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PubMed1986

A comparative study of free radical scavengers in cardioplegic solutions. Improved protection with peroxidase.

Oxygen-derived free radicals play an important role in the myocardial injury associated with ischemia and reperfusion. This study was designed to assess whether the protection afforded by a K+ rich, Mg2+ rich cardioplegic solution could be enhanced by the addition of free radical scavengers acting at different levels of the radical generating pathway. Forty isolated isovolumic rat hearts were divided into five groups (n = 8). Four groups of hearts were subjected to 90 minutes of normothermic cardioplegic arrest followed by 45 minutes of reperfusion. Hearts were given an initial bolus of either unmodified cardioplegic solution or cardioplegic solution enriched with superoxide dismutase (200,000 U/L) reduced glutathione (0.1 mmol/L), or peroxidase (6,000 U/L). One group of hearts was aerobically perfused throughout the experimental protocol and served as nonischemic controls. Based on comparisons of postreperfusion ventricular pressure development, maximal ventricular dP/dt, left ventricular compliance and coronary flow, peroxidase-containing cardioplegic solution afforded the best myocardial protection, with values of these indicators not significantly different from those of nonischemic perfused control heart. Glutathione afforded protection slightly inferior to that of peroxidase but still markedly better than in groups receiving superoxide dismutase or unmodified cardioplegic solution. This study confirms that cardioplegic protection can be enhanced by the addition of free radical scavengers, in particular peroxidase.

Animals↗
PubMed1986

Horseradish peroxidase-catalyzed conversion of iodine to iodide in presence of EDTA and H2O2.

EDTA (4 mM) blocks the oxidation of iodide to I-3 (increase of extinction at 353 nm) by H2O2 catalyzed by horseradish peroxidase, which is reversed by the addition of an equimolar concentration of Zn2+. Addition of suboptimal concentration of EDTA (2 mM) not only decreases the rate of forward reaction of I-3 formation but also causes loss of extinction of the same when I-3 is generated. The loss of extinction of I-3 is proportional to the enzyme concentration and is blocked by azide, the inhibitor of the peroxidase. EDTA also causes bleaching of nonenzymatically formed I-3 (from iodide and H2O2) only in the presence of horseradish peroxidase, and the effect is reversed by the equimolar concentration of Zn2+. Both the bleaching of I-3 by EDTA and reversal of EDTA effect by Zn2+ are sensitive to azide. The decrease of extinction of I-3 (formed by dissolving iodine in KI solution) is dependent on EDTA, H2O2, and horseradish peroxidase. Molecular iodine is also bleached but at a slower rate than I-3. Evidence is presented to show that this bleaching of I-3 is due to enzymatic conversion of I-3 to iodide in presence of EDTA and H2O2 and this involves pseudocatalatic degradation of H2O2 to O2.

Edetic Acid↗
PubMedDate not supplied

[Optimization of the use of horseradish peroxidase and its antibodies in immunoenzyme analysis].

The steady-state kinetics of peroxidation of 8 aromatic amines was studied. p-Phenylenediamine, o-dianisidine (o-DA) and 3,5,3',5'-tetramethylbenzidine were found to be optimal substrates of horse-radish peroxidase. The kinetics of oxidation of these substrates by horseradish peroxidase modified with three molecules of Strophanthin K was studied as well. Within the temperature range from 37 to 53 degrees C the inactivation rate constants were determined for peroxidase and its conjugate with Strophanthin K. The effect of sugars and polyols on thermal stability of the conjugate peroxidase-Strophanthin K was investigated. A comparative kinetic study was performed of oxidation of o-DA and its conjugate with dextran. The results obtained made a basis for an enzyme immunoassay of cardiac glycosides during their isolation from plant raw material.

Cardiac Glycosides↗
PubMedDate not supplied

Glutathione peroxidase: fact and fiction.

