Inhibition of activity was assessed by testing the effect of EDTA and Pefabloc® on protease activity. For the EDTA assay, 2 μL protease was added to the reaction buffer with no added CaCl2 (protease solution contributed with 63 μM CaCl2 from the maturation step) and 0–1 mM EDTA and incubated for 60 min. For the Pefabloc® assay, 5 μL matured protease was added to the reaction buffer with calcium in 0–5 mM Pefabloc®. Tris at pH 7.5 was used in the reaction buffer to reduce the auto-hydrolysis of Pefabloc®. The protease was incubated with Pefabloc for 2.5 h RT.
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