Horse radish peroxidase hydrogen peroxide

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... 

It has been suggested that N02 might be formed by the oxidation of nitrite by numerous biological oxidants. Thus, Shibata et al. [90] reported that horse radish peroxidase (HRP) + hydrogen peroxide oxidized nitrite by the following mechanism ... 

Hermann (2000) described a rapid automated method involving generation of a known amount of free radicals and the detection of the excess by photochemiluminescence. Test kits are available for determination of total water-soluble antioxidants, fat-soluble antioxidants and ascorbic acid. A luminometric method was developed for the determination of antioxidative activity and was subsequently applied to anthocyanin and betalaine colour concentrates (Kuchta et al., 1999). The method involved quantification of the interruption in luminescence from the hydrogen peroxide-horse radish peroxidase-luminol system in the presence of antioxidants. 

Torriero et al. [30] managed to estimate the concentration of lactate in untreated milk without the use of a microdialysis unit. Samples were fed into a reactor consisting of a rotating disc bearing lactate oxidase which by its motion ensured adequate mass transport of hydrogen peroxide to an enzyme electrode. The electrode consisted of horse-radish peroxidase immobilised over osmium on the surface of a glassy carbon electrode. Such an electrode can be poised at 0.0 Y so avoiding electrochemical... 

An even more indirect method which does not require the use of a fluorescence microscope is to use enzyme linked antibodies, e.g. the peroxidase anti-peroxidase (PAP) method of Stemberger et al. (1970). Here, after the antigen has been reacted with a rabbit antibody preparation this is conjugated to sheep anti-rabbit IgG which in turn is conjugated to a complex of rabbit anti-horse-radish peroxidase and horseradish peroxidase. This then reacts with diaminobenzidine and hydrogen peroxide when a brown colour indicates the presence of the antigen. 

Roulier et al. reported a sensitive and specific method for the measurement of choline and hydrogen peroxide in sea-water [45]. Choline was oxidized by choline oxidase to produce betaine and H202. The latter was used with horse-radish peroxidase to oxidize hydro-xyphenyl-propionic acid to produce a fluorescent diphenol end product. The resulting fluorescence at 410 nm (excitation at 320 nm) was proportional to the amount of H202, and could thus be used to measure the amount of choline present in the sample. Only 2-dimethyl aminoethanol interfered. The method was optimized, and used to determine 0 15 nM choline in coastal sea-water. 

In this test, oxygen from air is used for the enzymatic oxidation of (3-D-glucose in the presence of immobilized GOD to gluconic acid and hydrogen peroxide. Hydrogen peroxide can be determined by a second enzyme reaction. With horse radish peroxidase as catalyst o-phenylendiamine is oxidized by H2O2 to 2,3-diaminophenazine, which can be photometrically determined at 490 nm, thus establishing a quantitative relationship between active GOD sites and the intensity of the absorption band. 

Fig. 4.16. Fluctuation of product formation from dehydro-rhodamin [130 nM] by a single horse radish peroxidase molecule in the presence hydrogen peroxide [10p,M]. Observation by confocal epi-illumination (a) Autocorrelation function of product fluctuation (b) [39]...

The chemiluminescent reaction between Horse Radish Peroxidase (HRP)/ Alkaline Phosphatase (AP) and the luminol/CSPD/hydrogen peroxide substrate is used in a multianalytical ELISA approach to simultaneous analysis of different pesticides. The pesticides included in the present study were 2,4-D, Atrazine and Simazine. A novel variant of peroxidase (from transgenic tobacco, TOP) has also been investigated. 

Agner (148) has reported that crude tetanus and diphtheria toxins are rapidly detoxified by treatment with hydrogen peroxide and crystalline horse radish peroxidase or highly purified verdoperoxidase from leucocytes. More recently Agher (149) has demonstrated that highly purified diphtheria toxin is not inactivated by hydrogen peroxide and peroxidase unless an, as yet, unidentified dialyzable factor is present. This dialy-... 

Table XII, based partly on Table III of Millikan s review (137), summarizes the various constants, reduced to 20 and pH 7.4. In the case of sheep hemoglobin we have retained the values given by Roughton as nearly as possible rather than attempt to reconcile the equilibrium constant and the velocity constants on the assumption of independent hemes. This table includes rough, indirect estimates of constants for the oxygen reaction of the respiratory ferment (232) and the hydrogen peroxide reaction of catalase (67) as well as the recent elegant results of Chance (29) on the hydrogen peroxide reaction of horse-radish peroxidase.

Conn and Seki have reported that mitochondrial preparations from higher plants catalyze the oxidation of phenylpyruvic acid to benz-aldchyde, CO2 and other products, with the consumption of one molecule of oxygen per molecule of substrate (159a). The reaction is heat labile, and is inhibited by catalase as well as by cyanide and by catechol and hydroquinone, but all attempts to demonstrate hydrogen peroxide in the reaction mixture during the course of the reaction have been unsuccessful. A mixture of horse-radish peroxidase and manganous chloride also catalyzes the reaction. 

Horse-radish peroxidase on addition of hydrogen peroxide may give three spectroscopically very different compounds. A small amount (one to two moles hydrogen peroxide per mole peroxidase) first gives a greenish compound ( I ) (73,77) characterized by a sharp band in red at 655 to 658 mjLi. This is spontaneously converted to a red compound, first found by Keilin and Mann (47), with bands at 530 and 560 m/u. Keilin and Mann numbered this compound I, which we have called 11. A third red... 

The reaction velocity of this compound is probably comparatively slow, since Sumner and Gjessing (66) found, for horse-radish peroxidase, that increasing the hydrogen peroxide concentration above an optimal concentration gave a successive decrease in the amoimt of purpurogallin formed. However, it was not established whether the decrease was due to the formation of compound III or to destruction of the enzyme. 

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-... 

Peroxidase, another iron protoporphin enzyme, activates hydrogen peroxide so that it becomes a powerful oxidizing agent. It has been crystallized by Theorell (133) from horse-radish and is also found in many plant and animal cells. A peroxidase specific for the oxidation of cytochrome c by hydrogen peroxide has been found in yeast (1). 

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