The present knowledge of glutathione (GSH) peroxidase is briefly reviewed: GSH peroxidase has a molecular weight of about 85,000, consists of four apparently-identical subunits and contains four g atom of selenium/mol. The enzyme-bound selenium can undergo a substrate-induced redox change and is obviously essential for activity. In accordance with the assumption that a selenol group is reversibly oxidized during catalysis, ping-pong kinetics are observed. Limiting maximum velocities and Michaelis constants, indicating the formation of an enzyme-substrate complex, are not detectable. The enzyme is highly specific for GSH but reacts with many hydroperoxides. It can be deduced from the kinetic analysis of GSH peroxidase that in physiological conditions removal of hydroperoxide is largely independent of fluctuations in the cellular concentration of GSH. However, the system will abruptly collapse if the rate of hydroperoxide formation exceeds that of regeneration of GSH. By these considerations, the pathophysiological manifestation of disorders in GSH metabolism and pentose-phosphate shunt may be explained. With regard to its low specificity for hydroperoxides, GSH peroxidase could be involved in various metabolic events such as H2O2 removal in compartments low in catalase, hydroperoxide-mediated mutagenesis, protection of unsaturated lipids in biomembranes, prostaglandin biosynthesis, and regulation of prostacyclin formation.

Amino Acids↗
PubMed1985

Phenols as enhancers of the chemiluminescent horseradish peroxidase-luminol-hydrogen peroxide reaction: application in luminescence-monitored enzyme immunoassays.

Certain phenol derivatives, including p-iodophenol and p-phenylphenol, enhance light emission from the horseradish peroxidase-catalyzed oxidation of cyclic diacyl hydrazides such as luminol. The light emission decays slowly (glowing for several minutes) and its intensity may be greater than 1000-fold that of the unenhanced reaction. The enhanced system enables rapid, sensitive assay of peroxidase conjugates. We describe its application in immunoassays for human choriogonadotropin, digoxin, and factor VIII-related antigen. Luminescent quantification of peroxidase labels has been directly incorporated into immunoassays based on beads, tubes, or microtiter plates, used in conjunction with photodetectors such as photomultiplier tubes or instant photographic film. Enhancement with phenol derivatives exceeds that achieved with 6-hydroxybenzothiazole derivatives and depends on pH and enhancer concentration. Emission spectra of phenol-enhanced and unenhanced reactions are remarkably similar, suggesting that the enhancers do not act as more efficient emitters but exert their action earlier in the complex reaction between peroxidase, oxidant, and luminol.

Antigens↗
PubMed1986

Heme-linked ionizations in horseradish peroxidase detected by Raman difference spectroscopy.

Heme-linked ionizations of the acidic and basic isoenzymes of ferrous horseradish peroxidase influence both the Fe-histidine stretching mode and the oxidation-state marker line. First, Raman difference spectroscopy of horseradish peroxidase confirms earlier work showing that v(Fe-His) undergoes a transition in frequency with a pK that is characteristic of the enzyme's functional properties. The Fe-histidine mode shifts by about 2.5-3.0 cm-1 for horseradish peroxidase C and by about 6 cm-1 for the acidic isoenzyme. Further, we find that the oxidation-state marker line v4 also exhibits a transition with the same pK. For horseradish peroxidase C the shift in v4 is 0.4 cm-1 and the pK is 7.1 +/- .5, in good agreement with the pK found by other techniques. Shifts in these two Raman lines are correlated for the pK 7.1 transition and attain their highest frequency at low pH. The correlation is in marked contrast with R/T shifts in hemoglobins for which delta v(Fe-His) and delta v4 are also linearly related but shift in opposite directions. The shift in v4 suggests a mechanism for pH control of catalytic function based on ring pi-charge density effects on the energy of charge-depleted high oxidation-state intermediates. A second transition in v4 (delta v4 = 2.6 cm-1) with a pK of 10.0 is interpreted in terms of a change in ligation and spin state.

Heme↗
PubMed1986

Horseradish peroxidase-catalyzed oxidation of acetaminophen to intermediates that form polymers or conjugate with glutathione.

