Bio chem

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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 B7, vitamin H)
with a high degree of affinity and specificity. The dissociation constant of
avidin is measured to be KD ≈ 10-15 M, making it one of the strongest known
non-covalent bonds

In its tetrameric form, Avidin is estimated to be between 66-69 kDa in size.

10% of the molecular weight is attributed to carbohydrate content composed
of four to five mannose and three N-acetylglucosamine residues The
carbohydrate moieties of Avidin contain at least three unique oligosaccharide
structural types which are similar in structure and composition. 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

Discovery of Avidin

Avidin was first isolated from Chicken egg white by Esmond Emerson Snell

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. It was concluded that a component of the egg-white was sequestering
biotin which Snell verified in vitro using a yeast assay 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”. At the time the protein had been
tentatively named avidalbumin (literally, hungry albumin) by the involved
researchers at the University of Texas. The name of the protein was later
revised to Avidin based on its affinity for biotin (avid + biotin)

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. Soon after, researchers
Bayer and Wilchek developed new methods and reagents to biotinylate
antibodies and other biomolecules, 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 exhibits 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
(NeutraLite).

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. The modified avidin exhibits strong biotin
binding characteristics at pH 4 and releases biotin at a pH of 10 or higher. 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 KD ≈ 10-7M. This allows
elution from the Avidin matrix to occur under milder, non-denaturing
conditions, using low concentrations of biotin or low pH conditions.