Separation of human Glu-plasminogen, Lys-plasminogen and plasmin by high-performance affinity chromatography on Asahipak GS gel coupled with p-aminobenzamidine.
Human Glu-plasminogen, Lys-plasminogen and plasmin were effectively separated by high-performance affinity chromatography. The affinity adsorbent was prepared by using a micro-particulate polyvinyl alcohol gel (Asahipak GS-gel) as the supporting material and p-aminobenzamidine as the specific ligand. All of the active enzyme and proenzymes were adsorbed. Glu-plasminogen was eluted by changing the pH of the eluent and Lys-plasminogen by using an eluent containing 6-amino-hexanoic acid. This affinity adsorbent recognized the difference between these proenzyme species. For the elution of plasmin, addition of urea was necessary. Plasmin may have been adsorbed through a two-site interaction with the adsorbent. All proteins were eluted as sharp peaks and the time required for one cycle was about 1 h. Fluorimetric detection of eluted protein and on-line assay of enzyme activity using a fluorigenic substrate made it possible to analyse microgram amounts of proteins specifically.
D000818:Animals; D001550:Benzamidines; D002417:Cattle; D002846:Chromatography, Affinity; D005260:Female; D005341:Fibrinolysin / Q000302:isolation & purification*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010446:Peptide Fragments / Q000302:isolation & purification*; D010958:Plasminogen / Q000302:isolation & purification*; D011142:Polyvinyl Alcohol; D011247:Pregnancy; D013050:Spectrometry, Fluorescence
D001550:Benzamidines; D010446:Peptide Fragments; C018998:lysyl-plasminogen; C006500:4-aminobenzamidine; D010958:Plasminogen; D011142:Polyvinyl Alcohol; D005341:Fibrinolysin