High-performance affinity chromatography of plasmin and plasminogen on a hydrophilic vinyl-polymer gel coupled with p-aminobenzamidine.
p-Aminobenzamidine was covalently attached via a spacer moiety to a microparticulate hydrophilic vinyl-polymer gel (Toyopearl HW65S) and this affinity adsorbent was used for the separation of plasmin and plasminogen by high-performance affinity chromatography. Toyopearl HW65S was alkylated with chloroacetylglycylglycine in dimethyl sulphoxide using methylsulphinyl carbanion as a catalyst, then p-aminobenzamidine was coupled to the carboxyl group of glycylglycine to form an acid amide bond. A column packed with the adsorbent retained both plasmin and plasminogen. Plasminogen was eluted with 6-aminohexanoic acid, a haptenic compound for the lysine-binding sites of plasminogen. For the elution of plasmin, the coexistence of 6-aminohexanoic acid and leupeptin (a competitive inhibitor for plasmin) was necessary. The results indicate a two-site interaction of plasmin with the immobilized ligand, i.e., at the lysine-binding sites and the catalytic site. Fluorometric detection of eluted protein and on-line assay of plasmin activity using a fluorogenic substrate, peptidylmethylcoumarylamide, revealed that effective chromatographic separation of the enzyme could be achieved with high sensitivity (10 micrograms) within 1 h.
D000578:Amidines; D001550:Benzamidines; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005341:Fibrinolysin / Q000302:isolation & purification*; D007976:Leupeptins / Q000032:analysis; D010958:Plasminogen / Q000302:isolation & purification*; D013056:Spectrophotometry, Ultraviolet; D014361:Trypsin Inhibitors
D000578:Amidines; D001550:Benzamidines; D007976:Leupeptins; D014361:Trypsin Inhibitors; C006500:4-aminobenzamidine; D010958:Plasminogen; D005341:Fibrinolysin; C032854:leupeptin