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PubMed1978

Proton stoichiometry of the cytochrome c peroxidase mechanism as a function of pH.

The proton stoichiometry for the oxidation of cytochrome c peroxidase (ferrocytochrome c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) to cytochrome c peroxidase Compound I by H2O2, for the reduction of cytochrome c peroxidase Compound I to cytochrome c peroxidase Compound II by ferrocyanide, and for the reduction of cytochrome c peroxidase Compound II to the native enzyme by ferrocyanide has been determined as a function of pH between pH 4 and 8. The basic stoichiometry for the reaction is that no protons are required for the oxidation of the native enzyme to Compound I, while one proton is required for the reduction of Compound I to Compound II, and one proton is required for the reduction of Compound II to the native enzyme. Superimposed upon the basic stoichiometry is a contribution due to the perturbation of two ionizable groups in the enzyme by the redox reactions. The pKa values for the two groups are 4.9 +/- 0.3 and 5.7 +/- 0.2 in the native enzyme, 4.1 +/- 0.4 and 7.8 +/- 0.2 in Compound I, and 4.3 +/- 0.4 and 6.7 +/- 0.2 in Compound II.

Cytochrome-c Peroxidase↗
PubMed1981

The protoporphyrin-apoperoxidase complex as a horseradish peroxidase analog. A fluorimetric study of the heme pocket.

Similarity of the protein tertiary structures of the native horseradish peroxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) and protoporphyrin-apoperoxidase complex has been shown on the basis of identity of the tryptophan fluorescence parameter at pH 2.0-8.0 and of the circular dichroism spectra of the two proteins. Absorption and fluorescence spectra have been obtained for protoporphyrin in the complex in the pH range 7.0-1.6. A shift in the apparent pK by 4 units has been observed for protonation of the protoporphyrin pyrrolic ring in the complex. From this shift, the dielectric constant has been evaluated for the heme pocket of the peroxidase (approx. 20). Fluorescence quantum yield of protoporphyrin in the complex increased with pH decreasing from 5.0 to 3.5, whereas the spectrum pattern and fluorescence lifetime did not change. The ions, I- and [Fe(CN)6]-4, peroxidase substrates, did not quench the protoporphyrin fluorescence in the complex at about neutral pH, whereas the quenching markedly enhanced with lowering pH. The bimolecular constant for the I- -quenching of the porphyrin fluorescence on the complex showed a pH-dependence similar to that of the bimolecular rate constant for the reaction of peroxidase compound I with I-. Mechanism for I- oxidation at an acid pH in the presence of peroxidase has been proposed.

Apoenzymes↗
PubMed1975

Proton magnetic resonance studies of peroxidases from turnip and horseradish.

Proton NMR spectra at 270 MHz have been measured for horseradish peroxidase and turnip peroxidase isoenzymes (P1, P2, P3 and P7) in both their high spin ferric native states and as the low spin ferric cyanide complexes. Resonances of amino acids near the heme have been identified and used to investigate variations in the structure of the heme crevice amongst the enzymes. Ligand proton resonances have been resolved in spectra of the cyanide complexes of the peroxidases and these provide information on the heme electronic structure. The electronic structure of the heme and the tertiary structure of the heme crevice are essentially the same in the acidic turnip isoenzymes, P1, P2 and, to a lesser extent, P3 but differ in the basic turnip enzyme, P7. The heme electronic structure and nature of the iron ligands in peroxidases are discussed. Further evidence is presented for histidine as the proximal ligand. A heme-linked ionizable group with a pK of 6.5 has been detected by NMR in the cyanide complex of horseradish peroxidase.

Binding Sites↗
PubMed1977

Magnetic circular dichroism studies on acid and alkaline forms of horseradish peroxidase.

The heme vicinities of the acid and alkaline forms of native (Fd(III)) horseradish peroxidase were investigated in terms of the magnetic circular dichroism (MCD) spectroscopy. The MCD spectrum of the acid form of native horseradish peroxidase was characteristic of a ferric high spin heme group. The resemblance in the MCD spectrum between the acid form and acetato-iron (III)protoporphyrin IX dimethyl ester suggests that the heme iron of the acid form has the electronic structure similar to that in a pentocoordinated heme complex. The MCD spectra of native horseradish peroxidase did not shown any substantial pH dependence in the pH range from 5.20 to 9.00. The MCD spectral change indicated the pK value for the equilibrium between the acid and alkaline forms to be 11.0 which agrees with the results from other methods. The alkaline form of native horseradish peroxidase at pH 12.01 exhibited the MCD spectrum of a low spin complex. The near infrared MCD spectrum suggests that the alkaline form of native horseradish peroxidase has a 6th ligand somehow different from a normal nitrogen ligand such as histidine or lysine. It implicates that the alkaline form has an overall ligand field strength of between the low spin component of metmyoglobin hydroxide and metmyoglobin azide.

