Effect of pH on Sap-catalyzed kinin release from the HK-D4 peptide. HK-D4 (10 μM) was cleaved using 0.2 μM Saps (1–4, 8, and 9) in 50 mM citrate buffers (pH 3.0–5.0) or 25 mM phosphate buffers (pH 6.0–7.0) for 24 hours at 37°C. The reaction was stopped using HCl (0.33 M), and the samples were analyzed using HPLC as specified in Figure 1. The amounts of Met-Lys-bradykinin or des-Arg1-bradykinin (for Sap9) that formed were estimated based on the peak areas and are expressed relative to the maximum possible amount (calculated using the molarity of the reaction substrate). Data represent mean values from the analysis of three independent samples (three separate digests) ± the standard deviation.