The plasminogen activator and esterase activities of the two forms of urokinase

High and low mol. wt. urokinase (UK) purified to homogeneity by affinity chromatography, and partially purified commercial preparations with varying ratios of high to low mol. wt. forms were assayed for plasminogen activator activity by: physiologic clot lysis time using varying amounts of native and degraded plasminogen; plasminogen activator activity by protamine assay; and esterase activity on the synthetic peptide ester N-Ac-Gly-Lys-O-Me (AGLMe); as well as by active-site titration using p-nitrophenyl-p′-guanidino-benzoate (NPGB). The molar activity of both high and low mol. wt. UK by the three rate assays was about 1 × 10¹³ IU/mole active site and was not affected by molecular size or low mol. wt. impurities. Depending on the particular UK preparation and the type of plasminogen, one or more impurities may affect the fibrinolytic assay at limiting substrate concentrations. Gel filtration or affinity chromatography abolishes the effect. NPGB titration provides another rapid and simple means of estimating UK activity and relating all three rate assays to molar activities. Correlation of the results of protamine and AGLMe assays with those of the fibrinolytic assays was generally good, except for the tissue culture UK which showed at least 25% higher values by both the protamine and AGLMe assays. The protamine assay proved a useful alternative to fibrinolytic and synthetic substrate.