Purification and some properties of urokinase

A method for efficient extraction of urokinase from human urine was established by using polyacrylonitrile synthetic fiber as an adsorbent. By a combination of this method and known methods for purification of proteins, such as gel filtration and ion-exchange chromatography, urokinase with a specific activity of 224,000 International Units per mg of protein was obtained. This sample showed homogeneity by ultracentrifugation, moving-boundary electrophoresis at pH 4.8 and 9.0 and polyacrylamide gel disc electrophoresis at pH 4.0, but was separated into five active fractions by isoelectric focusing and polyacrylamide gel disc electrophoresis at pH 9.4. This sample showed a single precipitin line in double radial immunodiffusion and immunoelectrophoresis using rabbit anti-urokinase serum. This precipitin line fused with that of the International Standard preparation of urokinase and its immunological identity was established. The molecular weight of this sample was 33,000, agreeing with that of the International Standard preparation. Its optimal pH as a plasminogen activator was approximately 8.8.