Polymers horseradish peroxidase

It should be pointed out that the addition of substances, which could improve the biocompatibility of sol-gel processing and the functional characteristics of the silica matrix, is practiced rather widely. Polyethylene glycol) is one of such additives [110— 113]. Enzyme stabilization was favored by formation of polyelectrolyte complexes with polymers. For example, an increase in the lactate oxidase and glycolate oxidase activity and lifetime took place when they were combined with poly(N-vinylimida-zole) and poly(ethyleneimine), respectively, prior to their immobilization [87,114]. To improve the functional efficiency of entrapped horseradish peroxidase, a graft copolymer of polyvinylimidazole and polyvinylpyridine was added [115,116]. As shown in Refs. [117,118], the denaturation of calcium-binding proteins, cod III parvalbumin and oncomodulin, in the course of sol-gel processing could be decreased by complexation with calcium cations. 

Horseradish peroxidase (HRP) is an extracellular plant enzyme that acts in regulation of cell growth and differentiation, polymerization of cell wall components, and the oxidation of secondary metabolites essential for important pathogenic defense reactions. Because of these essential functions, and also because of its stability and ready availability, HRP has attracted considerable attention.13 It has been involved in a number of applications, such as diagnostic assays,14 biosensors,15 bioremediation,16 polymer synthesis,17 and other biotechnological processes.18 More applications in which HRP catalysis is translated into an electrochemical signal are likely to be developed in the near future. 

A horseradish peroxidase-osmium redox polymer-modified glassy carbon electrode (HRP-GCE) has also been applied to this analysis to improve sensitivity and reduce problems with faradic interference. Kato and colleagues (1996) employed this electrode in measurement of basal ACh in microdialysates using a precolumn enzyme reactor. This system was three to five times more sensitive than a conventional Pt electrode. ACh in rat hippocampus dialysate was quantitated at 9 5 fmol/15 pi (n = 8). ACh was analyzed in PC12 cells in a similar assay by Kim and colleagues (2004). No precolumn enzyme reactor was employed. 

Osborne and Yamamoto (1998) compared disposable, fihn carbon electrodes (PFCEs) and glassy carbon electrodes that were both modified with cast-coated Osmium-polyvinylpyrridine-wired horseradish peroxidase gel polymer (Os-gel-HRP). Sensitivities for ACh were 16 and 10 fmol/10 pi respectively. 

Kato T, Liu JK, Yamamoto K, Osborne PG, Niwa O. 1996. Detection of basal acetylcholine release in the microdialysis of rat frontal cortex by high- performance liquid chromatography using a horseradish peroxidase-osmium redox polymer electrode with pre-enzyme reactor. J Chromatogr B 682 162-166. 

Attachment of Hydroxycinnamic Acids to Structural Cell Wall Polymers. Peroxidase mediation may also result in binding the hydroxycinnamic acids to the plant cell wall polymers (66,67). For example, it was reported that peroxidases isolated from the cell walls of Pinus elliottii catalyze the formation of alkali-stable linkages between [2-14C] ferulic acid 1 and pine cell walls (66). Presumably this is a consequence of free-radical coupling of the phenoxy radical species (from ferulic acid 1) with other free-radical moieties on the lignin polymer. There is some additional indirect support for this hypothesis, since we have established that E-ferulic acid 1 is a good substrate for horseradish peroxidase with an apparent Km (77 /tM), which is approximately one fifth of that for E-coniferyl alcohol (400 /iM) (unpublished data). 

Alvarez S, Manolache S, Denes F (2003) Synthesis of polyaniline using horseradish peroxidase immobilized on plasma-functionalized polyethylene surfaces as initiator. J Appl Polym Sci 88(2) 369-379... 

Akkara JA, Senecal KJ, Kaplan DL (1991) Synthesis and characterization of polymers produced by horseradish peroxidase in dioxane. J Polym Sci A Polym Chem 29 1561-1574... 

Similarly, catechin polymers formed upon horseradish peroxidase-catalyzed oxidation of catechin or polycondensation of catechin with aldehydes prove much more efficient than catechin (at identical monomer concentration) at inhibiting XO and superoxide formation. A more detailed investigation with the catechin-acetaldehyde polycondensate (which is expected to form in wine because of the microbial oxidation of ethanol to acetaldehyde) shows that inhibition is noncompetitive to xanthine and likely occurs via binding to the FAD or Fe/S redox centers involved in electron transfers from the reduced molybdenum center to dioxygen with simultaneous production of superoxide. 

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 ... 

In contrast to the EPOS system, a modification of the highly sensitive two-step immunohistochemical EnVision system allows the detection of a broad spectrum of antigens in frozen sections in less than 13 min (Kammerer et al., 2001). In this study 38 out of 45 antibodies tested showed specific staining. In fact, the modified EnVision procedure allows the use of any suitable primary antibody, preferably monoclonal antibodies. Like the EPOS system, EnVision employs a dextran polymer coupled to horseradish peroxidase molecules for detection. No attempt was made to block endogenous peroxidase, nor was any antigen retrieval pretreatment used. Because of the very short incubation durations, a humid chamber is not required to avoid evaporation of immunoreagents. 

Acetylcholineesterase and choline oxidase Immobilization of horseradish peroxidase in the redox polymer poly (4-vinylpyridine-chlorobis-(2,2 -bipyridyl) osmium cross-linked by means of polyoxyethylene 400 diglycidyl ether on polished vitreous carbon electrodes. Response time of 30 s and maintained its sensitivity for 24 h at low substrate concentration. Responses were rectilinear up to 10 mM H202 for 100 pM choline with detection limit of 10 nM and 1 pM. [78]... 

Acetylcholineesterase and choline oxidase A glassy C electrode surface was modified with osmium poly (vinyl-pyridine) redox polymer containing horseradish peroxidase (Os-gel-HRP) and then coated with a co-immobilized layer of AChE and ChO. A 22 pL pre-reactor, in which ChO and catalase were immobilized on beads in series, was used to remove choline. The variation in extracellular concentration of ACh released from rat hippocampal tissue culture by electrical stimulation was observed continuously with the online biosensor combined with a microcapillary sampling probe. Measurement of ACh and Ch was carried out by using a split disc C film dual electrode. 

A BASJ ion-exchange microbore column (45 cm x 1 mm i.d.) 0.05 Sodium phosphate buffer of pH 8.5 containing 0.1 mM EDTA [60 pL/min]. Electrochemical at horseradish peroxidase osmium redox polymer-modified vitreous C electrodes at 0 mV versus Ag/AgCl. Rat frontal cortex dialysate samples [188]... 

Dordick JS (1987) Production of phenolic polymers catalyzed by horseradish peroxidase in organic solvents. Proc Mater Biotechnol Symp 225-239... 

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