Purification of urokinase by combined cation exchanger and affinity chromatographic cartridges

Crude urokinase from human urine processed through foam flotation and ammonium sulfate precipitation containing 720 National Health Institute Committee on Thrombolytic Agents U/mg activity was purified by an SP cation exchanger followed by a zinc-chelated affinity chromatographic cartridge. The cartridges were of a radial-flow type formed by using acrylic and cellulose composite matrices. The high rigidity of the matrix structure permits fast flow of protein solutions (liters per minute) and thus allows processing of a large volume of crude urokinase under low operating pressures. A greater than six-fold increase in specific enzyme activity of urokinase was achieved by adsorbing and eluting 1 l of a 3 mg/ml crude urokinase solution on an SP cartridge. The eluent was further purified by passing through a zinc-chelated affinity cartridge to achieve greater than a eighteen-fold increase in urokinase specific activity. This report demonstrates the combined use of a cation exchanger with zinc-chelated chromatographic cartridges in purifying urokinase on a relatively large scale. The relationship between the amount of zinc chelated in the matrix to its effect on urokinase purification is also discussed.