High-performance affinity chromatography of trypsins on Asahipak GS-gel coupled with p-aminobenzamidine

An adsorbent for high-performance affinity chromatography of trypsins was prepared, based on a micro-particulate polyvinyl alcohol gel for high-performance liquid chromatography, Asahipak GS-gel. After the hydroxyl groups had been activated with 1,1'-carbonyldiimidazole, 6-aminohexanoic acid was coupled as a spacer, then p-aminobenzamidine, a specific ligand for trypsin-family enzymes, was immobilized on the spacer. Fluorometric detection of eluted protein and on-line assay of enzyme activity using a fluorogenic substrate, peptidylmethylcoumarylamide, made it possible to attain very high sensitivity. Microgram amounts of bovine trypsin and Streptomyces griseus trypsin could easily be analyzed in a short time (less than 1 h).