MODIFYING BIOMOLECULES...

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BIOTINYLATION REAGENTS

Introduction

Cyanagen has a product line of amine-reactive BIOTINYLATION REAGENTS for modifying biomolecules such as proteins and amine-modified nucleic acids for Biotin/Streptavidin applications. These reagents are divided into two groups based on water solubility: NHS and sulfo-NHS.

Order-No Linkage formed Features MW     arm(Å)
B001N NHS-Biotin Membrane permeable 341.38    16.1
B002N NHS-LC-Biotin Membrane permeable 454.54    22.4
B101N Sulfo NHS-Biotin Water soluble 443.43    16.1
B102N Sulfo NHS-LC-Biotin Water soluble 556.60    22.4

Features

D-biotin is a small molecule (MW 244.3) present in minute amounts in every living cell as a growth factor (vitamin H or Coenzyme R). It plays an indispensable role in numerous carboxylation reactions. Biotin combines with a protein, Avidin (MW 60.000), present in raw egg white and becomes inactive. The dissociation constant of the egg white Avidin/Biotin complex is among the lowest known (Kd = 10-15 M).

A perfect fit occurs between the bicyclic ring of Biotin and a pocket of the tetrameric protein. 

The valeric acid side chain of Biotin is not directly involved in the bond. Thus, it can be activated or modified without altering or changing the binding affinity toward Avidin. An analogous interaction takes place between Biotin and the protein Streptavidin (MW 60.000, isolated from Streptomyces Avidinii).

Proteins or other molecules can be labeled with Biotin in a wide variety of methods. This process is called biotinylation. Since Biotin is a relatively small molecule, large macromolecules are hardly affected by the labeling. Biotinylation reagents are designed to form covalent bonds with common functional groups, in particular amino (-NH2), carboxylic (-COOH), aldehydes (-CHO) and thiols (–SH). The amine group is the most common target for modifying protein molecules because it is abundant in the majority of proteins due to the presence of lysine. Amine-reactive biotinylation reagents can be divided into two groups based on water solubility: NHS-esters and sulfo-NHS-esters.
NHS-esters of Biotin are water-insoluble and membrane-permeable because they do not possess a charged group. They may be used for biotinylating internal as well as external cellular components.

Sulfo-NHS-esters are water soluble and useful for applications that cannot tolerate organic solvents. Sulfo-NHS-esters of Biotin are recommended for use as cell surface biotinylation reagents.

Since the binding sites for Biotin are buried deep inside Avidin and Streptavidin (about 9 Ǻ below the protein surface) spacers improve both the accessibility and reaction rate of biotinylated compounds with respect to Avidin and Streptavidin, often enhancing the sensitivity of assay systems.

The most common spacer group is aminocaproic acid (LC), which increases the length of the side arm by about 9 Å.

 

Applications
  • Conjugated biopharmaceuticals for targeted delivery of cytotoxic agents
  • Enzyme-labeling of antibodies (monoclonal, polyclonal & fragments) for signal generation in diagnostic procedures
  • Enzyme-labeling of small molecules such as steroids and thyroid hormones for competitive immunoassays
  • Biotinylation of proteins for use with Streptavidin-based binding reagents
  • Coupling of small molecules to carrier proteins such as Albumin or gelatin, for use as immunogens or “coating” reagents
  • Coupling of proteins and small molecules to solid phases such as micro particles, beads and microtitre wells    
  1. O.Livnah, E.A.Bayer, M. Wilchek, J.L.Sussman, “Three dimensional structures of avidin and the avidin-biotin complex”, Proc. Natl. Acad. USA, Vol. 90, p. 5076 (1993).
  2. Kim, M.J. et al. (1995) Anal. Biochem. 231, 400-406
  3. Bayer, E.A. and Wilchek, M. (1980) Methods Biochem. Anal., J. Wiley & Sons (New York, NY), 26, 1-45
    Homogeneous assays for riboflavin mediated by the interaction between enzyme-biotin and avidin-riboflavin conjugates. The use of the avidin-biotin complex as a tool in molecular biology.