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At least 199 records · Page 11Linked to original sources
PubMed1991

Phospholipid hydroperoxide glutathione peroxidase is a selenoenzyme distinct from the classical glutathione peroxidase as evident from cDNA and amino acid sequencing.

The primary structure of phospholipid hydroperoxide glutathione peroxidase (PHGPx) was partially elucidated by sequencing peptides obtained by cyanogen bromide cleavage and tryptic digestion and by isolating and sequencing corresponding cDNA fragments covering about 75% of the total sequence. Based on these data PHGPx can be rated as a selenoprotein homologous, but poorly related to classical glutathione peroxidase (GPx). Peptide loops constituting the active site in GPx are, however, strongly conserved in PHGPx. This suggests that the mechanism of action involving an oxidation/reduction cycle of a selenocysteine residue is essentially identical in PHGPx and GPx.

Amino Acid Sequence↗
PubMed1986

The mechanism of peroxidase-mediated cytotoxicity. I. Comparison of horseradish peroxidase and lactoperoxidase.

The kinetics of the cytolytic activity expressed by lactoperoxidase and horseradish peroxidase toward erythrocytes in the presence of H2O2 and iodide have been investigated at physiological pH. The action of both enzymes was found to be very similar with respect to their kinetic mechanisms. Both enzymes showed saturation kinetics at higher enzyme concentrations under conditions where substrate concentrations were not limiting. Optimal concentrations of H2O2 and iodide were found to be 40 and 25 microM, respectively, for both enzymes. Higher concentrations of H2O2 inhibited the cytolytic activity. The pH dependence of the cytolytic reaction is also very similar for both enzymes, showing maximal activity at about pH 6.3. Moreover, the cytolytic activities of both enzymes were inhibited by tyrosine, tryptophan, cysteine, and to a lesser extent by histidine. We conclude from these data that the mechanisms of horseradish peroxidase and lactoperoxidase in promoting the lysis of erythrocytes are closely related if not identical.

Animals↗
PubMed1988

The coordination and spin states of yeast cytochrome c peroxidase and their implication to peroxidase mechanism.

Cytochrome c peroxidase, freshly prepared, contains a penta-coordinated heme iron and is fully reactive with hydroperoxides. On the other hand, the enzyme normally stored in frozen states invariably contains different amounts of an altered, aged species whose heme iron is hexa-coordinated. The aged enzyme reacts with hydroperoxides only after a slow conformation change leading to the formation of a reactive penta-coordinated state. Thus, the reactivity of cytochrome c peroxidase with hydroperoxides is strongly controlled by the coordination state of the heme iron. A penta-coordinated heme iron may be a prerequisite for rapid reactions of hydroperoxidases with hydroperoxides.

Binding Sites↗
PubMed1984

Binding of benzo(a)pyrene with peroxidase and its oxidation by peroxidase-H2O2 intermediate.

The oxidation of benzo(a)pyrene (BP) by horseradish peroxidase (HRP) (EC 1.11.1.7) was examined spectrophotometrically by the decomposition of peroxidase-H2O2 intermediate "compound II." The rate constant of the oxidation of BP was 9.5 X 10(4) M-1 sec-1. The oxidation of BP by HRP was inhibited at high BP concentrations, and the hydrogen donor (BP) inhibition constant, KA', was 1.48 microM. The association constant, Kassoc, of the formation of a complex of BP and HRP at 403 nm was 4.37 X 10(4) M-1. The oxidation products of BP have been identified as 1,6-, 3,6- and 6,12-quinone BP. These products showed no mutagenicity in the mutagenicity assay.

Benzo(a)pyrene↗
PubMed1997

Potential cancerostatic benfluron is metabolized by peroxidase: in vitro biotransformation of benfluron by horseradish peroxidase.

Horseradish peroxidase (HRP) was used to investigate whether benfluron (a potential cytostatic drug) can be biotransformed extra-hepatically by systems other than flavin-containing monooxygenase and cytochromes P450. Three types of incubation mixtures differing in buffers (Na-phosphate buffer 50 mmol/l, pH 6.8 and 8.4 and Tris-HCl buffer 25 mmol/l, pH 7.5) were tested. The amount of N-demethylated benfluron (demB) formed was significantly higher (up to 4 times in the Na-phosphate buffer, pH 8.4, and 5 times in the Na-phosphate buffer, pH 6.8, and in the Tris-HCl buffer, pH 7.5) compared to control experiments. The highest yields of demB were obtained with the moderately alkaline Na-phosphate buffer (50 mmol/l, pH 8.4). The concentration of demB increased during thirty minutes of incubation, and then remained constant through the end of two-hour incubation. The results support the hypothesis that benfluron can be metabolized extra-hepatically by N-demethylation reaction catalyzed by peroxidases.

