DNA; no peroxidase-derived adducts were detected in this tissue. In contrast, adducts eluting similarly to peroxidase-derived adducts were found in urinary tract tissues of this dog with the relative abundance of these adducts in the order urothelium greater than renal medulla greater than renal cortex, which correlates with the respective tissues' prostaglandin H synthase activity. N-(Deoxyguanosin-8-yl)-2-aminofluorene was detected in the renal tissues, but not in urothelium. For dog B, only the N-(deoxyguanosin-8-yl)-2-aminofluorene adduct was observed in all tissues examined, including the urothelium. However, total binding to liver, kidney, and bladder were two-, two-, and four-fold lower, respectively, than dog A. These data indicate that both prostaglandin H synthase-mediated activation and N-hydroxylation of 2-aminofluorene occur in vivo and may be subjected to pharmacodynamic considerations. Furthermore, the tissue distribution of the peroxidase-mediated 2-aminofluorene adducts suggests this process may also be of importance in the bladder-specific carcinogenicity of aromatic amines.

Animals↗
PubMedDate not supplied

Substrate specificity of rat sera IgG antibodies with peroxidase and oxidoreductase activities.

We have recently shown that intact IgGs from the sera of healthy Wistar rats oxidize 3,3'-diaminobenzidine (DAB) in the presence and in the absence of H(2)O(2) similar to horseradish peroxidase (HRP). Here we demonstrate for the first time that the peroxidase and oxidoreductase activities of IgGs can efficiently oxidize not only DAB but also o-phenylendiamine, phenol, p-dihydroquinone, alpha-naphthol, and NADH but, in contrast to HRP, cannot oxidize adrenalin. In contrast to IgGs, HRP cannot oxidize phenol, p-dihydroquinone, or alpha-naphthol in the absence of H(2)O(2). In contrast to plant and mammalian peroxidases, IgGs were more universal in their metal dependence. The specific wide repertoire of polyclonal peroxidase and oxidoreductase IgGs oxidizing various substances could play an important role in protecting the organism from oxidative stress and serve as an additional natural system destroying different toxic, carcinogenic, and mutagenic compounds.

Animals↗
PubMed1993

Glutathione peroxidase isolated from plasma reduces phospholipid hydroperoxides.

The reactivities of a selenoenzyme, glutathione peroxidase isolated from plasma, against phosphatidylcholine hydroperoxides and photoperoxidized erythrocyte ghosts have been investigated. Glutathione peroxidase isolated from plasma was found to catalyze the reduction of phosphatidylcholine hydroperoxides to the corresponding hydroxy derivatives. The enzyme is also capable of reducing the hydroperoxides in photoperoxidized ghosts. Thin layer chromatographic analysis of enzyme-treated ghosts revealed that plasma glutathione peroxidase can reduce phospholipid-derived hydroperoxides but cannot reduce cholesterol hydroperoxides. These studies demonstrate that the three known glutathione peroxidase (cellular, plasma, and phospholipid hydroperoxide) differ from each other in substrate specificity.

Erythrocyte Membrane↗
PubMed1996

The effects of calcium on the thermal stability and activity of manganese peroxidase.

The presence of micromolar Ca2+ efficiently prevented the thermal inactivation of manganese peroxidase from Phanerochaete chrysosporium. The amount of Ca2+ normally present in the enzyme decreased when the enzyme was thermally inactivated and EGTA increased the rate of inactivation. The inactivation kinetics were biphasic, suggesting a sequential two-step process. The rate of inactivation during the second, slower step corresponded to the rate of loss of heme from the enzyme. Thermally inactivated manganese peroxidase could be readily reactivated in the presence of excess Ca2+. However, as the time of thermal incubation increased and the amount of remaining heme decreased, the amount of enzyme activity recovered decreased. Therefore, while both steps of denaturation could be prevented by Ca2+, only one step could be reversed upon the addition of Ca2+. It is proposed that the first step of denaturation involves the loss of Ca2+ which causes conformational changes resulting in the loss of manganese peroxidase activity. The second step is believed to involve further structural loss and results in the loss of heme from the enzyme. It is concluded that manganese peroxidase is susceptible to thermal inactivation because it contains relatively labile Ca2+ ions required for stability and activity.

Basidiomycota↗
PubMed1997

Effect of calcium on the reversible thermal inactivation of lignin peroxidase.

This study investigated the effects of calcium on the thermal inactivation of lignin peroxidase from Phanerochaete chrysosporium. The monophasic loss of veratryl alcohol oxidase activity corresponded to the loss of calcium when the enzyme was thermally inactivated. Addition of calcium slowed and oxalate and EGTA increased the apparent inactivation rate. The thermally inactivated lignin peroxidase could be readily reactivated by addition of Ca2+. The amount of activity recovered was dependent on temperature, Ca2+ concentration, and incubation conditions. Enzyme activity could be recovered up to 95% of its original value by addition of Ca2+ when lignin peroxidase was depleted of Ca2+ by incubation with EGTA. Although heme absorbance decreased when the enzyme was thermally inactivated, the amount of iron in the enzyme did not change. Changes in the heme environment of the inactivated enzyme were suggested by changes in the electronic absorption in which the Soret band shifted from 408 to 410 nm, the absorption at 502 nm shifted to 532 nm, and the absorption at 634 nm disappeared upon inactivation. Upon the addition of Ca2+, the bands returned to the original wavelength. Therefore, it is proposed that the inactivation mechanism of lignin peroxidase is that the loss of calcium causes heme environmental changes resulting in the loss of enzyme activity.

Basidiomycota↗
PubMed2000

Function of glutathione peroxidase in endothelial cell vitality.

