Bp-13 PLA2: Purification and Neuromuscular Activity of a New Asp49 Toxin Isolated from Bothrops pauloensis Snake Venom

A new PLA2 (Bp-13) was purified from Bothrops pauloensis snake venom after a single chromatographic step of RP-HPLC on μ-Bondapak C-18. Amino acid analysis showed a high content of hydrophobic and basic amino acids and 14 half-cysteine residues. The N-terminal sequence showed a high degree of homology with basic Asp49 PLA2 myotoxins from other Bothrops venoms. Bp-13 showed allosteric enzymatic behavior and maximal activity at pH 8.1, 36°–45°C. Full Bp-13 PLA2 activity required Ca2+; its PLA2 activity was inhibited by Mg2+, Mn2+, Sr2+, and Cd2+ in the presence and absence of 1 mM Ca2+. In the mouse phrenic nerve-diaphragm (PND) preparation, the time for 50% paralysis was concentration-dependent (P < 0.05). Both the replacement of Ca2+ by Sr2+ and temperature lowering (24°C) inhibited the Bp-13 PLA2-induced twitch-tension blockade. Bp-13 PLA2 inhibited the contractile response to direct electrical stimulation in curarized mouse PND preparation corroborating its contracture effect. In biventer cervicis preparations, Bp-13 induced irreversible twitch-tension blockade and the KCl evoked contracture was partially, but significantly, inhibited (P > 0.05). The main effect of this new Asp49 PLA2 of Bothrops pauloensis venom is on muscle fiber sarcolemma, with avian preparation being less responsive than rodent preparation. The study enhances biochemical and pharmacological characterization of B. pauloensis venom.


Introduction
Phospholipase A 2 belongs to an expanding superfamily of enzymes that catalyzes ester bond hydrolysis at the sn-2 position of 1,2-diacyl-sn-3-phosphoglycerides and generates arachidonic acid. Depending on the molecular taxonomy, intracellular and secretory PLA 2 s are currently classified in six to twelve groups [1]. Secretory PLA 2 s are enzymes of 13-18 kDa with 5-8 disulfide bonds whose activity requires millimolar Ca 2+ concentration [2].
B. pauloensis is found in humid and cool regions in the central and Southwest of the state of São Paulo [15,16] and in seasonally dry savannas of the Brazilian Cerrado [17]. From the 292 notified accidents caused by Bothrops snakes, 18% (52 cases) were caused by B. pauloensis [18], thus evidencing that the study of the venom of this snake species can be of medical relevance. In this work, we describe the isolation and 2 Biochemistry Research International enzymatic characterization of a highly basic PLA 2 from the venom of B. pauloensis. We also investigated whether this isolated PLA 2 possesses neurotoxic activity. Solvents (HPLC grade), 4-nitro-3-octanoyloxy-benzoic acid, sequencing grade bovine pancreatic trypsin and other reagents were also obtained from Sigma Chemical Co. (St. Louis, MO, USA).

Reverse Phase HPLC (RP-HPLC).
Bp-13 PLA 2 from B. pauloensis venom was purified by reverse phase HPLC, according to the method described by Ponce-Soto et al. [19], with minor changes. Briefly, 5 mg of the whole venom was dissolved in 200 L of buffer A (0.1% TFA) and centrifuged at 4500 g; the supernatant was then applied to a -Bondapak C18 column (0.78 × 30 cm; Waters 991-PDA system), previously equilibrated in buffer A for 15 min. The protein elution was then conducted using a linear gradient (0-100%, v/v) of buffer B (66.5% acetonitrile in buffer A) at a constant flow rate of 1.0 mL/min. The chromatographic run was monitored at 280 nm of absorbance. The purity and PLA 2 activity were monitored according to Sections 2.3 and 2.6. All fractions eluted were lyophilized and then stored at −20 ∘ C for further biochemical and pharmacological assays.

