Snake venom and animals
Snake venoms were milked from D. acutus captured in Chongqing, China, and lyophilized for experimental use. Kunming mice (20 ± 2 g of body weight) were obtained from the Laboratory Animal Center of the Third Military Medical University. They were housed in temperature-controlled rooms and received water and food ad libitum until use.
Sephadex G-50, DEAE Sepharose Fast Flow and Hitrap Capto DEAE were purchased from GE Healthcare (USA). Protein MW Marker (Low) was obtained from TAKARA (Japan), ACN and Methanol from Fulltime Co. (China), and Bovine thrombin and fibrinogen from Biosharp (China). All other chemicals were of analytical grade.
Preparation of mouse ileum tissues
The preparation method of ileum tissues was modified as described in several reports [14,15,16]. Mice were killed by cervical dislocation and a segment of ileum approximately 15 cm long was removed from a distance of 2 cm from the ileo-caecal junction and kept in Krebs’ solution (118.4 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM CaCl2, 25.0 mM NaHCO3, and 11.1 mM glucose, pH 7.4) oxygenated with 95% O2 and 5% CO2. The mesentery and fatty tissues were removed and the lumen carefully flushed of its content with Krebs’ solution. Segments of ileum approximately 2 cm in length were dissected and mounted vertically in 10 ml water-jacketed organ baths containing Krebs’ solution kept at 37 °C and oxygenated with 95% O2 and 5% CO2. Changes in tissue tension were measured isometrically using force displacement transducer (Biopac, USA) and recorded on MP36 system (Biopac, USA). The tissues were slowly placed under a resting tension of 0.5 g (unless otherwise stated) and allowed to equilibrate for an at least 20 min period before the construction of the agonist or antagonist concentration-response curves. The active tension and rate of spontaneous tensions were continuously monitored for up to 90 min throughout the experiment. To avoid tachyphylaxis caused by the repeated use of the same ileum segment in each experiment, the used ileum segment was replaced with new one . In control experiments, the ileum segment was incubated with normal saline for at least 90 min without apparent decline in the parameters.
Isolation and purification of protein component
D. acutus venom (200 mg) was dissolved in 2.5 ml of 0.05 M Tris-HCl buffer (pH 8.4) overnight at room temperature, and centrifuged at 5000 rpm for 10 min at room temperature. The supernatant was loaded on a Sephadex G-50 column (1.1 cm × 100 cm) equilibrated with 0.05 M Tris-HCl buffer (pH 8.4), then eluted with the same buffer at an elution rate of 0.15 ml/min. The isolated fraction with the strongest inhibitory contractile response of ileum muscle was loaded on a DEAE Sepharose Fast Flow column(1.6 cm × 20 cm) equilibrated with 0.05 M Tris-HCl buffer (pH 8.4), and chromatographed with a linear gradient of 0 to 0.2 M NaCl in 0.05 M Tris-HCl buffer (pH 8.4) at an elution rate of 1.5 ml/min. The obtained fraction was pooled, desalted and concentrated, then applied to a Hitrap Capto DEAE column (0.7 cm × 2.5 cm) pre-equilibrated with 0.05 M Tris-HCl buffer (pH 7.4), and chromatographed with a linear gradient of 0 to 0.8 M NaCl in 0.05 M Tris-HCl buffer (pH 8.4) at an elution rate of 1.5 ml/min. The final active peak was manually collected, then desalted, lyophilized and stored at −20 °C.
The venom protein sample was applied to a C18 column (4.6 mm × 250 mm, ø 5 μm), and eluted using an acetonitrile-trifluoroacetic acid (TFA) gradient (buffer A: 0.1% TFA, buffer B: 80% acetonitrile-0.1% TFA; gradient: 0-30 min: 80% B, 30-35 min: 80–100% B) at a flow rate of 1 ml/min. The elution peaks were monitored at an absorbance of 215 nm. The major peak was collected and lyophilized for mass spectrometry and other studies.
Protein concentration was determined by the Lowry method  with BSA as a standard.
SDS-PAGE under reducing and non-reducing conditions were carried out according to Laemmli method .
MALDI-TOF mass spectrometry
Protein masses were determined by Matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Spectra were recorded and analyzed using an AB SCIEX instrument in a linear positive mode. The protein band of interest was sliced from 15% SDS-PAGE, and reduced, alkylated, then subjected to digestion with trypsin. The peptide mixtures were dried and analyzed with an ABI Voyager-DE Pro MALDI-TOF mass spectrometer. The peptide mass fingerprint (PMF) results were compared with the trypsin digest of protein of NCBInr database by using Mascot software 2.3.02.
Fibrino(geno)lytic activity assay
The hydrolytic activities of the purified venom protein on fibrinogen were evaluated by SDS–PAGE according to Rodrigues et al.  with some modifications. Different amounts of the purified venom protein (0.4 μg – 2.4 μg), or different mixtures of 2.4 μg of the purified venom protein with the different inhibitors (0.05 M PMSF, 0.05 M β-mercaptoethanol, 0.05 M EGTA and 0.05 M EDTA, respectively), were separately incubated with 20 mL of 10 mg/mL bovine fibrinogen (0.05 M PBS, pH 8.0) at 37 °C for 1 h. All the reactions were terminated with 10 mL of Tris–HCl buffer (0.05 M, pH 8.8) containing 10% (v/v) 2-mercaptoethanol, 2% (v/v) SDS, and 0.05% (w/v) bromophenol blue. The final reaction mixtures were analyzed by SDS–PAGE gels (12%, w/v).
Fibrinolytic activity was measured on fibrin plate. Fibrin plate was made of 8 mL of 0.4% fibrinogen, 8 mL of 1% agarose and thrombin (20 U) in 0.025 M Tris-HCl buffer (pH 7.4). After the wells (3 mm in diameter) were made in the plate, an aliquot volume (15 μL) of saline, Dacin (8 μg) and crude venom (20 μg), respectively, were added into the wells, then incubated at 37 °C for 12 h to visualize the transparent zones.
PLA2 activity assay
PLA2 activity was determined according to the methods reported by Habermann and Hardt  with some modifications. Briefly, one part of egg yolk was mixed with 3 parts of 0.85% (V/V) NaCl and centrifuged for 2 min at 2000 rpm, and the supernatant (egg yolk suspensions) was transferred into tubes for the following use. Agarose (0.15 g) was dissolved in 25 mL of 50 mM sodium acetate buffer (pH 7.5) in boiling water bath, then the solution was cooled down to 50 °C. The cooled agarose solution, egg yolk suspensions (500 μL) and 10 mM CaCl2 solution (250 μL) were fully mixed, finally was poured into Petri dishes. After the wells were punched in the plate, an aliquot volume (15 μL) of saline, purified venom protein (8 μg) solution and crude venom (20 μg) solution, respectively, were added into the wells, and incubated at 50 °C for 20 h to visualize the transparent zones.
According to the method , Kunming mice (18–20 g) received common feedstuff and water freely. To evaluate the hemorrhagic activity of purified venom protein, groups of 4 mice were injected intradermally on the dorsal region with the following dosages, respectively: a, 100 μL of 0.9% saline solution; b, 100 μL of saline solution containing 20 μg of D. acutus venom; c, 100 μL of saline solution containing 30 μg of purified venom protein; d, 100 μL of saline solution containing 10 μg of purified venom protein. Two hours after the injection the mice were sacrificed and the dorsal skin was sectioned for observation.
Data analyses were performed using the PRISM 5.0 software package. The results regarding biological activities were presented as means and standard deviation. Statistical analysis of significance was carried out by one-way or two-way ANOVA, The value of p < 0.05 was considered significant.