Hydrogen peroxide peroxidases and

Horseradish peroxidase, hydrogen peroxide and buffers were purchased from Sigma Chemical Company. Benzidine and alkyl substituted phenols were purchased from Aldrich Chemical Company. Solvents used were high performance liquid chromatography grade and were purchased from Caledon Laboratories. 

This enzyme Is widely distributed, more particularly in plants. Three important sources of the enzyme are horse-radish, turnips and milk. Peroxidase is capable of activating both hydrogen peroxide and a suitable substrate so that the latter is oxidised, although hydrogen peroxide alone may be incapable of affecting this change. It sometimes happens that hydrogen pcr-... 

In the most common method for chemiluminescent immunoassay (GLIA), after the immunological reaction and any necessary separation steps, the labeled compounds or complexes react with an oxidizer, eg, hydrogen peroxide, and an enzyme, eg, peroxidase, or a chelating agent such as hemin or metal... 

Reaction takes place ia aqueous solution with hydrogen peroxide and catalysts such as Cu(II), Cr(III), Co(II), ferricyanide, hernia, or peroxidase. Chemiluminescent reaction also takes place with oxygen and a strong base ia a dipolar aprotic solvent such as dimethyl sulfoxide. Under both conditions Qcis about 1% (light emission, 375—500 am) (105,107). 

Tamaoku and colleagues presented an efficient enzymatic photometric determination of hydrogen peroxide ffiat is essentially a color reaction resulting from the oxidative condensation of A/-ethyl-A/-(2-hydroxy-3-sulfopropyl)aniline derivatives wiffi 4-aminoantipyrine in the presence of hydrogen peroxide and peroxidase (82CPB2492). A similar calorimetric detection of hydrogen peroxide has been patented (83GEP3301470). 

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... 

Peroxidases are found in milk and in leukocytes, platelets, and other tissues involved in eicosanoid metabolism (Chapter 23). The prosthetic group is protoheme. In the reaction catalyzed by peroxidase, hydrogen peroxide is reduced at the expense of several substances that will act as electron acceptors, such as ascorbate, quinones, and cytochrome c. The reaction catalyzed by peroxidase is complex, but the overall reaction is as follows ... 

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. 

Other solutions to dealing with interferences in the detection of H O have included the use of a copperfll) diethyldithiocarbamate precolumn to oxidize the sample before it reaches the immobilized enzyme, as well as the use of a palladium/gold sputtered electrode which catalyzes the oxidation of hydrogen peroxide In addition, peroxidase has been used to catalyze the reaction between hydrogen peroxide and iodide ferrocyanide and organo-fluorine compounds Am-... 

Thiols are also important protection against lipid peroxidation. Glutathione (7-Glu-Cys-Gly) is used by several glutathione-dependent enzymes such as free-radical reductase (converts vitamin E radical to vitamin E), glutathione peroxidase (reduces hydrogen peroxide and lipid hydroperoxides to water and to the lipid alcohol, respectively), and others. In addition, the thiol group of many proteins is essential for function. Oxidation of the thiol of calcium ATPases impairs function and leads to increased intracellular calcium. Thiol derivatives such as the ovothiols (l-methyl-4-mercaptohistidines) (Shapiro, 1991) have been explored as therapeutics. 

Levels of a number of metabolites as well as a number of enzymes in body fluids are indicative of disease conditions. Many of the enzymatic reactions mentioned above have been used in solution clinical assays as well as in test strips.446,497-508 512-515 Assays for hydrogen peroxide and the enzyme peroxidase using NADH and a tetrazolium salt have been de-scribed.509,5io Assays of exogenous substances (e.g., drugs or their metabolites) also utilize this chemistry. The determination of alcohol using alcohol dehydrogenase is an example.511 As mentioned above, the assay of enzyme levels can also be achieved using tetrazolium salts.516-520... 

Peroxidases have been implicated in carcinogenesis by PAH, aromatic amines, and estrogens inter alia (6-9). These enzymes catalyze the reduction of hydrogen peroxide and organic... 

In the reaction of luminol, hydrogen peroxide, and horse radish peroxidase 122> the chemiluminescence intensity is proportional to the square of luminol radical concentration. The lifetime of these luminol radicals was found by ESR techniques to be about 10 sec. Titration studies revealed that luminol acts as two-electron donor during the reduction of a hydrogen peroxide-horseradish peroxidase complex. The enzyme is not involved in the reaction step leading directly to light emission. This step is formulated as... 

For example, peroxidase catalyzes the reaction of luminol derivatives with hydrogen peroxide and results in an increase of the CL reaction velocity and CL intensity. Therefore, intense CL can be obtained from the analyte labeled with luminol derivatives after HPLC separation, followed by reaction with peroxidase. 

Various hydroxyl and amino derivatives of aromatic compounds are oxidized by peroxidases in the presence of hydrogen peroxide, yielding neutral or cation free radicals. Thus the phenacetin metabolites p-phenetidine (4-ethoxyaniline) and acetaminophen (TV-acetyl-p-aminophenol) were oxidized by LPO or HRP into the 4-ethoxyaniline cation radical and neutral V-acetyl-4-aminophenoxyl radical, respectively [198,199]. In both cases free radicals were detected by using fast-flow ESR spectroscopy. Catechols, Dopa methyl ester (dihydrox-yphenylalanine methyl ester), and 6-hydroxy-Dopa (trihydroxyphenylalanine) were oxidized by LPO mainly to o-semiquinone free radicals [200]. Another catechol derivative adrenaline (epinephrine) was oxidized into adrenochrome in the reaction catalyzed by HRP [201], This reaction can proceed in the absence of hydrogen peroxide and accompanied by oxygen consumption. It was proposed that the oxidation of adrenaline was mediated by superoxide. HRP and LPO catalyzed the oxidation of Trolox C (an analog of a-tocopherol) into phenoxyl radical [202]. The formation of phenoxyl radicals was monitored by ESR spectroscopy, and the rate constants for the reaction of Compounds II with Trolox C were determined (Table 22.1). 

Glutathione peroxidase is a selenium-dependent enzyme, which rapidly detoxifies hydrogen peroxide and various hydroperoxides. Suttorp et al. [67] showed that the impairment of glutathione cycle resulted in an increase in the injury of pulmonary artery endothelial cells. Glutathione cycle protected against endothelial cell injury induced by 15-HPETE, an arachi-donate metabolite produced by 15-lipoxygenase-catalyzed oxidation [68]. 

There is as yet no known enzyme that breaks the C—F bond in fluoroacetic acid, FCH2COOH, or in related compounds. It is interesting to note, however, that in the course of our investigations2 on peroxidase-catalysed oxidations, we have effected an enzymic cleavage of the C—F bond in p-fluoroaniline.3 In acetate buffer (pH 4-5) and at room temperature, the amine was oxidized by hydrogen peroxide and peroxidase to give mainly the red crystalline 2-amino-5-p-fluoroanilinobenzoquinone di-p-fluoroanil (XX). 

A quite different approach came from Chance and others using heme enzymes (1947). Purified horseradish peroxidase has a characteristic absorption spectrum which was visibly altered in the presence of hydrogen peroxide. When an appropriate substrate was added it was oxidized by the hydrogen peroxide and the spectrum reverted to that of the original state of the enzyme. Similar studies were performed with catalase, showing that prosthetic groups in enzymes underwent reversible changes in the course of their reactions. 

Subrahmanyam VV, McGirr LG, O Brien PJ. 1990c. Peroxidase/hydrogen peroxide-or bone marrow homogenate/hydrogen peroxide-mediated activation of phenol and binding to protein. Xenobiotica 20 1369-1378. 

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