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

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Abstract
A number of biomolecules were coupled covalently by nucleophilic displacement to agarose preparations substituted with tosyl groups. In one series of experiments N6‐(6‐aminohexyl)‐adenosine 5′‐monophosphate and N6‐(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%.

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