Specificity of rPRCP activation of PK. Twenty nanomolar PK was incubated in HCB, pH 7.4, in microtiter plate cuvette wells precoated with 2 μg HK with either 10 nM rPRCP or 40 pM FXIIa in the absence or presence of 300 μM angiotensin II (AgII) or 0.2 mg/mL neutralizing antibody to FXIIa (Ab). (A) At the end of the incubation, the wells were washed, and the reactions were stopped by the addition of sample buffer for SDS-PAGE, reduced with 2% β-mercaptoethanol, boiled, and applied to 12% SDS-PAGE for electrophoresis followed by immunoblot with a polyclonal antibody to human PK. The formed kallikrein was detected by a secondary antibody conjugated with horseradish peroxidase and then by chemiluminescence and autoradiography. Numbers to the right of the gel represent molecular mass standards in kilodaltons. This experiment is representative of 1 of 3 experiments. (B) This experiment was performed simultaneously with the experiment depicted in panel A. At the end of the incubation, the wells were washed with HCB, pH 7.4, and then by 100 μL HCB, pH 7.4, containing 0.8 mM S2302 was added. The formed kallikrein was indicated by the hydrolysis of the substrate for 1 hour at 37°C. This experiment is the mean ± SEM of 3 experiments performed simultaneously with 3 PK cleavage experiments shown in panel A.