Horseradish peroxidase
The enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...

 horseradish peroxidase (HRP), found in horseradish
Horseradish is a perennial plant of the Brassicaceae family, which also includes mustard, wasabi, broccoli, and cabbages. The plant is probably native to south eastern Europe and the Arab World , but is popular around the world today...

, is used extensively in biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...

 applications primarily for its ability to amplify a weak signal and increase detectability of a target molecule.


Horseradish peroxidase is a 44,173.9-dalton glycoprotein with 4 lysine residues for conjugation to a labeled molecule. It produces a coloured, fluorimetric, or luminescent derivative of the labeled molecule when incubated with a proper substrate, allowing it to be detected and quantified.
HRP is often used in conjugates
Conjugated protein
A conjugated protein is a protein that functions in interaction with other chemical groups attached by covalent bonds or by weak interactions.Many proteins contain only amino acids and no other chemical groups, and they are called simple proteins...

 (molecules that have been joined genetically or chemically) to determine the presence of a molecular target. For example, an antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...

 conjugated to HRP may be used to detect a small amount of a specific protein in a western blot
Western blot
The western blot is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide...

. Here, the antibody provides the specificity to locate the protein of interest, and the HRP enzyme, in the presence of a substrate, produces a detectable signal. Horseradish peroxidase is also commonly used in techniques such as ELISA
Enzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...

 and Immunohistochemistry
Immunohistochemistry or IHC refers to the process of detecting antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots "immuno," in reference to antibodies used in the procedure, and...


Horseradish peroxidase is ideal in many respects for these applications because it is smaller, more stable, and less expensive than other popular alternatives such as alkaline phosphatase
Alkaline phosphatase
Alkaline phosphatase is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation...

. It also has a high turnover rate that allows generation of strong signals in a relatively short time span.

Moreover, "In recent years the technique of marking neurons with the enzyme horseradish peroxidase has become a major tool. In its brief history, this method has probably been used by more neurobiologists than have used the Golgi stain
Golgi's method
Golgi's method is a nervous tissue staining technique discovered by Italian physician and scientist Camillo Golgi in 1873. It was initially named the black reaction by Golgi, but it became better known as the Golgi stain or later, Golgi method.Golgi' staining was famously used by Spanish...

 since its discovery in 1870."


Alone, the HRP enzyme, or conjugates thereof, is of little value; its presence must be made visible using a substrate
Substrate (biochemistry)
In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate. In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or...

 that, when oxidized
Redox reactions describe all chemical reactions in which atoms have their oxidation state changed....

 by HRP using hydrogen peroxide
Hydrogen peroxide
Hydrogen peroxide is the simplest peroxide and an oxidizer. Hydrogen peroxide is a clear liquid, slightly more viscous than water. In dilute solution, it appears colorless. With its oxidizing properties, hydrogen peroxide is often used as a bleach or cleaning agent...

 as the oxidizing agent, yields a characteristic change that is detectable by spectrophotometric methods.

Numerous substrates for the horseradish peroxidase enzyme have been described and commercialized to exploit the desirable features of HRP. These substrates fall into several distinct categories. HRP catalyzes the conversion of chromogenic substrates (e.g., TMB
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...

3,3'-Diaminobenzidine is an organic compound. This derivative of benzidine is a precursor to polybenzimidazole fiber. As its water-soluble tetrahydrochloride, it has been used in immunohistochemical staining of nucleic acids and proteins.-Preparation:...

In biochemistry, 2,2'-azino-bis or ABTS is chemical compound used to observe the reaction kinetics of specific enzymes...

) into colored molecules, and produces light when acting on chemiluminescent substrates (e.g., SuperSignal, ECL).

Enhanced chemiluminescence (ECL)

Horseradish peroxidase catalyses the oxidation of luminol to 3-aminophthalate via several intermediates. The reaction is accompanied by emission of low-intensity light at 428 nm. However, in the presence of certain chemicals, the light emitted is enhanced up to 1000-fold, making the light easier to detect and increasing the sensitivity of the reaction. The enhancement of light emission is called enhanced chemiluminescence (ECL). Several enhancers can be used, but the most effective are modified phenols, especially p-iodophenol. The intensity of light is a measure of the number of enzyme molecules reacting and thus of the amount of hybrid.
ECL is simple to set up and is sensitive, detecting about 0.5 pg nucleic acid in Southern blots and in northern blot
Northern blot
The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA in a sample. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation,...

s. Detection by chemiluminescent substrates has several advantages over chromogenic substrates. The sensitivity is 10- to 100-fold greater, and quantitation of light emission is possible over a wide dynamic range, whereas that for coloured precipitates is much more limited, possibly over only one order of magnitude. Stripping filters are much easier when chemiluminescent substrates are used.

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