The two human umbilical vein endothelial cell-derived lines, ECRF24 and ECV304, differ in responsiveness to oxidative stress. In confluent monolayers of ECRF24, but not in ECV304, peroxides induce stress responses such as plasma membrane blebbing and nuclear condensation. The peroxide effect on ECRF24 was preceded by oxidation of reduced glutathione (GSH) and of NAD(P)H, and by oxidation of the redox-sensitive probe, chloromethyl 2',7'-dichlorofluorescin (DCFH). In monolayers of ECV304, peroxides induced only minimal oxidation of GSH, NAD(P)H and DCFH, which was associated with a greatly reduced GSH peroxidase activity in these cells. However, in spite of the absence of a blebbing response, ECV304 were more susceptible than ECRF24 to membrane lipid peroxidation and peroxide-induced necrosis. Only for ECV304, the culturing with high levels of polyunsaturated fatty acids increased lipid peroxidation and cellular death. Treatment of these cells with the GSH peroxidase mimic ebselen effectively reversed their decreased vitality. We conclude that, in peroxide-treated endothelial cells, cell death (necrosis) can result from lipid peroxidation by peroxide that has not been removed by GSH peroxidases, whereas extensive peroxidase activity may cause a stress response (blebbing). The data further identify ECV304 as a stress-sensitive cell line, where peroxides exert their effects independently of GSH oxidation.

Cell Line↗
PubMed1994

Stabilization of purified human 5-lipoxygenase with glutathione peroxidase and superoxide dismutase.

Human 5-lipoxygenase (5LO) becomes very unstable after purification. Commonly used methods for protein stabilization could not prevent this inactivation. However, addition of small amounts of glutathione peroxidase (0.15 micrograms/ml) and superoxide dismutase (1 microgram/ml) to the solution of purified 5LO (300-500 micrograms/ml) stabilized the enzyme during storage. The protected 5LO maintained full activity for at least 12 days at 25 degrees C, while 50% of the activity was lost within 10 h without protection. Glutathione peroxidase alone also preserved the activity of 5-lipoxygenase; however, the effect declined rapidly in the absence of superoxide dismutase. 2-Mercaptoethanol was the most efficient hydrogen donor substrate for glutathione peroxidase in the protection of 5LO. Catalase was less effective as a stabilizing agent, and ebselen, a synthetic glutathione peroxidase-mimicking compound, did not protect 5LO. Since many metal ion binding proteins are susceptible to H2O2 inactivation, this method could be useful also for the stabilization of other proteins.

Arachidonate 5-Lipoxygenase↗
PubMed2000

Fluorometric determination of mucin-type glycoproteins by the galactose oxidase-peroxidase method.

We developed a convenient and specific method for the determination of mucin-type glycoproteins using galactose oxidase and horseradish peroxidase on the basis of the contents of galactosyl and N-acetylgalactosaminyl residues in glycoproteins. Galactose and galactosamine residues released from glycoproteins after hydrolysis were oxidized with galactose oxidase and subsequently the resultant hydrogen peroxide was determined by a combination of horseradish peroxidase and 3-(p-hydroxyphenyl) propionic acid as a fluorogenic substrate. The contents of galactose/galactosamine residues in N- and O-glycans, as determined by the galactose oxidase-peroxidase method, were in good agreement with those described in the previous reports. We applied the present method to determine mucin-type glycoproteins secreted from rat gastric mucosa by stimulation with misoprostol, a prostaglandin E(1) analogue in vivo. Thus, the galactose oxidase-peroxidase method is useful for the determination of mucin-type glycoproteins in biological materials.

Animals↗
PubMed1993

Purification and partial amino acid sequence of a wound-inducible, developmentally regulated anionic peroxidase from soybean leaves.

We have isolated a peroxidase activity to electrophoretic homogeneity from the leaves of the soybean plant, Glycine max. The highly anionic peroxidase isozyme has an isoelectric point of 3.7 and a molecular mass of 32 kilodaltons. Partial amino acid sequence information confirms the identity of the enzyme as a peroxidase but shows significant deviation from other plant peroxidases in the distal histidine box. The enzyme is developmentally regulated as it begins to accumulate in 8-10 day old leaves and its level remains fairly constant thereafter. The expression of this isozyme is also responsive to environmental cues in that it accumulates in young (less than 8 day old) leaves as a consequence of mechanical wounding. The appearance of the isozyme begins approximately 8 hr post-wounding and continues to accumulate for 48 hr.

Amino Acid Sequence↗
PubMed1993

Identification of the heat shock protein of Neurospora crassa corresponding to the stress-inducible peroxidase.

Heat shock and other stress treatments, resulting in thermotolerance in Neurospora crassa cells, stimulate the induction of a peroxidase at a high level. The putative gene encoding this heat shock-induced peroxidase (hspp) has been cloned, using a cDNA clone of the manganese peroxidase of Phanerochaete chrysosporium, as a probe. Northern blot analysis of total RNA from heat-shocked cell showed the stress-dependent accumulation of a approximately 10 kb transcript. The identity of the HSP, corresponding to the heat-induced peroxidase, was established using hybrid-arrested in vitro translation.

Blotting, Northern↗
PubMed1995

Cloning of a new FRTL5-derived cell line stably expressing active human thyroid peroxidase.

A full-length cDNA clone for human thyroid peroxidase inserted into the mammalian cell expression vector pECE was stably transfected into the rat thyroid cell line FRTL5. Clones expressed immunologically and enzymatically assessed human thyroid peroxidase protein. Methimazole (25 microM) inhibited the thyroid peroxidase activity dose-dependently and this effect was completely antagonised by 100 microM NaI. Ethylenethiourea, metabolite of dithiocarbamate pesticides, inhibited the enzyme at 50 microM. Thus, we have obtained thyroidal cells stably expressing enzymatically active human thyroid peroxidase which can be pharmacologically modulated and studied.

Animals↗