(The) immobilized urokinase to sepharose 4B as a model system

Abstract

Urokinase (EC 3. 4. 99. 26), a plasminogen activator, converts plasminogen to plasmin which has the ability to dissolve the fibrin clot. Hence it is generally used for the clinical treatment of the various vascular diseases and for the preparation of the thromboresistant material such as artificial organ and suture material. Through the urokinase is used as the most frequently prescribed agent and also there is an increasing need to develope the better medical material with high antithrombogenicity, the maintenance of the enzyme activity over a long period $mbox{\underline{in}}$ $\mbox{\underline{vivo}}$ is most difficult to attain and the purification of the urokinase causes difficulties. Several attempts have been tried to improve such materials by using immobilization technique so far. However, the recovery of activity or steric hinderance after the immobilization causes many problems in the urokinase immobilization. In this study, therefore, the immobilization of the urokinase was carried out by various methods and also the effects of spacer molecule on the accessibility of substrate, a macromolecular plasminogen, to the coupled urokinase to macromolecular matrix were examined by interposing different length of hydrocarbon chain between enzyme and the matrix back-bone as a model study. The human plasminogen, substrate for urokinase, was purified from Human Cohn Fraction III. The Cohn III solution was applied to the lys-Sepharose affinity column and the plasminogen was dialyzed, followed by freeze-drying. The final products were used in the assay of the urokinase. The urokinase was immobilized directly to the CNBr-activated Sepharose 4B as well as to the Sepharose 4B which contained different length of hydrocarbon chain as spacers. For the direct immobilization of urokinase, Sepharose 4B was activated by CNBr, followed by coupling with urokinase at pH 7.5. For indirect coupling two methods were applied. Firstly, different length of aminoalkyl carboxylic acid...