Activation and pH-dependence profile of rAc-cathB-1.

• (A) Processing of rAc-cathB-1. Purified rAc-cathB-1 (0.2 mg/mL) was incubated with activation solution or reference solution in a 2:1 (v/v) ratio. Mixtures were incubated at 37 °C for 30 min, and the reaction was stopped by addition of pepstatin A (Sigma-Aldrich) to a final concentration of 1 mM. Samples were analyzed on a 12% SDS-PAGE gel followed by Coomassie brilliant blue staining. MW, molecular weight markers; purified rAc-cathB-1 incubated with activation solution and reference solution as indicated.
• (B) Enzymatic activity assay. Z-Arg-Arg-7-amido-4-methylcoumarin hydrochloride was used for studying the activity of pepsin-treated rAc-cathB-1 and the control. The fluorescence was measured with excitation and emission wavelengths of 355 and 460 nm, respectively, and data were presented as relative activities, where activity of the control was taken as 1.
• (C) The pH-dependence profile of activated rAc-cathB-1. The assay was performed with the fluorescent substrate at a final concentration of 50 μM. Fluorescence was measured and data were presented as relative activities of activated rAc-cathB-1, where the highest activity at the pH optimum was taken as 100%. Asterisk (*), P < 0.05.