Biotin is a water-soluble vitamin that is involved in fatty acid synthesis, gluconeogenesis and regulating cell signaling. It is a coenzyme required for the biological activity of five carboxylases (propionyl-CoA carboxylase, pyruvate carboxylase, methylcrotonyl-CoA carboxylase [MCC], acetyl-CoA carboxylase 1 and acetyl-CoA carboxylase 2; see Table 1.
The molecular structure of biotin is comprised of a ureido ring and a tetrahydrothiophene ring. Biotin plays a critical role in histone modification and gene regulation by covalently attaching to chromatin proteins.
Besides being a cofactor for apocarboxylases, biotin is also found in protein complexes such as ribosomes and cytoplasmic vesicles. It is involved in the regulation of protein expression by modifying the activity of transcription factors.
In mammals, biotin is incorporated into a number of different protein molecules via a process called biotinylation. This covalently bonded addition is catalyzed by an enzyme known as holocarboxylase synthetase. This process is essential for the biological activity of all mammalian biotin-dependent carboxylases and histones.
There are several reagents that can be used to label or detect biotin-containing proteins. These reagents include maleimide-containing reagents, which are highly reactive towards sulfhydryl groups at acidic to neutral pH.
A common reagent is EZ-Link biotin-BMCC (1-biotinamido-4-[4′-(maleimidomethyl)cyclohexane-carboxamido]butane). This reagent is very reactive towards sulfhydryl-containing amino acids at an acidic pH, but its cyclohexane ring stabilizes the spacer arm during conjugation and increases the length of the reaction.
Another reagent is desthiobiotin, which is non-sulfur containing and binds less tightly to avidin and streptavidin than biotin. However, desthiobiotin provides soft-release elution from avidin and streptavidin that minimizes the isolation of naturally biotinylated molecules that can interfere with results.