p ‐Toluenesulfonyl Chloride as an Activating Agent of Agarose for the Preparation of Immobilized Affinity Ligands and Proteins

A number of biomolecules were coupled covalently by nucleophilic displacement to agarose preparations substituted with tosyl groups. In one series of experiments N 6 ‐(6‐aminohexyl)‐adenosine 5′‐monophosphate and N 6 ‐(6‐aminohexyl)adenosine 2′,5′‐bisphosphatewere bound by their terminal amino groups to the polysaccharide support. It could be shown that from a mixture of lactate and 6‐phosphogluconate dehydrogenase the immobilized monophosphate showed bio‐affinity only for NAD + ‐dependent lactate dehydrogenase, whereas the immobilized bisphosphate showed affinity only for the NADP + ‐dependent 6‐phosphogluconate dehydrogenase. Furthermore, the immobilized monophosphate (5 μmol/g wet gel) was applied for the single‐step purification of lactate dehydrogenase from crude beef heart extract. To demonstrate the immobilization of proteins, soybean trypsin inhibitor (75 mg/g dry support) was immobilized to tosylated agarose, tested as affinity chromatography material and shown to bind 60 mg trypsin/g dry gel. Horseradish peroxidase and horse liver alcohol dehydrogenase were used as model enzymes. Although no optimization had been attempted, the former (approximately 70 mg/g dry support) had a coupling yield of approximately 18% with a specific activity (relative to soluble enzyme) of approximately 10%, whereas approximately 60% of alcohol dehydrogenase was coupled (approximately 100 mg/g dry support) with a specific activity of approximately 25%.