Horseradish peroxidase stability

Song HY, Yao JH, Liu JZ et al (2005) Effects of phthalic anhydride modification on horseradish peroxidase stability and structure. Enzyme Microb Technol 36 605-611... 

Garcia D, Ortega F, Marty JL (1998) Kinetics of thermal inactivation of horseradish peroxidase stabilizing effect of methoxypoly(ethylenglycol). Biotechnol Appl Biochem 27 49-54... 

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. 

Stone et al. use this method to simultaneously synthesize the silica and entrap the butyrylcholinesterase which retains all its activity after the process of encapsulation, a high enzyme loading (90 %) is reached and the stability is increased [168]. The method has been further developed to simultaneously entrap catalase and horseradish peroxidase with inorganic magnetic nanopartides [169] which will fadlitate the separation [170,171]. 

A.L. Crumbliss, J.Z. Stonehuerner, R.W. Henkens, J. Zhao, and J.P. O Daly, A carrageenan hydrogel stabilized colloidal gold multi-enzyme biosensor electrode utilizing immobilized horseradish peroxidase and cholesterol oxidase/cholesterol esterase to detect cholesterol in serum and whole blood. Biosens. Bioelectron. 8, 331-337 (1993). 

Veitch NC. 2004. Horseradish peroxidase a modem view of a classic enzyme. Phytochemistry 65 249—259. Wegrzyn TF, Farr JM, Hunter DC, Au J, Wohlers MW, Skinner MA, Stanley RA and Waterhouse DS. 2008. Stability of antioxidants in an apple polyphenol—milk model system. Food Chem 109 310-318. 

Freeman and Seitz [6] developed one of the first enzyme-based CL sensors with convincing performance. They immobilized horseradish peroxidase (HRP) at the end of an optical fiber and achieved a detection limit of 2 X 10 4 6 mol/L H202. Preuschoff et al. [23] developed a fiberoptic flow cell for H202 detection with long-term stability, suitable for fast FTA. Different peroxidases were covalently... 

The form of the enzyme with the greatest oxidation potential is known as horseradish peroxidase, compound 1 (HRP-I), which consists of a radical cation stabilized throughout the highly conjugated protoporphyrin IX ring system. In the presence of vindoline, HRP-I is reduced to HRP-H, an Fe(IV) form of the enzyme. The vindoline cation radical 55 thus formed eliminates a second elec-... 

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. 

The presence of calcium in horseradish peroxidase was demonstrated originally by Haschke and Friedhoff, working with the C and A (imspec-ified, but likely to have been predominantly A2) isoenzymes (209). HRP C and HRP A contain 2.0 0.13 and 1.4 0.19 moles calcium per mole enzyme, respectively, as determined by atomic absorption spectroscopy. Incubation in 6 M guanidinium hydrochloride and 10 mM EDTA for 4 hours at neutral pH and room temperature gave calcium-depleted enzymes with specific activities decreased by 40% and 15%, respectively. The thermal stability of calcium-depleted HRP C was also reduced compared to native enzyme. Reconstitution was successful only with calcium-depleted HRP C (209). It remains to be established whether this reflects true structural differences between the calcium binding sites of the two isoenz5unes, or is a consequence of the relatively harsh... 

The uses and applications of horseradish peroxidase in organic synthesis have been reviewed (260, 261). As a reagent it offers several advantages, including wide commercial availability, good stability under a range of conditions, and broad substrate specificity. Future developments are likely to focus on the increased use of site-directed mutants of HRP C to improve stereo- and enantiospecificity in reactions of interest. As yet these enzymes are not available commercially. 

Gao CY, Liu XY, Shen JC, Mohwald H. Spontaneous deposition of horseradish peroxidase into polyelectrolyte multilayer capsules to improve its activity and stability. Chem Commun 2002 1928-1929. 

Azevedo AM, Vojinovic V, Cabral JMS, Gibson TD, Eonseca LP (2004) Operational stability of immobilised horseradish peroxidase in mini-packed bed bioreactors. J Mol Cat B Enzym 28(2-3) 121-128... 

Wang, S.F., Chen, T., Zhang, Z.L., and Pang, D.W., Activity and stability of Horseradish Peroxidase in hydrophilic room temperature ionic liquid and its application in non-aqueous biosensing, Electrochem. Commun., 9, 1337-1342, 2007. 

