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PubMed1985

Peroxidase-catalyzed O-demethylation reactions. Quinone-imine formation from 9-methoxyellipticine derivatives.

Despite numerous reports on the N-demethylation reactions catalyzed by peroxidases, to our knowledge, O-demethylation reactions with the same enzymes seem to be still a questionable matter. Unexpectedly, a peroxidase system (horseradish peroxidase and hydrogen peroxide) is able to effect the O-demethylation of the cytotoxic agents 9-methoxyellipticine and N2-methyl-9-methoxyellipticinium acetate. The reaction leads directly to the formation of the corresponding quinone-imine derivatives with the concomitant formation of one molecule of methanol per molecule of methoxy compound. One hydrogen peroxide molecule is consumed during the process. Experiments in H218O-enriched water clearly indicate that 18O is nearly quantitatively incorporated in the carbonyl group of the generated quinone-imine compound with the concomitant elimination of the methoxy group as methanol. So this peroxidase-catalyzed apparent O-demethylation in fact implies an oxidative demethoxylation step. This enzymatic reaction exhibits normal Michaelis-Menten saturation kinetics. Like the 9-hydroxylated ellipticines, both the 9-methoxylated ellipticines show a good affinity for the peroxidase itself (Km approximately 10 microM) but are slowly transformed to the corresponding quinone-imines. The Vmax values for methoxylated ellipticines are 10(-1)-10(-3) lower than those for hydroxylated compounds. This new route for the in vitro formation of electrophilic derivatives from the cytotoxic 9-methoxyellipticine and N2-methyl-9-methoxyellipticinium might be considered as a novel possible metabolic pathway for these drugs, especially if we bear in mind the "bio-oxidative alkylation" process previously described for at least one of the corresponding hydroxylated ellipticine derivatives (see Bernadou, J., Meunier, B., Meunier, G., Auclair, C., and Paoletti, C. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1297-1301; and Monsarrat, B., Maftouh, M., Meunier, G., Dugué, B., Bernadou, J., Armand, J. P., Picard-Fraire, C., Meunier, B., and Paoletti, C. (1983) Biochem. Pharmacol. 32, 3887-3890).

Alkaloids↗

PubMed1979

Absorption and fluorescence of fluram-labelled lysozyme and peroxidase solutions.

The absorption spectra of fluram, lysozyme, horse-radish peroxidase, and mixtures of lysozyme + fluram and peroxidase + fluram and the fluorescence and fluorescence excitation spectra of the mixtures in 0.05 M phosphate buffer with 1 per cent dioxane are determined. Due to formation of a protein-fluram compound, the absorption spectra of the mixtures are not algebraic sums of the components. From the fluorescence intensities the number of bonding sites is found 6 in both cases. The fluorescence spectrum of the peroxidase-fluram compound has maxima at 305, 350, 400, 450 nm due to peroxidase and at 480 nm originating from fluram. In mixtures of 10(-5) M lysozyme + 10(-4) M fluram, 3/4 of the excitation energy is transferred from lysozyme to fluram within the compound under 280 nm excitation. Under similar conditions 4/5 of the excitation energy is transferred from peroxidase to fluram.

Binding Sites↗

PubMed1984

The crystal structure of fluoride-inhibited cytochrome c peroxidase.

The three-dimensional crystal structure of yeast cytochrome c peroxidase complexed with fluoride (F- or HF) has been determined by difference Fourier techniques and partially refined at 2.5-A resolution. Fluoride binding induces significant perturbations of the enzyme structure of the distal side of the heme. The major effect occurs at the active-site arginine residue (Arg-48) which moves about 2 A in order to optimize hydrogen-bonded interactions with the fluorine atom. A small readjustment of the distal histidine (His-52), about 0.5 A, is also seen upon fluoride binding. Additionally, a hydrogen-bonded network of 4 water molecules at the active site is reorganized. No significant movements are detectable in either the heme itself or in the proximal histidine ligand. These observations imply that movement of the Arg-48 side chain may play a key role in the enzymic mechanism of cytochrome c peroxidase. Although we cannot unequivocally determine whether fluoride is bound as HF or F-, the hydrogen-bonding pattern around the ligand points to the protonated form. Structural comparison suggests that there is a difference between the tautomeric state of the imidazole side chain of the distal histidine in cytochrome c peroxidase and of the similarly positioned distal histidine in the globins. This difference accounts for the observation that cytochrome c peroxidase preferentially binds the protonated form of ligands, whereas the globins bind the anionic form. The tautomer indicated by the peroxidase structure is the one required for acid base catalysis (Poulos, T.L., and Finzel, B. C. (1984) in Peptide and Protein Reviews (Decker, M., ed) in press).

