Purification and reaction mechanism of the primary inhibitor of plasmin from human plasma

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Abstract
The primary inhibitor of plasmin in human plasma was purified by a 4-step procedure involving fractional (NH4)2SO4 precipitation, ion-exchange chromatography on a column of DEAE-Sepharose CL-6B and affinity chromatography on a plasminogen-CH-Sepharose 4B column and a column of 6-aminohexanoic acid covalently coupled through the carboxylate function to AH-Sepharose 4B. No impurities in the final preparation could be detected when tested by immunoelectrophoresis against a range of specific antisera or against rabbit anti-human serum. On polyacrylamide-gel electrophoresis the inhibitor preparation showed a single band. The dissociation constant for the inhibitor-plasminogen complex was .apprx. 3 .mu.M at pH 7.8. Reactions of the inhibitor with human plasmin and bovine trypsin were studied. Comparison of the results confirms the hypothesis previously presented, that the reaction of the inhibitor with plasmin involves at least 2 steps, the initial rapid formation of an enzyme-inhibitor complex followed by a slow irreversible transition to another complex. The reaction of the inhibitor with trypsin apparently involves just a single, irreversible step, so that this reaction seems less complicated than that of the inhibitor with plasmin. The ways in which 6-aminohexanoic acid influences the reactions were studied. The same value for the dissociation constant (.apprx. 26 .mu.M) for 6-aminohexanoic acid was obtained for its effect on the reaction of the inhibitor with trypsin and for competitive inhibition for trypsin. The inhibitory effect of 6-aminohexanoic acid seems due to its blocking of the active site of trypsin. The inhibitory effects of L-lysine and 6-aminohexanoic acid on the inhibitor-plasmin reaction occur at concentrations much too low to affect the active site of plasmin. The possible dependence of the reaction of the inhibitor with plasmin on a 2nd site(s) on plasmin is discussed.