Naturally occurring interactions have been a great tool in a spectrum of labs, ranging from biotechnology and applied physics to medicine and biochemistry to achieve countless scientific milestones.
One such non-covalent interaction is avidin-biotin interaction which has significant applications in a variety of lab research, such as biochemical assays, affinity purification, diagnosis, and drug delivery.
Avidin is a basic glycoprotein (also known as tetrameric biotin-binding protein) isolated from egg whites of birds, reptiles, and amphibians. The avidin produced in chicken eggs represents a maximum of 0.05% of its total protein content.
Structurally, it has four identical subunits of about 68 kDa. Avidin binds biotin (also known as vitamin H) with high affinity and specificity through these homotetramers. 10% of the avidin molecular weight consists of carbohydrates, with 3-N-acetylglucosamine residues and 4-5 mannose.
The interaction of the protein with biotin has a dissociation constant of KD ≈ 10−15 M, making it one of the strongest non-covalent bonds — 103 to 106 times higher than an antigen-antibody interaction. Further, the protein has an isoelectric point (pI) of 10-10.5 — the closer the pI value to a neutral pH, the higher the chances of preventing non-specific binding.
A related protein to avidin, with a similar affinity for biotin, is streptavidin (produced as recombinant proteins). It’s found in the bacterium Streptomyces and has a dissociation constant of ≈10−14 mol/L.
The avidin-biotin complex has a wide range of applications in lab workflows, such as immunohistochemistry (IHC), ELISA, Western Blot, and many more.
In this article, we will cover the working mechanism of avidin, its interaction with biotin, and its applications in a variety of life sciences labs.
The tetrameric Avidin protein has a high affinity to binding four biotin molecules. It is stable and functional under a variety of pH and temperature conditions. A distinctive feature of avidin is that it can be conjugated to multiple proteins or modified in many ways without any effect on its functional roles. And, that’s why Avidin is highly useful for biotinylated protein detection and purification in a variety of conditions.
In the avidin-biotin complex (ABC)-based detection method, the avidin protein and some other biotinylated enzymes (such as Horseradish peroxidase (HRP) and Alkaline Phosphatase (AP) are complexed together to form a large macromolecule. The macromolecule has the binding affinity for biotinylated targets, such as primary and secondary biotinylated antibodies, peptides, lectins, nucleic acids, and other macromolecules.
When a chromogenic reagent or enzyme substrate is added to the reaction mixture, a colored precipitate forms. Multiple enzymes present in the complex amplify the obtained signals that help in detecting the target proteins.
Avidin is a tetrameric protein, made of four monomer units. Each avidin chain consists of 128 amino acids, arranged as eight-stranded antiparallel beta-barrel. The D-biotin binding sites are present in the inner region of the structure. Avidin’s secondary structure consists of beta sheets and extended beta turns (66%).
The monomeric subunits of avidin contain one binding site for the biotin molecule and one oligosaccharide modification. The amino acid residues, tryptophan, and lysine build the binding pocket of the avidin molecule that facilitates its binding with biotin.
Avidin has both insecticidal and antimicrobial activity. Additionally, it also has a bacteriostatic effect which has been observed against the bacterium Salmonella typhimurium. However, the activity is lost in the presence of biotin.
Researchers exploit chicken avidin and streptavidin in many research projects as probes and affinity matrices. For example, the insecticidal activity of the protein has been genetically engineered to impart resistance in host plants against the Colorado potato beetle. Moreover, the avidin-biotin interaction also serves as an important tool in developing highly sensitive nonradioactive detection and drug delivery systems.
Avidin is used in many lab assays for several purposes, such as immunohistochemistry, ELISA, and western blotting. In nanobiotechnology, avidin conjugated proteins are used to resolve acidic, basic, and neutral compounds.
Furthermore, it also has applications in direct serum injection assays to detect and purify drug enantiomers in biological samples.
NOTE: While performing these assays, it’s necessary to optimize blocking and wash conditions to avoid nonspecific binding (that result due to carbohydrate content and basic pI of avidin) and obtain quality assay results.
Avidin has a major application in protein detection and purification. Immobilized avidin matrices are used to immobilize small ligands and biotinylated proteins. The elution for this monomeric avidin column is achieved either by using biotin as a displacer or weak acid.
Furthermore, it is used for enantioseparation of acidic compounds, such as 2-aryl propionic acid derivatives, and can also be done using avidin matrices.
A range of life science industries and labs, such as biotechnology, molecular biology, and biochemistry, utilizes commercially available avidin for many lab workflows.
Many of them use products like antibody labeling reagents, cell-surface-protein biotinylation, purification kits, and new photoreactive biotinylation reagents to carry out their research work.In biotechnology, the avidin-biotin system has versatile applications in performing affinity purification and biochemical assays. It offers several advantages over other systems, such as efficient operation, weak signal amplification, high stability, and efficient use of highly diluted primary antibodies.
In biotech labs and industries, the avidin-biotin system is used for many purposes including medical research and manufacturing, clinical studies, biochemical assays, diagnostics, and nanoscale drug delivery.
For example, the oxidized natural avidin has major applications in drug delivery, considering it serves as a high-affinity receptor for a range of biotinylated drugs.
Avidin is also a major tool in tumor treatment. It’s used as a transport vehicle to transfer drugs, therapeutic genes, toxins, and radioisotopes to cancer tissues for their destruction. The protein can bind lectins, which are expressed on the tumor cell surface, through terminal mannose and N-acetylglucosamine. Thus, during the drug transfer, avidin binds to lectin and kills cancer cells.
The binding affinity of avidin with biotin has a spectrum of uses in biochemical assays, such as ELISA, pull-down assays, Western Blot, and ELISPOT.
The avidin-biotin system has also been used as an alternative assay system to radioimmunoassay systems. It was done by replacing radioiodine-labeled antibodies with biotinylated antibodies.
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Avidin is a tetrameric protein, whose high-affinity binding property with biotin is extensively exploited in a variety of research. In lab workflows, it forms a complex with biotinylated enzymes to bind target proteins, like biotinylated primary antibodies and secondary antibodies for their detection and purification.
Each monomer of the avidin can bind to one biotin and one carbohydrate, which makes the protein a major tool in biotech labs for many disease diagnosis and treatment procedures, such as Cancers.
The lab assays that frequently involve the use of avidin include ELISA, Western Blot, Immunohistochemistry, and many others. Scientists either prepare their own reaction mixture to perform these assays or utilize commercially available avidin kits to carry out the workflows.
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