title: Studies on the interaction of immobilized trypsin and specific ligands by quantitative affinity chromatography.
abstract: The interaction of beta-trypsin [EC 3.4.21.4] immobilized on Sepharose with competitive inhibitors was quantitatively studied by frontal affinity chromatography. Analysis of the dependence of the elution volume of an inhibitor on its initial concentration gave two important parameters. One is the dissociation constant (Kd) of the immobilized trypsin-inhibitor complex, and the other is the total amount of trypsin retaining binding ability that is present in the column (Bt). Experiments using benzamidine showed that frontal affinity chromatography is a theoretically simple system and can be treated in the same way as enzyme kinetics. The Kd value obtained for benzamidine was very similar to the Ki value. The Bt value corresponded to 73% of the immobilized trypsin. These results suggest that the function of trypsin was hardly affected by the immobilization. Results obtained with beta-naphthamidine were somewhat less satisfactory, probably due to a considerable non-specific interaction. In the case of proflavine, analysis was almost impossible due to extremely strong non-specific interaction. This procedure is very useful to analyze specific interactions if non-specific interactions are not significant. The basic features of frontal affinity chromatography are discussed.
mesh_terms: D000578:Amidines / Q000494:pharmacology; D001550:Benzamidines / Q000494:pharmacology; D002846:Chromatography, Affinity; D004800:Enzymes, Immobilized; D007976:Leupeptins / Q000494:pharmacology; D008024:Ligands; D009281:Naphthalenes / Q000494:pharmacology; D011370:Proflavine / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism*; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000494:pharmacology
chemical_list: D000578:Amidines; D001550:Benzamidines; D004800:Enzymes, Immobilized; D007976:Leupeptins; D008024:Ligands; D009281:Naphthalenes; D014360:Trypsin Inhibitor, Kunitz Soybean; D011370:Proflavine; D014357:Trypsin