Affinity membranes containing trypsin and papain-immobilized as affinity ligands were prepared from glass fiber filters. The filters were first treated with a concentrated solution of H2SO4 + H2O2 ('piranha' solution), and then modified by silanization to introduce epoxyl or amino functional groups, which were further modified to carboxyl or aniline moieties, respectively. Onto the modified membranes, bovine serum albumin, trypsin and papain were immobilized as ligands. Three immobilization methods based on glutaraldehyde, carbodiimide and diazotization were used and compared to conclude that the one based on glutaraldehyde was the most efficient. Using the papain-immobilized membranes, papain inhibitors from potato tubers were separated. (C) 2003 Elsevier Science B.V. All rights reserved.