Human Plasma Kallikrein: Purification, Enzyme Characterization and Quantitative Determination in Plasma

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Abstract
Human plasma kallikrein [kininogen, EC 3.4.21.8] was isolated from a plasma fraction related to Cohn fraction IV4 by affinity- and Sephadex G-150 chromatography yielding a material with 17.3 TAME[tosyl-L-arginine methyl ester]-U[units]/A280 unit. The preparation was characterized by immunological and enzymatic methods. Complex formation with .alpha.2-macroglobulin, .**GRAPHIC**. inactivator and aprotinin was demonstrated by immunoelectrophoresis. The bradykinin release from high-molecular weight kininogen and its inhibition by antibodies to kallikrein, AT [anti-thrombin] III and AT III-heparin complex were measured using a biological test system (rat uterus). Time dependent inactivation of kallikrein by AT III and AT III-heparin complex was shown by means of a synthetic kallikrein substrate: Bz-Pro-Phe-Arg-pNan [N-benzoyl-L-propyl-L-phenylalanyl-L-arginine-p-nitranilide]. The same substrate was used to measure the activation of prekallikrein in plasma by kaolin and factor XII a. Antibodies raised against kallikrein were shown to inhibit the reaction specifically. A quantitative determination of plasma prekallikrein by electroimmunodiffusion according to Laurell was developed: the plasma concentration in normal individuals was found to be 1.8-2.2 TAME-U/ml related to kallikrein activity; this corresponds approximately to 9-11 mg antigen/100 ml plasma.