Chapter 11 - Biotinylation Reagents

Abstract

The highly specific interaction of avidin with the small vitamin biotin can be a useful tool in designing assay, detection, and targeting systems for biological analytes. The extraordinary affinity of (strept)avidin's interaction with biotin allows biotin-containing molecules in complex mixtures to be discretely bound with (strept)avidin conjugates. If the (strept)avidin–biotin complex contains detection components, then the targeted analytes can be located or quantified. This assay concept is made possible through the ability of biotin to be covalently attached to other targeting molecules, such as antibodies. In this sense, biotin derivatives may be prepared, which contain reactive portions able to couple with particular functional groups in proteins and other molecules. This chapter specifically studies the biotin modification of secondary molecules, called biotinylation, which results in covalent derivatives containing one or more bicyclic biotin rings extending from the parent structure. These biotinylation sites are still capable of binding avidin or streptavidin with the specificity and nearly the same avidity of free biotin in solution. Biotinylation reagents also possess various cross-bridges or spacer groups built off the valeric acid side chain of the molecule. Since the binding sites for biotin on avidin and streptavidin are pockets buried about 9 Å beneath the surface of the proteins, spacers can affect the accessibility of biotinylated compounds for efficiently binding avidin or streptavidin conjugates.