Design of novel cationic ligands for the purification of trypsin-like proteases by affinity chromatography.

Abstract

A number of new cationic ligands have been designed and synthesized for the selective resolution and purification of the trypszin-like proteases. A series of ligands based on 4-[2'-methyl-4'-(2'',4''-dichloro-1'',3'',5''-triazin-6-ylamino ) phenylazo]benzamidine were able to bind to trypsin and the trypsin-like proteases, thrombin and urokinase, but bound pancreatic kallikrein only weakly. Ligands possessing a second cationic group (either 4-aminophenyltrimethylammonium or 4-aminobenzamidine) substituted onto the triazine ring displayed higher affinities than the parent compound for trypsin in solution but bound the enzyme weakly or not at all after immobilization. In contrast, these bis-cationic ligands bound pancreatic kallikrein in solution and following immobilization. The presence of the second cationic group was crucial, since its replacement by neutral or anionic groups led to loss of affinity for pancreatic kallikrein. One of the bis-cationic ligands was used to purify pancreatic kallikrein 9.5-fold from a crude pancreatic extract in 79% yield, to generate a product 99.9% free of contaminating trypsin activity.