A sensitive, coupled assay for plasminogen activator using a thiol ester substrate for plasmin.

In parallel with the increased interest during the past decade in plasminogen activator involvement in fibrinolysis, tumorigenesis, inflammatory responses, and the expression of hormonal regulation, there has been a rising interest in sensitive and precise methods for the specific assay of plasminogen activator. Several assays for plasminogen activator employ a direct assay These are remarkably sensitive methods, yet they suffer in comparison to the sensitivity of coupled Coupling the assay with plasminogen not only amplifies the sensitivity by the multiplicative effect of plasmin, but insures that only those proteases specific for plasminogen are assayed. The choice of substrate for plasmin is critical. In general, esters are superior to amides in both K,,, and kcof, but they suffer a major deficiency in that they are frequently unstable at pH 7-9, the optimum range for plasmin. Green and Shaws synthesized a thiol ester substrate, thiobenzyl benzyloxycarbonyl-L-lysinate (2-Lys-SBzl) , which combines high kcat with alkaline stability relative to the commonly used esters. In an effort to characterize the plasminogen activator from hepatoma tissue culture (HTC) and its hormonally controlled inhibitor, several of the known direct and coupled methods were found inadequate by reason of either low sensitivity or imprecision. Using 2-Lys-SBzl in a coupled approach we have developed an assay that is superior to the [1Z51]fibrinolytic assay. It is also extremely sensitive to plasminogen activator (-2 X lo-’? moles of urokinase) and can be used for routine analysis of purification as well as kinetic and binding studies.