Cytochrome-c Peroxidase↗

PubMed1982

A new staining method for hepatitis B surface antigen by horseradish peroxidase.

An affinity of horseradish peroxidase for hepatitis B surface antigen (HBsAg), irrespective of antigen-antibody conjugation, was disclosed incidentally, and a new staining method of HBsAg was established. The new technique is simple, inexpensive and available for formalin-fixed, paraffin embedded sections, and does not require fresh liver tissue. Compared with three other methods, involving Shikata's orcein staining, the conventional immunoperoxidase method, and the peroxidase-anti-peroxidase method, it showed high specificity and sensitivity, which were not lower than the other three methods. Conjugation of horseradish peroxidase with HBsAg was confirmed by affinity chromatography. Attention should be paid to the fact that there is a possibility of obtaining false positive results by immunoperoxidase methods, staining antigens other than HBsAg in HBsAg positive liver tissue because of antigen-antibody independent affinity of peroxidase for HBsAg.

Hepatitis B Surface Antigens↗

PubMed1981

Binding of arylazidocytochrome c to yeast cytochrome c peroxidase.

Cytochrome c derivatives modified with a photoactivatable arylazido group in selected lysine residues were irradiated in the presence of cytochrome c peroxidase (EC 1.11.1.5). A derivative modified at lysine 13 was able to cross-link to the enzyme and inhibit electron transfer activity. Complete inhibition of cytochrome c peroxidase activity was obtained when 1 mol of cytochrome c was covalently bound per mol of cytochrome c peroxidase. Chemical cleavage of the covalent complex has been used for a preliminary characterization of the site of cross-linking of cytochrome c to cytochrome c peroxidase. This linkage site was localized to the NH2 terminal part of cytochrome c peroxidase including residues 1-51.

Amino Acids↗

PubMed1984

The cytochrome c peroxidase-cytochrome c electron transfer complex. The role of histidine residues.

The histidine-selective reagent diethyl pyrocarbonate and dye-sensitized photooxidation have been used to study the functional role of histidines in cytochrome c peroxidase. Of the 6 histidines in cytochrome c peroxidase, 5 are modified by diethyl pyrocarbonate at alkaline pH and 4 by photooxidation. The sixth histidine serves as the proximal heme ligand and is unavailable for reaction. Both modification reactions result in the loss of enzymic activity. However, photooxidized peroxidase retains its ability to react with H2O2 and to form a 1:1 cytochrome c peroxidase-cytochrome c complex. It is, therefore, concluded that the extra histidine modified by diethyl pyrocarbonate is the catalytic site distal histidine, His 52. In the presence of cytochrome c, no enzymic activity is lost by photooxidation and a single histidine, His 181, is protected from oxidative destruction. This finding provides strong support for the hypothetical model of the cytochrome c peroxidase-cytochrome c complex in which His 181 lies near the center of the intermolecular interface where it seems to provide an important link in the electron transfer process.

Amino Acids↗

PubMed1982

Determination of selenium concentration and glutathione peroxidase activity in plasma and erythrocytes.

