Synthesis of glycidyl methacrylate-based matrix and its application in affinity chromatography of urokinase

Abstract

A number of glycidyl methacrylate (GMA) copolymers were synthesized with various pore-forming agents in different proportions using ethylene dimethacrylate (EDMA) as a crosslinking agent. One of greater pore diameter was chosen as the matrix for affinity chromatography. This was treated with ammonia liquor and reacted with succinic anhydride to extend the spacer arm. The p-aminobenzamidine was immobilized to the succinylated resin as the ligand in the presence of water-soluble carbodiimide. The resulting resin had a ligand density of 45.6 μmol g wt −1 resin and was used for purification of crude urokinase with a purity ranging from 125 to 221 IU mg −1. The optimal condition for the adsorption of urokinase was 0.05 M phosphate buffer (pH 7.5) containing 1.0 M NaCl. The optimal elution conditions were: First the contaminating proteins were eluted with Tween 80 solution, then urokinase was eluted with 0.2 M HAc (pH 2.6). The elution yield was 93.7% with a 46-fold purification.