Streptavidin biotin release

After molecules modified with sulfo-NHS-SS-biotin are allowed to interact with avidin or streptavidin probes, the complexes can be cleaved at the disulfide bridge by treatment with 50 mM DTT. Reduction releases the biotinylated molecule from the avidin or streptavidin capture reagent without breaking the (strept)avidin interaction. The use of disulfide biotinylation reagents... 

The on-bead assay was conducted according to Scheme 3.19, which shows the chain of events, which leads to a colorimetric response, when an oligosaccharide binds effectively to the B. purpurea lectin. The lectin was covalently linked to biotin, a small molecule with an extremely high affinity for streptavidin. The bead-lectin-biotin conjugates were then exposed to streptavidin, linked to the enzyme alkaline phosphatase. Alkaline phosphatase hydrolyses phosphate esters [e.g., 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 110]. When the 5-bromo-4-chloro-3-hydroxyindole (111) is released, in the presence of nitro blue tetrazolium (NBT), it forms a dark purple, insoluble dye, thus staining beads where there was a favorable binding interaction. 

The strong bond between biotin and avidin/streptavidin can only be released by extreme and often denaturating conditions. For a reversible binding based on this complex, o-desthiobiotin can be used instead of biotin (see Fig. 13). This conjugate can be separated at room temperature by exposure to an excess amount of biotin, respectively desthiobiotin [42]. 

Another variable to consider in choosing biotinylation reagents is the use of a biotin analog such as iminobiotin that has a moderated affinity constant in its binding of avidin or streptavidin (Section 3.1). Analogs may be useful if release of the avidin—... 

A receptor-mediated gene delivery system using streptavidin and biotin-derivatized, pegylated epidermal growth factor. /. Control. Release 83 109-119. 

Fig. 1. Preparation of ssDNA from biotinylated PCR-amplified DNA. (a) DNA is amplified using one primer that has a biotin molecule (B) at the 5 end and one primer that lacks biotin, (b) Double-stranded amplified DNA is captured on a streptavidin-coated magnetic bead via the biotin molecule, (c) The dsDNA is denatured, with the biotinylated strand remaining attached to the streptavidin-coated magnetic bead and the nonbiotinylated strand being released into the supernatant, (d) The nonbiotinylated strand is used as an ssDNA sequencing template.

One of the first smart streptavidins was developed by Morag et al. [32], NitroAvidin was created by nitrating the Tyr33 residue in vitro. This modification reduced the pfCa of the phenol group of streptavidin and created a pH-dependent biotin binding system. In this case, biotin is strongly bound at acidic pH (4-5) and released when the pH is raised and/or the complex is incubated in the presence of free biotin. 

Many foodstuffs contain a metabolic intermediate of biotin, biocytin s-N-biotinyUysine), which is cleaved in the intestinal tract by the enzyme biotinidase. Only free biotin can be resorbed in the proximal small intestine, a process, which can be blocked by avidin, a glycoprotein with a molar mass of ca. 70,000. Avidin occurs in greater amounts in egg-white, and forms with biotin an extraordinarily stable molecular complex (dissociation constant at 25 °C K = 10 M), which can be cleaved neither by acids nor by peptidases. Only irradiation or longer exposure to heat leads to denaturation of avidin and thereby the release of biotin. This is another reason why a breakfast egg ought to be cooked for at least AVi minutes. In this way avidin is denatured and loses its harmful effect. Similarly stable complexes are formed by biotin with neutravidin (de-glycosyl-ated avidin), streptavidin and stravidin from certain Streptomyces and Saccharo-myces species respectively. 

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