Horseradish peroxidase catalyzed the polymerization of acetaminophen. Addition of reduced glutathione (GSH) to reaction mixtures resulted in decreased polymerization and formation of minor amounts of GSH-acetaminophen conjugates. The conjugates were identified as 3-(glutathion-S-yl)acetaminophen and 3-(glutathion-S-yl)diacetaminophen. Horseradish peroxidase also catalyzed polymerization of synthetic 3-(glutathion-S-yl)acetaminophen to a dimer conjugate. In contrast to acetaminophen, 3-(glutathion-S-yl)acetaminophen oxidation was slowly catalyzed by horseradish peroxidase. However, in reaction mixtures containing equimolar concentrations of acetaminophen and synthetic 3-(glutathion-S-yl)acetaminophen, the formation of 3-(glutathion-S-yl)diacetaminophen and 3-(diglutathion-S-yl)diacetaminophen was rapid and accounted for approximately 95% of the products, whereas acetaminophen polymers accounted for only 5% of the products. These findings suggest that horseradish peroxidase catalyzed the one-electron oxidation of acetaminophen to N-acetyl-p-benzosemiquinone imine which preferentially polymerized rather than reacted with GSH. N-Acetyl-p-benzosemiquinone imine may also oxidize 3-(glutathion-S-yl)acetaminophen to form acetaminophen and 3-(glutathion-S-yl)-N-acetyl-p-benzosemiquinone imine. The data indicate that once this conjugate radical is formed it reacts with either N-acetyl-p-benzosemiquinone minine or 3-(glutathion-S-yl)-N-acetyl-p-benzosemiquinone imine via a radical termination mechanism.

Acetaminophen↗
PubMed1985

Peroxidase-catalyzed formation of triplet acetone and chemiluminescence from isobutyraldehyde and molecular oxygen.

It has been established that the horseradish peroxidase/O2/isobutyraldehyde (IBAL) system leads to triplet acetone and formic acid formation followed by phosphorescence of the triplet acetone (see, for example, Bechara, E.J.H., Faria Oliveira, O.M.M., Durán, N., Casadei de Baptista, R., and Cilento, G. (1979) Photochem. Photobiol. 30, 101-110). In this paper many of the mechanistic details are established. The reaction is initiated by the autoxidation of IBAL to form the peracid (CH3)2CHC = O(OOH). The peracid converts horseradish peroxidase into compound I which in turn is converted into compound II by abstracting the alcoholic hydrogen atom from the enol form of IBAL. This creates a free radical with two resonance forms. (Formula: see text) Addition of molecular oxygen to the latter resonance form creates a peroxy radical which abstracts a hydrogen atom near the active site of the enzyme. The newly formed alpha-peroxide in turn forms a dioxetane-type of intermediate which rapidly decomposes into triplet acetone and formic acid. Compound II reacts with the enol by the same pathway as compound I. Thus native horseradish peroxidase is regenerated. The hydrogen atom abstraction near the enzyme active site may occur directly from ethanol, present to solubilize IBAL or from a group on the enzyme, in which case ethanol participates in a repair mechanism. Phosphate buffer is necessary because it catalyzes the keto-enol conversion of IBAL. Thus horseradish peroxidase participates in a normal peroxidatic cycle. The only chain reaction is the uncatalyzed autoxidation of IBAL, most of which occurs prior to the mixing of IBAL with the oxygenated horseradish peroxidase solution.

Acetone↗
PubMed1979

Donor substrate specificity and thiol reduction of glutathione disulfide peroxidase.

By isolation of a mixed disulfide product of glutathione and cysteine, glutathione peroxidase was shown to be highly specific for only one donor substrate. Using the coupled assay of NADPH and yeast glutatione reductase, which is highly specific for flutathione disulfide, it was shown that the apparent inhibition of glutathione peroxidase by mercaptoethanol can be described kinetically and that it is competitive with glutathione. Also, when limiting amounts of hydroperoxide were present in the reaction mixture with mercaptoethanol or cysteine, the total amount of glutathione disulfide produced decreased as compared with that in a reaction mixture without mercaptoethanol or cysteine. This finding is consistent with enzymatic formation of mixed disulfides. Data presented suggest that the selenium in glutathione peroxidase was oxidized to a seleninic acid in the absence of glutathione. These results can be explained by a mechanism for glutathione peroxidase wherein the selenium atom is the only atom in the enzyme that undergoes oxidation reduction.

