AVIDIN

The Uses of AVIDIN

Avidin egg white was used in an assay using functionalized xenon as a biosensor to detect biotin-avidin binding. Egg white was used at 80 nmol.

The Uses of AVIDIN

Avidin forms an extremely strong complex with d-biotin (K_d ~ 10^-15). This activity has made both avidin and biotin extremely useful labels in immunochemical methods of detection and quantitation.

What are the applications of Application

Avidin is a biotin-binding Glycoprotein containing four identical subunits, frequently used to tag antibodies and other probes for immunodetection methods.

Definition

A protein occurring in egg white, where it constitutes approximately 0.2% of the total protein. It has the property of combining firmly with biotin and rendering it unavailable to organisms, since proteolytic enzymes do not destroy the avidin–biotin complex. Avidin loses its ability to combine with biotin when subjected to heat; hence cooked egg white does not lead to biotin deficiency.

General Description

Avidin is a homotetrameric glycoprotein found in the egg white of birds, reptiles and amphibians. Each subunit is 16 kDa, singly glycosylated and binds to a molecule of biotin with greater affinity and specificity. Recombinant avidin from corn is similar to avidin from egg white in terms of properties like isoelectric point (pI) and antigenic properties except for the molecular weight. Avidin from corn has low molecular weight than chicken egg-derived avidin. This might be due to less complex glycosylation composition in plants. Commercial production of avidin from corn has certain advantages in terms of availability of greater biomass and avoiding the co-purification of animal virus.

Enzyme inhibitor

This bacteriostatic egg-white glycoprotein (MW = 54.7 kDa; CAS 1405- 69-2; Abs10mg/mL = 15.5 at 282nm) contains four identical subunits, each containing a ultrahigh-affinity binding site (Kd ≈ 10?15 M) for biotin, a coenzyme essential in many carboxylation reactions. The avidin-biotin interaction is among the strongest known noncovalent interactions, making it the constant subject of biophysical inquiry. By binding ~15 μg biotin per mg, avidin (which makes up makes up ~0.05% of total egg protein, or around 1.8 mg/egg) deprives invading microorganisms of this vital coenzyme. Many microorganisms partly by-pass this effect by possessing enzymes that covalently link biotin to e-NH2 groups of active-site lysyl residues within biotin-dependent carboxylases. Upon cooking, biotin-free avidin is fully denatured, thus imparting no effect on those consuming cooked eggs. Applications in Cytology: The avidin-biotin interaction provides a simple and sensitive immunoenzymatic method to localize antigens (Ag) in formalin-fixed tissues, typically involving a biotin-labeled secondary antibody (Ab), followed by the addition of avidin-biotin peroxidase complex, and affording superior results over the use of unlabeled antibodies. The availability of biotin-binding sites in the Ab Ag complex is created by the incubation of a relative excess of avidin with biotin-labeled peroxidase. The oligomeric structure of avidin serves to bridge biotin-labeled peroxidase molecules to biotin-labeled peroxidase molecules. When compared on a mol-for-mol basis, the extreme affinity of avidin-biotin interactions rivals or exceeds that of nearly all known Ab-Ag interactions (See also Streptavidin). Biochemical Applications with Recombinant Fusion Proteins: In Escherichia coli, the biotin carboxy carrier protein (BCCP) is biotinylated by BirA, a biotin ligase that covalently attaches a biotin to the amino group of a lysine residue present within the recognition sequence within BCCP. A minimal biotinylation sequence has been found from screens of combinatorial peptide libraries; this 13-residue peptide (Sequence: GLNDIFEAQKIEW), along with a 13- residue long variant (Sequence: GLNDIFEAQKIEWHE), termed the AviTag?, have been identified as effective in vivo and in vitro substrates for the BirA enzyme. When targets proteins are fused to the AviTag and co-expressed in vivo along with BirA, they can be biotinylated in bacteria, yeast, insect, or mammalian cells. Furthermore, when recombinant proteins are fused to the AviTag and incubated in vitro with purified BirA, they can be biotinylated efficiently on the central lysine residue in the AviTag.

Purification Methods

Avidin is purified by chromatography of an ammonium acetate solution on CM-cellulose [Green Biochem J 101 774 1966]. It is also purified by affinity chromatography on 2-iminobiotin-6-aminohexyl-Sepharose 4B [Orr J Biol Chem 256 761 1981]. Itis a biotin-binding protein.