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|> 11.0||> 14.5||> 14.5|
(% w : w)
(% w : w)
|< 3.0||< 0.5||< 0.5|
Count in 1 mg
|<1||< 1||< 1|
|Appearance||white free flowing lyophilised powder,
free of any visible impurities
|Definition of |
|One Unit activity binds 1 µg of biotin|
|Origin||Egg proteins from hens|
Avidin vs Streptavidin
Avidin shows only 30% homology with streptavidin, however its secondary, tertiary and quaternary structures are identical.
The amino acid sequences of avidin (black) and streptavidin (blue).
Homologous residues/domains in both proteins are shown in bold type. Positively charged residues (Lys, Arg) are underlined. The RYD-containing sequence in streptavidin, which is homologous to that of fibronectin, appears in bold red. The N- and C-terminal sequences clipped by post-secretory processing (via proteolytic cleavage) on streptavidin are shown in purple. The position of the sugar residue, linked to Asn-17 of avidin in the typical NXT(S) type carbohydrate-containing consensus sequence, is identified by a star (*). Tyr-33 & -43, the nitrated amino acid residue in Nitro-Avidin & -Streptavidin, respectively, is similarly (*) identified. Cys-4 & 83 formed a disulfide bridge in native avidin; natural variant (Ile-34→Thr-34) in ~50% of the molecules.
Avidin - biotin system
Avidin, a egg protein, binds the water-soluble vitamin biotin (vitamin B7 or H) very tightly – irreversibly – and specifically, with an outstanding dissociation constant of ~10-15 M. The bond formation between biotin and Avidin is very rapid, and once formed, is unaffected by extremes of pH, temperature, organic solvents and other denaturing agents.
The protein, its stability and affinity for biotin, have been extensively investigated and characterized. It folds into a quaternary tetramer structure made of 4 subunits of identical amino acid composition and sequence, each containing 9 lysine residues.
The avidin-biotin system is used extensively as a mediator in a broad range of biological applications, including isolation, localization, cytochemistry, immunoassay and diagnostics (Wilchek & Bayer, 1990). The alkaline properties and oligosaccharide content of the avidin isolated from egg white – which at least appear to be responsible for some nonspecific interaction at biological levels – had originally given the preference to the use of the bacterial biotin-binding analog, ie Streptavidin. However, the nonspecific absorption properties of avidin have been corrected to the extent that the bioengineered neutral & deglycosylated avidin, ie NeutraLite Avidin, exhibits very low levels of nonspecific absorption to biological materials while at the same time leaving a large number of lysine residues readily available for chemical derivatization & conjugation.
The Wilchek & Bayer Group at the Weizmann Institute are to be credited with the merit & success of developing innovative molecular probing / targeting techniques – sometimes coined “bio-recognition” – using the Avidin-Biotin System (ABS).
The general approach in avidin-biotin technology
A target molecule in an experimental system is labeled with a biotinylated binder molecule. The biotinylated binder is in turn labeled with an avidin-conjugated probe. Alternatively, the biotinylated binder can be labeled with free avidin followed by a biotinylated probe.
The protein was discovered by Esmond Emerson Snell (1914–2003) but not yet isolated. In 1941, in collaboration with Paul Gyorgy a specialist of biotin (Vitamin H) , they confirmed that the isolated egg protein was the cause of biotin deficiency or “egg white injury”.. The researchers at the University of Texas named the protein : avidalbumin representing the concatenation of Avid(= hungry) + albumin.
Few years later, the protein was renamed as avidin representing the concatenation of avid + biotin.
Avidin is a 66-KDa glycoprotein synthesised in the oviduct of a hen and deposited in the albumen of its eggs. Depending of the littérature the molecular weight of avidin can be slightly different.
How many sugars has the avidin ?
