Source of algae
Specimens of the red marine alga Bryothamnion triquetrum were collected from the Atlantic Coast of Brazil (Pacheco Beach, Ceará) and kept in plastic bags at -20°C until processed.
Algae were thawed, rinsed with distilled water, cleaned of epiphytes, and ground to a fine powder under liquid nitrogen. The powder was stirred for 4 hours with 0.02 M phosphate buffer (pH 7.0), with 0.15 M NaCl, filtered through nylon tissue, and then centrifuged at 7,000 × g, for 30 min, at 4°C. The supernatant was acidified and remained under refrigeration for 4 hours. The precipitated pigments were removed by centrifugation and the supernatant (crude extract) was adjusted to pH 7.0, followed by ammonium sulphate precipitation (0–60%). Precipitated proteins were recovered by centrifugation, dialyzed in distilled water and applied onto a DEAE-cellulose column, equilibrated and eluted with 0.02 M phosphate buffer (pH 7.6), followed by elution with 1.0 M NaCl solution in the same buffer, at a flow rate of 30 mL/h. The unabsorbed fractions with haemagglutinating activity were pooled and rechromatographed in the same column. Active fractions containing lectins were dialyzed and lyophilised (DEAE fraction).
After that, approximately 5 mg of freeze-dried DEAE-fraction was dissolved in 250 μL of ammonium bicarbonate buffer (0.05 M; pH 7.9) and homogenised until complete dissolution, followed by clarification by centrifugation (4,500 × g for 2 min, at 27°C). The supernatant was recovered and injected onto an ion exchange HPLC column (Protein Pack SP 5PW, 0.75 × 10 cm, Waters), previously equilibrated with the same buffer used for dissolving the fraction. The elution was conducted using a discontinuous linear gradient of ammonium bicarbonate at a concentration of 1.0 M, pH 8.0, at constant flow rate of 1 mL/min.
The chromatography was monitored at 280 nm and the peaks were collected and the two fractions containing haemagglutinanting activity were lyophilised and than subjected to the final chromatographic steps of the Reverse Phase (RP-HPLC). The elution of samples was carried out using a linear gradient concentration of an aqueous solution of 66% acetonitrile. Samples were previously dissolved in buffer A of RP-HPLC (0.1% TFA) that was used to equilibrate the column. The fractions were immediately concentrated by ultrafiltration (AMICON), followed by dialysis with 5.0 mM ammonium bicarbonate solution (pH 7.8), sterilized by microfiltration (0.22 μm) and stored at 4°C for further use.
The electrophoresis was carried out following the Laemmli method . The degree of purity of fractions was assessed by discontinuous electrophoresis using a final acrylamide concentration of 12% in the running gels (1.0 M Tris-HCl, pH 8.8) and 5% in the stack gel (0.5 M Tris-HCl, pH 6.8). The electrophoretic run was carried out using a BIORAD electrophoretic system for SDS-PAGE. All samples and the molecular marker were treated with SDS and 1.0 M DTT and the run was conducted at 40 mA for both plates. After electrophoresis, samples were stained with Coomassie brilliant blue R-250.
The purified BTL-2 was dissolved in water and the solution was adjusted to a final concentration of 10 mM of protein. The sample was transferred to a 1 mm path-length quartz cuvette. Circular dichroism spectra in the wavelength range of 185–300 nm were obtained using a J715 spectropolarimeter (Jasco Corp., Japan) using a bandwidth of 1 nm and a response time of 1s. Data collection was performed at room temperature with a scanning speed of 100 nm/min; a total of nine scans were accumulated for the sample.
PLA2 from the rattlesnake, Crotalus durissussus casvella, was purified by a combination of chromatographic procedures and its molecular homogeneity was determined by SDS-PAGE. The whole venom was fractionated by HPLC molecular exclusion chromatography using a Superdex 75 column (1 × 60 cm). The active PLA2 fraction was then subjected to Reverse Phase HPLC in a C18 μ-Bondapack column (0.78 cm × 30 cm) (Waters 991-PDA system). The column was eluted with a linear gradient (0–66.5%, v/v) of acetonitrile (solvent B) at a flow rate of 2 mL/min, and absorbance was monitored at 280 nm. The fractions were collected manually, lyophilized and stored at -20°C.
