Physicochemical characterization of PEG-PPG conjugated human urokinase.

Human urokinase (UK) was conjugated with polyethylene glycol-polypropylene glycol (PEG-PPG) and its physicochemical properties were examined. PEG-PPG modification decreased the activity for plasminogen activation, but increased the half-life of this protein when injected intravenously in rabbits. Kinetic analysis of PEG-PPG conjugated UK (PEG-PPG-UK) revealed that the kcat for plasminogen activation decreased 1/5-fold with the increase of Km in comparison with that of UK, although these parameters for cleavage of synthetic substrate (S-2444) did not change. However, the inhibitor constant of PEG-PPG-UK for plasminogen activator inhibitor 1 (PAI 1) was equal to that of UK. Peptide mapping analysis revealed that PEG-PPG binding sites were mainly determined to be Lys 35, 46, 61, 98, 120 and 135 in A-chain and Lys 211, 300, 318, 338, 348, 383 and 404 in B-chain. In addition, the modification rates of A and B-chain were 37.8% and 19.8% on average, respectively.