Circular Dichroism↗
PubMed1980

A 19F nuclear magnetic resonance study of the interaction between cytochrome c and cytochrome c peroxidase.

The reaction between ferrocytochrome c and yeast cytochrome c peroxidase was studied using cytochrome c derivatives specifically trifluoroacetylated at single lysine amino groups. The only modifications that decreased the reaction rate were those of lysines immediately surrounding the heme crevice, lysines 13, 25, 79, and 87. Modification of lysines 22, 55, 88, and 99 had no effect on the reaction. The 19F chemical shifts of the cytochrome c derivatives trifluoroacetylated at lysines 13, 79, and 87 were not changed upon complex formation with cytochrome c peroxidase, indicating that no detectable conformational changes occurred. The cytochrome c trifluoroacetyl groups had the same T1 values in the paramagnetic fluorocytochrome c peroxidase complex as in the diamagnetic reduced form of the complex, indicating that they were more than 2.3 nm from the paramagnetic iron atom in cytochrome c peroxidase. This is consistent with a separation of at least 1.5-2.0 nm between the iron atom of cytochrome c and the iron atom of cytochrome c peroxidase.

Animals↗
PubMed1984

A chemical modification to make horseradish peroxidase soluble and active in benzene.

Horseradish peroxidase was modified with 2,4-bis(O- methoxypolyethylene glycol)-6-chloro-s-triazine. The modified peroxidase, in which 60% of the amino groups were coupled with polyethylene glycol, had 70% of the enzymic activity in aqueous solution and was found to be soluble in benzene. Since the modified peroxidase in benzene had an absorption spectrum similar to that of unmodified peroxidase in aqueous solution, the prosthetic group, protohaemin IX, remained with the apoprotein even in benzene. The modified peroxidase in benzene had 21% of the enzymic activity relative to that of unmodified enzyme in aqueous solution.

Benzene↗
PubMed1985

Detection of a catalytic intermediate of peroxidase in hog thyroid microsomes.

A catalytic intermediate, Compound II of peroxidase was detected spectrophotometrically in thyroid microsomes. From comparison with the spectral data on purified thyroid peroxidase, the content of the peroxidase was estimated to be 0.019 nmol per mg of the microsomal protein, being about one-eighth of the amount of cytochrome b5. It was concluded that thyroid peroxidase exhibits the same peroxidase activity for guaiacol or ascorbate in the free and the microsome-bound forms.

Animals↗
PubMed1992

Inhibition of the human leukocyte enzymes myeloperoxidase and eosinophil peroxidase by dapsone.

Dapsone (4,4'-diaminodiphenylsulfone) is an antimicrobial substance that also has anti-inflammatory activity, which has been attributed to inhibition of the leukocyte enzyme myeloperoxidase (MPO). We observed that dapsone was a much better inhibitor of the eosinophil peroxidase (EPO) in an assay that measured peroxidase-catalyzed oxidation of tetramethylbenzidine at pH 5.4. To clarify the specificity and pH-dependence of dapsone inhibition of the purified enzymes under more physiologic conditions, we studied peroxidase-catalyzed oxidation of chloride to the antimicrobial and cytotoxic agent hypochlorous acid. Taurine was added as a trap for hypochlorous acid, to prevent inactivation of the enzymes or chlorination of dapsone by hypochlorous acid. Dapsone was much more effective as an inhibitor of both MPO and EPO when chloride rather than tetramethylbenzidine was the substrate. Inhibition of both enzymes was greater at neutral pH than at acid pH (pH 7 vs pH 5), but EPO was more sensitive to inhibition than MPO regardless of pH. Inhibition was increased by lowering chloride, raising hydrogen peroxide, or lowering the enzyme concentration. Inhibition was accompanied by irreversible loss of enzyme activity, which was correlated with loss of the heme absorption spectrum, indicating chemical modification of the enzyme active site. EPO, but not MPO, was partially protected against inactivation by adding physiologic levels of bromide along with chloride. The results suggest that dapsone could prevent MPO- and EPO-mediated tissue injury at sites where the peroxidase enzymes are secreted and diluted into the neutral pH environment of the tissue interstitial space. Dapsone might not inhibit peroxidase-mediated antimicrobial activity, which occurs at high enzyme concentrations in the acid environment of phagolysosomes.

