Purification and partial characterization of a single-chain high-molecular-weight form of urokinase from human urine.

Affinity chromatography on fibrin/celite, a previously described technique for isolating plasminogen activators with a high fibrin affinity (S. Husain, B. Lipinski, and V. Gurewich, 1981, Proc. Nat. Acad. Sci. USA 78, 4265-4269), was performed on freshly voided urine. About 10-25% of the plasminogen activator activity present was adsorbed whereas less than 1% was adsorbed when urine was stored at room temperature for 12-24 h. The adsorbed activator was isolated by elution with arginine (0.1 M) and further purified by gel filtration. This plasminogen activator was similar in molecular weight (56,000) to the known high-molecular-weight form of urokinase but differed from it by its binding affinity for fibrin/celite, single-chain subunit structure, lower specific activity (approximately 20,000 IU/mg), and slower reaction with diisopropylfluorophosphate. The enzymatic activity of the purified activator was quenched by anti-urokinase antibody and therefore established it to be a new form of urokinase. The relationship of this high affinity, single-chain urokinase to the previously described forms of urokinase is unclear.