Bothrops pirajai venom was purchased from Bio-Agents Serpentarium in the city of Batatais (São Paulo, Brazil).
Leaves of Harpalyce brasiliana Benth. were collected at the Chapada do Araripe, Barbalha (Ceará, Brazil) by Prof. Edilberto Rocha Silveira. Botanical authentication was made by Prof. Edson P. Nunes of the Department of Biology, Federal University of Ceará. Voucher specimen (number: 32 525) has been deposited at the Prisco Bezerra Herbarium (EAC), Department de Biology, Federal University of Ceará, Fortaleza (Ceará, Brazil).
The mass spectra were obtained on a Hewlett-Packard 5971 mass spectrometer by electron impact ionization (70 eV). 1 H and 13C NMR spectra were recorded on a Bruker Avance DRX-500 (500 MHz for 1 H and 125 MHz for 13C); chemical shifts were expressedin scale and were referenced to residual DMSO (2.5 and 39.5 ppm). Silica Gel 60 (Merck, 70–230 mesh) was used for analytical TLC. Column chromatographies were performed over silica gel (Merck, 60 F254 230–400 mesh).
Extraction and isolation of harpalycin 2
Leaves of Harpalyce brasiliana were pulverized and extracted with EtOH at room temperature. The solvent was removed under reduced pressure which produced a dark viscous extract (HBFE). Liquid-liquid partition of a water suspension of HBFE (110 g) using petrol ether, CHCl3, EtOAc and n-BuOH yielded five fractions after solvent evaporation or lyophilization: HBFEEp (24.5 g), HBFEC (22.4 g), HBFEA (6.8 g), HBFEB (30.4 g) and HBFEAq (21.2 g).
Flash chromatography of HBFEC (12.0 g) using n-hexane and EtOAc as binary mixtures of increasing polarity afforded 30 fractions, which were pooled in 9 fractions after thin layer chromatography (TLC) analysis. HBFEC (10–12) presented a yellow precipitate, yielding 120.0 mg of a white amorphous solid (m.p. 206.9-208.9°C). Spectrometric analysis showed the structure of the isoflavone harpalycin 2. The fractions HBFEC (8–9) and HBFEC (13–17) were purified, using the same method, yielding more 200.0 mg of harpalycin 2 (HP-2).
Purification of PrTX-III
Bothrops pirajai venom was first fractioned in two consecutive chromatographic steps as described by Toyama et al.
. Approximately 20 mg of the lyophilized venom was dissolved in 250 μL of 0.05 M ammonium bicarbonate, pH 7.8 (Buffer A). After homogenization, venom solution was clarified by centrifugation at 10,000 rpm for 3 min. The supernatant was inserted into a Protein Pack SP 5PW column (0.78 x 7.0 cm) and the elution performed using a linear gradient of concentration between 0.05 and 1.0 M ammonium bicarbonate with 750 μL of 0.1% (v/v) trifluoroacetic acid (Buffer B). The fractions were collected, lyophilized and clarified by centrifugation and the supernatant inserted into a μ-Bondapack C18 column (0.78 cm x 30 cm) (Waters 991-PDA system). Elution of peaks proceeded with a linear gradient between 0% and 66.5% (v/v) acetonitrile (solvent B) in 0.1% (v/v) trifluoroacetic acid, at a flow rate of 2.0 mL/min. Absorbances were monitored at 280 nm. Fractions were collected, lyophilized and stored at −20°C. The PrTX-III fraction was identified by the retention time and the measurement of the catalytic activity (since PrTX-III is the activity isoform of B. pirajai venom). The purity degree of PrTX-III was evaluated by Tricine SDS-PAGE and by mass spectrometry on a MALDI-TOF mass spectrometer, as previously described
Measurement of sPLA2 activity
sPLA2 activity was measured following the protocols described by Lee et al.
 and modified by Toyama et al.
 for 96-well plate, using 4-nitro-3-octanoyloxy-benzoic acid (4N3OBA, manufactured by BIOMOL, USA) as the substrate. Enzyme activity was calculated based on the increase in absorbance after 20 min. All assays were performed using n = 12 and absorbances at 425 nm were measured using a SpectraMax 340 multiwell plate reader (Molecular Devices, Sunnyvale, CA). After the addition of sPLA2 (20 μg), the reaction mixture was incubated for 40 min at 37°C, and the absorbance read at 10 min intervals. For the estimation of the IC50 of harpalycin 2 for PrTX-III, different concentrations of HP-2 (5, 10, 20, 40 and 80 μg) were added to each well. The remaining enzymatic assay was conducted as described above. Harpalycin 2 was previously dissolved in DMSO 1%.
Incubation of sPLA2 with harpalycin 2 and purification of HP-2 treated sPLA2 (PrTX-III: HP-2) and amino acid analysis
The incubation of sPLA2 with HP-2 (w: w; 4:1), followed the procedures described by Iglesias et al.
. Briefly, HP-2 was dissolved in DMSO 1%. 250 μL of HP-2 solution was added to 1,000 μL of homogenized solution of PrTX-III. The mixed solution was incubated for 60 min in a water bath at 37°C. Samples of 200 μL of this mixture were loaded into a preparative reverse phase HPLC column to separate the treated enzyme (PrTX-III: HP-2) from HP-2. After column equilibration with buffer A (aqueous solution of 0.1% TFA), samples were eluted using a discontinuous gradient of buffer B (66.6% of acetonitrile in 0.1% TFA) at a constant flow rate of 2.0 mL/min. The chromatographic run was monitored at 214 nm for detection of PrTX-III, PrTX-III: HP-2 and HP-2. 1 nmol of purified protein (PrTX-III or PrTX-III: HP-2) was hydrolyzed with 6 N HCl (200 μL) in the presence of 10μL of phenol solution for prevention of unspecific amino acid oxidation. Amino acid hydrolysis was performed at 106°C for 24 h. After this time, the excess HCl was removed and the hydrolyzed amino acids were rehydrated with a solution of ethanol: water: triethylamine (v: v; 2:2:1). Post-column derivatization was performed with an aqueous solution of phenylisothiocyanate (ethanol: water: triethylamine: phenylisothiocyanate; v: v; 7:1:1:1). Samples and amino acid standards were derivatized using a PICO-TAG amino acid analyzer system (Waters, USA).