Animals↗
PubMed1984

Mechanisms of thyroid peroxidase- and lactoperoxidase-catalyzed reactions involving iodide.

In a previous communication we proposed a reaction scheme to explain our observation that thyroid peroxidase and lactoperoxidase degrade H2O2 catalatically in the presence of low concentrations of iodide. An essential feature of the scheme was the proposal that enzyme-bound hypoiodite, designated [EOI]-, is a common intermediate in various peroxidase-catalyzed reactions involving iodide. In the present investigation, we tested the validity of this scheme by studying the predictions that it makes concerning the formation of OH-, O2, I2, and organically bound iodine. Stoichiometric and kinetic measurements were made to correlate formation of these various products. Three different peroxidase-catalyzed reactions were studied: 1) oxidation of I- to I2; 2) iodide-dependent catalytic degradation of H2O2 to O2; and 3) iodination of tyrosine or thyroglobulin. Reaction 2 was also studied nonenzymatically using I2, for comparison with the enzyme-catalyzed reaction. In all three reactions, both the stoichiometric and kinetic results with thyroid peroxidase agreed closely with the predictions made by the proposed scheme. This was largely the case with lactoperoxidase also. However, in the case of lactoperoxidase-catalyzed iodination of tyrosine or thyroglobulin, we observed a marked discrepancy between initial rates of OH- release and iodination, inconsistent with the mechanism originally proposed for the iodination reaction. As a possible explanation for this kinetic discrepancy, we postulate that lactoperoxidase generates hypoiodous acid and that the latter is the active intermediate in the various reactions involving iodide.

Hydrogen Peroxide↗
PubMedDate not supplied

[Catalysis by enzymes entrapped into reversed micelles of surfactants in organic solvents. Peroxidase in the aerosol OT-water-octane system].

Spectral and catalytic parameters of peroxidase solubilized in the aerosol OT-water-octane system have been studied. The spectrum of peroxidase solubilized in octane with AOT reversed micelles, a degree of surfactant hydration being above 12, is actually identical to that of the enzyme aqueous solution. On the other hand, significant spectral changes have been detected when transferring the enzyme from water to the reversed micelle medium at low degrees of surfactant hydration, precisely [H2O]/[AOT] less than 12. The reversed micelle-entrapped peroxidase catalyses the oxidation of pyrogallol with hydrogen peroxide much more actively (at [H2O]/[surfactant] approximately 13) than that in aqueous solution. The entrapment of peroxidase into surfactant reversed micelles increases precisely the catalytic constant of the reaction, i.e. the virtual reactivity of the enzyme increases ten and hundred times depending on degrees of surfactant hydration and concentration. The systems of reversed micelles may be considered as models of biomembranes. Our findings hence show that enzymes in vivo can be much more catalytically active then it appears possible to reveal in conventional experiments in vitro in aqueous solutions.

Catalysis↗
PubMed1982

Import of proteins into mitochondria. Import and maturation of the mitochondrial intermembrane space enzymes cytochrome b2 and cytochrome c peroxidase in intact yeast cells.

The import of cytochrome b2 and cytochrome c peroxidase into mitochondria was investigated by pulse-chase experiments with intact yeast cells combined with subcellular fractionation. Import and processing of the precursors of these intermembrane space proteins is blocked by uncouplers of oxidative phosphorylation, indicating that an "energized" inner membrane is required. Cytochrome b2 is processed in two steps. The first step involves energy-dependent transport across both mitochondrial membranes and cleavage by a matrix-located protease to yield an intermediate which is smaller than the precursor, but larger than the mature protein. The second step involves conversion of the intermediate to the mature form. Whereas the precursor and the mature form are soluble, the intermediate is membrane-bound and exposed to the intermembrane space. The maturation of cytochrome c peroxidase is much slower than that of cytochrome b2. Proteolytic processing rather than import is rate-limiting since cytochrome c peroxidase precursor labeled during a 3-min pulse is already found attached to the outer face of the mitochondrial inner membrane. Import of cytochrome b2 and probably also of cytochrome c peroxidase thus involves energy-dependent transport to the matrix and cleavage by a matrix-localized protease. Maturation of cytochrome b2 proceeds in the sequence: soluble precursor leads to membrane-bound intermediate form leads to soluble mature form.