Amino Acid Analysis.
Amino acid analysis was performed on a Pico-Tag Analyzer (Waters Systems), as described by Heinrikson and Meredith [21], with minor changes. Bp-13 PLA 2 sample (30 g) was hydrolyzed at 105 ∘ C for 24 hours, in 6 M HCl (Pierce sequencing grade) containing 1% phenol (w/v). Hydrolyzates were reacted with 20 L of derivatized solution (ethanol : triethylamine : water : phenylisothiocyanate, 7 : 1 : 1 : 1, v/v) for one hour at room temperature. Afterwards, PTC-amino acids were identified and quantified by HPLC, by comparing their retention times and peak areas with those from a standard amino acid mixture (Sigma-Aldrich).

Mass
Spectrometry. Molecular mass of intact native and alkylated Bp-13 PLA 2 was analyzed by MALDI-TOF mass spectrometry using a Voyager-DE PRO MALDI-TOF apparatus (Applied Biosystems, Foster City, CA, USA) equipped with a pulsed nitrogen laser (337 nm, pulse with 4 ns). The amount of 1 L of sample in 0.1% TFA was mixed with 2 L of sinapinic acid matrix (3, 5-dimethoxy-4-hydroxycinnamic acid). The matrix was prepared with 30% acetonitrile and 0.1% TFA and its mass analyzed under the following conditions: 25 kV accelerating voltage, the laser fixed at 2890 J/cm 2 , 300 ns delay, and linear analysis mode [22].
For de novo sequencing of N-terminal, the first 52 amino acids from Bp-13 PLA 2 , alkylated tryptic peptides were fractionated by RP-HPLC, manually collected, lyophilized, and resuspended in 80% H 2 O, 20% acetonitrile and 0.1% TFA. One peptide was introduced separately into the mass spectrometer source using a syringe pump at a 500 nl/min flow rate. Before performing a tandem mass spectrum, an ESI/MS mass spectrum (TOF MS mode) was acquired for each HPLC fraction over the mass range of 400-2000 m/z, aiming to select the ion of interest. Subsequently, these ions were fragmented in the collision cell (TOF MS/MS mode). Different collision energies were used depending on the mass and charge state of the ions. The resulting product-ion spectra were acquired with the TOF analyzer and deconvoluted using the MassLynx-MaxEnt 3 algorithm (Waters). Singly charged spectra were manually processed using the PepSeq application included in MassLynx.
2.6. PLA 2 Activity. PLA 2 activity was measured using the assay described by Cho and Kézdy [23] and Holzer and Mackessy [24] modified for 96-well plates. The standard assay mixture contained 200 L of buffer (10 mM Tris-HCl, 10 mM CaCl 2 , and 100 mM NaCl, pH 8.0), 20 L of synthetic chromogenic substrate 4-nitro-3-(octanoyloxy) benzoic acid 3 mM, 20 L of water, and 20 L of PLA 2 fractions (1 mg/mL) or whole venom (1 mg/mL) in a final volume of 260 L. After adding the samples, the mixture was incubated for up to 40 min at 37 ∘ C, absorbance reading at intervals of 10 min. The enzyme activity, expressed as the initial velocity of the reaction ( 0 ), was calculated based on the increase of absorbance after 20 min.
The pH and optimal temperature of PLA 2 were determined by incubating the four reaction buffers with different pH ranging from 4 to 10 and at different temperatures, respectively. The effect of substrate concentration (40, 20, 10, 5, 2.5, 1.0, 0.5, 0.3, 0.2, and 0.1 mM) on enzyme activity was determined by measuring the increase of absorbance after 20 min in optimum pH and temperature. The effect of different concentration of Ca 2+ on Bp-13 PLA 2 enzymatic activity was tested by preincubating the enzyme with different ion concentrations (0.005, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 M) at 37 ∘ C for 30 minutes prior to standard experiment. Also, the effects of different divalent ions (Sr 2+ , Mg 2+ , Mn 2+ , and Cd 2+ , 5 mM) were tested in presence (1 mM) or absence of Ca 2+ . Finally the effect of urea (4 M) on the enzymatic activity was tested by preincubating Bp-13 for 30 minutes at 37 ∘ C.
All assays were done in triplicate and the absorbance at 425 nm was measured using a VersaMax 190 multiwell plate reader (Molecular Devices, Sunnyvale, CA, USA). (PND) and extensor digitorum longus (EDL) muscles isolated from mice and biventer cervicis muscle (BC) isolated from chick were mounted according to Bülbring [25] and Ginsborg and Warriner [26], respectively.
Some curarized PND preparations (d-tubocurarine, 10 M) were incubated with Bp-13 toxin at 1.42 M and 3.56 M concentrations and the twitch response was recorded under direct muscle stimulation at supramaximal pulses of 70 V, 0.1 Hz at 2 ms duration. The effect of divalent ions, Mg 2+ , Mn 2+ , Sr 2+ , and Cd 2+ (10 mM), on the Bp-13 PLA 2 activity was done by replacement of Ca 2+ (1.8 mM) in the nutritive Tyrode solution; replacement of Ca 2+ by Sr 2+ (4 mM) was also assayed. The effect of temperature (5 to 60 ∘ C) during 20 min was read at 425 nm.
The biventer cervicis (BC) preparations were suspended in a 5 mL organ bath containing Krebs solution (composition in mM: NaCl 118.6, KCl 4.69, CaCl 2 1.88, KH 2 PO 4 1.17, MgSO 4 1.17, NaHCO 3 25.0, and glucose 11.65), aerated with carbogen (95% O 2 -5% CO 2 ) at 37 ∘ C. A bipolar platinum ring electrode was placed around the muscle tendon, within which run the motor nerve trunk. Field stimulation using a Grass S48 stimulator set at 0.1 Hz, 0.2 ms, and 4-6 V was applied and the muscle contractions and contractures were recorded isometrically via force-displacement transducer coupled to a physiograph. The muscle responsiveness to exogenously applied acetylcholine (ACh, 110 M) and KCl (13.4 mM) was recorded in the absence of field stimulation both prior to toxin addition and at the end of the experiment (120 min). The BC preparation was stabilized for at least 15 min before addition of Bp-13 at concentration of 3.56 (50 g/mL) and 7.12 M (100 g/mL). The results were compared with control BC preparations incubated with Krebs solution alone.

