31] Streptokinase and staphylokinase

Abstract

This chapter presents a procedure for purification and two staged assay for streptokinase and staphylokinase. Assay method is based on the principle that streptokinase or staphylokinase forms a complex with plasminogen. The complex catalyzes the conversion of plasminogen to plasmin in the first stage of the assay. NaCl is added to a sufficient concentration to stop further plasminogen activation. The amount of plasmin generated in the first stage is measured in the second stage using the plasmin-specific substrate, D-Val-Leu-Lys-p-nitroanilide. The hydrolytic product p-nitroanilide is measured spectrophotometrically. In the streptokinase assay, Triton X-100 can be included in the first stage because it enhances the streptokinase activity. In the staphylokinase assay, it is best to use the Lys form of human plasminogen, because the staphylokinase exhibits a serious lag in activation rate when human Gluplasminogen is used. For protein purification suitable strain of S. aureus is chosen based on its high yield of fibrinolytic activity when the centrifuged culture supernatants are tested for plasminogen activation on canine fibrin plates. Purification steps involve: precipitation of supernatant and then adsorbed batchwise at room temperature to SP-Sephadex C-25 column. Further process includes chromatography on the DIP-canine plasmin-Sepharose column, final step involves affinity-column product to be resuspended in 0.05 M Tris and applied to a G-75 (superfine) column.