We determined selenium concentrations and activities of the selenoenzyme, glutathione peroxidase (EC 1.11.1.9), in the plasma and erythrocytes of 38 apparently healthy women. We determined selenium concentrations directly by polarized Zeeman-effect flameless atomic absorption spectroscopy. Within-run precision studies for the assays gave CVs of 5.6% for a mean erythrocyte selenium concentration of 149.9 (SD 8.3) microgram/L (n = 10) and 6.4% for a mean plasma selenium concentration of 97.3 (SD 6.2) microgram/L (n = 12). For the women, mean selenium concentrations were 141.4 (SD 14.3) microgram/L of erythrocytes [0.49 (SD 0.07) microgram/g of hemoglobin and 96.3 (SD 14.2) microgram/L of plasma. Glutathione peroxidase activities were measured by a modification of the method of Paglia and Valentine (J. Lab. Clin. Med. 70: 158--169, 1967). Within-run precision studies for the glutathione peroxidase assays gave CVs of 12.8% for mean erythrocyte glutathione peroxidase activity of 77.2 (SD 9.9) U/g of hemoglobin (n = 13), and 8.1% for mean plasma activity of 312.5 (SD 25.2) U/L (n = 11). Mean enzyme activity was 78.7 (SD 12.9) U/g of hemoglobin for erythrocytes and 424 (SD 40) U/L for plasma. Erythrocyte selenium concentrations and glutathione peroxidase activities were positively, but poorly, correlated (r = 0.41, p less than 0.01).

Adult↗

PubMed1977

[Catalytic properties and thermostability of horseradish peroxidase covalently bound with Sepharose by carbohydrate residues of the enzyme].

A procedure for isolation of stabilized preparations of peroxidase, covalently bound with AH-Sepharose 4B by the enzyme carbohydrate residues, which had been previously oxidized by iodine acid, is proposed. The catalytic properties and thermostability of soluble oxidized and immobilized peroxidase has been studied and a comparison of these properties with those of the native enzyme has been made. It has been demonstrated that the oxidation of the carbohydrate residues dose not affect eh Km value for soluble oxidized peroxidase, but leads to a decrease in the kcat approximately two times for the reaction of peroxidase exidation of o-dianizidine. In case of immobilized peroxidase the Km value for H2O2 does not change, whereas the Km value for o-dianizidine is decreased approximately 20 times. It is demostrated that the chemical structure of the matrix surface and in case of the same matrix used--the degree and type of modification of the functional groups of protein, have a strong effect on the stability of the immobilized enzyme.

Chemical Phenomena↗

PubMed1976

Reversible blood-brain barrier dysfunction to peroxidase after a small stab wound in the rat cerebral cortex.

Small stab wounds were made in the rat frontal lobe. The animals were injected with horseradish peroxidase intravenously at different times after the injury in order to study the extravasation of this tracer. There was a leakage of peroxidase into the brain during the first 3 days after the injury. The route of passage from the vessel lumen into the brain was through disrupted blood vessels in the injured region. Endothelial pinocytosis and formation of thin, trans-endothelial channel-like structures with or without a content of peroxidase were two other possible routes of passage across the blood vessels. Occasionally, badly damaged endothelial cells displayed a diffuse cytoplasmic distribution of peroxidase, indication a diffusion into and possibly across these injured cells. No widened tight junctions were seen. Thus, this study indicated four possible routes of passage of horseradish peroxidase across the endothelial cells: cellular gross damage with disrupture of the cells, diffusion across badly injured endothelial cells, possibly pinocytosis and formation of trans-endothelial channel-like structures. The cellular uptake of the tracer was vesicular in most neurons, astrocytes, oligodendrocytes and hematogeneous phagocytes. However, a diffuse distribution of the tracer was seen in some "dark" neurons near leaking vessels in the vicinity of the stab wound.

Animals↗

PubMed2000

The rational design of semisynthetic peroxidases.

A semisynthetic peroxidase was designed by exploiting the structural similarity of the active sites of vanadium dependent haloperoxidases and acid phosphatases. Incorporation of vanadate ion into the active site of phytase (E.C. 3.1.3.8), which mediates in vivo the hydrolysis of phosphate esters, leads to the formation of a semisynthetic peroxidase, which catalyzes the enantioselective oxidation of prochiral sulfides with H(2)O(2) affording the S-sulfoxide, e.g. in 66% ee at 100% conversion for thioanisole. Under reaction conditions the semi-synthetic vanadium peroxidase is stable for over 3 days with only a slight decrease in turnover frequency. Polar water-miscible cosolvents, such as methanol, dioxane, and dimethoxyethane, can be used in concentrations of 30% (v/v) at a small penalty in activity and enantioselectivity. Among the transition metal oxoanions that are known to be potent inhibitors, only vanadate resulted in a semisynthetic peroxidase when incorporated into phytase. A number of other acid phosphatases and hydrolases were tested for peroxidase activity, when incorporated with vanadate ion. Phytases from Aspergillus ficuum, A. fumigatus, and A. nidulans, sulfatase from Helix pomatia, and phospholipase D from cabbage catalyzed enantioselective oxygen transfer reactions when incorporated with vanadium. However, phytase from A. ficuum was unique in also catalyzing the enantioselective sulfoxidation, albeit at a lower rate, in the absence of vanadate ion.

