Human plasma kallikrein: purification, enzyme characterization and quantitative determination in plasma

Human plasma kallikrein was isolated from a plasma fraction related to Cohn fraction IV4 by affinity- and Sephadex G-150 chromotography yielding a material with 17.3 TAME-U/A280 unit. The preparation was characterized by immunological and enzymatic methods. Complex formation with alpha2-macroglobulin, C1-inactivator and aprotinin was demonstrated by immunoelectrophoresis. The bradykinin release from high-molecular weight kininogen and its inhibition by antibodies to kallikrein, AT 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. The same substrate was used to measure the activation of prekallikrein in plasma by kaolin and F 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.