Statistical Analyses.
Results were reported as mean ± SEM. Differences among means was assessed by one-way ANOVA and followed by Mann-Whitney test for comparison between two groups. Differences were considered statistically significant if < 0.05.

Neuromuscular Activity.
Assays to study the neuromuscular activity of Bp-13 PLA 2 were performed using avian BC preparation and rodent's PND and EDL preparations. The toxin induced a time-and concentration-dependent and irreversible twitch-tension blockade. In the PND preparations, the time needed for 50% paralysis in response to 7.12 M ( = 3), 3.56 M ( = 6), and 1.42 M ( = 3) of Bp-13 PLA 2 was 18 ± 1 min, 28 ± 3 min, and 120 ± 4 min, respectively ( < 0.05); Bp13 at 0.71 M concentration induced a 25% paralysis only after 120 ± 2 min relative to control ( < 0.05) (Figure 6(a)). The catalytic activity of Bp-13 PLA 2 was similar both in the presence of 1 or 10 mM in the nutritive bath of PND preparation. The addition of Mg 2+ , Mn 2+ , Sr 2+ , and Cd 2+ (10 mM) in the nutritive Tyrode solution in the absence of Ca 2+ or presence of 1 mM Ca 2+ showed significant loss of the catalytic activity of the toxin indicating that these divalent ions cannot replace the Ca 2+ for the development of the PLA 2 catalytic activity. The replacement of 1.8 mM Ca 2+ by 4 mM Sr 2+ in the Tyrode solution prevented the blocking effect of Bp13 PLA 2 (3.56 M) since the twitch-tension response showed an amplitude of 83.7 ± 14% after 120 min incubation which was not different from baseline of the control preparations ( Figure 6(b)). The finding indicates that the neuromuscular blocking effect of the Bp-13 is calcium-dependent.

