Enzymes horseradish peroxidase

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. 

Taraban, M.B., Leshina, T.V., Anderson, M.A., and Grissom, C.B., Magnetic field dependence of electron transfer and the role of electron spin in heme enzymes horseradish peroxidase, J. Am. Chem. Soc.,... 

Enzymes Horseradish peroxidase, alkaline phosphatase, /3-galactosidase... 

The enzyme horseradish peroxidase is a hemoprotein and the region of the Soret band exhibits large differences between the position and extinction coefficients of the uncombined and combined forms. Both forms were first studied by spectrophotometry, but the E—S complexes were 0 labile that they could not be examined extensively by any other spectroscopic method. Using rapid-scanning spectrophotometry and rapid mixing, Chance was able to distinguish the spectra of compound I and II and determine the various rate constants of the multistep reaction with rather poor precision. 

As indicator enzymes horseradish peroxidase (HRP or HRPO), alkaline phosphatase (AP), or /i-galactosidase, are favored, since they are relatively robust, have a high product-forming rate, are easy to purify, and are cheap. The most used colloids are from gold, silver, and iron, and iodine isotopes are mostly taken as radioactive labels in immunoassays. 

Peroxidases are haem proteins that are activated from the ferric state to one-electron oxidants by H202. They play a significant role in the generation of radicals from xenobiotics. The compound I state contains one oxidising equivalent as an oxoferryl-haem entity and the second as a porphyrin -radical cation. Upon the oxidation of a substrate the porphyrin radical is repaired, giving the compound II. Reduction of the oxoferryl haem back to the ferric state by a second substrate molecule completes the enzyme cycle. In addition to the classical peroxidases, several other haem proteins display pseudo-peroxidase activity. The plant enzyme horseradish peroxidase (HRP) is often employed in model systems. 

Immobilized enzymes are becoming increasingly important in commercial processes. In this experiment, students will trap molecules of the enzyme horseradish peroxidase within a polyacrylamide gel matrix. The reaction kinetics and thermal stability of the immobilized enzyme will be measured. This experiment introduces students to the use of enzymes in biotechnology. 

Four methods have been developed for enzyme immobilization (1) physical adsorption onto an inert, insoluble, solid support such as a polymer (2) chemical covalent attachment to an insoluble polymeric support (3) encapsulation within a membranous microsphere such as a liposome and (4) entrapment within a gel matrix. The choice of immobilization method is dependent on several factors, including the enzyme used, the process to be carried out, and the reaction conditions. In this experiment, an enzyme, horseradish peroxidase (donor H202 oxidoreductase EC 1.11.1.7), will be imprisoned within a polyacrylamide gel matrix. This method of entrapment has been chosen because it is rapid, inexpensive, and allows kinetic characterization of the immobilized enzyme. Immobilized peroxidase catalyzes a reaction that has commercial potential and interest, the reductive cleavage of hydrogen peroxide, H202, by an electron donor, AH2 ... 

Haga et al. developed another type of immunosensor by combining an enzyme membrane immunoassay and an enzyme sensor using oxygen electrodes (HI). In this assay antigen molecules (theophylline) are attached on the surface of the liposomes and an enzyme (horseradish peroxidase) is encapsulated in the sensitized liposome. When antibody (antitheophylline antibody) and complement are added, the enzyme is released by the liposome lysis. The enzyme activity with the NADH-NAD reaction can be determined by the oxygen electrode. When antigen is added, it competitively binds to antibodies, then liposome lysis and enzyme activity are decreased. The sensitivity of this method for theophylline determination was reported as 0.7 ng/ml. 

Studies in experimental and chemical neuroanatomy underwent then, as it frequently happens in scientific research, a sudden acceleration. Retrograde axonal transport was discovered on the basis of the finding that proteins, such as the enzyme horseradish peroxidase (HRP), are retrogradely transported from axon terminals to their parent neuronal cell bodies (Kristensson and Olsson, 1971). The modern era of neuroanatomy... 

A similar approach was used for the detection of monosaccharides [56] pyranose oxidase delivered a suitable oxidase-based sensor with the hydrogen peroxide being determined again via the direct electron transfer from the carbon paste electrode to the enzyme horseradish peroxidase. In aU bi-enz5mie systems, both the enzymes were entrapped within the carbon paste. These electrodes were used as detectors in a liquid chromatography system allowing the simultaneous determination not only of several carbohydrates but also of ethanol within approximately 20 min. Ethanol production and carbohydrate consumption were monitored on-line during a fermentation of P. stipitis for 16 h (see also Section 11.2.3). 

Figure 8 is a possible redox cycle occurring in an amperometric sensor for hydrogen peroxide involving enzyme-wiring of a typical enzyme (Horseradish peroxidase, HRP) with polyaniline. HRP immobilized on the electrode surface can be oxidized by H2O2 to compound I that contains an oxyferryl centre with the iron in the ferryl state (Fclv = O), and a porphyrin 7r cation radical, followed by further direct (mediatorless) electroreduction of compound I at the electrode surface to the initial HRP state [106], The electrode is considered as an electron donor. 

ILs have been used for immobilization of enzymes. Horseradish peroxidase (HRP) activity is evaluated in a [BMIM][BF ] sol-gel matrix [26]. Higher activity in [BMIM][PFg] is also observed when lipase from an unspecified Pseudomonas (PsL) is immobilized on methacryloxypropyl-modified mesoporous silica [27]. Similarly, IL-coated enzyme was found to completely retain the activity and easily reusable [28]. 

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