Fig 1.
Juvenile and adult Bothrops moojeni.
Specimens of B. moojeni snake at juvenile (A) and adult (B) stages. Photos by Sávio Stefanini Sant’Anna.
Table 1.
Data of age, size, and localization where the snakes or their parents came from.
Fig 2.
One-dimensional electrophoresis (1-DE, 15%) profiles of male and female B. moojeni at different ages.
The samples (20 μg) were applied to the gel under reducing conditions (β-mercaptoethanol) and the protein bands were stained with Coomassie Blue G-250, according to the manufacturer’s recommendation. Molecular weight markers (Dual Color Precision Plus, BioRad) are shown on the left side. PDE: phosphodiesterase; SVMP: snake venom metalloprotease; LAAO: L-amino acid oxidase; SVSP: snake venom serine protease; CRISP: cysteine-rich secretory protein; NGF: nerve growth factor; PLA2: phospholipase A2; CTL: C-type lectin.
Fig 3.
Venom profile of male B. moojeni at different ages by RP-HPLC.
Samples of 500 μg of lyophilized venom were dissolved in 0.1% trifluoroacetic acid (TFA; solution A) and submitted to RP-HPLC in a C18 column. Elution was carried out at 1 mL/min by applying a solution B (95% acetonitrile containing 0.1% TFA) gradient: 5% for 5 min; 5–25% for 10 min, 25–45% for 60 min, 45–70% for 10 min, 70–100% for 10 min, and 100% for 10 min. Absorbance was measured at 215 nm. *: marked peak that may be related to collagenolytic activity. DIS: disintegrin; PLA2: phospholipase A2; SVSP: snake venom serine protease; SVMP: snake venom metalloprotease.
Fig 4.
Venom profile of female B. moojeni at different ages by RP-HPLC.
Samples of 500 μg of lyophilized venom were dissolved in 0.1% trifluoroacetic acid (TFA; solution A) and submitted to RP-HPLC in a C18 column. Elution was carried out at 1 mL/min by applying a solution B (95% acetonitrile containing 0.1% TFA) gradient: 5% for 5 min; 5–25% for 10 min, 25–45% for 60 min, 45–70% for 10 min, 70–100% for 10 min, and 100% for 10 min. Absorbance was measured at 215 nm. *: marked peak that may be related to collagenolytic activity. DIS: disintegrin; PLA2: phospholipase A2; SVSP: snake venom serine protease; SVMP: snake venom metalloprotease.
Fig 5.
Overview of protein families’ abundances in male and female B. moojeni venoms at different ages.
All venom pools were subjected to LC-MS/MS, and proteins were identified, quantified, and assigned to each protein family expressed as percentage. CRISP: cystein-rich secretory protein; HYA: hyaluronidase; NUC: nucleotidase; VEGF: vascular endothelial growth factor; LAAO: L-amino acid oxidase; LZTS: leucine zipper tumor suppressor; CTL: C-type lectin; SVSP: snake venom serine protease; SVMP: snake venom metalloprotease; PLA2: phospholipase A2. Others: included families that accounted for < 1% in all venoms: bradykinin-potentiating peptide, calmodulin, disintegrin, flavin monoamine oxidase, glutaminyl-peptide cyclotransferase, Kunitz-type serine protease inhibitor, nerve growth factor, phosphodiesterase, phospholipase B, phospholipase A2 inhibitor, TNF receptor-associated factor 6, and waprin.
Table 2.
Relative abundance of the protein families obtained in each venom by LC-MS/MS.
Fig 6.
In vitro activities of the venom of male and female B. moojeni at different ages.
Proteolytic activity over collagen (A) and casein (B), PLA2 activity over the synthetic substrate 4-nitro-3-octanoyloxy-benzoic acid (C), LAAO activity with L-methionine (D), and coagulant activity over human plasma (E). Statistical data of all the groups is shown in S2 Table.
Fig 7.
In vivo assays of neonates and adults B. moojeni venom.
Hemorrhagic activity (A) was carried out by measuring the halos after intradermal injection of 20 μg venom. Median lethal dose (B) was determined by intraperitoneal injection of different doses of venom and the use of probit to obtain the values. The mice were observed for 6 consecutive hours and the number of deaths registered at each hour and 24 h after the injection of BmN (C) and Bm>4 (D) venoms.
Fig 8.
Immunointeraction between B. moojeni venoms and commercial antibothropic serum.
The interaction of the proteins in B. moojeni venom with the antivenom produced at Instituto Butantan was assayed by western blotting. The samples were submitted to 1-DE, the proteins were electrotransferred onto a PVDF membrane, and incubated with the antibothropic serum. SVMP: snake venom metalloprotease; LAAO: L-amino acid oxidase; SVSP: snake venom serine protease; CRISP: cysteine-rich secretory protein; NGF: nerve growth factor; PLA2: phospholipase A2; CTL: C-type lectin.