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activity occurred around 38 ∘ C ( Figure 6(c), insert). The neuromuscular blockade was prevented when the temperature incubation was set at 24 ∘ C; after 120 min, the twitch-tension response was 37.4% compared with the 90% seen at 37 ∘ C, Figure 6(c). The PND preparations previously treated with d-Tc (10 M) and under direct electrical stimulation showed that Bp-13 (1,42 and 3.56 M) was able to cause a significant contracture followed by blockade of the contractile response (73 ± 7% and 14 ± 6%, respectively, = 3-6, < 0.05, Figure 6(d)).
In the EDL preparations, the time needed for 50% paralysis at a 3.56 M Bp13 PLA 2 concentration was 120 min ± 2 min ( Figure 7). As displayed in the Figure 7, the contractile response of the EDL preparation was maintained steady during the 60 min period, regardless of whether the EDL was incubated in normal Tyrode solution or in Tyrode solution whose Ca 2+ (1.8 mM) was replaced by Sr 2+ (4 mM). The Bp-13 (3.56 M) addition caused a significant blockade of the twitch tension which achieved 90% after 80 min of toxin addition in the normal Tyrode solution. The replacement of 1.8 mM Ca 2+ by 4 mM Sr 2+ also prevented completely such neuromuscular blockade induced by the toxin (3.56 M) which was sustained until the end of observation (180 min, = 6, < 0.05). Similarly, the lowering of temperature to 24 ∘ C prevented the blockade of twitch tension in the EDL preparation (not shown).
In relation to avian preparations, it was shown that they exhibited lower sensibility to Bp-13 PLA 2 than the mammalian preparations. Bp-13 PLA 2 (3.56 and 7.12 M) induced an irreversible but mild decrease of the twitch tension of 21 ± 6% and 28 ± 2% after 120 min, respectively (Figure 8(a)). The response to acetylcholine (ACh, 110 M) was significant just when Bp-13 PLA 2 concentration was set at 7.12 M ( > 0.05). In contrast, Bp-13 PLA 2 regardless of the concentration, 3.56 or 7.12 M, did not interfere in the KCl (13.4 mM) induced contracture (Figure 8(b)).