Benzidines↗
PubMed1984

Localization of axonally transported label in chick retinal ganglion cell axons after intravitreal injections of wheat germ agglutinin conjugated to horseradish peroxidase.

We have studied the subcellular localization of peroxidase-labeled organelles after anterograde axonal transport by chick retinal ganglion cells that had been exposed 23-25 h earlier to wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). After intravitreal injection of WGA-HRP, we found in the optic tectum that 82% of labeled organelles were located within axons and axon terminals. The organelles included: tubules and cisternae of the smooth endoplasmic reticulum, hypolemmal cisternae, vesicles, dense bodies and multivesticular bodies. We also measured the distances between the centers of the labeled organelles and the plasma membrane of these profiles. The density of organelles (number of organelles/micron 2) was plotted as a function of distance from the plasma membrane. Irrespective of the dose of lectin-peroxidase injected, labeled organelles were most densely concentrated in a 30 nm wide annular zone centered 75 nm in from the plasma membrane. In axon terminals the labeled organelles were most concentrated 75-90 nm in from the plasma membrane. Assuming that the peroxidase label indicates the presence of WGA-HRP, we conclude that after anterograde axonal transport the lectin accumulates in lysosomal organelles and elements of the smooth endoplasmic reticulum. Therefore, in contrast to the more restricted localization of [125I]WGA as inferred from electron microscopic autoradiography after uptake and transport by the same cell type, WGA-HRP-labeled organelles are found more diffusely within the axoplasm, particularly in axon terminals. Furthermore, peroxidase-labeled organelles in dendritic, glial or neuronal cell bodies in the tectum were seen less frequently than expected based on evidence of frequent transfer to second cells after intravitreal injections of [125I]WGA. Thus, we infer that at these concentrations WGA labeled with HRP may not be transferred intercellularly as efficiently as even lower concentrations of iodinated WGA are apparently transferred.

Animals↗
PubMed1989

Enhancement of the peroxidase-mediated oxidation of butylated hydroxytoluene to a quinone methide by phenolic and amine compounds.

We have recently demonstrated that butylated hydroxyanisole (BHA) markedly stimulates the peroxidase-dependent oxidation of butylated hydroxytoluene (BHT) to the potentially toxic BHT-quinone methide. Using both horseradish peroxidase and prostaglandin H synthase we now report the ability of a wide variety of compounds to stimulate peroxidase-dependent activation of BHT. These compounds include several phenolic compounds commonly present in pharmacologic preparations or occurring naturally in foods. The ability of a given compound to stimulate BHT oxidation was found to depend on the type of radical it forms upon peroxidase oxidation. Compounds which have been shown to form phenoxy radicals or nitrogen-centered cation radicals were observed to enhance BHT oxidation. Conversely, compounds which are known to form peroxy radicals or semiquinone radicals either inhibited or had no effect on BHT oxidation. Compounds which enhanced BHT oxidation (monitored by covalent binding of [14C]BHT to protein) were also observed to stimulate the formation of BHT-quinone methide and stilbenequinone. This suggested a common mechanism of interaction of these compounds with BHT. The stimulation of BHT covalent binding by BHA was also seen in various human and animal tissues using either arachidonic acid or hydrogen peroxide as substrate. The possible toxicologic implications of the enhancement of peroxidase-catalyzed BHT oxidation to BHT-quinone methide are discussed.

Animals↗
PubMed1986

Peroxidase activity and estradiol receptors in human breast cancer.

In order to establish the relation between peroxidase activity in human breast carcinoma and its estrogen-dependency, estradiol receptors and their peroxidase activities were determined by a very sensitive fluorimetric method in 61 carcinomas. The geometrical mean of peroxidase activity in the 34 estradiol receptor negative carcinomas, 12.8 mU/g tissue, was significantly higher (p less than 0.05) than that of the 27 estradiol receptor positive cases, with a mean of 10.57 mU/g tissue. Necrosis and/or inflammation were found more frequently in the estradiol receptor negative carcinomas (p less than 0.01) than in the estradiol receptor positive. It is concluded that peroxidase activity is linked more to the typical biological behavior of the tumor than to tumoral estrogen-dependency. Thus, the estradiol receptor negative tumors, which showed a more aggressive behavior associated with frequent necrosis and intratumoral inflammatory signs, had a greater peroxidase activity.

