Peroxidase, horseradish

4 Peroxidase, Oxidase, and Superoxide Dismutase Horseradish Peroxidase.—Chlorine dioxide, CIO a, is formed by the disproportionation of chlorite ClOa catalysed by HRP, 

Azide binding to HRP is fast compared with normal haemoproteins, 

Morishima, S. Ogawa, T. Yonezawa, H. Nakatani, K. Hiromi, T. Sano, and T. Yasunaga, Biochem. Biophys. Res. Commun., 1978, 83, 724. 

The dependence of Ara on pH is complex and requires the presence of two ioniz-able groups. No kinetic isotope effects are noted in the [Fe(CN)s] reductions of HRP(i) and HRP(ii), which are affected by protonation of groups on the proteins with pATa values of 5.2 and 8.6 respectively. 

An enzyme-substrate complex has been detected in the oxidative dimerization of L-(-)-tyrosine by HRP(i) to give HRP(ii). The pH dependence of the reaction reveals an enzyme protonation at pATa 5.42 with a less reactive protonated form. The hydroperoxide of indole-3-acetic acid (lAA) is important in the autoxidation of lAA catalysed by HRP. Formation of HRP(i) by reaction with the hydroperoxide is easier for the neutral isoenzyme than for the acidic species at pH 4.4 and this determines the catalytic activity. HRP(ii) is detected as an intermediate in the reaction and it reacts with lAA to form radicals. The involvement of both HRP(i) and HRP(ii) is proposed in the autoxidation of 2-nitro-propane to acetone and HNO2, catalysed by HRP. 

HRP is a hemoprotein containing photohemin IX as its prosthetic group. The presence of the heme structure gives the enzyme its characteristic color and maximal absorptivity at 403 nm.The ratio of its absorbance in solution at 403 nm to its absorbance at 275 nm, called the RZ or Reinheitzahl ratio, can be used to approximate the purity of the enzyme. However, at least seven isoenzymes exist for HRP (Shannon et al., 1966 Kay et al., 1967 Strickland et al., 1968), and their RZ values vary from 2.50 to 4.19. Thus, unless the RZ ratio is precisely known or determined for the particular isoenzyme of HRP utilized in the preparation of an antibody-enzyme conjugate, subsequent measurement after crosslinking would yield questionable results in the determination of the amount of HRP present in the conjugate. 

The disadvantages associated with HRP are several. The enzyme only contains two available primary e-amine groups—extraordinarily low for most proteins—thus limiting its ability to be activated with amine-reactive heterobifunctionals. HRP is sensitive to the presence of many antibacterial agents, especially azide. It also is reversibly inhibited by cyanide and sulfide (Theorell, 1951). Finally, while the enzymatic activity of HRP is extremely high, its useful lifespan or practical substrate development time is somewhat limited. After about an hour of substrate turnover, in some situations its activity can be decreased severely. 

Nevertheless, HRP is by far the most popular enzyme used in antibody-enzyme conjugates. One survey of enzyme use stated that HRP is incorporated in about 80 percent of all antibody conjugates, most of them utilized in diagnostic assay systems. 

P-cresol and at least one other phenol, guaiacol, provide a different type of exception to the general mechanism Under certain conditions each of these phenols need only be present in only one-half molar equivalent 

Myeloperoxidase is the principal heme protein of the mammalian neutrophil functioning in the phagocytic process through its capacity at acid pH to peroxidize chloride ions to hypochlorous acid. Investigations of the structure of myeloperoxidase from various species have shown it to be a two-heme protein with a molecular weight in the region 135,000-150,000 (Agner, 1958, 1970 Harrison and Schultz, 1976 Harrison et al, 1977). 

The mechanism of action of the myeloperoxidase/H202/halide system is complex (Klebanoflf, 1975). The halide cofactor is oxidized by myeloperoxidase and hydrogen peroxide, and it is presumed that this leads to toxic agents. Recently interest has been focused on the possible role of singlet oxygen in the oxidation sequence (Rosen and Klebanoff, 1977). 

