The Isolation and Characterization of the S-Carboxymethyl β (Light) Chain Derivative of Human Plasmin

Abstract

Abstract The S-carboxymethyl β chain derivative from both moderately reduced urokinase-activated plasmin and extensively reduced streptokinase-activated plasmin was separated in a highly purified form by gel filtration through Sephadex G-200. Electrophoresis in urea-starch gel systems at pH 3.2 showed that the faster moving β electrophoretic component was the light chain derivative. The sedimentation coefficient, s20,w, of this derivative was found to be 1.4 S. The apparent molecular weight was found by sedimentation equilibrium analysis with absorption optics to be 25,700. The apparent molecular weight of the S-carboxymethyl heavy chain derivative was found to be approximately 57,200. Carboxyl-terminal amino acid analysis with carboxypeptidases A and B showed that asparagine was the carboxyl-terminal amino acid of the light chain and indicated that arginine must be the carboxyl-terminal amino acid of the heavy chain. Incorporation of 1.18 ± 0.08 moles of DFP-32P per mole of human plasmin indicated a single proteolytic site. Starch gel electrophoresis and Sephadex gel filtration experiments both showed that the proteolytic active site of human plasmin is located on the light chain. The amino acid compositions of the S-carboxymethyl light chain derivatives of both urokinase-activated plasmin and streptokinase-activated plasmin were found to be almost identical, confirming the specificity of the activation of human plasminogen to plasmin by both urokinase and streptokinase.