6-Phytase↗

PubMed2000

Cantaloupe melon peroxidase: characterization and effects of additives on activity.

Peroxidase in cantaloupe melon (Cucumis melo L. var. reticulatus Naud.), a fruit commonly fresh cut processed, was characterized to determine reaction pathway, optimal conditions for activity and effect of some additives on enzymatic action. Mn2+, CaCl2, NaNO2 and kinetin had partial inhibitory effects on enzyme activity. Activity was effectively inhibited by compounds capable of chelating peroxidase heme iron such as diethyldithiocarbamate and tiron, but unaffected by EDTA. Free radical scavenger, superoxide dismutase, also had no effect on reaction velocity. Enzymatic action was consistent with that of ascorbate peroxidase based on the relatively higher affinity for ascorbate over guaiacol. Optimum activity temperature was 50-55 degrees C. The enzyme was stable at temperatures below 40 degrees C and at 50 degrees C for up to 10 min. Over 90% of total activity was lost at 80 degrees C within 5 min. Broad pH optima, 5.5-7.5 at 50 degrees C and 6-7 at 30 degrees C, were obtained. Peroxidase activity in cantaloupe was higher than those in strawberry (Fragaria ananassa Duch.) and lettuce (Lactuca sativa L.), suggesting a relatively high oxidative stress in fresh cut cantaloupe. The potential use of ascorbate as an additive in fresh cut cantaloupe melon was demonstrated by its ability to preserve color in minimally processed fruits for 25 days at 4 degrees C, possibly as a result of an enhanced antioxidative action of the ascorbate-peroxidase complex and trace metal ion cofactors.

Color↗

PubMed1995

Sputum peroxidase activity correlates with the severity of lung disease in cystic fibrosis.

Patients with cystic fibrosis (CF) of the same age differ significantly in their degree of pulmonary disease. Based on preliminary observations, we postulated that the activity of myeloperoxidase would be significantly increased in patients with greater structural lung damage than in those with less lung damage. Acid extracts of weighed sputum samples were assayed for lactoferrin concentrations by ELISA. Activities of peroxidase, cathespsin G, and elastase (with and without proteinase 3) were determined by kinetic analysis using chromogenic substrates. The patients were divided into quartiles based on their Brasfield chest-radiograph score. Patients in the first quartile (least amount of structural lung abnormality) were compared to those in the fourth quartile. The concentration of lactoferrin, a specific (secondary) granule protein of neutrophils, did not differ between the two patient groups. However, the activities of the neutrophil primary granule proteins, peroxidase, elastase, and elastase plus proteinase 3, were significantly elevated in the group with the most structural lung abnormality. Sputum albumin concentration was used to estimate leakages of plasma proteins into the airways. Peroxidase activity, but not the activity of cathepsin G, of elastase, or of elastase plus proteinase 3, correlated significantly with albumin/g sputum in both quartile groups. To confirm the association of sputum peroxidase activity with differences in lung structure and to test its correlation with lung function, spirometry was performed in a second group of patients during the week prior to the time of sputum sampling. In this second group, increased sputum peroxidase activity was associated with worse Brasfield scores and with decreased percent-predicted forced expiratory volume in 1 sec.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1988

Induction of estrogen receptor, peroxidase activity, and epithelial abnormalities in the mouse uterovaginal epithelium after neonatal treatment with diethylstilbestrol.

