Distribution of bradykinin, Met-Lys-bradykinin, and des-Arg
-bradykinin in the Sap-digested LK samples. LK samples (1.5 μM) were digested using 0.03 μM Sap in the citrate buffer (pH 5.0) at 37°C for 6 hours. After sequentially stopping the reaction using pepstatin A and HCl, the obtained peptide mixtures were separated on the Luna C18 column in a TFA water-ACN binary gradient system. The fractions were collected at the retention times that correspond to the bradykinin, Met-Lys-bradykinin, and des-Arg1-bradykinin standards, evaporated to dryness, and redissolved in the assay buffer of the ELISA kit in order to quantitatively determine the kinin concentrations. The corresponding fractions, obtained from the HPLC separation of intact (undigested) LK served as the controls, and the kinin concentrations, determined in these fractions are subtracted from those in the Sap-digested LK samples. The corrected amount of each of the three kinins is expressed relative to the maximum possible amount of all kinins (as calculated using the molarity of LK in the sample). Results, obtained from two independent experiments (two LK-digests independently analyzed by HPLC for each Sap), with three replicate ELISA measurements (in three different wells) for each fraction obtained during each HPLC separation, are presented as the mean values ± standard deviation. Asterisks denote the statistical significance of the difference between the kinin levels in the Sap-treated and undigested LK samples (t-Student test, p < 0.05). The data plotted in the three panels are for bradykinin (A), Met-Lys-bradykinin (B), and des-Arg1-bradykinin (C).