The reactions occurred at 37 °C and were initiated by the addition of EP24.15 (7.5 ng), being monitored (λEM 420 nm and λEX 320 nm) in a spectrofluorophotometer (Victor 3™ Perkin–Elmer), as described [20]. The results were obtained in triplicate. The single peptide fraction containing inhibitory peptides was purified sequentially in the RP-HPLC system described above, but with a slower gradient (1.25% B/min), until reaching the pure peptide, and then subjected to mass spectrometric analyses. The peptide was analyzed by LC–MS/MS on a Synapt G1 mass spectrometer (Waters Co.). The peptide was resuspended in water and 2–5 μL injected onto a Symmetry
C18 trapping column (180 μm × 20 mm, Waters). see more The sample was desalted for 15 min and the trapped peptide was then separated by elution with a water/acetonitrile 0.1% formic acid gradient through a BEH 130 – C18 column (100 μm × 100 mm, Waters), as previously described [3]. Data was acquired in data-dependent mode and the peptide dissociated by Z-VAD-FMK clinical trial collisions with argon. The assays conditions included a flow rate of 600 nL/min, nanoflow capillary voltage of 3.5 kV, block temperature of 100 °C, and cone voltage of 100 V. The MS spectrum was analyzed manually from the ESI-MS/MS product ion mass spectra as previously described [19]. The peptides KEILG and KELLG were synthesized [1] with a purity grade greater than 95%. With the aim of determining
which peptide sequence was present in the venom, it was performed a RP-HPLC analysis as described above of a peptide mixture containing 20 μL of venom Peptide Pool, with 40 μM of KEILG and 40 μM of KELLG. This mixture was compared to the original Peptide Pool profile. The Ki was determined using seven concentrations of QFS and two concentrations of KELLG and KEILG peptides, maintaining the same EP24.15 concentration. Controls
without the peptides were also performed. The assay was carried out as described before. In order to analyze the mechanism of inhibition for both Acyl CoA dehydrogenase peptides, an Eadie–Hofstee plot was constructed and, based on the type of mechanism, the Ki was calculated as described [21]. After verifying the Peptide Pool inhibitory efficiency upon the QFS hydrolysis by EP24.15, the first step of purification using a C-18 reverse-phase was performed. Fourteen peptide peaks were obtained and submitted to peptidase screening, reaching a single one responsible for the inhibitory effect. This peak was submitted to the same purification method described before, but using a slower gradient, resulting in three new peaks, yet only one inhibited EP24.15 activity. For this reason, it was submitted to LC–MS/MS analyses, revealing the pentapeptide KEXXG (Fig. 1, panel A), where X could represent isoleucine/leucine. Two peptides were synthesized, KELLG and KEILG, to observe its performance at RP-HPLC and inhibition analyses.