Neonatal female NMRI mice were treated with daily doses of 10(-2) or 5 micrograms diethylstilbestrol (DES) on one or more of days 1-5 after birth. Using immunohistochemical techniques and a monoclonal antibody to the estrogen receptor, we demonstrated an estrogen-induced precocious appearance of receptor protein in the nuclei of the uterovaginal epithelium. High levels of peroxidase activity and a pronounced stromal infiltration with peroxidase positive cells occurred in the uterine cervix and upper vagina after estrogen treatment. This regional restriction in peroxidase activity was similar to the regional restriction for estrogen-induced epithelial abnormalities (heterotopic columnar epithelium, adenosis). A combined treatment with DES and corticosterone depressed peroxidase activity but not to the control level. The distribution of abnormal epithelium was similar in DES- and DES-corticosterone-treated females. The conclusion is that neonatal estrogen treatment induces an epithelial receptor for estrogen and a high level of cervical peroxidase activity, but the relationship between these parameters and the appearance of abnormal cervicovaginal epithelial changes could not be settled in the present study.

Animals↗

PubMed1998

Evidence for an unusual electronic structure of wheat germ peroxidase compound I.

Oxidized states of wheat germ peroxidase isozyme C2 (WGP C2) were investigated by means of electronic absorption spectroscopy. Addition of one molar equivalent of H2O2 to ferric WGP C2 led to the formation of an oxidized species with an absorption spectrum very similar to that of peroxidase compound II, with a Soret maximum at 411 nm and visible maxima at 523 and 553 nm. The transformation took place with an isosbestic point at 409 nm. Stopped flow spectroscopy showed no inflection points for the formation of this species when it was registered at 420 nm, and we could verify the persistence of the isosbestic point from 20 ms to 10 s. The oxidized species decays spontaneously to ferric enzyme in a double-exponential manner. By adding excess H2O2 to the system we obtained an inactive derivative identical to horseradish peroxidase P-670. In the presence of one equivalent of reducing substrate and excess H2O2 compound III was formed. The results so indicate that the species obtained in the reaction of WGP C2 with equimolecular amounts of H2O2 is compound I. The resulting compound I spectrum was identical to that of cytochrome c peroxidase, suggesting the formation of a protein radical rather than the typical pi cation radical, a feature which had not been described before for a plant peroxidase.

Enzyme Activation↗

PubMed2001

Isolation and characterization of two peroxidases from Cucumis sativus.

Two heme peroxidases of 35.2 and 36.5 kDa have been isolated from cucumber (Cucumis sativus) peelings and characterized through electronic and 1H NMR spectra in the pH range 3.5-10.5. Their spectroscopic and catalytic properties, which are closely similar, are characteristic of highly homologous isoenzymes. Both proteins, as isolated, exist as a mixture of two ferric forms containing a high-spin and a low-spin heme in an approximately 2:1 molar ratio. The latter form likely contains a hydroxide ion axially coordinated to the heme iron and is proposed to be the result of partial irreversible protein inactivation due to the purification procedure. Both proteins in the reduced form are fully high-spin. The high-spin ferric form is sensitive to two acid-base equilibria with apparent pKa values of approximately 5 and 8.5, which have been assigned to the distal histidine and the arginine adjacent to it, respectively. These equilibria also affect the catalytic activity and the interaction with inorganic anions such as azide and fluoride. The reactivity of both proteins is closely similar to that of other plant peroxidases, primarily horseradish peroxidase; however, they also show spectroscopic properties similar to those of cytosolic ascorbate peroxidase. Therefore, overall, these two species show molecular, spectroscopic and catalytic features which are rather peculiar among plant peroxidases.

Azides↗

PubMed1995

Determination of rate constants for rapid peroxidase reactions.

We describe an improved enzyme-monitored stopped-flow method for determining rate constants for peroxidase reactions that are too rapid to measure by conventional pseudo-first-order methods. Ascorbate will reduce many substrate radicals as rapidly as they are generated by a peroxidase. This ensures a constant substrate concentration during the peroxidase reaction. We investigated the reactions of horseradish peroxidase compound I (HRP-I) with three standard substrates (chlorpromazine (CPZ), 2,2'-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS), and p-methoxyphenol) in the presence and absence of ascorbate. The rate of reaction of CPZ with HRP-I is slow enough that it can be measured using pseudo-first-order conditions maintained by a minimum 10-fold excess of CPZ, or by the addition of ascorbate at very low CPZ concentrations. The same rate constant was obtained with either method. The rate of reaction of ABTS with HRP-I at lower pH (5.0-3.3) is rapid; consequently, we were unable to obtain rate constants using concentrations of ABTS which constitute pseudo-first-order conditions. However, using much lower ABTS concentrations with the addition of ascorbate, we obtained rate constants that ranged from 5 x 10(7) to 8 x 10(8) M-1 s-1. Hence, ascorbate provides a simplified way to maintain pseudo-first-order conditions for fast peroxidase reactions even at low substrate concentrations.

