A chromogenic enzymatic assay capable of detecting prourokinase-like material in plasma

We report a functional assay capable of quantifying prourokinase (ProUK)- like material in plasma where urokinase (UK) is also present. The assay involves inactivation of urokinase with a specific, active site directed irreversible inhibitor, dansyl-glutamyl glycyl arginine chloromethylketone (dansyl- GGACK). Excess inhibitor is subsequently quenched with dithiothreitol (DTT). The ProUK-like material in plasma is then converted to active urokinase with thermolysin, a proteolytic enzyme of bacterial origin. Alpha 2-macroglobulin in plasma inhibits thermolysin; however alpha 2-macroglobulin is inactivated with methylamine. The assay can detect as little as 20 ng of ProUK and is linear from 20 to 120 ng. The assay was applied to quantify the amount of ProUK-like material in plasma obtained from dog at various times after i.v. administration of 100,000 or 75,000 U/kg, of pro-urokinase.