In biochemistry, biotinylation is the process of covalently attaching biotin to a protein, nucleic acid or other molecule.
Biotinylation is rapid, specific and is unlikely to disturb the natural function of the molecule due to the small size of biotin (MW = 244.31 g/mol).
Biotin-binding to streptavidin and avidin is resistant to extremes of heat, pH and proteolysis, making capture of biotinylated molecules possible in a wide variety of environments.
Due to the strong affinity between biotin and streptavidin, the purification of biotinylated proteins has been a widely used approach to identify protein-protein interactions and post-translational events such as ubiquitylation[1] in molecular biology.
[2] Most chemical biotinylation reagents consist of a reactive group attached via a linker to the valeric acid side chain of biotin.
This biotinylation reaction can also go to completion, meaning that the product is generated with high uniformity and can be linked to streptavidin in a defined orientation e.g. for MHC multimers.
[4] The most common targets for modifying protein molecules are primary amine groups that are present as lysine side chain epsilon-amines and N-terminal α-amines.
The water solubility of sulfo-NHS-esters stems from their sulfonate group on the N-hydroxysuccinimide ring and eliminates the need to dissolve the reagent in an organic solvent.
The chemical reactions of NHS- and sulfo-NHS esters are essentially identical, in that they both react spontaneously with amines to form an amide bond.
Because the target for the ester is a deprotonated primary amine, the reaction is favored under basic conditions (above pH 7).
Carboxyl groups are found on the C-terminal ends of proteins and on glutamate and aspartate amino acid side chains.
Sodium periodate oxidizes the sialic acids on glycoproteins to aldehydes to form these stable linkages at pH 4–6.
[13] Upon the standard deprotection, the conjugates obtained can be purified using reverse-phase or anion-exchange HPLC Photoactivatable biotinylation reagents are ideal when primary amines, sulfhydryls, carboxyls and carbohydrates are not available for labeling.
These reagents rely on aryl azides, which become activated by ultraviolet light (UV; >350 nm), which then react at C-H and N-H bonds.
Biotinylation of red blood cells has been used as a means of determining total blood volume without the use of radiolabels such as chromium 51, allowing volume determinations in low birth weight infants and pregnant women who could not otherwise be exposed to the required doses of radioactivity.
When biotinylated proteins or other molecules are introduced, the biotin displaces the dye, resulting in a change in absorbance at 500 nm.