The Peptide Chains of Human Plasmin

Abstract

Abstract Human plasminogen and plasmin were found to be monomers by physical measurements. The cleavage of a single disulfide bond in both the proenzyme and enzyme, at pH 9, by reduction with 2-mercaptoethanol in 8 m urea, resulted in complete loss of proteolytic activity. At pH 3, activity losses paralleled the number of disulfide bonds cleaved with complete loss in activity only after total cleavage of all the disulfide bonds (21 to 22). The cleavage of the single interchain disulfide bond of plasmin at pH 9 resulted in the formation of two major chains (α and β) which were first demonstrated by starch gel electrophoresis in 8 m urea-2-mercaptoethanol systems at pH 3. S-Sulfo and S-carboxymethyl derivatives of plasmin were prepared; they were also found to contain two major chains on electrophoresis in starch gels. Sedimentation velocity measurements of both proenzyme and enzyme and their S-carboxymethyl derivatives, in urea-2-mercaptoethanol systems, confirmed the presence of plasmin chains. The amino-terminal amino acid residue of plasminogen and one plasmin chain was found to be lysine, and the amino-terminal amino acid residue of the second plasmin chain was found to be valine. The carboxyl-terminal amino acid sequence of plasminogen and one plasmin chain was found to be Leu-Asn-COOH, and the carboxyl-terminal amino acid sequence of the second plasmin chain was found to be Lys-Arg-COOH. The activation of human plasminogen monomer to plasmin monomer by urokinase is probably due to the cleavage of a single arginyl-valine bond by the activator, which results in a two-chain molecule held together by a single disulfide bond. The spontaneous proteolytic activity of human plasminogen preparations is probably due to contaminating plasmin. It is suggested that the activation of human plasminogen by other activators, such as streptokinase, trypsin, and pig heart activator, may involve a similar hydrolytic cleavage of an arginyl-valine bond.