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 N6-(6-aminohexyl)-adenosine 5'-monophosphate and N6-(6-aminohexyl)adenosine 2',5'-bisphosphate were 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 mumol/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%.
D002846:Chromatography, Affinity / Q000379:methods*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D007202:Indicators and Reagents; D007770:L-Lactate Dehydrogenase / Q000302:isolation & purification; D008024:Ligands; D010734:Phosphogluconate Dehydrogenase / Q000302:isolation & purification; D011134:Polysaccharides; D011506:Proteins / Q000302:isolation & purification; D012685:Sepharose; D014105:Tosyl Compounds
D004800:Enzymes, Immobilized; D007202:Indicators and Reagents; D008024:Ligands; D011134:Polysaccharides; D011506:Proteins; D014105:Tosyl Compounds; D012685:Sepharose; D007770:L-Lactate Dehydrogenase; D010734:Phosphogluconate Dehydrogenase