The proteolytic activity of O. bauri crude venom on azocasein was determined as 102 U/µg. When evaluating the effect at various pH the venom presented higher and optimal activity in pH 8.0, with a significant loss in acidic (4.0; 5.0 and 6.0) and basic (11.0) pH (Figure 2A). The effect of temperature in the proteolytic activity showed high activities between 25 °C and 37 °C, with optimal activity at 37 °C and significant reduction at higher temperatures (Figure 2B). In this way, the following experiments were performed at 37 °C for one hour.

Concerning the effect of inhibitors, the proteolytic activity was significantly reduced after pre-incubation with aprotinin, leupeptin and EDTA. However, aprotinin allowed the highest reduction of the activity (45%) when compared to other inhibitors as leupeptin (29%) and EDTA (9%) (Figure 2C). In contrast, the effect of ions as Ca²⁺, Mg²⁺, Zn²⁺ and Cu²⁺ did not show any change of the proteolytic activity (data not shown).

The Zymogram method was used to determine the nature and the molecular weight of the gelatinolytic enzyme present in the venom of O. bauri. Figure 3 shows that the crude venom presented six proteins having gelatinolytic activity, with apparent molecular masses of 17, 20, 26, 29, 43 and 48 kDa (Figure 3A). When the effect of different buffers and pH (4.0–10.0) in the gelatinolytic activity was evaluated, we observed increased renaturation of proteases with buffer containing CaCl₂ and NaCl in the presence of the chemicals CHAPS and EDTA (Figure 3B) and with optimal pH of 8.0 (Figure 3C).

Temperature was also critical for gelatin proteolysis induced by the O. bauri crude venom, as we demonstrate that its activity was maintained between 25 °C and 37 °C after 30 min of reaction. However, this activity was impaired when the temperature increased above 56 °C (Figure 4A). Enzyme stability was also evaluated, and showed that the O. bauri venom was constant until the 20th day of incubation at 4 °C (Figure 4B).

Crude venom of O. bauri showed a time-dependent fibrinogenolytic activity. The enzymes completely degraded bovine fibrinogen α-chain at a concentration of 5 μg and with 30 min of incubation. However, degradation of fibrinogen β-chain was observed with longer incubation time (720 min) and on the other hand, the enzymes did not showed any activity over fibrinogen γ-chain (Figure 5).

The hemolytic activity of O. bauri crude venom was verified in different concentrations, reaching a maximal hemolytic activity (around 100% lysis) from the concentration from 60 to 180 µg/mL (p < 0.01) (Figure 6A).

Viability of HeLa cells and murine bone marrow-derived macrophages (BMDM) in the presence of different concentrations of O. bauri crude venom (Figure 6B) was above 63% and 85%, respectively, even when the highest concentrations (30 and 60 µg/mL) were used.

The crude extract of O. bauri was also evaluated for hemorrhagic and coagulant activities. There was no formation of minimum hemorrhagic lesion (above 10 mm of diameter) in Swiss mice inoculated intradermally with the crude venom, even using high concentration (50 µg). However, the O. bauri venom was able to coagulate bovine plasma in about 15 sec when compared to the positive control containing 0.2 M CaCl₂ (coagulation in about 2 min) (data not shown).

Crude venom of O. bauri caused defibrinogenation when administered intraperitoneally to mice, making the plasma uncoagulable. Animals treated with the venom promoted blood clotting after 4.3 min while the control animals had average clotting time of 1.6 min (p < 0.01) (data not shown).

The antimicrobial activity of the O. bauri crude venom was also examined and the results were measured by the zones of bacterial growth inhibition around each of the disks, comparing with positive controls. O. bauri crude venom presented antimicrobial activity against both Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria in the concentration of 15 μg/disk, with inhibition of bacterial growth in 62.5% and 72.7%, respectively, when compared to positive controls (Table 1).

Effect of the O. bauri crude venom on T. gondii infection and replication in HeLa cells was verified and shown in Figure 7. The pretreatment of T. gondii tachyzoites with O. bauri venom before infection of HeLa cells showed a dose-response inhibitory curve that reached up to 83% of inhibition and showed an IC₅₀ of 12.2 µg/mL for the infection index (Figure 7A). Concerning the inhibition of intracellular parasite replication, the pretreatment of T. gondii tachyzoites with O. bauri before infection of HeLa cells showed a dose-dependent inhibition, reaching rates of 68% and IC₅₀ of 35.1 µg/mL (Figure 7B).

Proteolytic activity of the O. bauri venom was determined using azocasein (Sigma-Aldrich) as substrate [44] with modifications. Aliquots of 1 µg of venom were added to a mixture of 500 µL of 50 mM Tris-HCl (Sigma-Aldrich) pH 6.8 and 500 µL of 2% azocasein solution (w/v). As negative control, 500 µL of saline solution were added to 500 µL of 2% azocasein solution. After 1 h of incubation at 37 °C the reaction was stopped by adding 100 µL of 15% trichloroacetic acid (TCA, Sigma-Aldrich) and the samples were centrifuged at 10,000× g for 10 min. One unit of activity was defined as an increase of 0.01 in absorbance units at 405 nm, and the results were expressed as specific activity units (U/mg).

