1% trifluoroacetic acid (TFA) in water (solvent A), and acetonitrile and solvent A (9:1) as solvent B. The separations were performed at a flow rate of 1 mL/min using a Shim-pack VP-ODS C-18 column (4.6 × 150 mm) and a 20–60% gradient of solvent B over 20 min. In all cases, elution was followed by ultraviolet absorption (214 nm). The scissile bonds in the peptides were determined by mass spectrometry analyses. The peptide fragments were detected by scanning from m/z 100 to m/z 1300 using an Esquire 3000 Plus Ion trap Mass Spectrometer with ESI and esquire CONTROL

software (Bruker Daltonics, MA, USA). Purified 18O-labeled or unlabeled oxidized W derivatives were dissolved in a mixture of 0.01% formic acid:acetonitrile (1:1) and infused into the mass (direct infusion pump) spectrometer at a flow rate of 240 μL/h. The skimmer voltage of the capillary was 40 kV, the dry gas was kept at 5.0 L/min, and the source learn more temperature was maintained at 300 °C. After defining the natural peptides that were hydrolyzed by BjV, the ability of the other venoms to hydrolyze

angiotensin I (65 μM) was analyzed using 4 μL of each MAPK Inhibitor Library in vitro one (B. alternatus [5.74 mg/mL], B. jararacussu [3.11 mg/mL], B. moojeni [0.86 mg/mL] and B. neuwiedi [0.11 mg/mL]). The scissile bonds found in angiotensin-I produced by these venoms were deduced by internal standardization of the HPLC system, using the results obtained with B. jararaca as reference. The ability of the antibothropic serum to neutralize the venoms proteolytic activities was estimated by incubating Thalidomide it with Bothrops spp. venoms. Samples of Bothrops venoms were incubated, at room temperature, in the presence and absence of the antibothropic serum. The residual proteolytic activities of the venoms were measured as described above, using both FRETs substrates. The volume of the antibothropic serum and the pre-incubation time for serum neutralization of the proteolytic activities were established by using the B. jararaca venom. After establishing the best conditions to neutralize the metallo- and serine peptidases from the B. jararaca venom, the other Bothops spp venoms were tested (B. alternatus, B. jararacussu, B. moojeni and B. neuwiedi). The venoms were

used in volumes of 2.0 μL when the Abz-Metal was utilized as substrate and 0.2 μL for the kinetics with the Abz-Serine (see concentration on 2.5). For the maximum blocking effect of the proteolytic activity, the venoms were incubated with 10 μL of the antibothropic serum for 30 min at room temperature. After this period, 5 μM of each substrate was added and the residual activity was measured as described above. The experiments were made in triplicate. The same concentrations of Bothrops spp venoms described in the angiotensin-I degrading assays were utilized to determine the neutralizing potential of the commercial serum. Thus, after a pre-incubation time (venoms and antivenom), 65 μM of angiotensin I was added and after 1 h more samples were analyzed by HPLC reverse-phase.