New fluorogenic peptide substrates for plasmin.
Fluorogenic peptides, peptidyl-4-methylcoumaryl-7-amides (MCA), containing COOH-terminal lysine residues, were newly synthesized and tested as substrates for plasmin. Among six peptidyl-MCA's, Boc-Val-Leu-Lys-MCA and Boc-Glu-Lys-Lys-MCA were found to be useful for the specific and sensitive assay of plasmin. The Km values estimated from Line-weaver-Burk plots for these substrates using human and bovine plasmins were in the region of 10(-4) M. Boc-Glu-Lys-Lys-MCA was slightly hydrolyzed by bovine plasma kallikrein, and Boc-Val-Leu-Lys-MCA was slightly hydrolyzed by human and hog urinary kallikreins and hog pancreatic kallikrein. However, both of the fluorogenic peptides were essentially unaffected by urokinase, alpha-thrombin, Factor Xa, Factor IXa, Factor XIa, and Factor XIIa. It was confirmed that plasmin hydrolyzed Boc-Glu-Lys-Lys-MCA, cleaving the lysyl-MCA bond, but not the lysyl-lysyl bond. These fluorogenic peptides were resistant to human plasmin activated by streptokinase. Boc-Glu-Lys-Lys-MCA was not hydrolyzed by human plasmin or plasminogen in the presence of more than a 5-fold molar excess of streptokinase. The sensitivity of Boc-Val-Leu-Lys- of more than a 5-fold molar excess of streptokinase. The sensitivity of Boc-Val-Leu-Lys-MCA to human plasmin was also reduced, but plasmin retained 35% of the maximum activity even in the presence of a 20-fold molar excess of streptokinase. These results suggest that streptokinase-plasmin complex has essentially no activity towards Boc-Glu-Lys-Lys-MCA.
D000818:Animals; D002417:Cattle; D003374:Coumarins / Q000138:chemical synthesis; D004789:Enzyme Activation; D005341:Fibrinolysin / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D010455:Peptides / Q000138:chemical synthesis; D010958:Plasminogen / Q000378:metabolism; D013300:Streptokinase / Q000378:metabolism; D013379:Substrate Specificity; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D003374:Coumarins; D010455:Peptides; D010958:Plasminogen; D013300:Streptokinase; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator