Preparation of high-capacity affinity adsorbents using formyl carriers and their use for low- and high-performance liquid affinity chromatography of trypsin-family proteases

Abstract

To prepare stable and high-capacity affinity adsorbents for trypsin-family proteases, high concentrations of m-aminobenzamidine (ABA) were immobilized by reductive amination with sodium cyanoborohydride on two types of formyl carriers I and II having different spacer lengths. Formyl carriers I were prepared by periodate oxidation of glucose carriers obtained by coupling glucose to epoxy-activated carriers. Formyl carriers II were prepared by periodate oxidation of glyceryl carriers obtained by hydrolysis of epoxy-activated carriers. High concentrations of ABA (15–21 μmol per g wet gel) were efficiently immobilized on both types of carriers, and formyl groups remaining on the adsorbents were converted into hydroxymethyl groups by reduction with NaBH 4. Two types of adsorbents prepared with Sepharose gel were successfully used for affinity chromatography of bovine trypsin, streptomyces griseus trypsin and serine proteases in urine, and exhibited high adsorption capacities, e.g., about 30 mg bovine trypsin per ml gel. The adsorbents prepared with Toyopearl gel (Fractogel TSK) were successfully used for high-performance liquid chromatography of bovine trypsin, though they exhibited lower adsorption capacities than those prepared with Sepharose gels.