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.
The affinity chromatography of human enterokinase using p-aminobenzamidine as the ligand [Grant, D.A.W. & Hermon-Taylor, J. (1976) Biochem. J. 155, 243-254] has been reassessed and the optimal conditions for the synthesis and operation of the derivatised 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 favourable position to bind enterokinase was on the external surface of the bead. Trypsin 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 unlabelled Sepharose 4B indicated that there was more than one 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; two populations of intestinal aminopeptidase were identified. Substitution of aniline for p-aminobenzamidine abolished specific enterokinase adsorption and improved the purification procedure by further removal of onon-specifically adsorbed contaminants.
D000578:Amidines; D000626:Aminopeptidases / Q000302:isolation & purification; D000814:Aniline Compounds; D001550:Benzamidines; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods; D004386:Duodenum / Q000201:enzymology; D010450:Endopeptidases / Q000302:isolation & purification*; D004765:Enteropeptidase / Q000302:isolation & purification*; D006033:Glycylglycine / Q000031:analogs & derivatives; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D014357:Trypsin; D000520:alpha-Glucosidases / Q000302:isolation & purification
D000578:Amidines; D000814:Aniline Compounds; D001550:Benzamidines; D008024:Ligands; D006033:Glycylglycine; C006500:4-aminobenzamidine; D000520:alpha-Glucosidases; D010450:Endopeptidases; D000626:Aminopeptidases; D014357:Trypsin; D004765:Enteropeptidase