=Biological Overview=

Avidin is a tetrameric protein produced in the oviducts of birds, reptiles and amphibians which is deposited in the whites of their eggs. In chicken egg white, avidin makes up approximately 0.05% of total protein (approximately 1.8 mg per egg). The tetrameric protein contains four identical subunits (homotetramer) each of which can bind to biotin (Vitamin H) with a high degree of affinity and specificity. The dissociation constant of avidin is measured to be "K"D ≈ 10-15 M, making it one of the strongest known non-covalent bonds [Green, 1963] .

In its tetrameric form, Avidin is estimated to be between 66-69 kDa in size [Korpela, 1984] . 10% of the molecular weight is attributed to carbohydrate content composed of four to five mannose and three N-acetylglucosamine residues [Green, 1975] . The carbohydrate moieties of Avidin contain at least three unique oligosaccharide structural types which are similar in structure and composition [Bruch & White, 1982] . A non-glycosylated form of Avidin has been isolated from commercially prepared product however; it is not conclusive whether the non-glycosylated form occurs naturally or is a product of the manufacturing process [Hiller et al., 1987] .

Discovery of Avidin

Avidin was first discovered by Esmond Emerson Snell (1914-2003). The route to discovery began with the observation that chicks on a diet of raw egg-white were deficient in biotin, despite availability of the vitamin in their diet [Eakin et al., 1940] . It was concluded that a component of the egg-white was sequestering biotin [Eakin et al., 1940] which Snell verified "in vitro" using a yeast assay [Snell et al., 1940 ] . Snell later isolated the component of egg white responsible for biotin binding and in collaboration with Paul Gyorgy, confirmed that the isolated egg protein was the cause of biotin deficiency or “egg white injury” [Gyorgy, 1941] . At the time the protein had been tentatively named avidalbumin (literally, hungry albumin) by the involved researchers at the University of Texas [Gyorgy, 1941] . The name of the protein was later revised to Avidin based on its affinity for biotin (avid + biotin) [Kresge et al., 2004] .

Applications of Avidin

Research in the 1970s helped establish the Avidin-biotin system as a powerful tool in biological sciences. Aware of the strength and specificity of the Avidin-biotin complex, researchers began to exploit Avidin as a probe and affinity matrix in numerous research projects [Hofmann & Kiso, 1976] [Bayer et al, 1976] [Angerer et al., 1976] [Heffegeness & Ash, 1977] . Soon after, researchers Bayer and Wilchek developed new methods and reagents to biotinylate antibodies and other biomolecules [Bayer et al, 1985] [Wilchek et al., 1986] , allowing the transfer of the Avidin-biotin system to a range of biotechnological applications. Today Avidin is used in applications ranging from research and diagnostics to medical devices and pharmaceuticals.

Avidin's affinity for biotin is exploited in wide ranging biochemical assays, including western blot, ELISA, ELISPOT and pull-down assays. Avidin immobilized onto solid supports is also used as purification media to capture biotin-labelled protein or nucleic acid molecules. For example, cell surface proteins can be specifically labelled with membrane impermeable biotin reagent, then specifically captured using an avidin-based support.

Modified Forms of Avidin

As a basically charged glycoprotein, avidin is purported to exhibit non-specific binding in some applications. Neutravidin, a deglycosylated avidin with modified arginines, exhibits a more neutral pI and is available as an alternative to native avidin where problems of non-specific binding arise. Deglycosylated, neutral forms of avidin are available through Sigma-Aldrich (Extravidin), Thermo Scientific (NeutrAvidin), Invitrogen (NeutrAvidin) and Belovo (NeutraLiteTM).

Given the strength of the avidin-biotin bond, dissociation of the avidin-biotin complex requires extreme conditions which cause protein denaturation. The non-reversible nature of the avidin-biotin complex can limit avidin’s application in affinity chromatography applications where release of the captured ligand is desirable. Researchers have created an avidin with reversible binding characteristics through nitration or iodination of the binding site tyrosine [Morag et al., 1996] . The modified avidin exhibits strong biotin binding characteristics at pH 4 and releases biotin at a pH of 10 or higher [Morag et al., 1996] . A monomeric form of Avidin with a reduced affinity for biotin is also employed in many commercially available affinity resins. The monomeric Avidin is created by treatment of immobilized native Avidin with urea or guanidine HCl (6-8M) giving it a lower dissociation "K"D ≈ 10-7M [Kohanski & Lane, 1990] . This allows elution from the Avidin matrix to occur under milder, non-denaturing conditions, using low concentrations of biotin or low pH conditions.

ee also




* Angerer, L. et al., (1976) An electron microscope study of the relative positions of the 4S and ribosomal RNA genes in HeLa cells mitochondrial DNA. Cell. 9, 81-90.
* Bayer, EA. et al., (1985). 3-(N-Maleimido-propionyl)biocytin: a versatile thiol-specific biotinylating reagent. Anal. Biochem., 149, 529-536.
* Bayer, EA., et al., (1976) Preparation of ferritin-avidin conjugates by reductive alkylation for use in electron microscopic cytochemistry. J. Histochem. Cytochem. 24, 933-939.
* Bruch, R. & White, H. (1982). Compositional and structural heterogeneity of Avidin glycopeptides. Biochemistry, 21, 5334-5341.
* Eakin, E. et al., (1940). Egg-white injury in chicks and its relationship to a deficiency of vitamin H (biotin). Science, 92, 224
* Green, N. (1963). The Use of [14C] Biotin for Kinetic Studies and for Assay. Biochem J, 89, 585-591
* Green, N. (1975). "Avidin," Advances in Protein Chemistry 29, 85-133.
* Gyorgy, P. (1941). Egg-white injury as the result of non-absorption or inactivation of biotin. Science, 93, 447-478.
* Heggeness, MH. & Ash, JF. (1977) Use of the Avidin-biotin complex for the localization of actin and myosin with fluorescence microscopy. J. Cell. Biol. 73, 783-788.
* Hiller., Y et al., (1987). Biotin binding to avidin. Biochem. J., 248, 167-171.
* Hofmann, K., & Kiso, Y. (1976) An approach to the targeted attachment of peptides and proteins to solid supports. Proc. Natl. Acad. Sci. USA. 73, 3516-3518.
* Kohanski, R & Lane, M. (1990) Methods in Enzymology. 183, 194.
* Korpela, J. (1984). Avidin, a high affinity biotin-binding protein as a tool and subject of biological research. Med. Bio. 62, 5-26.
* Kresge, N. et al., (2004). The Discovery of Avidin by Esmond E. Snell. J. Bio. Chem. 279, e5
* Morag, E. et al., (1996) Reversibility of biotin-binding by selective modification of tyrosine in Avidin. Biochem. J. 316, 193-199.
* Snell, E. et al., (1940). A quantitative test for biotin and observations regarding its occurrence and properties. J. Am. Chem. Soc., 62, 175-178.
* Wilchek, M. et al., (1986). p-Diazobenzoyl biocytin--a new biotinylating reagent for the labeling of tyrosines and histidines in proteins. Biochem. Biophys. Res. Commun. 138, 872-879.

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