Affinity chromatography spacer arm

Succinic anhydride also is a convenient extender for creating spacer arms on chromatography supports. Supports derivatized with amine-terminal spacers may be succinylated to totally block the amine functionalities and form terminal carboxylic acid linkers for coupling amine-containing affinity ligands (Cuatrecasas, 1970). 

Figure 6.14 Schematic representation of the principle of biospecific affinity chromatography. The chosen affinity ligand is chemically attached to the support matrix (agarose bead) via a suitable spacer arm. Only those ligands in solution that exhibit biospecific affinity for the immobilized species will be retained...

Other methods that are related to affinity chromatography include hydrophobic interaction chromatography and thiophilic adsorption. The former is based on the interactions of proteins, peptides, and nucleic acids with short nonpolar chains on a support. This was first described in 1972 [113,114] following work that examined the role of spacer arms on the nonspecific adsorption of affinity columns [114]. Thiophilic adsorption, also known as covalent or chemisorption chromatography, makes use of immobilized thiol groups for solute retention [115]. Applications of this method include the analysis of sulfhydryl-containing peptides or proteins and mercurated polynucleotides [116]. 

An example of a successful application of affinity chromatography is the isolation of the enzyme cytidine deaminase from cells of E. coli. Cytidine was linked covalently via long spacer arms to the agarose beads as in the following diagram ... 

Affinity chromatography combines the analytical and chemical capacities of chemically bonded stationary phases and immobilized enzymes. Technology and methodology of both techniques are joined in the development of affinity stationary phases. Since steric requirements are even more determining than in simple immobilized enzyme systems, spacer molecules have great importance in these modifications. Commonly used spacer arms are summarized in figure 8.3. 

Affinity chromatography, first described by Cuatrecasas et al.,U9 utilizes the ability of a protein or another biopolymer to recognize a natural or synthetic ligand. The affinity chromatography sorbent consists of a porous matrix itself on which a ligand is chemically immobilized directly or by means of a spacer arm. 

Figure 15.1 Ligand-protein interaction in affinity chromatography. A diagrammatic representation of the interaction between matrix-bound ligand and protein showing the necessity of a spacer arm joining the ligand to the matrix.

Fig. 9.1. Schematic illustration of the elements involved in bio-affinity chromatography. The solute (Lt) is retained on the stationary phase (S) by specific interaction with the ligand (Ln). The ligand is covalently attached to a spacer arm (SP) which is in turn attached to the stationary phase. Elution as shown here is achieved by specific ligand competition by another solute (Lt ). Alternatively the ligand-ligate complex can be disrupted by reversible denaturation using low pH solvent or mild chaotropic salts, e.g.

Chemical proteomics consists of the classical drug-affinity chromatography and modern high-resolution MS analysis for protein identification [3, 11]. The procedure typically involves immobilization of the compound of interest to a solid support through a spacer arm, and the affinity matrix is then used to purify specific interacting proteins from cellular lysate. The complex proteomic mixture is then proteolytically digested, and the resulting peptides are sequenced... 

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