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Horseradish peroxidase is a 44,173.9-dalton glycoprotein with six lysine residues which can be conjugated to a labeled molecule. It produces a coloured, fluorimetric [6] or luminescent derivative of the labeled molecule when incubated with a proper substrate, allowing it to be detected and quantified.
3,3′,5,5′-Tetramethylbenzidine or TMB is a chromogenic substrate used in staining procedures in immunohistochemistry as well as being a visualising reagent used in enzyme-linked immunosorbent assays . [1] TMB is a white solid that forms a pale blue-green liquid in solution with ethyl acetate.
Horseradish peroxidase has an accessible active site, and many compounds can reach the site of the reaction. On the other hand, for an enzyme such as cytochrome c peroxidase , the compounds that donate electrons are very specific, due to a very narrow active site.
It is commonly used as a substrate with hydrogen peroxide for a peroxidase enzyme (such as horseradish peroxidase) or alone with blue multicopper oxidase enzymes (such as laccase or bilirubin oxidase). Its use allows the reaction kinetics of peroxidases themselves to be followed.
A suitable alternative to radioimmunoassay would substitute a nonradioactive signal in place of the radioactive signal. When enzymes (such as horseradish peroxidase) react with appropriate substrates (such as ABTS or TMB), a change in color occurs, which is used as a signal. However, the signal has to be associated with the presence of an ...
Horseradish peroxidase is also capable of oxidizing these substrates, but its heme is not covalently bound and becomes damaged during turnover. [4] A specific vanadium bromoperoxidase in marine organisms (fungi, bacteria, microalgae, perhaps other eukaryotes) uses vanadate and hydrogen peroxide to brominate electrophilic organics. [5]
Some examples of substrates used (also known as chromogens) are AEC (3-Amino-9-EthylCarbazole), or DAB (3,3'-Diaminobenzidine). Use of one of these reagents after exposure to the necessary enzyme (e.g., horseradish peroxidase conjugated to an antibody reagent) produces a positive immunoreaction product.
According to Peroxidase substrate only H 2 O 2 This is contradiction and needs editorial interaction. Somogyi26 17:07, 22 October 2012 (UTC) []. Peroxidases have two substrates: one is the oxidant (hydrogen peroxide in this case; it gets reduced to water in the process) and the other is a reductant (most commonly a dye in lab application; it gets oxidized and gives color) which can be one of ...