Sugar heterogeneity of avidin. The avidin carbohydrate contains at least three distinct oligosaccharide structural types of similar size as shown above; N-linked glycan at Asn-17 consists of GlcNAc(beta1-2)Man(alpha1-3)[GlcNAc(beta1-4)][Man(alpha1-?) Man(alpha1-6)]Man(beta1-4) GlcNAc(beta1-4)GlcNAc. Some terminal galactose may even be present on some glycans.
What is the Iso electric point of avidin
Avidin is highly cationic with an isoelectric point (pI) of about 10.5. Glycosylation occurs at the Asn-17 residue, in a typical NXT(S) or Asn-Xxx-Thr(Ser) type carbohydrate-containing consensus sequence. Oligosaccharide components (heterogeneous structures composed largely of mannose (Man) and N-acetylglucosamine (GlcNac)) and positively charged amino acid residues (Lys & Arg) can interact nonspecifically with lectins and negatively charged cell surfaces and nucleic acids, thereby potentially causing nonspecific bindings in diagnostic and therapeutic applications1.
A avidin derivative enables to avoid this non specific bonding.
Bioengineering methods have been developed to suppress such nonspecific bindings. The result: NeutraLite Avidin , a nonspecific binding-free form of native avidin, in which the signal/background ratio can be customarily adjusted (see Poly -NeutraLite avidin or Oligomerized neutralite avidin) and the biotin-specific binding can be made reversible (see Nitroavidin, Avidin Monomer, Iminobiotin) to accommodate an ever larger range of applications.
How avidin binds the biotin ?
The biotin-binding site is a deep, pear-shaped pocket, whose volume as well as three-dimensional structure and orientation of the residues participating in hydrogen bonding with the vitamin are predetermined as complementary to that of the incoming vitamin (Livnah et al., 1993; Rosano et al., 1999). In the absence of biotin, the biotin-binding pocket contains five molecules of water that mimic the structure of biotin in the binding site until biotin is bound (Rosano et al., 1999).
In the apo-protein (without ligand) the vitamin-binding pocket is fairly open due to the flexible L3,4-loop, thus allowing fast access of biotin to the binding site.
When biotin is bound, it is buried inside the central pocket of the β-barrelstructured protein with the vitamin’s bicyclic ring at the bottom of the cavity. The vitamin is trapped by conformational readjustments of the protein primarily involving the stiffened L3,4-loop, but also L5,6. During the process, three amino acid residues of L3,4 contribute additional interactions with biotin (Rosano et al.,1999). Thus, the high affinity of biotin to avidin stems from an extremely slow dissociation rate (Green, 1963a; Green, 1990).
(Reference Avidin as an Alternative Scaffold S.LEHTONEN UNIVERSITY OF TAMPERE)
- This high affinity is mainly explained by shape complementarity : This interaction is the most is the most important in the binding by affinity in regards of the 2 others. It has been shown by a X-ray study (Livnah et al., 1993; Pugliese et al., 1993; Rosano et al., 1999).
- Five Hydrogen bonds are formed with an ureido-ring and with valeryl side chain of biotin, respectively (Livnah et al., 1993).
- Five aromatic residues form represents the Hydrophobic interaction
What is the avidin process purification ?
In 25.000 kg (25 tons) of albumen from 800.000 eggs, there is 1,2 kg of avidin available for extraction at 100% yield extraction. The current yield is about 30% so a production batch is about 500 gr of freeze dried product.
At e-Proteins, the purification process is based on a proven technology in use for more than 30 years. It has been developed by Belovo Chemicals (Bastogne, Belgium) in the 90’s, a world wild egg technology leader. In 2012 e-Proteins has launched an R&D project to improve and to optimise this process in order to offer to its customer a highest quality. Today our product presents a >99% of purity and can be produced with a endotoxin free certificate ( batch size 50 gr) .
What is the avidin market ?
Avidin is used in diagnostic kits , Cell culture for blocking the biotin and in pharmaceutical application. All of those markets have their certification ’s requirements and their quality’s requirements. e-Proteins is able to reach all of those certifications including the virus inactivation when required.