Affinity of trypsin for amidine derivatives immobilized on dextran-coated silica supports

The pancreas contains two very analogous enzymes: trypsin and chymotrypsin. These two enzymes are very similar in their physicochemical characteristics and are therefore quite difficult to separate by classical purification procedures. They constitute a good model for affinity chromatography. It was previously demonstrated that amidine derivatives are able to interact strongly and specifically with these serine proteases and are often used as ligand in affinity chromatography. To understand the trypsin interaction mechanism, we synthesized different amidines and immobilised them with or without spacer arm on silica beads previously coated by dextran substituted with a calculated amount of positively charged diethylaminoethyl functions, in order to minimize the non-specific interactions of silanol groups of the silica material. First the affinity constant and the adsorption capacity of these supports for trypsin were determined in batch procedures, then they were used in affinity chromatography. The effects of ionic strength, pH and competitive inhibitors on proteins desorption were also studied. Last, to demonstrate the importance of passivation, the chromatographic performances of dextran-coated silica phases and a commercial support grafted with the same amidine were compared.