Peroxidase complex with peroxides

Several catalases, including the type B catalase-peroxidases, seem to show true substrate saturation at much lower levels of peroxide than originally observed for the mammalian enzyme (in the range of a few millimolar). This means that the limiting maximal turnover is less and the lifetime of the putative Michaelis-Menten intermediate (with the redox equivalent of two molecules of peroxide bound) is much longer. The extended scheme for catalase in Fig. 2B shows that relationships between free enzyme and compound I, and the presumed rate-limiting ternary complex with least stability or fastest decay in eukaryotic enzymes of type A and greatest stability or slowest decay in prokaryotic type B enzymes. 

Cytochrome-c peroxidase forms a stable complex with hydrogen peroxide (Yonetani, 1965 Yonetani and Ray, 1965) with an absorption maximum at 419 nm, whereas that of the free enzyme is at 407 nm. 

The third and fourth steps differ according to the modification applied. In the first version, purified rabbit antiserum against peroxidase (anti-PO) reacts with above complex as an antigen with antigenic determinants of anti-rabbit-IgG. Then peroxidase (PO) reacts as antigen with bound anti-PO. In the last step bound peroxidase reacts with diaminobenzidine (DAB) and hydrogen peroxide. The excess of hydrogen-peroxide is detected with the osmium tctroxide. 

Chemiluminescence offers yet another sensitive detection system, which is easily implemented with simple instrumentation, but suffers to some extent from background interference in complex matrices. A recent example of an enzyme immunoassay with chemiluminescence as the detection system is the assay for 8-oxoguanine in DNA, which uses a secondary antibody conjugated with peroxidase-anti-peroxidase complex and a substrate solution containing hydrogen peroxide, luminol and p-iodophenol. 

Schiff base complexes have been extensively used to mimic the molecular reaction of bromo-peroxidases by treating the pre-formed complex with hydrogen peroxide. Decreasing the... 

The catalytic activity of peroxidase is intimately connected with its ability to form complexes with hydrogen peroxide. Three such complexes are formed, depending on the experimental conditions and they are interrelated ... 

The value of fc2, the velocity constant for the decomposition of the primary complex into peroxidase and peroxide cannot be obtained by direct measurement. Measurements of the equilibrium constant for the dissociation of the primary and secondary complexes with methyl hydroperoxide gave values of 3.2 X 10-6 M. and 3 X 10-7 M. from which fc2 may be estimated to be 2.2 sec.-1 or 3.4 sec.-1 from the known formation velocity constants. 

It has been shown above that the catalytic action of catalase and peroxidase is intimately connected with the ability of these hemoproteins to form complexes with hydrogen peroxide (or alkyl peroxides). By choosing the experimental conditions the existence of three different complexes can be demonstrated spectroscopically. The chemical nature of these complexes is as yet unknown, and mechanisms have been represented as bimolecular reactions between substrate and complex. 

As the drug enters a cell, it forms superoxide anions. These free radicals (e.g., hydrogen peroxide) normally are destroyed by glutathione peroxidase however, this enzyme, and the required glutathione, is of limited concentration in cardiac cells. Thus, the radicals complex with available I c ions, forming highly reactive radicals that rapidly cause lipid peroxidation and extensive mitochondrial destruction. 

The oxidation of iodide is performed by enzymes of the peroxidase type (Morrison, 1980), mainly lactoperoxidase, but also by horseradish peroxidase, microperoxidase and chloroperoxidase, which are hemoproteins with tetrapyr-rolic cores chelating iron. These enzymes are active in the presence of small amounts of hydrogen peroxide or glucose oxidase. They form a first complex with H2O2 that... 

Peroxidase forms a number of complexes with hydrogen peroxide. The activation mechanism of substrate molecules in the active center of peroxidase involves the formation of a ternary complex heme-H20-donor of electrons. The role of the iron ion consists of transferring electrons from the donor to the hydrogen peroxide molecule. Peroxidase is endowed, moreover, with the oxidase function which increases in the presence of divalent manganese ions. 

A different method of S3mthesizing polyanUine was reported [77]. It uses an enzyme, horseradish peroxidase, in the presence of hydrogen peroxide to polymerize aniline. To prevent reactions at the ortho positions of the phenyl rings that yield insoluble branched materials, a polyelectrolyte template, like sulfonated polystyrene, was used. The polyelectrolyte aligns the monomers, dopes the polyaniline to the conducting form, and forms an irreversible complex with the polyaniline to keep it water-soluble [77]. The conductivity of the complex increases with increasing polyaniline to sulfonated polystyrene molar ratios. Conductivities of0.005 S/cm are obtained with the pure complex and increase to 0.15 S/cm after additimial doping by exposure to HCl vapor [77]. 

The reaction cycle of the catalases (Fig. 6), like that of the peroxidases, begiris with the high-spin ferric state (7i) which reacts with a molecule of hydrogen peroxide to form the Compound I intermediate (14). Next, however, oxidation of a second hydrogen peroxide molecule yields dioxygen, with the concomitant return of catalase Compound I to the native resting state. Catalases can be made to produce a Compound II intermediate that is generally described as an Fe =0 complex like Compound II of the peroxidases. 

Finally, the similarity between the peroxidases, especially chloroperoxidase, and cytochrome P-450 in their reactivity with peroxide suggests that the active oxygen species in P-450 may also be a ferryl species, i.e., an oxo iron (TV) complex. Evidence both for [193] and against [194] the involvement of a Compound I-type intermediate in the reaction cycle of P-450 has appeared. As of yet, there exists no direct evidence for the nature of the intermediates beyond oxy-P-450 in the reaction cycle. 

Glassy carbon electrodes have been modified with a hydrophilic, permeable film of horseradish peroxidase (HRP) covalently bound to a polyvinylpyridine polymer complexed with osmium to enhance the detection of hydrogen peroxide. Vreeke et al demonstrated that such a system could be used to quantify hydrogen peroxide (reduction at 0.0 V, SCE) produced from complex coupled reactions in their assay of NADH ... 

Separationless immunoassays in whole blood present a number of challenges. Hydrogen peroxide, the substrate of the commonly used immunolabehng peroxidases, is eliminated by catalase and catalase-hke blood constituents, and proteins may quickly foul the sensors. Additionally, mass transport both to and within the sensors defines the time required for separationless immunoassays. The problems are alleviated in a redox hydrogel-based separationless immunoassay. The redox polymer used is a copolymer of acrylamide and A-vinylimidazole complexed with osmium-4,4 -dimethyl-2,2 -bipyridine and treated with hydrazine to provide... 

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