Preservation of thrombolytic activity of urokinase modified with monomethoxypoly(ethylene glycol)

A method is described to modify urokinase by covalent binding of monomethoxypoly(ethylene glycol) (mPEG) without impairing its catalytic ac tivity towards high molecular weight substrates. The urokinase active site is protected by an inhibitor, benzamidine, bound to Sepharose during the mPEG modification in order to avoid binding mPEG chains to the active site or to the surrounding area. The mPEG modified urokinase had increased activity towards small molecular weight substrates (acetyl-Gly-methyl ester) as com pared to the unmodified enzyme, while the activity towards the high molecular weight plasminogen and the insoluble substrate fibrin clot was preserved. This did not occur when the enzyme was modified in the absence of active site pro tection. The polymer modification increased the enzyme's thermostability and the stability in plasma in vitro and prolonged in vivo retention after in travenous injection in rats.