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.
Kajihara
J
J
JCR Pharmaceuticals Co., Ltd., Kobe, Japan.
Shibata
K
K
Nakano
Y
Y
Nishimuro
S
S
Kato
K
K
D000818:Animals; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D006207:Half-Life; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008970:Molecular Weight; D010449:Peptide Mapping; D010958:Plasminogen / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000493:pharmacokinetics; D011108:Polymers / Q000737:chemistry* / Q000493:pharmacokinetics; D011409:Propylene Glycols / Q000737:chemistry* / Q000493:pharmacokinetics; D011817:Rabbits; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics / Q000652:urine
D011108:Polymers; D011409:Propylene Glycols; C012504:polypropylene glycol; D011092:Polyethylene Glycols; D010958:Plasminogen; D014568:Urokinase-Type Plasminogen Activator