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. 2014 Aug 21;10(8):e1004324. doi: 10.1371/journal.ppat.1004324

Figure 4. rAPRc activation product displays optimal activity at pH 6 and is strongly inhibited by specific HIV-1 PR inhibitors.

Figure 4

The effect of pH, class-specific and HIV-1 PR specific inhibitors on the proteolytic activity of rAPRc activation product was evaluated using the synthetic fluorogenic substrate (MCA)Lys-Ala-Leu-Ile-Pro-Ser-Tyr-Lys-Trp-Ser-Lys(DNP). (A) Activity at different pH values. Activated rAPRc was incubated with the substrate at 37°C in buffers ranging between pH 4 and pH 9 containing 100 mM NaCl (50 mM sodium acetate pH 4.0, 5.0, 5.5 and 6.0 and 50 mM Tris-HCl pH 7.0, 8.0 and 9.0). (B) and (C) To test the effect different compounds, the protease was pre-incubated in the presence of each inhibitor for 10 minutes at room temperature in 50 mM sodium acetate pH 6.0 containing 100 mM NaCl before adding the substrate. The rate of substrate hydrolysis (RFU/sec) was monitored for 3 hours and the relative activity normalized by setting the maximum activity at 100%. The error bars represent standard deviation of the mean.