Biotin-binding sites

FIG. 6 Successive coupling of two different biotinylated compounds with the DNA-STV conjugates 2 [33]. In a first step, a macromolecular functional component (FC, represented by the shaded ellipse), such as a biotinylated enzyme or oligonucleotide, is coupled. In a second step, a biotinylated low-molecular-weight modulator M, represented by the shaded sphere) is coupled to the remaining free biotin-binding sites. The modulator is used to modify the conjugate s hybridization properties or to supplement its functionality. 

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. 

Since a biotinylated molecule potentially is able to interact with (strept)avidin at its biotin binding sites just as strongly as biotin in solution, the degree of biotinylation may be determined using the HABA method as well. Comparison of the response of a biotinylated protein, for example, with a standard curve of various biotin concentrations allows calculation of the molar ratio of biotin incorporation. 

Gitlin, G., Bayer, E.A., and Wilchek, M. (1987) Studies on the biotin-binding site of avidin. Lysine residues involved in the active site. Biochem. /. 242, 923-926. 

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. 

It may be possible to build up a two- or three-dimensional architecture composed of proteins using avidin and biotin-labeled enzymes as building blocks. If we use enzymes tagged with more than two biotin residues, an enzyme multilayer illustrated in Figure 10 would be constructed because avidin contains four biotin-binding sites per molecule. Fortunately, the biotin-binding sites are located in two pairs on the opposed faces of the avidin molecule. Thus, the use of biotin-labeled enzyme... 

Green, N M (1963) Avidin 3. The nature of the biotin binding site Biochem J 89, 599-609... 

Avidin conjugates of oligodeoxynucleotides are hybrid molecules that not only provide multiple biotin binding sites, but can also be targeted to complimentary DNA or RNA sequences, by annealing interactions. Such conjugates are useful for the construction of macromolecular assemblies with a wide vari-... 

Streptavidin is another biotin binding protein isolated from Streptomyces avidinii that can overcome some of the nonspecificities of avidin (Chaiet and Wolf, 1964). Similar to avidin, streptavidin contains four subunits, each with a single biotin binding site. After some postsecretory modifications, the intact tetrameric protein has a molecular mass of about 60,000 D, slightly less than that of avidin (Bayer et al., 1986, 1989). 

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