Purification of streptokinase by affinity chromatography using human plasminogen

Abstract

A method for preparing a respectable quality streptokinase in a convenient manner was described. Streptokinase is important for enzyme theraphy, because it lyses blood clots by activating plasminogen to the fibrinolytic enzyme, plasmin. For the crystallography to determine its tertiary structure, the preparation of pure streptokinase is needed. Human plasminogen was purified from Cohn fraction III by the affinity chromatography with Llysine-substituted Sepharose CL-4B. This plasminogen was used as a ligand for streptokinase affinity chromatography. PBS (pH7.4) and Glycine (pH2.5) were used as washing and elution buffer, respectively. To obtain a single peak, $\varepsilon$-amino-N-caproic acid ($\varepsilon$-ACA) was added to both washing and elution buffers. The function of $\varepsilon$-ACA is the prevention of streptokinase fragmentation during the plasminogen activation. To study the role of Gly 24 in streptokinase, mutant streptokinaess, His 24, Ala 24 and Glu 24, were purified and tested for specific activity and protease activity with various artificial substrates. The specific activity of Ala-SK was about 76.6\% of Gly-SK, and His-SK and Glu-SK lost nearly all activator activity. And any protease activity against substrates tested so far has not been detected in wild-type and mutant streptokinases.