Animals↗
PubMed2000

Substrate specificity of lignin peroxidase and a S168W variant of manganese peroxidase.

Lignin peroxidase (LiP) and manganese peroxidase (MnP) are structurally similar heme-containing enzymes secreted by white-rot fungi. Unlike MnP, which is only specific for Mn(2+), LiP has broad substrate specificity, but it is not known if this versatility is due to multiple substrate-binding sites. We report here that a S168W variant of MnP from Phanerochaete chrysosporium not only retained full Mn(2+) oxidase activity, but also, unlike native or recombinant MnP, oxidized a multitude of LiP substrates, including small molecule and polymeric substrates. The kinetics of oxidation of most nonpolymeric substrates by the MnP variant and LiP were similar. The stoichiometries for veratryl alcohol oxidation by these two enzymes were identical. Some readily oxidizable substrates, such as guaiacol and ferrocyanide, were oxidized by MnP S168W and LiP both specifically and nonspecifically while recombinant MnP oxidized these substrates only nonspecifically. The functional similarities between this MnP variant and LiP provide evidence for the broad substrate specificity of a single oxidation site near the surface tryptophan.

Amino Acid Sequence↗
PubMed2002

Ca2+ and the bacterial peroxidases: the cytochrome c peroxidase from Pseudomonas stutzeri.

The production of cytochrome c peroxidase (CCP) from Pseudomonas ( Ps.) stutzeri (ATCC 11607) was optimized by adjusting the composition of the growth medium and aeration of the culture. The protein was isolated and characterized biochemically and spectroscopically in the oxidized and mixed valence forms. The activity of Ps. stutzeri CCP was studied using two different ferrocytochromes as electron donors: Ps. stutzeri cytochrome c(551) (the physiological electron donor) and horse heart cytochrome c. These electron donors interact differently with Ps. stutzeri CCP, exhibiting different ionic strength dependence. The CCP from Paracoccus ( Pa.) denitrificans was proposed to have two different Ca(2+) binding sites: one usually occupied (site I) and the other either empty or partially occupied in the oxidized enzyme (site II). The Ps. stutzeri enzyme was purified in a form with tightly bound Ca(2+). The affinity for Ca(2+) in the mixed valence enzyme is so high that Ca(2+) returns to it from the EGTA which was added to empty the site in the oxidized enzyme. Molecular mass determination by ultracentrifugation and behavior on gel filtration chromatography have revealed that this CCP is isolated as an active dimer, in contrast to the Pa. denitrificans CCP which requires added Ca(2+) for formation of the dimer and also for activation of the enzyme. This is consistent with the proposal that Ca(2+) in the bacterial peroxidases influences the monomer/dimer equilibrium and the transition to the active form of the enzyme. Additional Ca(2+)does affect both the kinetics of oxidation of horse heart cytochrome c (but not cytochrome c(551)) and higher aggregation states of the enzyme. This suggests the presence of a superficial Ca(2+)binding site of low affinity.

Animals↗
PubMed1991

Partition of free and monoclonal-antibody-bound horseradish peroxidase in a two-phase aqueous polymer system--novel procedure for the determination of the apparent binding constant of monoclonal antibody to horseradish peroxidase.

The principle that the antigen and the antibody prefer different phases in an aqueous two-phase system is the analytical basis of the work presented here. The antigen horseradish peroxidase, which is bound to a monoclonal antibody (mAb), is separated from free Ag in an aqueous phase system (polyethylene glycol (PEG)/dextran) as a function of the concentration of mAb. The plot of the partition coefficient kappa of horseradish peroxidase versus the concentration of mAb yields a sigmoidal curve similar to the curve obtained by enzyme-linked immunosorbent assay (ELISA). Comparing the plots normally used for ELISA in order to determine the apparent binding constant of mAb and the number of epitopes on the Ag we derived a relationship between the difference in partitioning of the free Ag and the bound Ag (delta kappa) and the concentration of mAb. The new linear plot of reciprocal delta kappa versus reciprocal concentration of mAb gives the apparent binding constant of mAb, which is evaluated from the slope. From the intercept at the ordinate the maximum difference of the partition coefficient of the free and bound antigen is derived and the apparent partition coefficient of the free monoclonal antibody can be calculated.

