Retention behavior of very large biomolecules in ion-exchange chromatography.
Hepatitis B virus surface antigen (HBsAg) particles were efficiently adsorbed (retained) on a Sulfate-cellulose (S-C) bead column, and then desorbed with sodium chloride solutions (0.5-3.0 M). The HBsAg particles were not efficiently retained onto either sulfopropyl-agarose (SP-A) or quaternary amine-agarose (Q-A) at pH 4.5, 6 and 8. The size-exclusion curve showed that proteins of molecular mass higher than ca. 20,000 cannot penetrate into the pores of S-C beads. The dynamic binding capacity (DBC) values of lysozyme (ca. 7 mg/ml-gel) and of gamma-globulin (ca. 3 mg/ml gel) for S-C did not depend on the flow velocity while the DBC of gamma-globulin for SP-A decreased sharply with an increase in flow velocity. These results indicated that very large molecules are adsorbed only onto the surface of S-C, which resulted in fast adsorption-desorption rates although the equilibrium adsorption capacity is lower than conventional porous gel beads. Because of the rapid adsorption rate, the DBC values of gamma-globulin for S-C at high flow-rate regions are similar to those for SP-A. Bovine serum albumin was not adsorbed onto S-C. As this can not be explained by a simple electrostatic interaction mechanism, molecular recognition of S-C might be different from the agarose-based ion-exchange beads.
Yamamoto
S
S
Department of Chemical Engineering, Yamaguchi University, Tokiwadai, Ube, Japan. shu-yama@po.cc.yamaguchi-u.ac.jp
Miyagawa
E
E
D002240:Carbohydrate Sequence; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006514:Hepatitis B Surface Antigens / Q000737:chemistry*; D009113:Muramidase / Q000737:chemistry*; D005719:gamma-Globulins / Q000737:chemistry*
D006514:Hepatitis B Surface Antigens; D005719:gamma-Globulins; D009113:Muramidase