An affinity adsorbent derived from aminopropyl silica for serine protease chromatography

Abstract

A silica‐based adsorbent for affinity chromatography of serine protease was prepared by bonding p‐aminobenzamidine (pABZ) to aminopropyl silica. Silanization of silica with both γ‐aminopropyltrimethoxysilane and γ‐amino‐propyltriethoxysilane under anhydrous conditions led to a monolayer density of primary amino groups. The aminopropyl silica was converted to primary hydroxylcontaining silica via diazotization, and consequently activated with p‐nitrophenyl chloroformate and immobilized with ligand pABZ. The resultant silica–pABZ has a ligand density of 13.8 μmol g−1. Pyridine was indicated by the data to increase the reactivity of the chloroformate toward the hydroxyl group more efficiently than 4‐dimethylaminopyridine. Two stages of adsorption were found in batch adsorption of trypsin with an equilibrium adsorption isotherm of the Langmuir type. When the chromatographic column packed with this silica–p ABZ was operated under a higher flow rate (2·33 cm3 min−1) and with 3 g dm−3 of crude urokinase as the influence, the yield was 55%. Both the flow rate and the concentration of the crude protein were shown, by measuring the dynamic binding capacity from chromatographic experiments, to be the factors which influenced the chromatographic efficiency.