Horseradish peroxidase substrate oxidation

In this study, the kinetics of horseradish peroxidase-catalyzed oxidation of p-cresol (4-methylphenol) is evaluated in a number of representative water-miscible organic solvents. Cresol is one of the most common phenols used in the phenolic resin industry (X) and is an excellent substrate of peroxidase (JL2.). The stoichiometry of peroxidase catalysis is described in Equation 1. The predominant products in aqueous solutions are... 

Figure 6. ESR spectra (25°C) of radicals from the horseradish peroxidase/HjOj oxidation of peroxidase substrates, a, 2mM menadione (hydroquinone form), I mM H2O2, 1.6 pM peroxidase, pH 7.5 b, 1 mM hydroquinone, 0.5 mM H2O2, 0.1 pM peroxidase, pH 6.5 c, 8 mM 3,4-dihydroxyphenyIalanine (dopa), 0.175 mM H2O2 0.04 pM peroxidase, 0.225 M Zn, pH 4.5. From [83,120], with permission.

Two enzymes are commonly used in the ELISA methods, horseradish peroxidase or alkaline-phosphatase, with little difference in the sensitivity of the two. The yellow color produced by the alkaline-phosphatase may be more difficult to see visually at low levels than the blue or green color produced by the horseradish peroxidase. The major problem with the horseradish peroxidase is that an oxidizing agent is necessary for color development, whereas a substrate tablet is all that is needed for the alkaline-phosphatase color development. Alkaline-phosphatase is used in the ball kit, whereas horseradish peroxidase is utilized in the other ELISA kits. Alkaline-phosphatase is preferable because there is no problem with the substrate, whereas with the horseradish peroxidase the oxidizing agent is subject to deterioration. 

Extensive studies have established that the catalytic cycle for the reduction of hydroperoxides by horseradish peroxidase is the one depicted in Figure 6 (38). The resting enzyme interacts with the peroxide to form an enzyme-substrate complex that decomposes to alcohol and an iron-oxo complex that is two oxidizing equivalents above the resting state of the enzyme. For catalytic turnover to occur the iron-oxo complex must be reduced. The two electrons are furnished by reducing substrates either by electron transfer from substrate to enzyme or by oxygen transfer from enzyme to substrate. Substrate oxidation by the iron-oxo complex supports continuous hydroperoxide reduction. When either reducing substrate or hydroperoxide is exhausted, the catalytic cycle stops. 

Akhavan-Tafti et al. have developed a new class of peroxidase substrates that produce CL upon enzymatic oxidation. Horseradish peroxidase (HRP) is... 

The classic oxidizing systems of human myeloperoxidase and horseradish peroxidase were exploited for their well-known abilities to oxidize phenolic substrates. Under conditions of incubations, the following oxidation pathway was defined (155). Peroxidases are first converted to the oxidized... 

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. 

A quite different approach came from Chance and others using heme enzymes (1947). Purified horseradish peroxidase has a characteristic absorption spectrum which was visibly altered in the presence of hydrogen peroxide. When an appropriate substrate was added it was oxidized by the hydrogen peroxide and the spectrum reverted to that of the original state of the enzyme. Similar studies were performed with catalase, showing that prosthetic groups in enzymes underwent reversible changes in the course of their reactions. 

Horseradish peroxidase catalyzes the cleavage of hydroperoxide substrates forming active oxygen, which oxidizes molecules resulting in a colored product. For application in Western blots the reaction product must be insoluble in aqueous buffer solutions. 

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. 

Simultaneous detection of three antigens within one tissue section became possible by employing an additional peroxidase substrate such as the Vector VIP Substrate kit (Vector Lab, Burlingham, CA) (Pujic et al., 1998). This substrate is oxidized by horseradish peroxidase and yields a rose-colored final reaction product which differs in color from that... 

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. 

Horseradish peroxidase (HRP) is a hemeprotein which catalyses the oxidation of a large variety of inorganic and organic substrates (Dunford and Stillman, 1976). Chloroperoxidase (CPO) is a versatile heme enzyme since it shares similar properties with classical peroxidases and P-450 monooxygenases and also catalyses the oxidative halogenation of organic substrates (Blake and Hager, 1990). 

Redox potential is a catalytically relevant property of heme peroxidases, as in theory, sets the limit for the oxidative ability of the enzyme. An inverse correlation was found between the activity and the redox potential of methoxybenzenes and methoxy-substituted benzyl alcohols for lignin peroxidase (LiP) and horseradish peroxidase (HRP) [42, 43]. These enzymes were able to catalyze the oxidation of methoxybenzenes with redox potential as high as 1.45 V and 1.12 V, respectively [42]. In the case of methoxy-substituted benzyl alcohols, the maximum substrate redox potential was 1.39 V for both enzymes [43]. This type of correlation has allowed ranking enzymes from the more oxidant to the less oxidant. The inverse... 

Gilabert MA, Hiner ANP, Garcia-Ruiz PA et al (2004) Differential substrate behavior of phenol and aniline derivatives during oxidation by horseradish peroxidase kinetic evidence for a two-step mechanism. Biochim Biophys Acta 1699 235-243... 

Certain fluorescent compounds, such as the coumarin derivative, scopoletin, can act as hydrogen donors in the oxidative reaction catalysed by horseradish peroxidase (Udenfriend, 1969), an enzyme that exhibits substrate specificity for hydrogen peroxide... 

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