2-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.

NHS-iminobiotin can be used to label amine-containing molecules, creating amide linkages. 

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). 

NHS-iminobiotin is insoluble in aqueous solution. It can be dissolved in organic solvent (DMF) prior to addition of a small aliquot to a buffered reaction medium. Don t exceed 10 percent DMF in the reaction to avoid protein precipitation problems. Optimal conditions for protein derivatization include non-amine-containing buffers at a pH of 7-9. The following... 

Add 100 pi of the NHS-iminobiotin solution to each ml of the antibody solution. Mix well to dissolve. Note Some turbidity may be present in the reaction due to incomplete dissolution of the NHS-iminobiotin. The solution may look cloudy or have a microparticulate suspension present. This is normal for many water-insoluble reagents when added to an aqueous solution in an organic solvent. As the reaction takes place, the NHS-iminobiotin will be driven into solution, both by coupling to the protein and by hydrolysis of the NHS ester. 

Remove unreacted NHS-iminobiotin and reaction by-products by dialysis or gel filtration... 

Hofmann, K., Wood, S.W., Brinton, C.C., Montibeller, J.A., and Finn, F.M. (1980) Iminobiotin affinity columns and their application to retrieval of streptavidin. Proc. Natl Acad. Sci. USA 77, 4666 -668. 

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—... 

Zeheb, R., Chang, V., and Orr, G. A. (1983) An analytical method for the selective retrieval of iminobiotin-derivatized plasma membrane proteins. Anal. Biochem. 129, 156—161. 

---