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).
D000327:Adsorption; D000578:Amidines / Q000302:isolation & purification*; D000818:Animals; D001550:Benzamidines / Q000302:isolation & purification*; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D013305:Streptomyces griseus / Q000201:enzymology; D014357:Trypsin / Q000032:analysis*
D000578:Amidines; D001550:Benzamidines; C006500:4-aminobenzamidine; D014357:Trypsin