Avidin bound complex

The avidin-biotin complex, known for its extremely high affinity (Green, 1975), has been studied experimentally more extensively than most other protein-ligand systems. The adhesion forces between avidin and biotin have been measured directly by AFM experiments (Florin et al., 1994 Moy et al., 1994b Moy et al., 1994a). SMD simulations were performed on the entire tetramer of avidin with four biotins bound to investigate the microscopic detail of nnbinding of biotin from avidin (Izrailev et al., 1997). 

The following procedure is based on the Vectastain ABC kit from Vector (suppliers appendix). It uses an avidin-biotin complex to attach horseradish peroxidase (HRP) or alkaline phosphatase (AP) to the biotinylated secondary antibody. Avidin-biotin systems are capable of extremely high sensitivity because multiple reporter enzymes are bound to each secondary antibody. In addition, the detergent Tween 20 is a popular alternative to protein blocking agents when using nitrocellulose or PVDF membranes. 

Streptavidin (from Streptomyces avidinii) and avidin (from chicken egg) both possess four binding sites for biotin. The biotin molecule is easily conjugated to antibodies and enzymes. In the avidin-biotin complex (ABC) method secondary antibodies are conjugated to biotin and function as links between tissue-bound primary antibodies and an avidin-biotin-peroxidase complex (5). 

Dietary biotin bound to avidin (Section 11.6) is unavailable, but intravenously administered avidin-biotin is biologically active. Cells in culture are not inhibited by the addibon of avidin to the culmre medium, and can take up the avidin-biotin complex by pinocytosis followed by lysosomal hydrolysis, releasing free biotin. Unlike other B vitamins, for which concentrative uptake into tissues is achieved by facilitated diffusion, followed by metabolic trapping, the incorporation of biotin into enzymes is slow and cannot be considered part of the uptake process. 

Fig. 4.16 SERS spectra of avidin bound to gold-coated wing right) and the avidin-biotin complex bound to gold-coated wing left) (Reproduced with permission from [40])...

Avidin-biotin complex (ABC) uses a reagent made from avidin, biotin, and HRPfor a much larger increase in detection sensitivity (Hsu et al., 1981). ABC is based on the molecular complex that is made by mixing HRP-bound biotin with an excess of unlabeled avidin (Fig. 7.6). The proportions of biotin HRP and avidin are critical for developing the complex size that is needed for immunocytochemistry. The vendor. Vector Laboratories, produces an ABC reagent that is easy to use and consistent. 

Fig. 7.6 Avidin-biotin complex (ABC). To increase the number of HRP enzymes bound to a 1° antibody an avidin-biotin complex (ABC) is used. To make the complex, first multiple biotins are bound to HRP and then this is incubated with a dilute avidin. All of the HRP-biotin reagents bind to avidin, generating complexes with unbound avidin still available. This reagent is now used in ABC immunocytochemistry...

The procedure begins with the standard ABC protocol, where biotin-labeled 2° antibody is bound with the avidin-biotin complex (ABC). This adds a significant number of HRP molecules for each L antibody. To start the TSA method, incubate tissue with biotin-labeled tyramide with H2O2 the HRP enzymes generate activated tyramide (Fig. 7.10). The activated tyramide then binds to the available tyrosine amino acids on cellular proteins near the antigen, on the antibodies, and even on... 

The reactivity of biotin with several reagents can be exploited in some chemical assays like its reaction with diazo derivatives, or the reaction of the ureido ring with p-dimethylaminocinnamaldehyde in an acidic medium, but the sensitivity of these assays is relatively poor. Several methods described for biotin assay involve a competitive complex formation between biotin and avidin bound with a chromophore/ fluorophore probe. In these assays, biotin, which has a higher affinity for avidin, quantitatively displaces the probe from the complex. 

This technique has been developed by Aizawa and coworkers. The goal is to build a convenient and specific detector using an enzymatic activity as signal. In the case of biotin determination, avidin coupled with catalase is bound to a membrane bearing covalently linked HABA residues. Addition of biotin destroys quantitatively the HABA-avidin-catalase complex. Washing the membrane and measurement of the remaining catalase activity afforded a sensitive (0.5 ng) and convenient titration of biotin (95). 

In another example, ligands can be biotinylated with a cleavable biotinylation reagent and then incubated with receptor molecules. The resulting complex can be isolated by affinity chromatography on immobilized (strept)avidin. Final purification of the ligand-receptor can be accomplished by cleaving the biotin modification sites while the complex is still bound to the support. The receptor complex thus can be eluted from the column without the usual harsh conditions required to break the avidin-biotin interaction. 

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