Biotin peptide is an essential coenzyme for carboxylase enzymes, involved in fatty acid and isoleucine synthesis and gluconeogenesis. It has a chemical structure that comprises a ureido ring joined to a tetrahydrothiophene ring.
The biotin molecule is water soluble, and its solubility in solution is regulated by conjugation to a poly(ethylene glycol) chain. Increasing the length of this spacer arm increases the flexibility of the biotinylation reaction. Typical spacer arms are 3-6 amino acid residues long, and they vary between biotinylation reagents. The Thermo Scientific EZ-Link NHS-PEG4-Biotin reagent has a four-ethylene glycol chain (PEG) added to the spacer arm to increase reactivity and flexibility of the biotinylation reaction.
Using an avidin-biotin complex assay, biotinylation reactions are usually detected using the 4’-hydroxyazobenzene-2-carboxylic acid (HABA) dye, which noncovalently binds to biotin in the absence of protein. Biotin binding to HABA results in a change in HABA absorbance at 500 nm, which is proportional to the number of biotin molecules bound to the protein.
Sulfhydryl-reactive groups are the most commonly used reagents for labeling proteins with biotin. Maleimide groups such as those found on BMCC-Biotin are highly reactive towards sulfhydryl groups at acidic to neutral pH. Iodoacetyl and pyridyl disulfide groups have similar reactivity but require a higher pH to react. These sulfhydryl-reactive groups can be reduced to the corresponding thiol-biotin disulfide by reagents such as HPDP-Biotin, resulting in labeling of both the protein and the biotinylation reagent itself. These cleavage reaction products are then easily analyzed for biotinylation status by immunoblotting.