Molecular exclusion chromatography
A molecular exclusion chromatography was initially performed using an AP-1 column (Waters, 1x60 cm) previous packed with Superdex 75 (GE Healthcare Pharmacia) as previously described by Oliveira et al.
. For molecular exclusion chromatography of the PrTX-III and PrTX-III: HP-2 as well as the protein marker, 1 mg of the protein sample was dissolved in the same buffer used for equilibration of the chromatographic column and sample elution (Potassium phosphate buffer 0.05 M, pH 7.5). Samples were dissolved in 250 μL of this buffer and then centrifuged at 4,500 g for 5 minutes. 200 μL of supernatant was recovered and taken at 37°C for 60 minutes before injection into the column. Injections of 25 μL of each sample were carried out through the column; and elution of fractions was performed under isocratic condition with constant flow rate of 0.2 mL/min and monitored at 280 nm.
The molecular mass of PrTX-III and PrTX-III: HP-2 were determined by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry using a Voyager-DE PRO MALDI-TOF mass spectrometer (Applied Biosystems®, Life TechnologiesTM, USA). One microliter of samples (PrTX-III and PrTX-III: HP-2) in 0.1% TFA was mixed with 2 μL of the matrix a-cyano-4-hydroxycinnamic acid, 50% acetonitrile, and 0.1% TFA (v/v). The matrix was prepared with 30% acetonitrile and 0.1% TFA (v/v). The equipment conditions were as follows: accelerating voltage of 25 kV, laser fixed at 2,890 μJ/com2, delay of 300 ns and linear analysis mode.
Circular dichroism spectroscopy
Purified enzymes - native and HP-2 treated PrTX-III – were dissolved in a 10 mM sodium phosphate buffer (pH 7.4) and the final protein concentrations were adjusted to 8.7 mM. After centrifugation at 4,000 g for 5 min, samples were transferred to a 1 mm pathlength quartz cuvette. Circular dichroism spectra in the wavelength range of 185–300 nm were acquired in-house with a J720 spectropolarimeter (Jasco©, Japan) using a bandwidth of 1 nm and a response time of 1 s. Data collection was performed at room temperature with a scanning speed of 100 nm/min. Nine scans were taken for each sample and all spectra were corrected by subtraction of buffer blanks.
The relative intrinsic fluorescence intensity of native PrTX-III or HP-2 treated PrTX-III (PrTX-III: HP-2) was monitored with a spectrofluorimeter (Shimadzu©, Japan). 2.0 mL of the reaction mixtures, consisting of 100 mM Tris–HCl buffer (pH 7.4), sPLA2 (200 μg/mL) and 5 mM CaCl2, were put into a 10 mm pathlength quartz cuvette. Fluorescence was measured at between 300 and 450 nm after excitation at 280 nm.
Platelet aggregation studies
The platelet aggregation activities were conducted as described by Oliveira et al.
 and dos Santos et al.
. Venous blood was collected with informed consent from healthy volunteers who formally denied taking any medication in the previous 14 days. All experiments using human material were carried out according to Helsinki Declaration and were approved by the Ethical Committee for Human Research of State University of Campinas under the no. 0318.104.22.168.000-09. 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 at 200 g for 10 min and the washed platelet solution (WP) was obtained from the residue by centrifugation of citrated blood at 1,500 g for 20 min. The platelets were left for 1 hour 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 and 3 minutes after to the addition, the aggregation was recorded for 5–10 min by using an aggregometer (Payton Scientific Inc., USA). Aggregation experiments were performed with 10 μg of PrTX-III and PrTX-III: HP-2., PrTX-III: aristolochic acid or PrTX-III: p-bromophenacyl bromide. In order to elucidate HP-2 mode of action, 10 μL of HP-2 (5 mg/mL), AACOCF3 (1 mM) and INDO (1 mM) were added 5 minutes before the addition of native PrTX-III (10 μg) or arachidonic acid (AA, 50 mM).
The structural optimizations of the harpalycin 2 (HP-2), aristolochic acid (Aris Ac) and p-bromophenacyl bromide (p-BPB) ligands were initially achieved using the AM1 method
 implemented in the BioMedCache program
 with default values for the convergence criteria. Docking calculations were performed with the GOLD 4.0 program
 to obtain the in silico affinity of the ligands with respect to the PrTX-III target. The tridimensional coordinates of the target were taken from the RCSB Protein Data Bank (PDB), under the PDB code 1GMZ, as a dimeric quaternary structure. The “A” chain was chosen for all the calculations. The docking calculations were performed to consider the flexibility of all the ligands and the flexibility of the target, in order to represent the induced fit generated by the presence of non-native ligands, using the following approach: the residues Phe5, Ile9, Phe18, Tyr21, Val22, Tyr27, His47, Asp48, Lys60 and Phe96 were configured in such a way that their side-chain torsions were considered active during the calculations. The active site was defined as all the atoms within a radius of 8.0 Å from the co-crystallized isopropyl alcohol (IPA).
Results were expressed as mean ± S.E.M. and analyzed by ANOVA followed by Dunnett’s test using GraphPad Prism® 5.0 with significance set at p < 0.05*.