Coupling of Antibodies with Biotin
The avidin-biotin bond is the strongest known biological interaction between a ligand and a protein (Kd = 1.3 × 1(105 M at pH 5) (1). The affinity is so high that the avidin-biotin complex is extremely resistant to any type of denaturing agent (2). Biotin Fig. 1) is a small, hydrophobic molecule that functions as a coenzyme of carboxylases (3). It is present in all living cells. Avidin is a tetrameric glycoprotein of 66,000-68,000 mol wt, found in egg albumin and in avian tissues. The interaction between avidin and biotin occurs rapidly, and the stability of the complex has prompted its use for in situ attachment of labels in a broad variety of applications, including immunoassays, DNA hybridization (4-6), and localization of antigens in cells and tissues (7). Avidin has an isoelectric point of 10.5. Because of its positively charged residues and its oligosaccharide component, consisting mostly of mannose and glucosamine (8), avidin can interact nonspecifically with negative charges on cell surfaces and nucleic acids, or with membrane sugar receptors. At times, this causes background problems in histochemical and cytochemical applications. Streptavidin, a near-neutral, biotin binding protein (9) isolated from the culture medium of Streptomyces avidinii, is a tetrameric nonglycosylated analog of avidin with a mol wt of about 60,000. Like avidin, each molecule of streptavidin binds four molecules of biotin, with a similar dissociation constant. The two proteins have about 33% sequence homology, and tryptophan residues seem to be involved in their biotin binding sites (10,11). In general, streptavidin gives less background problems than avidin. This protein, however, contains a tripeptide sequence Arg-Tyr-Asp (RYD) that apparently mimics the binding sequence of fibronectin Arg-Gly-Asp (RGD), a universal recognition domain of the extracellular matrix that specifically promotes cell adhesion. Consequently, the streptavidin-cell-surface interaction causes high background in certain applications (12).
KeywordsCarbohydrate DMSO Aldehyde Lysine Thiol
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