Indirect active-site titration of plasminogen activators

A method for the determination of the molar concentration of active tissue plasminogen activator (TPA) and urokinase (UK) has been developed. The method employs the principle of back-titration of a calibrated trypsin standard with a calibrated standard solution of a chloromethyl ketone inhibitor reactive with both trypsin and activator. Both trypsin and chloromethyl ketone standards are calibrated using a guanidinobenzoyl ester active-site titrant. Less than 20 ng of activator can be accurately determined by this method. The method is used to assay commercial samples of TPA and UK, to calculate the specific activity of such samples, and to determine kinetic constants of plasma activator inhibitor.