Adult↗
PubMed1987

Monoclonal anti-peroxidase isotype switch variants. Applications in studies of protein A binding and characterization of rat monoclonal antibodies.

A series of heavy chain isotype switch variants was derived from a hybridoma cell line secreting monoclonal antibodies specific for horseradish peroxidase. By the combined use of sensitive isotype-specific ELISAs and sequential sublining IgG2b, IgG2a, IgE and Iga anti-peroxidase-producing variants were successively isolated out of IgG1-secreting parental cells. The anti-peroxidase isotype variant antibodies are particularly appropriate for use in studies of the influence of heavy chain isotype in the effector functions of immunoglobulins. The use of variant antibodies with specificity for an enzyme favors their application in immunoassays because an enzyme-conjugated second antibody is not needed. Here we describe two applications of the anti-peroxidase switch variants. First, the variants are compared with respect to their affinity for Staphylococcus protein A. While IgG1 anti-peroxidase showed weak binding, both IgG2 variants strongly bound to protein A, whereas IgE and IgA variants had no affinity for protein A. Next, the switch variants were used to determine the isotype specificity of rat monoclonal antibodies generated to murine IgE.

Animals↗
PubMed1988

Peroxidase-induced enhancement of chemiluminescence by murine peritoneal macrophages.

A number of substances have been shown to enhance the respiratory burst (RB) of macrophages. Many of these substances are not normally found in vivo. The present study suggests that a group of enzymes characterized as peroxidases have the ability to significantly enhance the RB and concomitant phagocytosis by murine peritoneal macrophages. Horseradish peroxidase (HRP), lactoperoxidase (LPO), and microperoxidase (MPO) can significantly augment these functions. Both resident and thioglycollate-induced macrophages exhibited enhanced chemiluminescence (CL) upon exposure to HRP, however, the effect was more pronounced with the latter. The increase in CL was correlated with an increase in production of superoxide, which was measured by reduction of cytochrome c. Horseradish peroxidase immobilized on an inert carrier, was capable of enhancing the RB suggesting that it does not have to enter the cell in order to function. Hemin, hematoheme and hematoporphyrin had little effect on macrophage stimulated CL. All of the peroxidases tested caused increased phagocytosis of opsonized zymosan. These studies indicate that peroxidases are capable of stimulating the RB, phagocytosis and possibly other macrophage functions.

Animals↗
PubMed1985

Physicochemical characterization of the alkaline denaturation of cytochrome c peroxidase.

The alkaline denaturation of cytochrome c peroxidase and apocytochrome c peroxidase was investigated by analytical ultracentrifugation, gel-filtration chromatography, and circular dichroism. The results indicate that both cytochrome c peroxidase and the apoenzyme undergo extensive structural modifications upon exposure to alkaline pH, including dimer formation. The midpoint of the transition for dimer formation in the native enzyme occurs at pH 9.6 +/- 0.1, while loss of tertiary and secondary structure occurs with transition midpoints at pH 10.3 +/- 0.1 and pH 11.3 +/- 0.1, respectively. Studies performed in the presence of dithiothreitol and with carboxymethylated cytochrome c peroxidase indicate that dimer formation occurs via a disulfide crosslink involving the single cysteine residue in the enzyme. Denaturation of cytochrome c peroxidase in the presence of guanidine hydrochloride gave results similar to those obtained for the alkaline denaturation.

Apoproteins↗
PubMed1985

Unique cyanide nitrogen-15 nuclear magnetic resonance chemical shift values for cyano-peroxidase complexes. Relevance to the heme active-site structure and mechanism of peroxide activation.

