Enzymatic assays for native plasmin, plasminogen and plasminogen activators in bovine milk

Rapid and sensitive assays for plasmin, plasminogen and plasminogen activat ors (PA) were developed and applied to bovine milk. The reaction medium was clarified by addition of a dissolving agent after hydrolysis of a fluoresc ent substrate specific for plasmin. This final step enabled the use of larg er sample amount with higher substrate concentration than other methods, an d avoided previous sample preparation. The use of 4 g gelatin/l in buffers preserved plasmin activity, thus avoiding risks of overestimation of the as says results. Sensitivity, detection level, repeatability and analysis run time of plasmin and plasminogen assay were improved over previous enzymatic methods with synthetic substrates. The PA assay was assessed by measuring conversion of exogenous plasminogen into plasmin. A new kinetic approach wa s used to enable the direct determination of global PA activities on raw mi lk samples without interference from indigenous plasmin.