FIGURE 7.

Selective inhibition of PR3 in biological samples. A, inhibition of PR3 in a representative CF sputum supernatant. The volume of CF sputum was adjusted to give final concentrations of PR3 and HNE of ∼1 nm. Diluted samples were incubated with compound 5 (Cp5, 50 nm final) for 20 min at 37 °C. The residual activities of PR3 and HNE were measured as described in Fig. 3. Purified PR3 (1 nm) and HNE (1 nm) were used as controls. B, selective labeling of PR3 activity in CF sputum. CF volumes were adjusted to contain 4 or 20 ng PR3 (10 and 50 nm final) and incubated for 20 min at 37 °C with compound 5 (150 nm final), and the products were revealed with extravidin peroxidase. C, Western blot analysis of CF sputa with anti-PR3 antibodies showing active/inactive/degraded PR3 and additional bands due to antibodies interacting with unrelated CF sputum proteins. D, inhibition of PR3 in concentrated (10×) urine from three patients with bladder cancer. Proteolytically active PR3 in unconcentrated urine was ∼0.1–1 nm based on the rate of substrate hydrolysis. E, selective labeling of PR3 activity in concentrated (10×) urine (U) samples. F, Western blot analysis of concentrated urine samples (10×) using anti-PR3 antibodies.