To study the effect of pH on azocaseinolytic activity, 1 µg of venom was added to 500 µL of 2% azocasein solution buffered with 500 µL of the following buffers at various pH ranges: 0.2 M sodium acetate (Sigma-Aldrich) pH 4.0 and pH 5.0; 0.2 M sodium phosphate (Sigma-Aldrich) pH 6.0; 0.2 M Tris-HCl pH 7.0 and pH 9.0; 0.2 M sodium borate (Sigma-Aldrich) pH 10.0; 0.2 M phosphate sodium (Sigma-Aldrich) pH 11.0.

The effect of temperature on the azoproteolytic activity was verified by preheating for 15 min 1 µg of venom at temperatures ranging from 25 °C to 75 °C, following incubation with 2% azocasein solution. The reactions were stopped by adding 100 µL of 15% TCA, followed by centrifugation and the absorbance read in a spectrophotometer at 405 nm.

The stability of enzymes of the O. bauri venom was evaluated on the basis of its proteolytic activity on azocasein in the presence of different protease inhibitors as aprotinin (serine proteases, Sigma-Aldrich), leupeptin (cysteine proteases, Sigma-Aldrich) and EDTA (metalloproteases, Sigma-Aldrich) and bivalent ions (Ca²⁺, Mg²⁺, Zn²⁺ and Cu²⁺), all reagents at concentration of 5 mM. Aliquots of 1 µg of venom and 5 µL of inhibitors or ions were preincubated for 15 min and then solubilized in 2% azocasein solution. After 1 h of incubation the reaction was stopped and the enzymatic activity determined as above described.

The technique described by [45], with some modifications, was employed, using gelatin as substrate. Crude venom samples (5 µg) were separated by 12% SDS-PAGE containing 1% of the gelatin substrate (Sigma-Aldrich). Subsequent to the electrophoresis, the gel was washed twice for 30 min at room temperature in 2.5% Triton X-100 (Sigma-Aldrich) to remove the SDS and incubated at 37 °C for 18 h in one of the following buffers: 0.05 M sodium citrate pH 4.0, pH 5.0 and pH 6.0; 0.05 M Tris-HCl pH 7.0, pH 8.0, pH 9.0 and pH 10.0; and in the presence of ions and other chemicals as 50 mM Tris-HCl pH 8.0; 50 mM Tris-HCl and 10 mM CaCl₂ (Sigma-Aldrich) pH 8.0; 50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl₂, 0.002% CHAPS (Sigma-Aldrich) and 10 mM EDTA pH 8.0; and 50 mM Tris-HCl, 1 mM CaCl₂ and 1 mM ZnSO₄ (Sigma-Aldrich) pH 8.0. The gels were stained with R-250 Coomassie blue and gelatin proteolysis activity detected as colorless bands in the otherwise blue gel.

The thermal effect on the gelatin proteolysis activity of the O. bauri venom was investigated at temperatures from 25 °C to 75 °C. First, aliquots (20 µg) were incubated at different temperatures (25 °C, 37 °C, 56 °C, 65 °C and 75 °C) for 30 min and applied to the gel of gelatinase activity. After electrophoresis, the gel was incubated with 50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl₂, 0.002% CHAPS and 10 mM EDTA (pH 8.0) for 18 h and stained with Coomassie blue.

To analyze the enzyme stability, 200 µL of the O. bauri venom (stock solution at 500 µg/mL) was incubated at 4 °C in intervals from 2 to 20 days. At each day of incubation, aliquots of 10 µL (5 µg) were removed and applied to the gel of gelatinase activity as above described.

The fibrinogenolytic activity of the O. bauri venom was determined in SDS-PAGE according to the methodology of [46], with modifications. Briefly, 25 µL of bovine fibrinogen (stock solution at 3 mg/mL, Sigma-Aldrich) were incubated with 5 µg of the venom at 37 °C. At different time intervals (30, 60, 120, 720 and 1440 min), aliquots were collected and the reaction was stopped by adding SDS sample buffer. The hydrolysis profile was followed by SDS-PAGE at 12% gel [43].

Red blood cells of Swiss mice were used to evaluate the hemolytic activity of the crude venom of O. bauri according to [47] with modifications. After collected the red blood cells were washed twice with 0.9% NaCl (w/v) and 0.5% erythrocytes (v/v) were incubated at 37 °C in the presence of venom (32 to 0.06 µg) for 1 h. Samples were then centrifuged (450× g for 5 min), and the absorbance of the supernatants was measured at 540 nm. The absorbance measured from lysed red blood cells in presence of 1% (v/v) Triton X-100 was considered as 100%.