Animals↗
PubMed1992

Thyroid peroxidase glycosylation: the location and nature of the N-linked oligosaccharide units in porcine thyroid peroxidase.

Highly purified, trypsin/detergent-solubilized thyroid peroxidase (TPO), prepared from pig thyroid tissue, was subjected to reduction and alkylation followed by trypsin digestion. The resulting peptides were fractionated using HPLC. Corresponding carbohydrate positive regions from three separate HPLC experiments were pooled and further chromatography was carried out to yield purified peptide suitable for sequence analysis and complete carbohydrate composition analysis. Four of the five putative sites for N-linked glycosylation were found to carry oligosaccharide units in which mannose and glucosamine were the sole or predominant sugars. Three of the four glycosylations occur at asparagine residues which are likely to be at beta turns or bends. The fifth putative glycosylation site could not be confirmed and may either be poorly glycosylated or escape glycosylation. All of the confirmed glycosylated sites occur in the N-terminal third of the TPO polypeptide chain, in the portion of the molecule believed to be extracellular. The isolation of at least two chromatographic forms of glycopeptide derived from each of the confirmed sites suggests microheterogeneity in the structure of the oligosaccharide units of thyroid peroxidase similar to that observed in many other glycoproteins.

Amino Acid Sequence↗
PubMed1984

Serotonin-containing projections to the thalamus in the rat revealed by a horseradish peroxidase and peroxidase antiperoxidase double-staining technique.

The retrograde transport of horseradish peroxidase (HRP) has been used in combination with peroxidase antiperoxidase (PAP) immunocytochemistry in order to investigate serotonin-containing projections to the thalamus of the rat. Sections were histochemically stained to reveal retrogradely transported HRP and then PAP immunostained using a monoclonal anti-serotonin (5-HT) antibody. Following HRP injections into the ventral thalamus, retrogradely labelled cells were observed in a number of sites in the brainstem and including areas known to be rich in 5-HT-containing neurons. At rostral levels of the dorsal raphe nucleus, retrogradely labelled cells were observed both on the midline and in a distinct lateral group extending diffusely into the periaqueductal gray (PAG). In both of these areas many 5-HT-immunoreactive HRP retrogradely labelled neurons were observed. However, except for the most rostral levels of the dorsal raphe nucleus, such double-labelled cells represented only a small proportion of the total population of 5-HT-immunoreactive neurons. In the lateral group, the retrograde labelling was mainly unilateral to the injection site but some contralateral labelling was also seen. At caudal levels of the dorsal raphe nucleus, retrogradely labelled cells were observed predominantly in the lateral group. At the level of the dorsolateral tegmental nucleus, few 5-HT or 5-HT/HRP labelled cells were observed in the lateral group, although HRP retrogradely labelled neurons were present. Double-stained cells were detected also in the medial raphe nucleus (corresponding to the B8 cell group according to the nomenclature of Dahlström and Fuxe), among the fibres of the medial lemniscus (B9), and in nucleus raphe pontis (B5).

Animals↗
PubMed1992

Evidence for a new extracellular peroxidase. Manganese-inhibited peroxidase from the white-rot fungus Bjerkandera sp. BOS 55.

A novel enzyme activity was detected in the extracellular fluid of Bjerkandera sp. BOS 55. The purified enzyme could oxidize several compounds, such as Phenol red, 2,6-dimethoxyphenol (DMP), Poly R-478, ABTS and guaiacol, with H2O2 as an electron acceptor. In contrast, veratryl alcohol was not a substrate. This enzyme also had the capacity to oxidize DMP in the absence of H2O2. With some substrates, a strong inhibition of the peroxidative activity by Mn2+ was observed. Phenol red oxidation was inhibited by 84% with only 1 mM of this metal ion. Because DMP oxidation by this enzyme is only slightly inhibited by Mn2+, this substrate should not be used in assays to detect manganese peroxidase. The enzyme is tentatively named 'Manganese-Inhibited Peroxidase'.