Amino acid sequence
Three milligrams of purified protein were dissolved in 200 μl of 6 M guanidine chloride (Merck, Darmstadt, Germany) containing 0.4 mM Tris-HCl and 2 mM EDTA (final pH 8.15). Nitrogen was flushed over the top of the protein solution for 15 min, which was then reduced with DTT (6 M, 200 μl) and carboxymethylated with 14C-iodoacetic acid and cold iodoacetic acid. Nitrogen was again flushed over the surface of the solution and the reaction tube sealed. This solution was incubated in the dark at 37°C for 1 h and desalting was performed on a Sephadex G 25 column (0.7 × 12 cm) in 1 mM acetic acid buffer. The eluted reduced and carboxymethylated (RC) protein was lyophilised and stored at -20°C. The RC lectins were then purified and the N-terminal region sequenced.
For the RC-PLA2, we determined the complete amino acid sequence of the protein by sequencing the N-terminal region and the peptides from enzyme-treated PLA2. One sample of RC-protein was digested with Staphylococcus aureus protease V8 for 16 hours at 37°C, using an enzyme -substrate ratio of 1:30. The reaction was stopped by lyophilisation. A second sample of RC-PLA2 was digested with Clostripain for 8 hours at 37°C and then lyophilised ; the products were separated by Reverse Phase HPLC using a Waters PDA 991 system with a C-18 μ-Bondapack column.
Peptide peaks were isolated using a linear gradient (0–100% of acetonitrile in 0.1% TFA (V/V)). A third sample (2 mg) was cleaved with a 15-fold molar excess of cyanogens bromide (CNBr) over methionine residues in 70% formic acid (4 ml) under nitrogen for 24 hours at room temperature. The reaction mixture was then diluted with 40 mL of water and lyophilised. Excess reagents were removed by gel filtration on a Sephadex G-25 column (1 × 20 cm) equilibrated with 10% acetic acid. The CNBr peptide fragments were separated by Reverse Phase HPLC, using an analytical μ-Bondapack C18 column (0.39 × 30 cm; Waters) with 0.1% TFA as solvent A and acetonitrile containing 30% of solvent A (solvent B). The elution profile was monitored at 214 nm. Analysis of the amino acid sequence of the RC-protein, as well as that of the enzymatically-digested fragments were performed with an Applied Biosystems model Procise f gas-liquid protein sequencer. The phenylthiohydantoin (PTH) derivatives of the amino acids were identified with an Applied Biosystems model 450 microgradient PTH-analyser.
PLA2 activity was measured . The standard assay mixture contained 200 μL of buffer (10 mM Tris-HCl, 10 mM CaCl2, 100 mM NaCl, pH 8.0), 20 μL of 2.98 mM substrate (4-nitro-3-octanoyloxy-benzoic acid; 4N3OBA), 20 μL of water, and 20 μL of PLA2 (1 mg/mL) in a final volume of 260 μL. After the addition of PLA2, the mixture was incubated for 20 min at 37°C. The increase in absorbance at 425 nm, due to product formation, was monitored during this period and the activity was expressed as nmols of product formed nmols/min/mg. The inhibitory effect of BTL-2 (1 mg/mL) on PLA2 activity was investigated by incubating the two proteins at 37°C for 30 min prior to assaying the enzyme activity using the SpectraMax 340 multiwell plate reader (Molecular Devices).