Biological Transport↗
PubMed1983

Control of the transfer of oxidizing equivalents between heme iron and free radical site in yeast cytochrome c peroxidase.

A procedure has been developed for obtaining yeast cytochrome c peroxidase with the heme iron in the Fe(IV) state, without the concomitant formation of the protein free radical that occurs in the ES compound resulting from the oxidation of ferric peroxidase with hydrogen peroxide. In this procedure, ferrous peroxidase, prepared either by photochemical reduction or by trapping the dithionite-reduced enzyme with carbon monoxide, is oxidized with a stoichiometric amount of hydrogen peroxide. The resulting Fe(IV) enzyme oxidizes ferrocyanide monophasically, with a rate constant of 4 x 10(3) M-1 S-1. The optical spectrum of the free radical was obtained as the difference between the spectra of the ES and Fe(IV) compounds. EPR spectra of ES compound prepared with [16O]-and [17O]hydrogen peroxide are identical, demonstrating that no fragment of the oxidant is associated with the free radical. The heme in the Fe(IV) enzyme is stable and does not oxidize the free radical site either intra- or intermolecularly. On the other hand, previous results from the presteady state kinetics of reduction and reductive titrations of the ES compound with ferrocyanide imply that the heme and free radical sites exchange oxidizing equivalents, in particular that the radical site once reduced can be reoxidized, either intra- or intermolecularly, by the ferryl heme. To resolve these contradictions, we propose a catalytic mechanism for cytochrome c peroxidase in which the radical site can exist in two conformations having very different reduction potentials and in which a significant flow of oxidizing equivalents between heme and free radical sites occurs only (i) during the hydrogen peroxide oxidation of the resting Fe(III) enzyme to form compound ES and (ii) within the initial transient intermediate formed upon the one-electron reduction of this oxidized product.

Cytochrome-c Peroxidase↗
PubMed1984

Iodination and oxidation of thyroglobulin catalyzed by thyroid peroxidase.

The kinetics of iodination and oxidation of hog thyroglobulin were studied with purified hog thyroid peroxidase and the results were compared with the reactions of free tyrosine. From Lineweaver-Burk plots and on the basis of a value of 0.83 for delta epsilon mM at 289 nm/iodine atom incorporated, the rate constant for transfer of an assumed enzyme-bound iodinium cation to thyroglobulin was estimated to be 6.7 X 10(7) and 2.3 X 10(7) M-1 s-1 in native (iodine content = 1.0%) and more iodinated (iodine content = 1.2%) thyroglobulins, respectively. This iodine-transferring reaction was stimulated by iodothyronines, similarly as observed in the reaction with free tyrosine. The iodination of thyroglobulin was inhibited by GSH, the inhibition being competitive with thyroglobulin. Thyroglobulin was oxidized in the presence of a thyroid peroxidase system without giving any appreciable change in absorbance around 300 nm. From stopped flow data, the oxidation was concluded to occur by way of two-electron transfer and the rate constant for the reaction of thyroid peroxidase Compound I with thyroglobulin was estimated to be 1.0 X 10(7) M-1 s-1. The stopped flow kinetic pattern was similar to that observed on the reaction with free tyrosine and monoiodotyrosine. About 6 mol of hydrogen peroxide were consumed per mol of thyroglobulin. Thyroid peroxidase catalyzed thyroglobulin-mediated oxidation of GSH, but lactoperoxidase did not.

Animals↗
PubMed1982

Isolation and characterization of horseradish peroxidase compound X.