Chromatography, Gel↗
PubMed1994

Participation of Mn(II) in the catalysis of laccase, manganese peroxidase and lignin peroxidase from Phelbia radiata.

Oxidation capacities of laccase, manganese peroxidase (MnP) and lignin peroxidase (LiP) from Phlebia radiata were compared using non-phenolic (veratryl alcohol and ABTS) and phenolic (syringaldazine, vanillalacetone and Phenol red) compounds as reducing substrates. The effect of Mn(II) on enzyme reactions was also studied. Highest specific activities were recorded with laccase in the oxidation of phenolic compounds or ABTS and irrespective of Mn(II) concentration. LiP and MnP oxidized all these substrates but only the catalysis of MnP was dependent upon Mn(II). Only LiP clearly oxidized veratryl alcohol. However, Mn(II) interfered with this reaction by repressing veratraldehyde formation. These results point to multiple participation of manganese ions, either as a reducing (Mn(II)) or oxidizing (Mn(III)) agent in the enzymatic reactions.

Benzothiazoles↗
PubMed2005

Nano-assembly of manganese peroxidase and lignin peroxidase from P. chrysosporium for biocatalysis in aqueous and non-aqueous media.

Development, characterization, and activity studies of nano-assemblies of lignin peroxidase (LiP), and manganese peroxidase (MnP) from Phanerochaete chrysosporium on flat surfaces as well as colloidal particles have been investigated. These assemblies of LiP and MnP were fabricated with polyelectrolytes-poly(ethylenimine) (PEI), poly(dimethyldiallylammonium chloride) (PDDA), and poly(allylamine) (PAH)-using a layer-by-layer self-assembly technique (LbL). Characterization of these assemblies on flat surfaces was monitored using quartz crystal microbalance (QCM), while assemblies on microparticles such as melamine formaldehyde (MF) were carried out with zeta potential analyzer (ZPA). A unique dynamic adsorption-desorption of the enzyme layers is observed during the assembly. All the nano-assemblies of LiP and MnP can effectively oxidize veratryl alcohol (VA) to its aldehyde for an extended period of time. The effect of different polyions and the number of polyion layers on the activities of LiP and MnP nano-assembly was also examined. It is observed that drying of enzyme layer during the assembly and the use of non-aqueous media, such as acetone can significantly reduce the activity of the enzymes. Enzyme activity reaches a minimum when the concentration of acetone is increased to 30%; however, the activity can be restored to its original value by increasing the concentration of aqueous media. Preliminary studies using assemblies of LiP and MnP on MF microparticles further demonstrate the feasibility of developing potential systems for degradation of environmental pollutants.

Adsorption↗
PubMed1991

Enhanced axial symmetry at the Fe(3+)-heme center of peroxidase by ascorbate: a basis for the ascorbate-dependent peroxidase action.

In the absence of its substrate hydrogen peroxide, peroxidase exhibits perturbations in its Fe(3+)-heme center, when incubated with ascorbic acid. The electron paramagnetic pattern sprang towards a higher g-value side, denoting a sharpening of the rhombic axial symmetry around the heme-center. The interpretation is that the ascorbate dependent peroxidase action starts with the formation of an Fe(3+)-ascorbate charge transfer complex intermediate.

Ascorbic Acid↗
PubMed1999

Inactivation of Coprinus cinereus peroxidase by 4-chloroaniline during turnover: comparison with horseradish peroxidase and bovine lactoperoxidase.

