Avidin iminobiotin

Avidin-biotin Avidin-desthiobiotin Avidin-iminobiotin... 

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. 

NHS-iminobiotin can be used to label amine-containing molecules with an iminobiotin tag, providing reversible-binding potential with avidin or streptavidin. The NHS ester reacts with proteins and other amine-containing molecules to create stable amide bond derivatives (Figure 11.6). An iminobiotinylated molecule then can be used to target and purify other... 

Figure 11.5 At pH 4, the protonated form of iminobiotin does not interact with the binding sites on avidin or streptavidin. At pH 11, the imino group is unprotonated and regains binding capability toward these proteins.

The iminobiotin-avidin interaction also can be utilized in the opposite approach. Immobilized iminobiotin affinity columns can be used to purify avidin- or streptavidin-containing complexes under mild elution conditions (Hofmann et al., 1980). 

Orr, G.A. (1981) The use of the 2-iminobiotin—Avidin interaction for the selective retrieval of labeled plasma membrane components./. Biol. Chem. 256, 761-766. 

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—... 

Inoue H, Sato K, Anzai J (2005) Disintegration of layer-by-layer assemblies composed of 2-iminobiotin-labeled poly(ethyleneimine) and avidin. Biomacromolecules 6 27-29... 

The application of biotinylated receptor substrates is another approach, incubating the labeled substrate with the receptors prior to isolation on an avidin-coated support. In such cases, biotinylation with a cleavable biotinylation reagent such as Sulfo-NHS-SS-biotin or NHS-Iminobiotin would be essential for recovery of the isolated receptor. Alternatively, the receptor could be recovered by substrate competition. Perhaps one of the major drawbacks to the application of affinity techniques is the relative low molecular weight or small size of the receptor substrates, making them difficult ligands to immobilize. However, affinity procedures have been applied to the purification of a number of different receptors although relatively little work has been reported on those involved in the processing of neurotransmittors, neuropeptides, and hormones [1,2]. 

A simple and economical way to obtain complexes of optimum activity consists of the use of small column of iminobiotin saturated with avidin according to Section 3.2.1.3 (crude extract from egg whites can be used directly). One of the binding sites of avidin is occupied by the iminobiotin while the other remains free for reaction with the subsequently added biotinylated POase. Avidin is then added to saturate additional free biotin groups on POase. Complexes are then eluted from the column with 50 mM ammonium acetate (pH 4.0). 

The application of biotinylated receptor substrates is a useful approach, incubating the biotinylated substrates with their specific receptors prior to isolation on an avidin-coated support.In such cases, biotinylation with a cleavable reagent such as Sulfo-NHS-SS-biotin or NHS-Iminobiotin is essential for recovery of the isolated receptor. Alternatively, the receptor can be recovered by substrate competition. 

Raphael, M. R Rappole, C. A. Kurihara, L. K. Christodoulides, J. A. Qadri, S. N. Byers, J. M. Iminobiotin binding induces large fluorescent enhancements in avidin and streptavidin fluorescent conjugates and exhibits diverging pH-dependent binding affinities. J. Fluoresc. 2011,21,647-652. 

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