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. 

In this section, the thermal stability of acrylamide gel-immobilized peroxidase will be compared to that of the free enzyme. The free enzyme is assayed in the following manner Dilute 1 mL of the stock horseradish peroxidase (0.1 mg/mL, 15 units/mL) with 299 mL of glass-distilled water. Add 1.0 mL of this diluted enzyme to each of two test tubes. Place one of the tubes in a 60°C water bath for exactly 4 minutes. Allow the other tube to sit at room temperature for the same time interval. Cool the higher-temperature tube to room temperature by placing in a water bath. To each tube add 2.0 mL of the aminoantipyrine-phenol stock solution and 2.0 mL of the H202 solution and mix well. Allow the tubes to sit at room temperature for exactly 3 minutes then immediately read the sl5l0 for each. Record the results in your notebook. 

Thus, glycoproteins such as horseradish peroxidase, glucose oxidase, or most antibody molecules can be activated for conjugation by brief treatment with periodate. Cross-linking with an amine-containing protein takes place under alkaline pH conditions through the formation of Schiff base intermediates. These relatively labile intermediates can be stabilized by reduction to a secondary amine linkage with sodium cyanoborohydride (Fig. 303). 

First examples of the amperometric detection of H202 accomplished in such a range were based on the use of an enzyme, namely horseradish peroxidase (HRP), a prototypical hemeprotein peroxidase, which catalyses the reduction of H202 and due to its peculiar structure, allows direct electron transfer between its active site and the electrode surface at low applied potential [14 17]. This approach, although it shows good sensitivity and accuracy, suffers from some important shortcomings such as low stability and the limited binding of HRP to solid surfaces. 

Chemiluminescence of oxidized luminol has been the basis of several lumino-metric methods of estimation of TAC (Table 1). The mostcommon is to measure the induction time of the reaction. Often the chemiluminescence is first induced by an oxidant and then attenuated by addition of a sample, and the time to recover the initial fluorescence is measured. The enhanced chemiluminescent assay introduced a decade ago is based on the oxidation of luminol by perborate or by hydrogen peroxide in a reaction catalyzed by horseradish peroxidase. Enhancement (and stabilization) of luminescence is achieved by addition of p-iodophenol. The original procedure used a commercial reagent kit (ECL Anti-oxidant Detection Pack... 

Ozaki S, Inada Y, Watanabe Y (1998) Characterization of polyethylene glycolated horseradish peroxidase in organic solvents Generation and stabilization of a transient catalytic intermediate at low temperatures. J Am Chem Soc 120 8020-8025... 

Ferrari RP, Laurenti E, Casella L, Poli S (1993) Oxidation of catechols and catecholemines by horseradish peroxidase and lactoperoxidase ESR spin stabilization approach combined with optical methods. Spectrochim Acta 49A 1261-1267... 

Ryan BJ, O Fagain C (2007) Effects of single mutations on the stability of horseradish peroxidase to hydrogen peroxide. Biochimie 89 1029-1032... 

Morawski B, Quan S, Arnold FH (2001) Functional expression and stabilization of horseradish peroxidase by directed evolution in Saccharomyces cerevisiae. Biotechnol Bioeng 76 99-107... 

Song H-Y, Liu J-Z, Weng L-P, Ji L-N (2009) Activity, stability, and unfolding of reconstituted horseradish peroxidase with modified heme. J Mol Catal B Enzym 57 48-54... 

Mogharrab N, Ghourchian H, Amininasab M (2007) Structural stabilization and functional improvement of horseradish peroxidase upon modification of accessible lysines experiments and simulation. Biophys J 92 1192-1203... 

Chattopadhyay K, Mazumdar S (1999) Structural and conformational stability of horseradish peroxidase effect of temperature and pH. Biochemistry 39 263-270... 

In the first report about immobilization of peroxidases on mesoporous materials, Takahashi and coworkers shed light on different parameters that affect the process. Using horseradish peroxidase (HRP) as a model, the authors reported that higher stability to temperature and organic solvent, important variables on industrial processes, were obtained when the size of the pore match the size of the enzyme, in such a way that the encapsulated enzyme was located in a restricted space that slowed down its free movement, preventing its denaturation [4],... 

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