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At least 19 recordsLinked to original sources

PubMed1979

Comparison between peroxidase-conjugated antigen or antibody and peroxidase-anti-peroxidase complex in a postembedding procedure.

The staining efficiency of peroxidase labeled immunoglobulin conjugate, used either as antigen or as antibody, has been compared with that of peroxidase-anti-peroxidase complex (PAP) on ultrathin sections of araldite embedded material. The conjugate gave positive results in a two layer method as well as in a three layer method when used as antibody. No staining was observed when it was used as antigen. The conjugation seemed to impair the antigenic reactivity of immunoglobulin. The conjugate when used as antibody in the three layer method gave approximately the same staining efficiency as PAP.

Animals↗

PubMed1976

Peroxidase and fluorescein isothiocyanate as antibody markers. A quantitative comparison of two peroxidase conjugates prepared with glutaraldehyde or periodate anda fluorescein conjugate.

Batches of rabbit anti-human immunoglobulin G antibodies were labeled either with horseradish peroxidase, using the two-step glutaraldehyde method or the periodate method, or with fluorescein isothiocyanate (FITC). The peroxidase conjugates were isolated by chromatography using two different gel types. The five types of conjugates thus obtained were standardized to the same amount of rabbit immunoglobulin G. The antibody activity, as estimated by means of single radial immunodiffusion and passive hemagglutination, and the enzyme activity, determined with orthodianisidine, were compared. The ultimate dilutions and absolute amounts of the five conjugates giving positive reactions were determined in direct and indirect immunohistochemical tests, using both cryostat sections of skin and the agarose bead model system. It appeared that during the peroxidase conjugation procedures there was a considerable loss of abtibody and enzyme activity, whereas in the FITC conjugation procedure the antibody activity remained intact. Neverthe less, peroxidase conjugates prepared with glutaraldehyde still gave positive staining reactions in equal or somewhat higher dilutions than the fluorescin conjugate did. The peroxidase conjugates prepared with periodate could not be diluted to the same extent. For the detection of antibodies by indirect immunohistochemical methods, the peroxidase conjugate, prepared with glutaraldehyde, was comparable to the FITC conjugate. The peroxidase conjugate, prepared with periodate, was less effective.

Aldehydes↗

PubMed1992

1H NMR investigation of manganese peroxidase from Phanerochaete chrysosporium. A comparison with other peroxidases.

1H NMR spectra at 200- and 600-MHz of manganese peroxidase from Phanerochaete chrysosporium and of its cyanide derivative are reported. The spectrum of the native protein is very similar to that of other peroxidases. The assignment of the spectrum of the cyanide derivative has been performed through 1D NOE, 2D NOESY, and COSY experiments. This protein is very similar to lignin peroxidase, the only meaningful difference being the shift of H delta 2 of the proximal histidine. The spectra of the cyanide derivative of these two proteins are compared with those of horseradish peroxidase and cytochrome c peroxidase. The shift pattern of the protons of the proximal histidine is discussed relative to the structural properties which affect the Fe3+/Fe2+ redox potential.

Chemical Phenomena↗

PubMed1978

[Chemical modification of lysine epsilon-NH2-groups in horseradish peroxidase. Its effect on enzyme stability. Temperature dependence of thermo-inactivation constants for native and modified peroxidase].

Thermostability of horseradish peroxidase modified by acetic, propionic, butyric, valeric and succinic anhydrides and trinitrobenzolsulfonic acid (TNBS) is studied within the temperature range of 56-80 degrees C. Acylation of 4 amino groups and arylation of 3 amino groups with TNBS are found to stabilize the enzyme, while modification of 6 groups decreases the enzyme stability. Chemical modification of peroxidase does not change its pH-dependence with respect to enzyme thermostability. Thermodynamic activation parameters of irreversible thermoinactivation are determined for native and modified peroxidase. Native peroxidase has deltaH not equal to = 30+/-1 kcal/mole and deltaS not equal to = 14 e. e.; modified by acid anhydrides peroxidase has deltaH not equal to within 64-87 kcal/mole and deltaS not equal to within 110-178 e. e. depending on the nature of a modifying agent. The effect of the structure of a radical introduced into the enzyme molecule, and of a number of modified epsilon-amino groups on thermoinactivation deltaH not equal to and deltaS not equal to values is discussed.

Acylation↗

PubMed1992

Peroxidase and coupling activities of thyroid peroxidase in benign and malignant thyroid tumor tissues.

