Avidin, biotin binding affinity

Q Yang, X-Y Liu, M Hara, P Lundahl, J Miyake. Quantitative affinity chromatographic studies of mitochondrial cytochrome c binding to bacterial photosynthetic reaction center, reconstituted in liposome membranes and immobilized by detergent dialysis and avidin-biotin binding. Anal Chem 280 94-102, 2000. 

Avidin (from egg white) [1405-69-2] Mr -70,000. Purified by chromatography of an ammonium acetate soln on CM-cellulose [Green Biochem J 101 774 1966]. Also purified by affinity chromatography on 2-iminobiotin-6-aminohexyl-Sepharose 4B [Orr 7 Bio/C/iew 256 761 1981]. It is a biotin binding protein. 

Nitration of the tyrosine rings in the four binding pockets of avidin or streptavidin can be done to increase the steric hinderance within the biotin binding sites (Morag et al., 1996). This process yields chromogenic proteins that have reduced binding affinity for biotin, thus allowing elution of biotinylated molecules under mild conditions. 

Avidin-biotin complex (ABC) is based on the high affinity that streptavidin (from Streptomyces avidinii) and avidin (from chicken egg) have for biotin. Biotin is a naturally occurring vitamin. One mole avidin will bind four moles biotin. ABC method affords a several-fold higher antigen detectability than those achieved in the standard indirect method. 

Strepavidin A 60 kD extracellular protein of Streptomyces avidinii with four high-affinity biotin binding sites. Unlike avidin, streptavidin has a near neutral isoelectric point and is free of carbohydrate side chains. 

The low-molecular-weight vitamin biotin is easily conjugated to antibodies and enzyme markers. Up to 150 biotin molecules can be attached to one antibody molecule, and the strong affinity of the biotin for the glycoprotein avidin allows its use as complex-ing secondary reagents. Biotin labeling of the primary (direct) or secondary (indirect) antibody can be used in the avidin-biotin methods. In the labeled avidin method the tracer is attached directly to the avidin molecule. In the avidin-biotin bridge method a biotinylated enzyme such as peroxidase is allowed to bind after attachment of avidin to the biotin-labeled antibody. 

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