Interaction of trypsin with immobilized p-aminobenzamidine derivatives studied by means of affinity electrophoresis

Abstract

The strength of the interaction of trypsin with immobilized p-aminobenzamidine derivatives was studied by affinity electrophoresis on polyacrylamide gel in Trisdiethylbarbituric acid buffer (pH 8). p-Aminobenzamidine was coupled to two kinds of soluble macromolecular carrier: (i) periodate-oxidized Dextran T-500 and (ii) a synthetic copolymer of N-(2-hydroxypropyl)methacrylamide and 4-nitrophenyl esters of 6-(methacroyl)aminohexanoic acid. The enzyme inhibitor was attached to the periodate oxidized dextran either directly or through glycine, 6-aminohexanoic acid or 12-aminododecanoic acid spacer. The strength of the interaction of trypsin with immobilized p-aminobenzamidine did not depend on the type of macromolecular carrier that the ligand was bound to but it did depend on the length of the spacer arm. The dissociation constants of the trypsin-immobilized p-aminobenzamidine complexes decreased with increasing length of the spacer arm.