Affinity spacer arms

Compounds 13 and 20 were obtained in five and seven steps respectively from the intermediate alcohol 6. They, as well as the acetylated derivative 12, were tested in vivo i and proved to be, as their free acids, gratuitous inducers of pectinases in Erwinia chiysanthemi. These results provide evidence that the hydroxyl function in C-5 is not required for the recognition between inducers and the KdgR repressor protein. Tharefore we can now envisage the preparation of affinity columns, by immoWlizing inducers on suppc rts using a spacer arm boimd to the C-5 hydroxyl. Such columns could then be used for the purification of the repressor protein. 

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 16.1 The general design of an ICAT reagent consists of a biotinylation compound with a spacer arm containing stable isotope substitutions. The reactive group is used to label proteins or peptides at particular functional groups and the biotin affinity tag is used to isolate labeled molecules using immobilized (strept)avidin.

Figure 16.4 A more advanced ICAT design uses an acid-cleavable spacer arm to facilitate elution of labeled peptides from a (strept)avidin affinity column. The use of 14C isotopes instead of deuterium labels permits precise reverse phase separations prior to mass spec that show no elution peak time differences between isotope-labeled and normal atom-labeled peptides.

Figure 16.5 A catch-and-release ICAT design incorporates a gem-methyl group and an isopropyl group on either side of a disulfide bond within its spacer arm. The hindered disulfide permits the use of standard reducing gel electrophoresis conditions using DTT without reduction. After purification on a (strept)avidin affinity column, however, the disulfide group can be cleaved with TCEP, which provides recovery of the labeled peptides prior to mass spec separation.

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]. 

Compounds which have diamine groups such as hexanediamine, propanediamine and ethylenediamine are the most preferred spacer arms used in affinity cromatography. Some other examples of spacer arms are shown in Table 3 [19]. 

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 ... 

Suitable affinity resins are CNBr-activated Sepharose and similarly activated jV-hydroxysuccimmide ester-based gels with spacer arms. These react with free amino groups on the peptide. If the peptide contains many lysine residues, alternative coupling systems may be used, for example, carbodiimide-activated agaroses. In our experience, however, even peptides with internal lysines make good immunoadsorbents, and we routmely use CNBr-activated Sepharose. 

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. 

Typical molecules that are specifically recognized by antibodies are antigens. Therefore the immobilization of the antigen on a chromatographic matrix is also a means to purify selectively the antibody. To that end, the first requirement is the availability of the antigen for its chemical immobilization on a solid phase. If it is available, an effective affinity sorbent can be prepared by its chemical immobilization on a solid matrix. For small-size antigen molecules, a spacer arm may be required for a good accessibility to the active site of the antibody.204... 

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