The mechanism of activation of human plasminogen by streptokinase was examined, and several properties of the two types of plasmin [EC 3.4.4.14] formed were compared.Human plasminogen was prepared as follows: isoelectric precipitation of human plasma, at pH5.S, treatment of the euglobulin fraction with 0.03M ethylenediamine-tetraacetic acid (EDTA)* at pH4.8, dissolution of EDTA-treated euglobulin fraction in 0.1 m lysine at pH8.0 and precipitation of inert protein at pH5.5 to 5.6, ammonium sulfate fractionation of the supernatant at 0.18 to 0.3 saturation, and gradient elution from a DEAE-Sephadex column. This preparation of human plasminogen was homogeneous by ultracentrifugal analysis, disc-electrophoresis and amino-terminal determination. Its sedimentation coefficient was 5.4 at pH7.4 and 4.8 at pH9.0. The purification was 800 to 1000 fold from plasma. An amino-tcrminal lysine residue was found in human plasminogen. The mechanism of activation was examined with this preparation.When the amount of human plasminogen was constant and preincubation is omitted, the activation of human plasminogen was a function of the amount of streptokinase added. When plasminogen was activated with a trace of streptokinase, activation increased with the time of preincubation. From these results, two mechanisms of activation of human plasminogen by streptokinase were proposed. The first is due to the formation of a plasminogen-streptokinase complex, and the second is due to catalytic transformation by the cleavage of the plasminogen molecule. A large amount of streptokinase was added to human plasminogen, and the mixture was subjected to starch gel electrophoresis. Human plasminogen and human plasmin activated by a trace of streptokinase showed an identical single band which moved toward the cathode, whereas plasmin activated by a large amount of streptokinase appeared as a sharp band at the origin, while streptokinase migrated to the anode. A mixture of human plasminogen and a large amount of streptokinase was chromatographed on a DEAE-Sephadex column in the presence of 8 m urea. Although only slight esterolytic activity was observed without streptokinase in fraction 55 to 70, addition of streptokinase to these fractions resulted in strong esterolytic activity. These findings indicate that these fractions contained human plasminogen. These results confirm the idea that the activation of human plasminogen by a large amount of streptokinase is due to the formation of a plasminogen-streptokinase complex.In human plasmin which had been activated by a trace of streptokinase, a new amino-terminal valine was found besides the amino-terminal lysine residue. This supports the idea that cleavage of the plasminogen molecule occurs during its activation by a trace of streptokinase.Plasmins activated by these two mechanisms showed similar esterolytic and caseino-lytic activities. However, plasmin activated by complex formation showed far more plasminogen activating, and bovine fibrinogen hydrolyzing activities than plasmin activated by catalytic conversion. © 1969 BY THE JOURNAL OF BIOCHEMISTRY.
