Horseradish peroxidase
The enzyme horseradish peroxidase (HRP), found in horseradish, is used extensively in biochemistry applications primarily for its ability to amplify a weak signal and increase detectability of a target molecule.
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[edit] Applications
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 (molecules that have been joined genetically or chemically) to determine the presence of a molecular target. For example, an antibody conjugated to HRP may be used to detect a small amount of a specific protein in a western blot. 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.[1] Horseradish peroxidase is also commonly used in techniques such as ELISA and Immunohistochemistry due to its monomeric nature and the ease with which it produces coloure products. Peroxidase, a heam-containing oxidoreductase, is a commercially important enzyme which catalyses the reductive cleavage of hydrogen peroxide by an electron donor.
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. 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 since its discovery in 1870."[2]
[edit] Substrates
Alone, the HRP enzyme, or conjugates thereof, is of little value; its presence must be made visible using a substrate that, when oxidized by HRP using hydrogen peroxide as the oxidizing agent, yields a characteristic change that is detectable by spectrophotometric methods.[3]
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, DAB, ABTS) into colored molecules, and produces light when acting on chemiluminescent substrates (e.g., SuperSignal, ECL).
[edit] Structure
The structure of the enzyme was first solved by X-ray crystallography in 1997[4] and has since has been solved several times with various substrates [5]. It is an all alpha-helical protein which binds heme as a Cofactor_(biochemistry).
[edit] 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 blots. 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.
[edit] References
- ^ Chau YP, Lu KS (1995). "Investigation of the blood-ganglion barrier properties in rat sympathetic ganglia by using lanthanum ion and horseradish peroxidase as tracers". Acta Anatomica (Basel) 153 (2): 135–144. doi:10.1159/000313647. PMID 8560966.
- ^ Quoted in "Cell Marking with Horseradish Peroxidase" adapted from D. Purves and J.W. Lichtman, Principles of Neural Development. Sinauer Associates, Inc., Sunderland, 1985.
- ^ N.C.Veitch (2004). "Horseradish peroxidase: a modern view of a classic enzyme". Phytochemistry 65 (3): 249–259. doi:10.1016/j.phytochem.2003.10.022. PMID 14751298.
- ^ Crystal structure of horseradish peroxidase C at 2.15 A resolution. Gajhede, M. Schuller, D.J. Henriksen, A. Smith, A.T. Poulos, T.L. NAT.STRUCT.BIOL. vol:4, pag:1032-1038 (1997), PMID 9406554
- ^ http://www.ebi.ac.uk/pdbe-apps/widgets/unipdb?uniprot=P00433
[edit] External links
- HRP Reference Centre (The site of BBI Enzymes, a manufacturer of HRP).
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