SOLID AND FLUID-PHASE ASSAYS FOR BACTERIAL PLASMINOGEN ACTIVATORS

Certain bacteria secrete or possess membrane bound proteins which are capable of activating host plasminogen to form the serine protease, plasmin. Plasmin can play a central role in the pathogenesis of microbial disease, particularly with regard to dissemination, modifying host structures and modulating the complement cascade. To determine if organisms produce proteins which have the capacity to activate plasminogen, the following experimental approaches are presented. Assays can be performed on cells grown on agar (caseinolytic assay), or on crude culture supernatants resolved by SDS-PAGE (fibrinolytic assay). For increased sensitivity and specificity, the plasmin-specific chromogenic substrate, H-D-valyl-leucyl-lysine-para-nitroanilide can be used not only to analyze in liquid phase the presence of plasminogen activators in culture supernatants and column effluents but also in a solid phase assay on products transferred to nitrocellulose. The latter method also permits direct immunological comparisons and the ability to test for inhibitors.