Optimisation of Conditions for the Affinity Chromatography of Human Enterokinase on Immobilised p-Aminobenzamidine. Improvement of the Preparative Procedure by Inclusion of Negative Affinity Chromatography with Glycylglycyl-aniline

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Abstract
The affinity chromatography of human enterokinase [EC 3.4.21.9] using p-aminobenzamidine as the ligand was reassessed and the optimal conditions for the synthesis and operation of he derivatized gel defined. Satisfactory adsorbants were only produced using high concentrations of both CNBr and spacer-arm in the initial coupling slurry. Under these conditions it seemed likely that the majority of the ligand in a sterically favorable position to bind enterokinase was on the external surface of the bead. Trypsin [EC 3.4.21.4] binding to the adsorbant was not so critically dependent on the synthetic conditions and correlated closely with the degree of substitution. Dilution of the adsorbant with unlabeled Sepharose 4B indicated that there was more than 1 binding site per enterokinase molecule. The highest affinity was presumably for the active site, with adsorption supported by secondary interactions with spacer-arm or gel matrix not necessarily on the same bead. Maximal resolution was obtained by prolonged washing of the gel after loading; 2 populations of intestinal aminopeptidase [EC 3.4.11.2] were identified. Substitution of aniline for p-aminobenzamidine abolished specific enterokinase adsorption and improved the purification procedure by further removal of non-specifically adsorbed contaminants.