Myeloperoxidase isolated from tumor cells contains tightly bound porphyrins that are difficult to remove. The ESR spectra of the purified material exhibits a high-spin heme signal indicating a rhombic symmetry, — 6.92, Qy = 5.07, — 1.95 (Bakkenist et al, 1978). Upon acidification 

Bovine liver catalase is a tetrameric enzyme which catalyzes the decomposition of hydrogen peroxide. It has a molecular weight of 250,000 with 

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It should be noted that a number of different enzyme preparations can now be purchased directly from manufacturing chemists. It must be emphasised that the activity of an enzyme, whether purchased or prepared in the laboratory, may vary between rather wide limits. The activity is dependent on the source of the enzyme, the presence of poisons and also on the temperature. It appears, for example, that the quality of horseradish peroxidase depends upon the season of the year at which the root is obtained from the ground. It cannot be expected therefore that all the experiments described below will work always with the precision characteristic of an organic reaction proceeding under accurately known conditions. 

L-phenylalanine L-amino acid oxidase and horseradish peroxidase (E) I-... 

The emission yield from the horseradish peroxidase (HRP)-catalyzed luminol oxidations can be kicreased as much as a thousandfold upon addition of substituted phenols, eg, -iodophenol, -phenylphenol, or 6-hydroxybenzothiazole (119). Enhanced chemiluminescence, as this phenomenon is termed, has been the basis for several very sensitive immunometric assays that surpass the sensitivity of radioassay (120) techniques and has also been developed for detection of nucleic acid probes ia dot-slot. Southern, and Northern blot formats (121). 

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

Other specific discovery assays have been used such as differential inhibition of a vancomycin resistant S. aureus strain and its susceptible parent, and an assay based on antagonism of the antibacterial activity by N,A/-diacetyl-L-Lys-D-Ala-D-Ala [24570-39-6] a tripeptide analogue of the dalbaheptides receptor. AppHcation of this latter test to 1936 cultures (90) led to the isolation of 42 dalbaheptides, six of which, including kibdelin (Table 3), parvodicin (Table 3), and actinoidin A2 (68) were novel. A colorimetric assay based on competition between horseradish peroxidase bound teicoplanin and the... 

Catalytic oxidation of isobutyraldehyde with air at 30—50°C gives isobutyric acid [79-31-2] ia 95% yield (5). Certain enzymes, such as horseradish peroxidase, cataly2e the reaction of isobutyraldehyde with molecular oxygen to form triplet-state acetone and formic acid with simultaneous chemiluminescence (6). 

A method of detecting herbicides is proposed the photosynthetic herbicides act by binding to Photosystem II (PS II), a multiunit chlorophyll-protein complex which plays a vital role in photosynthesis. The inhibition of PS II causes a reduced photoinduced production of hydrogen peroxide, which can be measured by a chemiluminescence reaction with luminol and the enzyme horseradish peroxidase (HRP). The sensing device proposed combines the production and detection of hydrogen peroxide in a single flow assay by combining all the individual steps in a compact, portable device that utilises micro-fluidic components. 

According to Reichl et al. (2000), the oxidation of pholasin by compound I or II of horseradish peroxidase induces an intense light emission, whereas native horseradish peroxidase shows only a small effect. The luminescence of pholasin by native myeloperoxidase (verdoperoxidase) is diminished by preincubation with catalase, which is interpreted as the result of the removal of a trace amount of naturally occurring H2O2 in the buffer (about 10-8 M) that forms compound I... 

H2O2 was 2.9 x 10-6 M. A requirement for O2 could not be detected, suggesting that 02 is not involved in the luminescence reaction. The luciferase was found to be a peroxidase that catalyzes peroxidation of the luciferin, and it can be substituted with horseradish peroxidase. 

One of the most used systems involves use of horseradish peroxidase, a 3-diketone (mosl commonly 2,4-pentandione), and hydrogen peroxide." " " Since these enzymes contain iron(II), initiation may involve decomposition of hydrogen peroxide by a redox reaction with formation of hydroxy radicals. However, the proposed initiation mechanism- involves a catalytic cycle with enzyme activation by hydrogen peroxide and oxidation of the [3-diketone to give a species which initiates polymerization. Some influence of the enzyme on tacticity and molecular... 

Neuroanatomists have taken advantage of the phenomenon of fast retrograde transport to locate remote nerve cell bodies in the CNS of an experimental animal that are connected to an identified axonal fiber tract whose origin is uncertain. The tracer material [purified horseradish peroxidase (HRP) enzyme] is injected in the region of the axon terminals, where it is taken up by endocytosis and then is carried by retrograde axonal transport over a period of several hours to days back to the nerve cell body. The animal is sacrificed, and the enzyme tracer is localized by staining thin sections of the brain for peroxidase activity. 

