Binding of Bovine Plasminogen to Immobilized Casein and its Activation Thereon

Binding and activation of plasminogen to immobilized casein was studied using a type of ELISA performed as follows: casein was adsorbed to the wells of a microtiter plate. Plasminogen was then poured into the wells and its binding assessed by adding successively monoclonal antibody specific jot plasminogen, phosphatase conjugate goat anti-mouse antibodies and phosphatase substrate. It was found that the amount of plasminogen bound depended on the casein type. Lysine binding sites, as well as electrostatic forces, were involved in plasminogen binding to casein and the amount of plasminogen bound depended on casein type. The role of electrostatic forces in binding to immobilized casein seemed to be more important than in binding to casein micelles of milk. Bound plasminogen was converted to plasmin by urokinase without dissociation from the matrix and the plasmin thus generated was active while bound. However, it was not possible to activate more than approximately 80% of bound plasminogen, and only 65% of plasminogen in milk, suggesting that the plasminogen concentration enzymatically determined in milk after activation by urokinase may be underestimated when compared to the immunological results. It was possible to inhibit the plasmin activity by aprotinin, but the enzyme was protected from inhibition by SBTI. Dissociation of plasminogen from the immobilized casein was obtained only at pH values below 4.6. No dissociation was observed with either high concentration of epsilon-amino caproic acid or 2 m NaCl. The conditions. for activation of plasminogen and inhibition of plasmin seemed to be similar to the conditions encountered in milk. It was concluded that the method developed may be a useful tool to study the various pathways involved in activation and inhibition of the plasminogen-plasmin complex.