The coupling activity of thyroid peroxidase (TPO) in thyroid glands from patients with benign adenoma, papillary carcinoma, and diffuse goiter (Graves' disease) was measured for the first time, in addition to the peroxidase activity of these tissues. The peroxidase activity of TPO in the mitochondria-microsomes fraction was measured with guaiacol or iodide as the second substrate. In the case of papillary carcinoma, the mean protein-based specific activity obtained by the guaiacol assay was about 1/7 of that of diffuse goiter. The iodide oxidation activity of carcinoma was very low, about 1/25 [corrected] of that in diffuse goiter and 1/70 of that in adenoma. The peroxidase activity in adenoma was almost similar in the guaiacol oxidation assay and approximately one half in the iodide oxidation assay as compared with that in diffuse goiter. There was a close correlation between the guaiacol and iodide oxidation assays in individual patients with adenoma and diffuse goiter, but not in patients with papillary carcinoma. The coupling activity of TPO was measured with thyroglobulin purified from pooled toxic diffuse goiters and chemically iodinated to contain little additional T3 and T4. The specific coupling activity of TPO in mitochondria-microsomes from carcinoma was significantly lower (about 1/5) than that of diffuse goiter, and the activity in adenoma was not significantly different (about 1/2) from that of diffuse goiter. The data of coupling activities has a close correlation with that of peroxidase activities in individual patients with adenoma but not in patients with carcinoma. Based on these findings, the qualitative abnormality of TPO and its relation to the cold 123I scintigram in thyroid tumors are discussed.

Adenoma↗

PubMed1978

pH titration study of cytochrome c peroxidase and apocytochrome c peroxidase.

A pH titration study of cytochrome c peroxidase and apocytochrome c peroxidase was carried out at 25 degrees C and 0.1 M ionic strength. The net charge on cytochrome c peroxidase due to proton association and dissociation varies from +32 at pH 2 to --50.2 at pH 12, while that of apocytochrome c peroxidase varies between +24.5 at pH 3 to --48 at pH 12. The apoprotein tented to aggregate below pH 3. Between pH 4 and 8, the titration behavior of both the native enzyme and the apoenzyme are consistent with the semi-empirical Linderstrøm-Lang theory. Between pH 9 and 12, the titration behavior of both the holo- and apoproteins suggest they assume a more extended conformation which reduces the electrostatic interaction charged groups on the surface. In the acid region, between pH 4 and 3, a similar transition occurs in which the protein expands 40% based on the electrostatic factor of the Linderstrøm-Lang theory.

Apoenzymes↗

PubMed1992

Vanadium effect on the activity of horseradish peroxidase, catalase, glutathione peroxidase, and superoxide dismutase in vitro.

The effect of vanadium (V) on the activity of horseradish peroxidase, catalase, glutathione peroxidase, and superoxide dismutase has been studied. A competitive inhibition pattern was evident for vanadate ions on the activity of horseradish peroxidase (Ki = 41.2 microM). No significant inhibitory effects were found when V(V) was tested with catalase and when either V(IV) or V(V) were assayed with glutathione peroxidase. For the latter, the effect of V on the different components of the reaction system was investigated. V(V) did not significantly affect SOD activity when assayed with the sulfite method, which is devoid of interferences with V(V); however, there was an apparent inhibitory dose-response pattern for either V(IV) or V(V) using the pyrogallol assay, owing to an interference of pyrogallol with the metal. Besides, no significant binding of V(IV) or V(V) to the enzyme could be demonstrated. The lack of a direct inhibitory effect of V on the activity of the main antioxidant enzymes suggests that many biological and toxicological effects of V may be mediated more by oxidative reactions of the metal or of its complexes with physiologically relevant biomolecules than by a direct modulation of enzymatic activities.

Catalase↗

PubMed1992

Homology modeling of a heme protein, lignin peroxidase, from the crystal structure of cytochrome c peroxidase.

A 3-dimensional model of lignin peroxidase (LiP) was constructed based on its sequence homology with other peroxidases, particularly cytochrome c peroxidase, the only protein with a known crystal structure in the peroxidase family. The construction of initial conformations of insertions and deletions was assisted by secondary structure predictions, amphipathic helix predictions, and consideration of the specific protein environment. A succession of molecular dynamics simulations of these regions with surrounding residues as constraints were carried out to relax the bond lengths and angles. Full protein molecular dynamics simulations with explicit consideration of bound waters were performed to relax the geometry and to identify dynamically flexible regions of the successive models for further refinement. Among the important functionally relevant structural features predicted are: (i) four disulfide bonds are predicted to be formed between Cys3 and Cys15, Cys14 and Cys285, Cys34 and Cys120 and Cys249 and Cys317; (ii) a glycosylation site, Asn257, was located on the surface; (iii) Glu40 was predicted to form a salt bridge with Arg43 on the distal side of the heme and was considered as a possible origin for the pH dependence of compound I formation; and (iv) two candidate substrate binding sites with a cluster of surface aromatic residues and flexible backbones were found in the refined model, consistent with the nature of known substrates of LiP. Based on these predicted structural features of the model, further theoretical and experimental studies are proposed to continue to elucidate the structure and function of LiP.