Fujiyama K. Takemura H. Shinmyo A. Okada H. Takano M. (1990) Growth-stimulation of tobacco plant introduced the horseradish peroxidase gene prxCla / / Gene. V. 89. P. 163-169. 

In hiphasic water/1L mixtures, the latter can he used as immohilization systems. This idea was used for the synthesis of conducting polyanihne hy IL-immohilized horseradish peroxidase [68]. Tuning the IL hydrophohicity hy changing the anionic component allowed the increase in the yield of the product hy altering the affinity of the product to the IL. After completion of the reaction, the IL phase was separated, facilitating the recovery of the enzyme. 

As a result of the micellar environment, enzymes and proteins acquire novel conformational and/or dynamic properties, which has led to an interesting research perspective from both the biophysical and the biotechnological points of view [173-175], From the comparison of some properties of catalase and horseradish peroxidase solubilized in wa-ter/AOT/n-heptane microemulsions with those in an aqueous solution of AOT it was ascertained that the secondary structure of catalase significantly changes in the presence of an aqueous micellar solution of AOT, whereas in AOT/n-heptane reverse micelles it does not change. On the other hand, AOT has no effect on horseradish peroxidase in aqueous solution, whereas slight changes in the secondary structure of horseradish peroxidase in AOT/n-heptane reverse micelles occur [176],... 

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

Martmez-Parra, J. and Munoz, R., An approach to the characterization of betanine oxidation catalyzed by horseradish peroxidase, J. Agric. Food Chem., 45, 2984, 1997. 

Wasserman, B.P, Eiberger, L.L., and Guilfoy, M.P., Effect of hydrogen peroxide and phenolic compounds on horseradish peroxidase-catalyzed decolorization of betalain pigments, J. Food Sci., 49, 536, 557, 1984. 

Martmez-Parra, J. and Munoz, R., An approach to the characterization of betanine oxidation catalyzed by horseradish peroxidase, J. Agric. Food Chem., 45, 2984, 1997. Martmez-Parra, J. and Munoz, R., Characterization of betacyanin oxidation catalyzed by a peroxidase from Beta vulgaris L. roots, J. Agric. Food Chem., 49, 4064, 2001. Ashie, l.N.A. Simpson, B.K., and Smith, J.P., Mechanisms for controlling enzymatic reactions in foods, Crit. Rev. Food Sci. Nutr., 36, 1, 1996. 

Conventional ion-selective electrodes have been used as detectors for immunoassays. Antibody binding measurements can be made with hapten-selective electrodes such as the trimethylphenylammonium ion electrode Enzyme immunoassays in which the enzyme label catalyzes the production of a product that is detected by an ion-selective or gas-sensing electrode take advantage of the amplification effect of enzyme catalysis in order to reach lower detection limits. Systems for hepatitis B surface antigen and estradiol use horseradish peroxidase as the enzyme label and... 

HRA Histamine-releasing activity HRAN Neutrophil-derived histamine-releasing activity HRf Homologous-restriction factor HRF Histamine-releasing factor HRP Horseradish peroxidase HSA Human serum albumin HSP Heat-shock protein HS-PG Heparan sulphate protet ycan... 

Cardosi MR 1994. Hydrogen peroxide-sensitive electrode based on horseradish peroxidase-modified platinized carbon. Electroanalysis 6 89-96. 

Berglund GI, Carlsson GH, Smith AT, Szoke H, Henriksen A, Hajdu J. 2002. The catal3dic pathway of horseradish peroxidase at high resolution. Nature 417 463. 

Jacobs, B.L. Foote, S.L. and Bloom, F.E. Differential projections of neurons within the dorsal raphe nucleus of the rat A horseradish peroxidase (HRP) study. Brain Res 147 149-153, 1978. 

Secondary antibody and determination. A secondary antibody labeled with an enzyme is added which binds to the primary antibody that is bound to the coating antigen. If the primary antibody were produced in a rabbit, an appropriate secondary antibody would be goat anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP) (or another enzyme label). Excess secondary antibody is washed away. An appropriate substrate solution is added that will produce a colored or fluorescent product after enzymatic conversion. The amount of enzyme product formed is directly proportional to the amount of first antibody bound to the coating antigen on the plate and is inversely proportional to the amount of analyte in the standards. 

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