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.