HPLC molecular exclusion chromatography of BTL-2 andBTL-2-PLA2
One milligram of PLA2 from C. d. cascavella was dissolved in 0.5 mL of 0.05 M phosphate buffer, pH 7.5, and applied to a Protein-Pack TSK gel 3000 column (0.8 cm × 30 cm), previously equilibrated with the same buffer before use. BTL-2 and PLA2 (1:1) were preincubated together for 30 minutes in phosphate buffer. Separately, molecular mass markers (bovine serum albumin, 66 kDa; egg album, 45 kDa; carbonic anhydrase, 29 kDa, and lysozyme 14 kDa) were used to estimate the molecular weight of PLA2, BTL-2 and PLA2:BTL-2. All molecular weight markers were dissolved in phosphate buffer at a concentration of 2 mg/mL. The proteins were eluted at a flow rate of 0.2 mL/min and the absorbance profile was monitored at 214 nm.
Platelet aggregation studies
Sample collection and aggregation studies
Venous blood was collected with informed consent from healthy volunteers who denied taking any medication in the previous 14 days. Blood was collected by a two-syringe technique using polypropylene syringes and 19-gauge needles, and immediately transferred into polypropylene tubes containing 1/10 of final volume of 3.8% trisodium citrate.
After removing the platelet-rich plasma (PRP), the remaining blood was prepared by centrifugation of citrated blood at 200 × g for 10 min and washed platelets (WP) were obtained from the residue by centrifugation of citrated blood at 1500 × g for 20 min. The platelets were left for 1 h at room temperature to recover their sensitivity to aggregating agents. Platelet counts were performed on a Coulter S Plus (Coulter Electronics, Hialeah, FL) or by phase-contrast microscopy. Platelet aggregation was carried out using 400 μL of the washed platelets solution in a cuvette and kept at 37°C with constant stirring.
The desired concentration of protein was added 3 minutes prior to the addition of platelet aggregation inducers (thrombin for washed platelets). Subsequently, the aggregation was recorded for 5–10 min by using an aggregometer (Payton Scientific Instruments, Inc, Buffalo, NY). Aggregation experiments were performed in two concentrations (1 μg and 3 μg) of BTL-2, PLA2, and PLA2 incubated with BTL-2 (v/v) in a final volume of 20 μL, which was previously incubated (30 min) at room temperature.
Paw oedema assay
Male Wistar rats (120–150 g) were anaesthetised with halothane (inhaled). The rat paw oedema was induced by a single subplantar injection of 100 μL of BTL-2 (10 μg/paw), PLA2 (10 μg/paw), and PLA2 incubated with BTL-2 (v/v). Paw volume was measured immediately before the injection of the samples and at selected time intervals thereafter (15, 30, 60, and 120 minutes) using a hydroplethysmometer (model 7150, Ugo Basile, Italy). The compound 48/80 (C48/80) was incubated with BTL-D2 to show that the lectin was not acting on the tissues to decrease the action of the PLA2. All samples were dissolved in sterile saline solution (0.9%). Results were expressed as the increase in paw volume (mL) and calculated by subtracting the basal volume.
Xanthomonas axonopodis pv.passiflorae (Xap) and Clavibacter michiganensis subsp. michiganensis (Cmm) were harvested from fresh agar plates and suspended in distilled and sterilized water (A600 nm = absorbance, corresponding to 3 × 108 CFU/mL). Aliquots of this suspension were diluted to 3 × 103 CFU/mL and incubated with 100 μL of BTL-2 (1 mg/mL) for 30 min at 37°C. We then determined the survival on Agar Nutrient (Difco) Plate (n = 5).
Prediction of the three-dimensional structure of PLA2
Amino acid sequence similarity and alignment searches were carried out to compare the PLA2 isolated from Crotalus durissus cascavella venom against the Protein Data Bank using BLASTP. The 3D structure of PLA2 was determined by comparative homology modelling. Using computer graphics, the amino acid sequence of PLA2 was fitted to the high-resolution X-ray structure of the homologous 1BJJ_A. This allowed the construction of a model, which was then subjected to energy minimization and molecular dynamic simulations.
The results were reported as the means ± SEM of n experiments. The significance of differences between means was assessed by analysis of variance, followed by a Dunnett's test when several experimental groups were compared with the control group. The confidence limit for significance was 5% (p > 0.05).