Horseradish peroxidase
The enzyme horseradish peroxidase (HRP), found in horseradish, is used extensively in molecular biology applications primarily for its ability to amplify a weak signal and increase detectability of a target molecule.
Applications
It is a 44,000 dalton glycoprotein with 4 lysine residue for conjugation to a labelled molecule. It produces a colour, fluorimetric or luminescent derivative of the labelled molecule 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.
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]
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) colored molecules while the HRP products of chemiluminescent substrates (e.g. SuperSignal, ECL) generate light.
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. 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.
External links
- Horseradish+peroxidase at the U.S. National Library of Medicine Medical Subject Headings (MeSH)
.svg){kind=small} | This biochemistry article is a stub. You can help Wikipedia by expanding it. |