Cyanide ion has been utilized to probe the heme environment of the ferric states of horseradish peroxidase, lactoperoxidase and chloroperoxidase. The 15N-NMR signal for cyanide bound to these enzymes is located in the downfield region from 578 to 412 ppm (with respect to the nitrate ion reference). The corresponding signal for met-forms of hemoglobin, myoglobin and cytochrome c is much further downfield in the 1047-847 ppm region. The signal position for peroxidases is quite invariant with pH in the physiological ranges. The upfield bias for peroxidase chemical shifts must reflect unique trans iron(III) ligand types and/or proximal-group hydrogen bonding or steric effects. Model compound studies reveal a significant upfield cyanide 15N shift with addition of agents capable of hydrogen-bonding to the coordinated cyanide ion. An even more striking upfield shift of 277 ppm is associated with deprotonation of a trans imidazole residue. The distinctive chemical shifts observed for the cyano ligand in peroxidases support the hypothesis that a distal hydrogen-bonding network and perhaps a polar, basic trans ligand are essential for O-O bond activation by peroxidases.

Animals↗
PubMed1987

A proton NMR study of the non-covalent complex of horse cytochrome c and yeast cytochrome-c peroxidase and its comparison with other interacting protein complexes.

Cytochrome-c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) forms a noncovalent 1:1 complex with horse cytochrome c in low ionic strength solution that is detectable by proton NMR spectroscopy. When the entire proton hyperfine-shifted spectrum is considered only five hyperfine resonances exhibit unambiguously detectable shifts: the heme 8-CH3 and 3-CH3 resonances, single proton resonances near 19 ppm and -4 ppm and the methionine-80 methyl group. These shifts are very similar to those observed for the covalently crosslinked complex of cytochrome-c peroxidase and horse cytochrome c, but different from those reported for cytochrome c complexes with flavodoxin and cytochrome b5. By comparison with the shifts reported for lysine-13-modified cytochrome c we conclude that the results reported here support the Poulos-Kraut proposed structure for the molecular redox complex between cytochrome-c peroxidase and cytochrome c. These results indicate that the principal site of interaction with cytochrome-c peroxidase is the exposed heme edge of horse cytochrome c, in proximity to lysine-13 and the heme pyrrole II. The noncovalent cytochrome-c peroxidase-cytochrome c complex exists in the rapid-exchange time limit even at 500 mHz proton frequency. Our data provide an improved estimate of the minimum off-rate for exchanging cytochrome c as 1133 (+/- 120) s-1 at 23 degrees C.

Animals↗
PubMed1987

A covalent complex between horse heart cytochrome c and yeast cytochrome c peroxidase: kinetic properties.

The kinetic properties of a 1:1 covalent complex between horse-heart cytochrome c and yeast cytochrome c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) have been investigated by transient-state and steady-state kinetic techniques. Evidence for heterogeneity in the complex is presented. About 50% of the complex reacts with hydrogen peroxide with a rate 20-40% faster than that of native enzyme; 20% of the complex exists in a conformation which does not react with hydrogen peroxide but converts to the reactive form at a rate of 20 +/- 5 s-1; 30% of the complex does not react with hydrogen peroxide to form the oxidized enzyme intermediate, cytochrome c peroxidase Compound I. Intramolecular electron transfer between covalently bound ferrocytochrome c and an oxidized site in cytochrome c peroxidase Compound I is too fast to measure, but a lower limit of 600 s-1 can be estimated at 5 degrees C in a 10 mM potassium phosphate buffer at pH 7.5. Free ferrocytochrome c reduces cytochrome c peroxidase Compound I covalently bound to ferricytochrome c at a rate 10(-4) to 10(-5)-times slower than for free Compound I. The transient-state ferrocytochrome c reduction rates of Compound I covalently linked to ferricytochrome c are about 70-times too slow to account for the steady-state catalytic properties of the 1:1 covalent complex. This indicates that hydrogen peroxide can interact with the 1:1 complex at sites other than the heme of cytochrome c peroxidase, generating additional species capable of oxidizing free ferrocytochrome c.

Ascorbic Acid↗
PubMed1982

Human milk peroxidase is derived from milk leukocytes.

Peroxidase enzymes present in human colostrum, saliva, polymorphonuclear leukocytes, and bovine milk were compared with respect to their molecular exclusion chromatographic behavior and immunological cross-reactivity. Human milk peroxidase gave an elution profile similar to myeloperoxidase derived from blood polymorphonuclear leukocytes. Human salivary peroxidase reacted with an antibody directed against bovine lactoperoxidase, but with the same antibody preparation no reaction was detected either with human milk peroxidase or leukocyte myeloperoxidase. We conclude that the peroxidase enzyme in human milk is different from the human salivary and the bovine enzymes and is probably derived from milk leukocytes.

Colostrum↗