Amino Acid Sequence↗

PubMed1979

Amino acid sequence studies of horseradish peroxidase. Amino and carboxyl termini, cyanogen bromide and tryptic fragments, the complete sequence, and some structural characteristics of horseradish peroxidase C.

Horseradish peroxidase C dominates quantitatively among the isoperoxidases of horseradish root and has an isoelectric point close to 9. It consists of a hemin prosthetic group, 2 Ca2+ and 308 amino acid residues, including 4 disulfide bridges, in a single polypeptide chain that carries 8 neutral carbohydrate side-chains. The molecular weight of the polypeptide chain is 33890. Assuming an average carbohydrate composition of (GlcNAc)2, Man3, Fuc, Xyl for each carbohydrate chain, the molecular weight of native horseradish peroxidase C is close to 44 000. Cyanogen bromide fragments of reduced and carboxymethylated apo-peroxidase were purified by a combination of gel filtration and isoelectric focusing in urea, and cystine-containing tryptic fragments of apo-peroxidase were purified by gel filtration followed by disulfide cleavage and rechromatography at the initial conditions. The present paper discusses (a) isoelectric points and charge distribution within the native protein, the apoprotein and the cyanogen bromide fragments, (b) a buried pyrrolidonecarboxylyl amino terminus, (c) heterogeneity at the carboxyl terminus, and (d) a possible domain structure, likely from partial tryptic digestion.

Amino Acid Sequence↗

PubMed1978

Rapid preparation of peroxidase: anti-peroxidase complexes for immunocytochemical use.

Soluble immune complexes of horseradish peroxidase and antibody to peroxidase (PAP) have been widely used in the 'unlabelled antibody' method for the immunocytochemical detection of cellular antigens. This paper describes a simple and rapid method for preparation of these complexes by column chromatography of a mixture of the enzyme and the IgG fraction of antiperoxidase antiserum on Sephacryl S-200. The material eluting in the void column consists of stable soluble PAP complexes, with a molar peroxidase: antiperoxidase ratio of 0.8 and a molecular weight of approximately 400,000. When tested immunocytochemically this material gives identical results to those obtained with conventionally prepared PAP.

Animals↗

PubMed1977

[Peroxidases of high molecular weight identified as the membrane peroxidases in lentils].

Peroxidases extracted from lentil roots are separated in two peaks by gel chromatography on Sephadex G-100 or on Bio Gel A-5 M. On both resins, the first peak of extremely large molecular weight is demonstrated to be an association of some peroxidases with microsomes. These enzymes can be detached from membranes by NaCl. Starch gel electrophoresis shows that isoperoxidases associated electrostatically to microsomes are basic peroxidases apparently not different from those of the soluble fraction.

Cell Membrane↗

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↗

PubMed1992

cAMP-mediated differential regulation of lignin peroxidase and manganese-dependent peroxidase production in the white-rot basidiomycete Phanerochaete chrysosporium.

Lignin peroxidases (LIPs) and manganese-dependent peroxidases (MNPs) are major components of the lignin-degrading enzyme system of Phanerochaete chrysosporium and typically appear during secondary metabolism. The involvement of cAMP in the regulation of production of LIPs and MNPs was investigated in this study. Production of LIPs and MNPs was preceded by a sharp rise in intracellular cAMP concentration. Addition of atropine, theophylline, or histamine to cultures resulted in a drop in intracellular cAMP concentration and a concomitant inhibition of production of LIPs only or of both LIPs and MNPs, depending on the concentration of the inhibitor added. These results were independently confirmed by fast protein liquid chromatographic profiles of the LIPs and MNPs in the extracellular fluid of the inhibitor-treated and untreated control cultures. LIP production was generally more sensitive to the inhibitors than MNP production. Northern blot analyses showed that the inhibitors affect the production of LIPs and MNPs at the level of transcription. Furthermore, LIP and MNP gene expression appears to be differentially regulated depending on the intracellular concentration of cAMP. These results show that cAMP plays a key role in the regulation of production of LIPs and MNPs in P. chrysosporium.

Atropine↗

PubMed1975

The location of bacterial antigens on sections of Bacillus cereus by use of the soluble peroxidase--anti-peroxidase complex and unlabelled antibody.

