Peroxidase enzyme

The colour reactions with the enzyme peroxidase and HjO permit all the above amines to be distinguished from one another in very dilute solution (P- 523)-... 

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... 

An interesting further example of a homolytic aromatic reaction involves the oxidation of phenols in basic solution with one-electron oxidising agents (e.g. Fe(m), H20 + the enzyme peroxidase) ... 

The electrochemical rate constants for hydrogen peroxide reduction have been found to be dependent on the amount of Prussian blue deposited, confirming that H202 penetrates the films, and the inner layers of the polycrystal take part in the catalysis. For 4-6 nmol cm 2 of Prussian blue the electrochemical rate constant exceeds 0.01cm s-1 [12], which corresponds to the bi-molecular rate constant of kcat = 3 X 103 L mol 1s 1 [114], The rate constant of hydrogen peroxide reduction by ferrocyanide catalyzed by enzyme peroxidase was 2 X 104 L mol 1 s 1 [116]. Thus, the activity of the natural enzyme peroxidase is of a similar order of magnitude as the catalytic activity of our Prussian blue-based electrocatalyst. Due to the high catalytic activity and selectivity, which are comparable with biocatalysis, we were able to denote the specially deposited Prussian blue as an artificial peroxidase [114, 117]. 

It is important to compare the catalytic properties of Prussian blue with known hydrogen peroxide transducers. Table 13.2 presents the catalytic parameters, which are of major importance for analytical chemistry selectivity and catalytic activity. It is seen that platinum, which is still considered as the universal transducer, possesses rather low catalytic activity in both H202 oxidation and reduction. Moreover, it is nearly impossible to measure hydrogen peroxide by its reduction on platinum, because the rate of oxygen reduction is ten times higher. The situation is drastically improved in case of enzyme peroxidase electrodes. However, the absolute records of both catalytic activity... 

Two additional systems were exploited in order to confirm the involvement of free-radical processes during vindoline oxidations. These were the enzyme peroxidase and photochemistry. Horseradish peroxidase (HRP) oxidized both vindoline and 16-O-acetylvindoline in the presence of hydrogen peroxide. Vindoline was converted to the enamine dimer 59 (78). During the reaction, the following sequence of redox reactions occurs ... 

Incubate sections for 30 min with (strept)avidin conjugated with a fluorophore or with any enzyme (peroxidase or alkaline phosphatase). For localization of low-density antigens (<10K molecules/cell), VECTASTAIN ABC reagent (http //www.vectorlabs.com/) is preferable. 

When using an enzyme (peroxidase or alkaline phosphatase) marker, incubate sections in an enzyme substrate solution until desired stain intensity develops (see Sect. 2.3). A fluorophore label can be directly visualized in a fluorescent microscope. 

The PAP method was pioneered by Sternberger in 1979 (1). The method uses an immunological sandwich amplification and the enzyme peroxidase to effect a signal. The unique feature of this procedure is the enzyme/antibody solution, the PAP immune complex. The horseradish peroxidase enzyme, itself an immunogenic protein, is used to inoculate a given species, and a polyclonal immune response is generated against the enzyme. This antiserum is harvested and placed in solution with the enzyme so that immune complexes form that... 

A typical chemical system is the oxidative decarboxylation of malonic acid catalyzed by cerium ions and bromine, the so-called Zhabotinsky reaction this reaction in a given domain leads to the evolution of sustained oscillations and chemical waves. Furthermore, these states have been observed in a number of enzyme systems. The simplest case is the reaction catalyzed by the enzyme peroxidase. The reaction kinetics display either steady states, bistability, or oscillations. A more complex system is the ubiquitous process of glycolysis catalyzed by a sequence of coordinated enzyme reactions. In a given domain the process readily exhibits continuous oscillations of chemical concentrations and fluxes, which can be recorded by spectroscopic and electrometric techniques. The source of the periodicity is the enzyme phosphofructokinase, which catalyzes the phosphorylation of fructose-6-phosphate by ATP, resulting in the formation of fructose-1,6 biphosphate and ADP. The overall activity of the octameric enzyme is described by an allosteric model with fructose-6-phosphate, ATP, and AMP as controlling ligands. 

Another hormone that can be affected by phenolics is ethylene. Coumaric acid is necessary as a cofactor for the biosynthesis of ethylene [33]. On the other hand, caffeic acid inhibits an enzyme (peroxidase type) for which the cofactor is /7-coumaric acid. Therefore, the balance between /7-coumaric and caffeic acid can in theory influence the regulation of ethylene biosynthesis [6]. 

Among all enzymes, peroxidases are the most widely used and commercial kits for labelling and are already available [28]. 

If the activity of the first enzyme (glucose oxidase) is to be measured accurately, the second enzyme (peroxidase) and its co-substrates or coenzymes... 

Liu ZM, Dubremetz JF, Richard V et al (2005) Useful method for the spacial localization determination of enzyme (peroxidase) distribution on microfiltration membrane. J Membr Sci 2005 2-7... 

Oxygen gas is essential for all life as it oxygenates haemoglobin, which then transfers oxygen via the blood throughout the body. Sometimes small amounts of peroxide radicals, 022, are formed. The enzymes peroxidase and catalase in the blood destroy the peroxides as they are harmful to all living tissues... 

However, the same basic principles can be applied (with appropriate modifications) to the labeling of antigens with other enzymes (peroxidase, alkaline phosphatase, -galactosidase). This is particularly true for those labeling methods involving the use of biotinylated antigens, since biotinylated enzymes as well as avidin-enzyme conjugates are available from numerous commercial sources. 

In the glucose oxidase system, dissolved oxygen concentration as well as glucose levels will influence dehvery response requiring close control of mass-transfer limitations. For both systems containing a protein component, stabihty factors may limit operational hfetime. This may be particularly severe in the case of glucose oxidase where the reaction product H2O2 will accelerate enzyme denaturation unless it is rapidly removed by diffusion or reaction with a second enzyme (peroxidase or catalase). 

The phenoloxidase enzymes (peroxidases, tyrosinases, and laccases) are known to occur extracellularly in soils (16,17), and have been considered to play a role in catalyzing the coupling of aromatic amines to humic substances (3). Peroxidases have been studied most extensively. They use hydrogen peroxide to promote the one electron oxidation of aromatic amines and of phenolic moieties within humic substances to form free radicals. (12,18). 

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