Fig. 1
a Chromatographic profile of the crude venom of B. atrox on a Sephacryl S-200 molecular exclusion column. Elution was performed with 0.2 M ammonium bicarbonate buffer (AMBIC), pH 8.0, collecting 3 mL/tube at a flow rate of 20 mL/hour. b 12 % SDS-PAGE. Lanes: 1 – tube 74 (S1a), 2 – tube 82 (S1b), 3 – tube 92 (S2), 4 – tube 99 (S2 valley), 5 – molecular mass standard (260, 140, 100, 70, 50, 40, 35, 25, 15, 10 kDa), 6 – tube 103 (S3), 7 – tube 106 (S3), 8 – tube 115 (S4), 9 – tube 124 (S4), 10 – tube 145 (S5)
Fig. 2
a Chromatographic profile of fraction S3 on a DEAE Sepharose anion exchange column. Elution was initiated with 0.05 AMBIC, pH 7.8, followed by a gradient of AMBIC from 0.05 M to 0.5 M, pH 7.8, and finally 1 M AMBIC, pH 7.8. Fractions of 3 mL/tube were collected at a flow rate of 30 mL/hour. b12 % SDS-PAGE. Lanes: 1 – tube 15 (D1), 2 – tube 28 (D2), 3 – tube 36 (D3), 4 – tube 40 (D3), 5 – molecular mass standard (260, 140, 100, 70, 50, 40, 35, 25, 15, 10 kDa), 6 – tube 44 (D3-D4 valley), 7 – tube 49 (D4), 8 – tube 55 (D4), 9 – tube 71 (D5a), 10 – tube 77 (D5b)
Fig. 3
a Chromatographic profile of fraction D3 on a C18 reverse phase column. Elution was performed using a RP-HPLC system at a flow rate of 0.5 mL/minute with a linear concentration gradient of 0-100 % solvent B (70 % acetonitrile and 0.1 % TFA) in ten column volumes. b 12 % SDS-PAGE. Lanes: 1 – fraction 1; 2 – fraction 2; 3 – fraction 3; 4 – fraction 4; 5 – fraction 5; 6 – fraction 6; 7 – molecular mass standard (116, 66.4, 45, 35, 25, 18, 4, 14.4 kDa)
Fig. 4
a Chromatographic profile of fraction 6 on a C18 reverse phase column. Elution was performed using a RP-HPLC system at a flow rate of 0.5 mL/minute using a segmented concentration gradient of 0-60 % solvent B in three column volumes, 60-80 % in five column volumes and 80-100 % in one column volume. b 12 % SDS-PAGE. Lanes: 1 – PLA 2 , 2 – molecular mass standard (116, 66.4, 45, 35, 25, 18.4, 14.4 kDa)
Fig. 5
a Evaluation of the purity level of fraction D4 after Vivaspin® 20 on a C18 reverse phase column. After having been subjected to ultrafiltration on a Vivaspin® 20 system, fraction D4 was evaluated by RP-HPLC using a C18 column with a linear concentration gradient of 0-100 % solvent B in five column volumes. b 12 % SDS-PAGE. Lanes: 1 – MP, 2 – molecular mass standard (260, 140, 100, 70, 50, 40, 35, 25, 15, 10 kDa)
Fig. 6
Isoelectric focusing of the toxins isolated from B. atrox venom on a 7 % polyacrylamide gel. Samples: (1) isoelectric focusing standard (IEF Standards, pI ranges from 4.45 to 9.6, Bio-Rad, USA), (2) PLA 2 (pI ~6.5) and (3) MP (pI ~7.4). The pI of samples was calculated from the curve of pH versus the migration distance in the gel
Fig. 7
Comparison of the partial amino acid sequence obtained for the MP from B. atrox venom with two other P-I metalloproteases previously isolated from the same venom: Batroxase and atroxlysin-I. Multiple sequence alignment was made using the program ClustalX v. 2.0.11. (*) indicates positions with fully conserved amino acid residues
Fig. 8
Fibrin(ogen)olytic activity of the MP isolated from B. atrox venom.a Evaluation of the fibrinolytic activity on fibrin gel. Samples were applied on plates containing fibrin gels, and incubation was performed at 37 °C for 24 h. Samples (halo diameter): 1. PBS (0 mm), 2. B. atrox venom 20 μg (15 mm), 3. MP 4 μg (15 mm), 4. MP 6 μg (15.5 mm), 5. MP 8 μg (16 mm), 6. MP 10 μg (16.5 mm). b Evaluation of the fibrinogenolytic activity by SDS-PAGE. Samples were incubated at 37 °C for 1 h and then evaluated on 10 % polyacrylamide gel under denaturing conditions. Lanes: 1 Molecular mass standard (260, 140, 100, 70, 50, 40, 35, 25, 15 kDa), 2. MP 5 μg, 3. Fibrinogen 25 μg, 4. Fibrinogen 25 μg + MP 1 μg
Fig. 9
Evaluation of the hemorrhagic activity of the MP from B. atroxvenom. Samples (n = 2) were injected intradermally into the back of BALB/c mice, and after three hours, the animals were euthanized and had their skins removed to allow the observation of the presence or absence of hemorrhagic halos. Samples (halo diameter): (a) PBS (0 mm), (b) MP 5 μg (10 mm, determined as the MHD), (c) MP 10 μg (16 mm), (d) MP 20 μg (24 mm), (e) MP 5 μg + PLA 2 1:1 (12 mm), (f) MP 5 μg + 5 mM EDTA (0 mm)
Fig. 10
Multiple alignment of the N-terminal sequence of the PLA 2 fromB. atrox venom with other acidic PLA 2 s from snake venoms. The highlighted amino acid residues are part of disulfide bonds (yellow) and the catalytic site (red). GI numbers (from top to bottom, respectively): 129420, 528050010, 387537880, 387537878, 123907441, 3914270, 23396779, 458601843 and 403399514. (*) indicates positions with fully conserved amino acid residues; (:) indicates conservation of the following groups with high score: K/R/Q/H, S/A, K/N/D, F/L/V/I, E/D/N/Q, T/S/A, I/M/L; (.) indicates conservation of the following groups with lowest score: N/R/G, G/D/N, A/V/T, Q/K/E/R, S/K/G/A, D/K/H, C/S, T/P