One of the most popular methods of noncovalent conjugation is to make use of the natural strong binding of avidin or its derivative streptavidin to biotin. Each avidin molecule contains a maximum of four biotin binding sites thereby increasing the strength of their interaction with biotin. Depending on the functionality present on the biotinylation compounds, specific reactive groups on antibodies may be modified to create a avidin binding site. Amines, carboxylates, sulfhydryls, and carbohydrate groups can be specifically targeted for biotinylation through the appropriate choice of biotin derivative.
Avidin is a glycoprotein found in egg whites that contains four identical subunits of 16,400 Da each. The subunits each contain one binding site for biotin also known as vitamin H. The biotin interaction with avidin is the strongest noncovalent affinity known, exhibiting a dissociation constant of about 1.3×10-15M. Tryptophan and lysine residues in each subunit are involved in forming the binding pocket. Biotin bound to avidin, is a very stable complex and resistant to degradation even in the presence of up to 8M guanidine. The strength of this noncovalent avidin-biotin interaction along with its resistance to degradation makes it extraordinarily useful in conjugation chemistry. The biospecificity of the interaction between avidin and biotin is similar to the antibody-antigen or the receptor-ligand recognition. The only disadvantage of using avidin is its tendency to bind nonspecifically with components other than biotin due to its high isoelectric point and carbohydrate content. These nonspecific interactions can lead to elevated background signals.
Streptavidin also binds to biotin similar to that of protein avidin, but it is of bacterial origin and originates from streptomyces avidinii. Like avidin, streptavidin also consist of four subunits, each with a single biotin binding site. But, due to certain structural difference in the amino acid sequence and lack of carbohydrates it can overcome the nonspecific binding and background signal deficiencies of avidin. Both avidin and streptavidin can tolerate a wide range of buffer, pH and chemical modification.
Biotinylated PE when inserted into a liposome can bind to the biotin biological target, avidin, through non covalent interactions. Using avidin or streptavidin as a bridging molecule, biotinylated antibodies can be linked to biotinylated liposomes. Streptavidin is sometimes conjugated directly to antibodies thus requiring only a single binding event to link antibodies to liposomes. Since biotin is a relatively small molecule, coupling it to macromolecules usually can be done without disturbing the activity or binding capability of the targeting molecule. Antibodies can be modified to contain one or more biotins to be able to strongly interact with either avidin or streptavidin.
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