Horseradish peroxidase modeling

Choi, Y.J., Chae, H.J., and Kim, E.Y., Steady-state oxidation model by horseradish peroxidase for the estimation of the non-inactivation zone in the enzymatic removal of pentachlorophenol, J. Biosci. Bioeng., 88, 368-373, 1999. 

I. M.C.M., Reversible formation of high-valent-iron-oxo prophyrin intermediates in heme-based catalysis revisiting the kinetic model for horseradish peroxidase, Inorg. Chim. Acta, 275/276, 98-105, 1998. 

Based on IgG-bearing beads, a chemiluminescent immuno-biochip has been also realized for the model detection of human IgG. Biotin-labeled antihuman IgG were used in a competitive assay, in conjunction with peroxidase labelled streptavidin59. In that case, the planar glassy carbon electrode served only as a support for the sensing layer since the light signal came from the biocatalytic activity of horseradish peroxidase. Free antigen could then be detected with a detection limit of 25 pg (108 molecules) and up to 15 ng. 

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. 

Horseradish peroxidase, as the name implies, is derived from a plant not from humans or animals however, it is readily available and often used as a model to study peroxidase oxidations (42). The classic substrates are phenols, which are oxidized to phenoxy radicals, but aromatic amines are also good substrates. 

Zhao, D., Gilfoyle, D. J., Smith, A. T., and Loew, G. H. (1996) Refinement of 3D models of horseradish peroxidase isoenzyme C predictions of 2D NMR assignments and substrate binding sites. Proteins 26, 204-216. 

The present volume is a non-thematic issue and includes seven contributions. The first chapter byAndreja Bakac presents a detailed account of the activation of dioxygen by transition metal complexes and the important role of atom transfer and free radical chemistry in aqueous solution. The second contribution comes from Jose Olabe, an expert in the field of pentacyanoferrate complexes, in which he describes the redox reactivity of coordinated ligands in such complexes. The third chapter deals with the activation of carbon dioxide and carbonato complexes as models for carbonic anhydrase, and comes from Anadi Dash and collaborators. This is followed by a contribution from Sasha Ryabov on the transition metal chemistry of glucose oxidase, horseradish peroxidase and related enzymes. In chapter five Alexandra Masarwa and Dan Meyerstein present a detailed report on the properties of transition metal complexes containing metal-carbon bonds in aqueous solution. Ivana Ivanovic and Katarina Andjelkovic describe the importance of hepta-coordination in complexes of 3d transition metals in the subsequent contribution. The final chapter by Sally Brooker and co-workers is devoted to the application of lanthanide complexes as luminescent biolabels, an exciting new area of development. 

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. 

Commercially available horseradish peroxidase (crystalline) will substitute for luriferase in the foregoing reaction. In addition, a compound of known structure. 5-amino-2, 3-dihvdro-l, 4-phthalazinedione (also known as luminol), will substitute for luciferin. The mechanisms appear to be the same regardless of the way in which the crosses are made. Thus, a model bioluminescent system is available and can be used as a sensitivity assay for H2O2 at neutral pH. The identification of luciferase as a peroxidase is of interest since this represents the only demonstration of a bioluminescent system in which the catalytic nature of a luciferase molecule has been defined. 

The rates of asymmetric sulfoxidation of thioanisole in nearly anhydrous (99.7%) isopropyl alcohol and methanol catalyzed by horseradish peroxidase (HRP) were determined to be tens to hundreds of times faster than in water under otherwise identical conditions (Dai, 2000). Similar effects were observed with other hemo-proteins. This dramatic activation is due to a much higher substrate solubility in organic solvents than in water and occurs even though the intrinsic reactivity of HRP in isopropyl alcohol and in methanol is hundreds of times lower than in water. In addition, the rates of spontaneous oxidation of the model prochiral substrate thioanisole in several organic solvents was observed to be some 100- to 1000-fold slower than in water. This renders peroxidase-catalyzed asymmetric sulf-oxidations synthetically attractive. 

Buchanan ID, Nicell JA, Wagner M. Reactor models for horseradish-peroxidase-catalyzed aromatic removal. J Environ Eng 1998 124(9) 794-802. 

Buchanan ID, Nicell JA. Model development for horseradish peroxidase-catalyzed removal of aqueous phenol. Biotechnol Bioeng 1997 54(3) 251-261. 

Wu Y, Taylor KE, Biswas N, Bewtra JK. A model for the protective effect of additives on the activity of horseradish peroxidase in the removal of phenol. Enzyme Microb Tech 1998 22 315-322. 

An example of the immobilization of antibodies on channel surfaces was presented by Eteshola and Leckband [395]. A microfluidic sensor chip was developed to quantify a model analyte (sheep IgM) with sensitivities down to 17 nM. This was achieved by first immobilizing a layer of bovine serum albumine (BSA) onto the channel wall, followed by specific adsorption of protein A to which the primary antibody for IgM was coupled covalently. This antibody could capture IgM, which was detected with the secondary antibody, labeled with horseradish peroxidase (Scheme 4.91). This enzyme catalyzes the conversion of the fluorogenic substrate 3-(p-hydroxyphenyl)propioni c acid into a fluorophore, which was quantified off-chip with a spectrofluorometer. The measured fluorescence signal was proportional to the analyte concentration in the test sample. 

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],... 

Several of the proteins with ferryl intermediates have been crystalised at sufficient resolution to allow the elucidation of their 3-dimensional structure. These include cytochrome c peroxidase [95], horseradish peroxidase [96], catalase [97], myeloperoxidase [98], ribonucleotide reductase [99], cytochrome P-450 [100] and myoglobin [101]. Of these only cytochrome c peroxidase has proved stable enough to crystallise with the iron in the ferryl form [26]. High-resolution structures exist for small FeIV model compounds, both in the presence [102] and absence [7,8] of an Fe=0 bond. These compounds can have sulphur, nitrogen and chloride ligation to the iron and the iron can be five [7,8] or six [8] coordinate. 

Mossbauer spectra has been extensively used to probe the structure of the iron nucleus in biological FeIV=0 compounds. These include horseradish peroxidase compoundl[134,180,181], horseradish peroxidase compound II [182,183], horseradish peroxidase compound X [181], Japanese-radish peroxidase compounds I and II [184], chloroperoxidase compound I [185], cytochrome c peroxidase compound I [186] and ferryl myoglobin [183]. Examples of Mossbauer spectra attributed to non-porphyrin-bound FeIV are only available from synthetic model compounds. These include compounds with [130] and without [4-8] an FeIV=0 bond. 

One of the variables in the structures of the porphyrins present in heme proteins is the presence or absence of vinyl substituents on the periphery of the macrocycle. For example, b hemes have vinyl substituents whereas c hemes do not. Because of the sensitivity of such vinyl substituents during synthetic transformations, it has often been desirable to use octa-alkyl porphyrins in model studies of the spectroscopic properties of heme systems. The development of improved methods for the preparation of octa-alkyl porphyrins has likewise increased the availability of such porphyrins for model studies (20, 21). To assess the effect that replacement of the two vinyl substituents in protoporphyrin IX with alkyl (ethyl) groups has on the MCD properties of the heme system, an extensive and systematic study of the MCD properties of mesoheme IX-reconstituted myoglobin and horseradish peroxidase in comparison with the spectra of the native protoheme-bound proteins has been carried out (22). The structures of these two porphyrins are shown in Figure 3. 

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