Affinity Chromatography of Trypsin and Related Enzymes

I. Preparation and Characteristics of an Affinity Adsorbent Containing Tryptic Peptides from Protamine as Ligands

Abstract

An adsorbent for the affinity chromatography of trypsin [EC 3. 4. 21. 4] (AP Sepharose) was prepared. The ligand was a mixture of oligopeptides (mainly di-and tripeptides) containing L-arginine as carboxyl termini, and was obtained from a tryptic digest of protamine. Trypsin was adsorbed at relatively low pH (7-4), but was not adsorbed at the optimum pH of catalysis (8.2). This was clearly explained on the basis of the pH dependence of the interaction of trypsin with its products. Inactivated trypsin, trypsinogen, and chymotrypsin were not adsorbed. The adsorption of active trypsin was interferred with by either benzamidine or urea. From these observations, it is evident that AP Sepharose is an affinity adsorbent. AP Sepharose was useful for purification of commercial bovine trypsin. A preliminary application for the purification of Streptomyces griseus trypsin was also successful.