Immobilization - Supported Ligands

The reactions used for coupling affinity ligands to nanoparticles or microparticles basically are the same as those used for bioconjugation of molecules or for immobilization of ligands onto surfaces or chromatography supports. However, with particles, size can be a major factor in how a reaction is performed and in its resultant reaction kinetics. Since particle types can vary from the low nanometer diameter to the micron size, there are dramatic differences in how such particles behave in solution and how the density of reactive groups or functional groups affects reactions. 

Fig. 7. Schematic representation of the non-covalent immobilization of ligands to a dendrimer support and the actual supramolecular dendritic complex containing 32 phosphine ligands 21).

Haloacetyl Method The haloacetyl method uses supports that contain lodoacetyl or bromoacetyl groups for the immobilization of ligands through sulfhydryl residues. These supports are usually prepared via the reaction of an amine-containing material with iodoacetic or bromoacetic acid in the presence of ethyldimethylaminopropyl carbodii-mide (EDC) at pH 4 to 5. EDC reacts with the carboxylic acid in iodo- or bromoacetic acid... 

Selection of the matrix used to immobilize a ligand requires consideration of several properties. The stationary supports used in gel exclusion chromatography are found to be quite suitable for affinity chromatography because (1) they are physically and chemically stable under most experimental conditions, (2) they are relatively free of nonspecific adsorption effects, (3) they have satisfactory flow characteristics, (4) they are available with very large pore sizes, and (5) they have reactive functional groups to which an appropriate ligand may be attached. 

Solid-supported ligands provide an easy means of recycling the expensive Cinchona alkaloids. Until now, the immobilized Cinchona alkaloid ligands used in the AA process have been attached to different solid supports at the DHQ or DHQD moiety. 

Affinity chromatography is a form of adsorption chromatography, wherein the solid support surface has been functionalized with an immobilized compound (ligand) that complements the solute (ligate) to be separated from a complex mixture. It is an important technique for difficult biological separations. Here, it exploits the unique ability of proteins to specifically bind molecules noncovalently. 

K. S., and Rahman, S. (1994). Preferential antagonism of the interactions of the integrin a,npj with immobilized glycoprotein ligands by snake-venom RGD (Arg- Gly-Asp) proteins. Evidence supporting a functional role for the amino acid residues flanking the tripeptide RGD in determining the inhibitory properties of snake-venom RGD proteins. Biochem J. 5(34 929-936. 

Fig. 4.7.3. Schematic drawing of effects of concentration of immobilized affinity ligands and of uneven support surface on nonspecific and specific sorption.

Substances isolated Affinity ligands Solid supports or immobilized affinity ligands Refs. 

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