Discussion
The presence of Bp-10 and Bp-11 (K49 PLA 2 homologous Bnsp 6 and Bnsp7) [8], Bp-14 and Bp-15 (Asp49 PLA 2 NeuTX-I and NeuTX-II) [12], and Bp-12 (Lys49 PLA 2 ) [11] has been already demonstrated in the B. pauloensis snake crude venom. Interestingly, another Asp49 PLA 2 , the Bp13, of the same Bothrops species venom was now isolated and characterized biochemically and pharmacologically. Such a diversity of PLA 2 isoforms in the venom of a same species evidences the necessity of developing efficient methodologies to purify and identify different isoforms in venom fractions otherwise considered homogeneous. The RP-HPLC was more suitable to purify Bp-13 PLA 2 than other conventional methods previously described for other toxins of the same venom, since it required just a single chromatographic step [19,22,27].
The purity of Bp-13 PLA 2 was confirmed by rechromatography on an analytical RP-HPLC -Bondapak C18 column. SDS-PAGE showed the monomeric nature of Bp-13 and a relative molecular mass of ∼14 kDa and it was confirmed by MALDI-TOF mass spectrometry with a molecular mass of 14035.628 Da. MALDI-TOF/MS has a precision in measuring protein molecular mass of 0.1% Da; thus, this characteristic allows us to demonstrate that Bp13 is another PLA 2 isoform present in the Bothrops pauloensis venom, as was for other bothropic PLA 2 isoforms [19,22,[27][28][29][30][31][32].
The amino acid composition of Bp-13 PLA 2 suggests that this PLA 2 is a basic protein because it possesses more basic residues (Arg, His, and Lys, total 19) than acid residues (Asx/Gnx, total 13), 14 half-Cys, and also because this protein showed high content of residues Tyr, Pro, Gly, and Lys which was a composition featured by other catalytic active bothropic myotoxins such as the 6-1 PLA 2 and 6-2 PLA 2 isoforms from B. jararacussu orthe BaTX, a basic PLA 2 from B. alternatus [22,27].
Bp-13 PLA 2 herein analyzed showed the presence of some important mutations in N-terminal sequence (up to the 52nd residue). Thus, Bp-13 PLA 2 shows K7 -> Q7, N13 -> T13, F20 -> Y20, R34 -> G34, and F46 -> Y46, which are strategic positions for expression of the catalytic activity. The presence of K7 in Bp-13 PLA 2 shows that this residue can contribute to keep hydrophobic cavity conformation of the N-terminal region. The N-terminal channel present in PLA 2 enzymes is highly conserved and provides access to the lipid substrate to     the PLA 2 catalytic site. Also, Bp-13 shows at position 13 the lack of Thr residue usually found in other PLA 2 s enzymes; however, both BnpTX-I (A13 -> T13) and NeuTx-I (A13 -> T13) also showed mutation in this position for polar residue, suggesting that this position is not as well conserved and that it could be a structural feature for the PLA 2 of B. pauloensis. The same was observed for the position 16, but in this particular case this position conserved its hydrophobic nature. Despite the change, Bp-13 still maintains catalytic activity and indicates that these residues have no important role in Bp-13 activity [33,36].
Breithaupt [37] reported that Crotalus PLA 2 shows classic Michaelis-Menten behavior. However, PLA 2 activity of Bp-13 from B. pauloensis shows a discrete allosteric-like behavior, and this activity is enhanced by the presence of even low Ca 2+ concentrations. The PLA 2 from C. durissus terrificus venom exhibits a typical PLA 2 activity, since it hydrolyzes synthetic substrates at position 2 and preferentially attacks substrates in their micellar state [24,37]. Similarly, Bp-13 PLA 2 exhibits allosteric behavior with a max of 11.6 nmol/min and a of 11.8 mM.
However, at low concentrations of the synthetic substrate, Bp-13 PLA 2 showed a sigmoidal kinetic behavior; this phenomenon was observed for other Crotalinae PLA 2 s [2,22,[38][39][40], suggesting an allosteric behavior for these enzymes. The allosteric term was originally used for enzymes with altered kinetic properties in the presence of ligands (effectors) that do not show any structural similarity to the substrate. Allosteric enzymes show a number of properties that distinguish them from the nonallosteric ones. Sigmoidal kinetics in the velocity substrate curve, the existence of effectors, and a polymeric structure are some of the properties of a genuine allosteric enzyme. In the case of Crotalinae D49-PLA 2 , SDS-PAGE without reducing agents showed a weak band at ∼28 kDa [2,40] indicating that some molecule populations of these enzymes exist in a dimeric form, which could be responsible for the observed enzymatic behavior. Bp-13 PLA 2 was resistant to heat and acid like PLA 2 s from Crotalus d. cascavella [38,41], C. d. collilineatus, and Lachesis muta muta [42] venoms; Bp-13 optimal activity was at pH 8.3 and was inactivated at pH higher than 9, like the PLA 2 from C. mitchelli pyrrhus [24], C. d. terrificus [43], and B. neuwiedi [44] snake venoms. The maximal enzymatic activity occurred at ∼38 ∘ C and persisted at 60 ∘ C, indicating a heatstable enzyme.
Pharmacologically, as for other types of PLA 2 , the activity of Bp-13 PLA 2 was shown to be completely Ca 2+ -dependent [45]. The coincubation of Bp-13 PLA 2 with other divalent ions (Mg 2+ , Mn 2+ , Sr 2+ , and Cd 2+ ) in the presence of 1 mM Ca 2+ or in Ca 2+ absence reduced or abolished the enzymatic activity.