Figure 2.

Size-exclusion chromatography and preliminary characterization of PR2. PR2 after purification and in vitro folding was applied to the preequilibrated column (Superdex-75, 1.6 × 60 cm) at a flow rate of 1.4 mL/min at room temperature. Traces of the absorbance monitored at 280 (solid) and 260 nm (dotted) are shown for the entire chromatogram. Insets: (A) Susceptibility of PR2 to autoproteolysis as a function of pH. PR2 (1.5 μg/μL) was incubated for 2 h at room temperature at the pH values indicated. Note that lane 4 (pH 3.1) shows significant autoproteolytic cleavage products. (B) Catalytic activity of PR2 monitored as a function of time maintained at room temperature in 20 mM sodium phosphate buffer at pH 6.0 and 50 mM sodium chloride. Aliquots were assayed at pH 5.0 (see “Materials and Methods”) at the time intervals indicated. (C) Dependence of k_cat/K_m on pH for PR2 determined at a protein concentration of 0.14 μM at 28°C. The line is a theoretical curve for pK_a values of 3.8 and 5.0.