Comparisons of the generation of bradykinin, Met-Lys-bradykinin, and des-Arg
-bradykinin in LK samples that were digested using a mixture of Sap9 and other Saps vs digestion with single Saps. LK samples (1.5 μM) were digested with either 0.03 μM individual Saps or mixtures of 0.03 μM Sap9 with equimolar amounts of other Saps in the citrate buffer (pH 5.0) at 37°C for 6 hours. After sequentially stopping the reaction with pepstatin A and HCl, the obtained peptide mixtures were separated on the Luna C18 column in a TFA water-ACN binary gradient system. Fractions were collected at the retention times that correspond to the bradykinin (B), Met-Lys-bradykinin (M), and des-Arg1-bradykinin standards (D), evaporated to dryness, and redissolved in the assay buffer of the ELISA kit in order to quantitatively determine the kinin concentrations. The results are corrected by subtracting the data, obtained for the undigested LK sample as specified in Figure 5. The amount of each of the three kinins is expressed relative to the maximum possible amount of all kinins (calculated using the molarity of LK in the sample). For each Sap, the light gray bar represents the sum of kinin amounts in two independent samples, treated separately with this Sap and Sap9 while the dark gray bar represents the value for sample of LK that was treated with a mixture of this Sap and Sap9. All data bars represent the mean values of six determinations (averaged as specified in Figure 5) ± standard deviation. Asterisks denote the statistical significance (t-Student test, p < 0.05) of the difference between the indicated kinin levels (generated by a mixture of Sap9 and other Sap vs digestion with two Saps separately).