The peroxidase from Coprinus cinereus (CPX) catalyzed oxidative oligomerization of 4-chloroaniline (4-CA) forming several products: N-(4-chlorophenyl)-benzoquinone monoamine (dimer D), 4,4'-dichloroazobenzene (dimer E); 2-(4-chloroanilino)-N-(4-chlorophenyl)-benzoquinone (trimer F); 2-amino-5-chlorobenzoquinone-di-4-chloroanil (trimer G); 2-(4-chloroanilino)-5-hydroxybenzoquinone-di-4-chloroanil (tetramer H) and 2-amino-5-(-4-chlroanilino)-benzoquinone-di-4-chloroanil (tetramer 1). In the presence of 4-CA and H2O2, CPX was irreversibly inactivated within 10 min. Inactivation of CPX in the presence of H2O2 was a time-dependent, first-order process when the concentration of 4-CA was varied between 0 and 2.5 mM. The apparent dissociation constant (Ki) for CPX and 4-CA was 0.71 mM. The pseudo-first order rate constant for inactivation (k(inact)), was 1.15 x 10(-2) s(-1). Covalent incorporation of 20 mole 14C-4-CA per mole of inactivated CPX was observed. The partition ratio was about 2200 when either 4-CA or H2O2 was used as the limiting substrate. These results show that 4-CA is a metabolically activated inactivator (i.e. a suicide substrate). Unmodified heme and hydroxymethyl heme were isolated from native, 4-CA-inactivated and H2O2-incubated CPX. Inactivation resulted in significant losses in both heme contents. Analysis of tryptic peptides from 4-CA-inactivated CPX by MALDI-TOF/ MS and UV-VIS spectrophotometry suggested that trimer G and tetramer H were the major 4-CA derivatives that were covalently bound, including to a peptide (MGDAGF-SPDEVVDLLAAHSLASQEGLNSAIFR) containing the heme binding site. These studies show that heme destruction and covalent modification of the polypeptide chain are both important for the inactivation of CPX. These results were compared with similar studies on 4-CA-inactivated horseradish peroxidase (HRP) and bovine lactoperoxidase (LPO) during the oxidation of 4-CA.

Amino Acid Sequence↗
PubMed2001

Solid-state production of lignin peroxidase (LiP) and manganese peroxidase (MnP) by Phanerochaete chrysosporium using steam-exploded straw as substrate.

In the used media mainly consisting of steam-exploded wheat straw, the straw, which could replace expensive veratryl alcohol, might act not only as nutrient, but also as inducer of lignin enzymes. The activities of the enzymes lignin peroxidase (LiP) and manganese peroxidase (MnP) in solid-state fermentation (SSF) were far higher than in submerged fermentation (SmF). Under optimal conditions of SSF, the maximum activities of the enzymes Lip and MnP were 2600 and 1375 U/L, respectively. Thus, this would pave the way for production and application of lignin enzymes on a large scale.

Benzyl Alcohols↗
PubMed1997

Factors controlling the substrate specificity of peroxidases: kinetics and thermodynamics of the reaction of horseradish peroxidase compound I with phenols and indole-3-acetic acids.

The rates of oxidation of reducing substrates by heme peroxidases have previously been thought to be controlled only by their ease of oxidation. In the present study, we have compared the kinetics and thermodynamics of the oxidation of indole-3-acetic acid and derivatives and of phenols by horseradish peroxidase. Different dependencies of the reaction rates on the thermodynamic driving force reveal substrate specificity controlled by the enzyme-substrate complexes dissociation constants (Michaelis-Menten constants) and by the reorganization energies of electron-transfer within those complexes.

Catalysis↗
PubMed2004

Aryl thiol substrate 3-carboxy-4-nitrobenzenethiol strongly stimulating thiol peroxidase activity of glutathione peroxidase mimic 2, 2'-ditellurobis(2-deoxy-beta-cyclodextrin).

Artificial glutathione peroxidase (GPx) model 2, 2'-ditellurobis(2-deoxy-beta-cyclodextrin) (2-TeCD) which has the desirable properties exhibited high substrate specificity and remarkably catalytic efficiency when 3-carboxy-4-nitrobenzenethiol (ArSH) was used as a preferential thiol substrate. The complexation of ArSH with beta-cyclodextrin was investigated through UV spectral titrations, fluorescence spectroscopy, 1H NMR and molecular simulation, and these results indicated that ArSH fits well to the size of the cavity of beta-cyclodextrin. Furthermore, 2-TeCD was found to catalyze the reduction of cumene peroxide (CuOOH) by ArSH 200,000-fold more efficiently than diphenyl diselenide (PhSeSePh). Its steady-state kinetics was studied and the second rate constant kmax/KArSH was found to be 1.05 x 10(7) M(-1) min(-1) and similar to that of natural GPx. Moreover, the kinetic data revealed that the catalytic efficiency of 2-TeCD depended strongly upon the competitive recognition of both substrates for 2-TeCD. The catalytic mechanism of 2-TeCD catalysis agreed well with a ping-pong mechanism, in analogy with natural GPx, and might exert its thiol peroxidase activity via tellurol, tellurenic acid, and tellurosulfide.

Benzene Derivatives↗