Horseradish peroxidase substrate

Petersen KH. Novel horseradish peroxidase substrates for use in immunohistochemistry. J Immunol Methods 2009 340(1) 86-9. 

Table 16.3-8. Selected hydroxylation reactions of phenols catalyzed by horseradish peroxidase. Substrate Product Literature...

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

Secondary antibody and determination. A secondary antibody labeled with an enzyme is added which binds to the primary antibody that is bound to the coating antigen. If the primary antibody were produced in a rabbit, an appropriate secondary antibody would be goat anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP) (or another enzyme label). Excess secondary antibody is washed away. An appropriate substrate solution is added that will produce a colored or fluorescent product after enzymatic conversion. The amount of enzyme product formed is directly proportional to the amount of first antibody bound to the coating antigen on the plate and is inversely proportional to the amount of analyte in the standards. 

Rodrfguez-Lopez, J.N., Gilabert, M.A., Tudela, J., Thorneley, R.N.R, and Garcfa-Canovas, R, Reactivity of horseradish peroxidase compound II toward substrates kinetic evidence for a two-step mechanism,... 

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. 

Horseradish peroxidase (HRP) is a member of the large class of peroxidases, which are enzymes defined as oxidoreductases using hydroperoxide as electron acceptor. HRP has been widely used for the construction of amperometric biosensor for the determination of H202 and small organic and inorganic substrates. 

Figure 4.3. The catalytic cycle of horseradish peroxidase with ferulate as reducing substrate. The rate constants Ki, K2, and K3 represent the rate of compound I formation, rate of compound I reduction, and rate of compound II reduction, respectively.

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. 

The choice of enzyme is governed by the availability of substrate and the type of detector. Several substrates have been developed for use with horseradish peroxidase o-phenylene diamine (OPD), 2,2-azino-di(3-ethylbenzo-thiazoline-6-sulphonate) (ABTS) and 5,5 -tetramethylbenzidine hydrochloride (TMB). 

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. 

Enzymes can be linked to immunoassay reagents to amplify detection by the use of fluorogenic substrates. Enzyme-linked fluoroimmunoassays (ELFIAs) are very similar to photometric EIAs in format and workflow. EIAs are widely used, and many commercial ELFIA assays and systems are available/15 The most commonly used enzymes in ELFIAs are horseradish peroxidase, alkaline phosphatase, and fi-D-... 

If antibodies conjugated to fluorochromes are not desirable, enzyme-labeled antibodies can also be used. In this case, in the presence of a substrate and a chromagen, an enzyme provides the indicator system necessary to visuahze the location of the antibody (Carson, 1997). Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the enzymes most commonly conjugated to antibodies not labeled by... 

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