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.