A spectrophotometric assay for the determination of the catalytic efficiency of plasminogen activators using a slowly hydrolyzed plasmin substrate.
A simple spectrophotometric assay for the determination of the catalytic efficiency and activity of plasminogen activators is presented. The assay system contains activator, plasminogen, and the chromogenic substrate N-benzoyl-L-arginine-p-nitroanilide (BAPA). Plasmin production is monitored continuously by the hydrolysis of BAPA under non-steady-state, first-order conditions with respect to plasminogen. Apparent catalytic efficiency constants are calculated from the values obtained for the apparent first-order rate constant of activation. The results obtained with the present method were compared with the catalytic efficiency determined through the measurement of kcat and Km, using a different system, under steady-state conditions. Tissue plasminogen activator in the absence and presence of fibrinogen and high-molecular-weight urokinase were used as model activators. Potential applications are discussed.
Castro
M J
MJ
Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854, USA.
Kingston
I B
IB
Anderson
S
S
D001586:Benzoylarginine Nitroanilide / Q000378:metabolism; D002384:Catalysis; D005340:Fibrinogen / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010958:Plasminogen / Q000378:metabolism*; D013053:Spectrophotometry / Q000379:methods*; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D005340:Fibrinogen; D010958:Plasminogen; D001586:Benzoylarginine Nitroanilide; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator