p-Toluenesulfonyl Chloride as an Activating Agent of Agarose for the Preparation of Immobilized Affinity Ligands and Proteins. Optimization of Conditions for Activation and Coupling.

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Abstract
A new immobilization technique suitable for the binding of biomolecules to polysaccharide supports, such as agarose beads, is described. It involves treatment of agarose with p-toluenesulfonyl chloride (tosylchloride) with the formation of p-toluenesulfonic ester (tosylation). This reaction is rapid and can be controlled to give a range of substitutions up to very high levels. The tosylated agarose obtained shows excellent long-term stability and the swelling properties of the gel are similar to those of untreated agarose, indicating that no cross-linking of the support occurs during tosylation. The degree of substitution of agarose is easily monitored by UV measurement of the appearance of p-toluenesulfonate. Addition of nucleophiles, such as amino or mercapto group-containing compounds, leads to efficient displacement of the tosyl groups. With .beta.-alanine and .beta.-mercaptopropionic acid as model compounds, the degree of coupling was investigated as a function of time, temperature and pH. High degrees of substitution (in the range 6-10 .mu.mol/g wet gel) were obtained within 10 h at pH 8-10 and room temperature. Higher yields were obtained on prolonged reaction time, more alkaline conditions and higher temperature. It was concluded from titration data that ligands were coupled without the introduction of any additional charged groupings and that on coupling of thiol compounds uncharged linkages were formed between ligand and support. Coupling in organic solvent of ligands not soluble in water gave high yields of coupled product. Some results of the coupling of biomolecules to agarose are given, e.g., analogs of s''-AMP and 2''-ADP as affinity ligands for the purification of lactate dehydrogenase and G-phosphogluconate dehydrogenase and the immobilization of soybean trypsin inhibitor, horseradish peroxidase and horse liver alcohol dehydrogenase.