Inhibitors of rPRCP. HK (2 μg/100 μL) was linked to cuvette wells in 0.1 M Na₂CO₃, pH 9.6, overnight at 4°C. After washing unbound HK, the cuvette wells were blocked with 1% gelatin for 1 hour at 37°C. After washing, rPRCP (10 nM) and PK (20 nM) were added. (A) Ability of rPRCP to activate PK bound to HK in the absence or presence of 100 μM leupeptin, 1 mM DFP, 1 mM EDTA, 1 mM bradykinin, 300 μM angiotensin II, 1 mM bradykinin 1-5, or 300 μM angiotensin 1-7 is shown. When the rPRCP was treated with DFP, it was dialyzed overnight before being incubated with PK bound to HK. rPRCP lane: combination of HK + PK + rPRCP added to microtiter cuvette wells. HK + PK lane: proteins added alone to microtiter cuvette wells without rPRCP. Empty vector lane: material from the culture supernatant of cells transfected with vector alone. (B) Ability of rPRCP (10 nM) to activate PK bound to HK was also examined in the absence or presence of increasing concentrations of goat IgG (IgG), goat anti-PRCP (Anti-PRCP), Z-Pro-Prolinal, or corn trypsin inhibitor (CTI). In both panels, after 1 hour of incubation at 37°C, the wells were washed and 0.8 mM S2302 was added. The amount of kallikrein activity on the microtiter plate well surface was measured by hydrolysis of S2302 and was monitored at 405 nm for 1 hour at 37°C. The error bars present the mean ± SEM of 3 or more experiments.