Horseradish peroxidase reacts with sodium [36Clll] chlorite at pH 10.7 to form a 36Cl-labeled horseradish peroxidase intermediate. The optical absorption spectrum of this intermediate is quite stable and very similar to that of horseradish peroxidase Compound II. The intermediate can be separated from small molecules by chromatography on a Sephadex G-10 column. After fractionation, 65 to 93% of 36Cl in the reaction mixture remains associated with horseradish peroxidase. The remainder of 36Cl-labeled enzyme reacts with 5,5-dimethyl-2-chloro-1,3-cyclohexanedione (monochlorodimedone) at pH 4 to transfer 36Cl from the enzyme to the halogen acceptor molecule. [36C]5,5-Dimethyl-2,2-dichloro-1,3-cyclohexanedione (dichlorodimedone) was established as the major product of the transfer reaction by co-crystallization of the enzymic product with authentic dichlorodimedone and by thin layer chromatography. A chlorine oxide ligand on a ferryl heme iron protoporphyrin IX is proposed for the structure of Compound X.

Chemical Phenomena↗
PubMedDate not supplied

[A new view of estradiol biotransformation in the body. Estradiol metabolism in the erythrocytes via the peroxidase reaction].

The study has established that in the presence of horseradish peroxidase, estradiol was subject to oxidative destruction under the action of hydrogen peroxide via the peroxidase reaction. The stoichiometric ratio of the hydrogen peroxide consumption to estradiol oxidation is 1:1 in this peroxidase reaction. Estradiol peroxidation was found to be catalyzed by methemoglobin by the same order as in case of horseradish peroxidase. Based on these results and the fact that estradiol is bound to erythrocytes and penetrates inside, it is concluded that erythrocytic estradiol is metabolically converted via its peroxidation.

Animals↗
PubMed1978

[Immunochemical characteristics of the peroxidases of several mouse tissues].

Enzymes catalyzing peroxidase reaction of a lysosomal fraction in bone marrow, leucocytes, spleen, thyroid gland, stomach, kidney, heart, lungs, brain and skeletal muscle of mice were investigated by immunochemical methods. A high level of peroxidase activity was discovered in leucocytes, bone marrow, spleen, heart and lung, a lower activity appeared to be characteristic of liver, thyroid gland and kidney. The peroxidase activities in brain, skeletal muscle and stomach were low. The reaction of immunoprecipitation with myeloperoxidase-specific antiserum revealed considerable antigenic distinctions between the enzymes catalysing peroxidase reaction in various tissues of mice.

Animals↗
PubMed1996

Brain superoxide dismutase, catalase, and glutathione peroxidase activities in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a fatal paralytic disorder of unknown cause. Recent evidence implicated the role of free radicals in the death of motor neurons in this disease. To investigate this hypothesis further, we measured the activity of the main free radical scavenging enzymes copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, and glutathione peroxidase in postmortem brain samples from 9 patients with sporadic amyotrophic lateral sclerosis and from 9 control subjects. We examined samples from the precentral gyrus of the cerebral cortex, a region affected in amyotrophic lateral sclerosis, and from the cerebellar cortex, a region not affected. The two groups did not differ in age or postmortem delay. In the precentral gyrus from amyotrophic lateral sclerosis samples, glutathione peroxidase activity as measured by spectrophotometric assay (13.8 +/- 2.6 nmol/min/mg protein [mean +/- standard error of mean]) was reduced significantly compared to the activity in the precentral gyrus from control samples (22.7 +/- 0.5 nmol/min/mg protein). In contrast, glutathione peroxidase activity was not significantly altered in the cerebellar cortex from amyotrophic lateral sclerosis patients compared to controls. Copper/zinc superoxide dismutase, manganese superoxide dismutase (corrected or not corrected for citrate synthase), and catalase were not significantly altered in the precentral gyrus or cerebellar cortex in the patient samples. This study indicated that glutathione peroxidase activity is reduced in a brain region affected in amyotrophic lateral sclerosis, thus suggesting that free radicals may be implicated in the pathogenesis of the disease.

Amyotrophic Lateral Sclerosis↗