The location of antigens on sections of bacteria using the soluble peroxidase-anti-peroxidase complex in conjunction with unlabelled antibody is described. Using this technique, spore antigens have been detected in the cytoplasm of vegetative cells during forespore septum formation and subsequent stages of sporulation. Antigenic sites were first associated with poly-beta-hydroxybutyric acid granules and subsequently were found in increasing quantities in the cytoplasm of the sporangium. Vegetative cell antigens were located on the cell wall and in the cortical region during sporulation. During germination antigens were located in the cortical region, and during outgrowth on the cell wall. These findings are discussed in the light of existing biochemical data.

Antibodies, Bacterial↗

PubMed1979

Immunohistochemical localization of procollagens. I. Light microscopic distribution of procollagen I, III and IV antigenicity in the rat incisor tooth by the indirect peroxidase-anti-peroxidase method.

Frozen sections of the growing end of the rat incisor tooth were exposed to antisera or affinity prepared antibodies against partially purified type I, II, or IV procollagen in the hope of detecting the location of the corresponding antigens by the peroxidase-anti-peroxidase technique. The distribution of immunostaining was similar with antisera as with purified antibodies of a given type, but differed for each type; that is, predentin, odontoblasts, pulp and periodontal tissue were the sites of type I; blood vessel walls, pulp and periodontal tissue, of type III; and basement membranes, of type IV antigenicity. It was demonstrated, at least in cases of type I and III, that immunostaining detected the corresponding procollagens and related substances, but not the corresponding collagens. The interpretation of these observations is that: 1) odontoblasts elaborate procollagen I for release to predentin and subsequent transformation to dentinal collagen I; 2) pulp and periodontal cells produce procollagens I and III which presumably become collagens I and III respectively, while the adventitial cells of blood vessels give rise to collagen III; and 3) procollagen IV is associated with basement membranes and, occasionally, adjacent cells.

Animals↗

PubMed1978

A comparison of methods using diaminobenzidine (DAB) to localize peroxidases in erythrocytes, neutrophils, and peroxidase-antiperoxidase complex.

Reactions using diaminobenzidine (DAB) to localize the enzyme peroxidase in neutrophils and peroxidase-antiperoxidase (PAP) complex during immunological staining are usually performed in Tris-HCl or phosphate buffer at pH 7.2-7.6. However, DAB solutions at pH 7.2-7.6 often demonstrate erythrocyte pseudoperoxidase as well. By lowering the pH of the DAB solutions, it is possible to selectively suppress the reactivity of pseudoperoxidase while maintaining optimal reactions in neutrophils and PAP complex. For this purpose we recommend ammonium acetate-citric acid buffer at pH 5.5 (pH 5.0-6.0) containing 44 mg DAB per 100 ml buffer and 0.003%-0.03% with respect to H2O2.

3,3'-Diaminobenzidine↗

PubMed1978

Demonstration of the Fc-receptor of blood cells by soluble peroxidase-anti-peroxidase (PAP) complexes.

The Fc-receptor of normal human leukocytes, of CLL-cells, and of hematopoietic cell lines was demonstrated with soluble peroxidase-anti-peroxidase (PAP) complexes. In about 9% of normal lymphocytes an almost continuous, strong labeling of the cell membrane was established. Some of these lymphocytes were characterized by a peculiar uniform fine structure. The percentage of PAP-labeled monocytes was in the range of 25%, neutrophils nearly 100%, eosinophils 0%, CLL-cells 10%. Labeled portions of the membrane were interiorized from monocytes. The lymphoid cell-line Daudi established from a Burkitt's lymphoma appeared almost negative, the cell line K562 established from a myeloid leukemia in 75% of the cells strongly positive. PAP-labeling was not influenced by preincubation with trypsine or with neuraminidase; it was negative when PAP-F(ab)2 was used. Results of PAP-labeling were not always in agreement with EA-rosettes or with agg-Ig.

Binding Sites↗

PubMed1978

Neutralization tests for dengue and Japanese encephalitis viruses by the focus reduction method using peroxidase-anti-peroxidase staining.

Neutralization tests were made on 4 types of dengue (DEN) virus and Japanese encephalitis (JE) virus by incubation of serially diluted antisera and constant amounts of the viruses and then focus assay of surviving virus infectivity with peroxidase-anti-peroxidase (PAP) staining. Neutralization reactions were virtually completed in 2 hr on incubation of serum-virus mixtures at 28 C. A straight regression line was obtained on a probit chart by plotting the focus reduction rates at various dilutions of a given serum against the logarithm of the serum dilution used in the test. The slopes of the probit regression lines for the neutralization for DEN types 1 and 3 were similar, but differed somewhat from those for DEN type 2 and type 4. The slope of the line for JE virus was quite different from those for DEN viruses. Using these relations, the fifty percent focus reduction titer (FR50) of neutralizing antibodies of a given serum could be estimated from the focus reduction rates at several dilutions of the test serum when the latter was between 25-75% of the value of the control.

Animals↗