Ascorbic Acid↗

PubMed1995

Purification of major lignin peroxidase isoenzymes from Phanerochaete chrysosporium by chromatofocusing.

The basidiomycete Phanerochaete chrysosporium produces several isoforms of lignin peroxidase, which catalyzes the oxidative depolymerization of lignin To date, ion-exchange chromatography and preparative isoelectric focusing (IEF) have been commonly used for isolation of lignin peroxidase isoenzymes. In this work we have purified major lignin peroxidases to high purity by a one-step chromatographic method, chromatofocusing. The purified isoenzymes were identified by analytical IEF using isoenzymes purified by preparative IEF as standards. The specific activities and spectral properties of the isoenzymes were comparable with the previously published data. The predominant isoenzyme under the growth conditions used was LiP 4.65. Almost 50% of the lignin peroxidase activity applied into the column was recovered in the LiP 4.65 fraction. The total recovery of the lignin peroxidase activity was over 80%.

Chromatography, Ion Exchange↗

PubMed1995

Potential molecular targets mediating chemical teratogenesis: in vitro peroxidase-catalyzed phenytoin metabolism and oxidative damage to proteins and lipids in murine maternal hepatic microsomes and embryonic 9000g supernatant.

Phenytoin and related proteratogens may be bioactivated by peroxidases to a reactive free radical intermediate that initiates teratogenesis. This study evaluated the potential molecular targets mediating phenytoin teratogenicity. In vitro phenytoin-induced oxidative tissue damage following metabolic activation by prostaglandin H synthase (PHS) and lipoxygenases (LPOs) was quantified in both maternal hepatic microsomes and embryonic 9000g supernatant (S-9) from CD-1 mice, using lipid peroxidation and protein oxidation and degradation as indices. Phenytoin (0-0.2 mM) and the PHS/LPO cofactor arachidonic acid (AA, 0-1.0 mM) were incubated with maternal hepatic microsomes or embryonic S-9 (2 mg) for 0-60 min. Lipid peroxidation was measured by the formation of thiobarbituric acid-reactive substance, protein oxidation quantified by the formation of carbonyl groups in proteins, and protein degradation measured by the release of primary amines. Phenytoin was cooxidized by peroxidases in both maternal hepatic microsomes and embryonic S-9 to initiate oxidative damage to tissue lipids and proteins. The lipid peroxidation and protein oxidation and degradation were dependent on incubation time, AA concentration, and phenytoin concentration (p < 0.05), indicating that peroxidases were involved in phenytoin metabolic activation. Phenytoin-initiated oxidative tissue damage in maternal hepatic microsomes was inhibited by the cyclooxygenase inhibitor indomethacin, confirming that PHS was involved in the reactions. Phenytoin-induced oxidative damage in embryonic S-9 was not reduced by indomethacin, but was by nordihydroguaiaretic acid, an antioxidant and LPO inhibitor, indicating that additional enzymes containing peroxidase activity, such as LPO, may play a substantial role in phenytoin metabolic activation in embryonic tissues. Phenytoin-initiated lipid peroxidation and protein oxidation were early events, preceding protein degradation (10 min versus 60 min), and a lower phenytoin concentration was needed for lipid peroxidation (0.04 mM) and protein oxidation (0.01 mM) than for protein degradation (0.08 mM), suggesting that phenytoin-initiated protein and lipid oxidation may be two potential causes for protein cleavage. These results provide an in vitro basis for the in vivo evidence that embryonic lipid and protein may constitute important molecular targets mediating phenytoin teratogenicity, wherein peroxidase-catalyzed metabolic activation initiates early macromolecular oxidation by reactive oxygen species.

Animals↗