Cytotoxicity of O. bauri crude venom was assessed by determining cellular viability using MTT assay as previously described [48]. HeLa cells and BMDM from Swiss mice were cultured separately in 96-well plates (1 × 10⁵ cells/well) in triplicate, in the presence of O. bauri crude venom in different concentrations (0.03, 0.1, 0.3, 1, 3, 10, 30, 60, 90 and 180 µg/mL). As controls, cells were incubated with complete RPMI medium alone. After 24 h of incubation at 37 °C and 5% CO₂, cells were washed and pulsed with 10 µL of thiazolyl blue at 5 mg/mL in 90 µL of complete RPMI medium 4 h prior to the end of the culture. Formazan particles were solubilized in 10% sodium dodecyl sulfate (SDS) and 50% N,N-dimethyl formamide (Sigma-Aldrich). The optical density was read after 30 min at 570 nm in a plate reader (Titertek Multiskan Plus, Flow Laboratories, McLean, VA, USA). Results were expressed as percentage of cell viability in relation to controls.

The hemorrhagic activity was assessed according to [36]. Samples containing 50 μg of the crude venom of O. bauri were prepared in 0.9% NaCl and injected intradermally into the dorsal skin of Swiss mice, and saline solution alone was used as negative control. Three hours after the injection, the animals were sacrificed by cervical dislocation and the dorsal skin was removed. The minimum hemorrhagic dose (MHD) was defined as the amount of protein that induced a hemorrhagic lesion of 10 mm of diameter, as calculated using the perpendicular major diameters of the hemorrhagic spot.

The coagulant activity of venom was assessed on citrated bovine plasma as described by Denson et al. [49], with modifications. Samples of 20 µg of the crude venom of O. bauri were added to aliquots of 200 µL of bovine plasma and incubated at 37 °C. The activity was characterized by the immediate appearance of fibrin network compared with the clotting time of the control containing 0.2 M CaCl₂.

The defibrinating activity of venom was tested by the method of [50], with modifications. The activity was assessed by intraperitoneal injection of 2 μg/g body weight of mice of the O. bauri crude venom in 100 μL of saline solution into male Swiss mice (18–22 g), using three mice per group; control animals received 200 μL of saline solution. After one hour, the animals were anesthetized and submitted to cardiac puncture. Blood was placed in tubes and kept at 25–30 °C until clotting occurred. The minimum defibrinating dose (MDD) was defined as the amount of venom able to prevent coagulation.

The antimicrobial activity of the O. bauri venom was performed by the disk diffusion susceptibility method according to Yagmur et al. [51], with modifications, by applying a bacterial inoculum of approximately 2 × 10⁸ CFU/mL to the surface of a large (150 mm diameter) Mueller-Hinton agar plate. Bacteria specimens tested included a Gram-positive, Staphylococcus aureus (ATCC 25923) and a Gram-negative, Escherichia coli (ATCC 25922). Paper filter disks (0.5 mm diameter) were prepared with crude venom of O. bauri in the following concentrations: 15, 10, 5, 2.5, 1.25, 0.6 and 0.3 μg per disk unit; and placed on the inoculated agar surface. Commercially-prepared disks were used as positive control for S. aureus (Oxacillin; 1 μg, Laborclin, Pinhais, Brazil) and E. coli (Ampicillin; 10 μg, Laborclin). Sterile water disks as negative control were applied to both bacteria. Plates were incubated for 16–24 h at 35 °C prior to determination of results by measuring the zones of growth inhibition around each of the disks.

The antiparasitic activity of the O. bauri venom was verified on in vitro T. gondii infection following the protocols of de Oliveira et al. [52]. HeLa cells were cultured on 13-mm round glass coverslips into 24-well plates (1 × 10⁵ cells/well/200 µL) for 24 h at 37 °C and 5% CO₂. T. gondii tachyzoites (RH strain) were obtained from previously infected HeLa cells, washed in RPMI medium and pretreated for 1 h at 37 °C and 5% CO₂ with crude venom of O. bauri in different concentrations (0.3, 1, 3, 10, 30 and 60 µg/mL) or with medium alone (control). Next, parasites were washed and incubated with HeLa cell monolayers on coverslips at 2:1 (parasite: host cell) rate of infection (2 × 10⁵ tachyzoites/well/200 µL) for 24 h at 37 °C and 5% CO₂. Cells were washed with 0.9% NaCl to remove non-adherent parasites, fixed in 10% buffered formalin for 2 h and stained with 1% toluidine blue (Sigma-Aldrich) for 5 s. Coverslips were mounted on glass slides and cells were examined under a light microscope with regards to T. gondii infection index (percentage of infected cells per 100 examined cells) and parasite intracellular replication (mean number of parasites per cell in 100 infected cells).

Results were expressed as percentages of inhibition of infection as well as of parasite intracellular replication for each treatment in relation to controls. The median inhibitory concentration (IC50) of venom was calculated by extrapolation of the corresponding dose-curve response on a log linear plot employing the portions of the curve that transected the 50% response point [53].