A Novel Fast and Efficient Approach to Purify the Thrombin-like Enzyme from Two Bothrops -genus Snake Venoms

: Snake venoms are important sources of complex substances with a variety of pharmacological activities. Among them serine proteinases (SVSPs) have important effects on the hemostatic system influencing the hemodynamic of human or animal blood. Bothrops genus-snake venoms are rich in the thrombin-like enzyme, a type of SVSPs, with great interest to produce medicine. Therefore, the aim of this work was to describe a rapid, only two-step chromatographic-procedure developed to perform a faster purification of SVSPs from Bothrops alternatus and Bothrops moojeni venoms. As a result, two groups of serine proteinases respectively BaIII-4 - 8 and BmIII-2 - 5, were isolated and their molecular masses estimated by mass spectrometry and SDS-PAGE under denaturing conditions. The SVTLEs isolated from B. alternatus (BaIII-3 - 8) and B. moojeni (BmIII-2 - 5) fractions displayed apparent molecular mass around 30-40 kDa which closely relates to SVTLEs from other Bothrops species, as well their amino acid partial sequence triptych ions. Analysis of the alignment of the amino acid residue sequences of the N-terminal of the isolated proteins revealed a high level of identity with other SVTLEs. These enzymes coagulated plasma and showed fibrinogenolytic activity in blood. These SVTLEs isolated can be considered α-fibrinogenase mainly due to the fact that they hydrolyze the Aα chain fibrinogen. B. moojeni SVTLE showed greater activity than those from B. alternatus isolated. This new purification alternative approach developed was faster and more economical than the traditional process currently used. Faster purification and improved extraction yield can provide new insights into these enzymes including the use as a candidate molecule in the production of new drugs.


Introduction
Bothrops snake venoms contain a large variety of proteins and peptides affecting the hemostatic system; these proteins are classified as coagulant, anticoagulant or fibrinolytic factors [4,44,46].Among these, a group of serine proteinases enzymes convert fibrinogen into fibrin by cleaving fibrinopeptides A and/or B. As this activity resembles the activity of thrombin, these venom components are commonly named "thrombin-like" enzymes (SVTLEs) [41,43].These proteins are good tools to study molecular details of activation of specific factors involved in coagulation and fibrinolytic cascades.They are also useful in treating various thrombotic and hemostatic conditions after a medicine developed [9,28].In addition to affecting the hemostatic system these proteins affect platelet aggregation, complement system, blood pressure and nervous system [23,34].
The SVTLEs share many biochemical and structural properties such as a conserved catalytic triad (Ser195, His57, Asp102), and their three-dimensional structure is highly conserved.Their structure, taken as a whole, is made of two β-barrels constituted of six-strands and separated by the catalytic residues, and of a C-terminal helical segment [41].Moreover, they are glycoproteins with a single chain and molecular mass between 28 to 60 kDa and they are active on thrombin-specific synthetic substrates.SVTLEs have a high degree of homology among them, approximately 60 to 68%, but show less than 40% homology with human endogenous thrombin [38].
Bothrops snakes have their venom richly comnposed of SVTLE, several of which have already been purified and characterized: B. atrox [45], B. alternatus [11], B. pauloensis [10], B. moojeni [4], B. jararacussu [19], B. jararaca [42], B. brazili [46] and recently B. pictus [43].The isolation of SVTLEs is of great interest to the scientific community due to the possibility of their use in the treatment of thrombotic diseases and as anticoagulants, in addition to their relevance as a target for the bioprospecting of new products [25].
Within this context, in the early 1990s, the Center for the Study of Venoms and Venomous Animals (CEVAP) at São Paulo State University (UNESP), Brazil initiated an innovative research for the production of a new fibrin sealant from snake venom serine proteinases.The sealant was made from the replacement of bovine/human thrombin by thrombin-like gyroxin, purified from the venom of Crotalus durissus terrificus (Southamerican Rattlesnake) [6,8,13,18,20,33].Curiously, a systematic review about heterologous biopolymers with hemostatic, adhesive, sealant, scaffold, and drug delivery properties, the one produced by CEVAP/UNESP has been the only one found so far [1,7,12,14].In addition to gyroxin, other thrombin-like enzymes especially from Bothrops-genus could be employed in the composition of this unique biopolymer after being purifying and standardizing the biological activities.
The SVTLEs purification processes that have been reported so far involve three or more chromatographic steps and, in several cases, their performance and recovery are considered low [17,24,26].Consequently, to obtain a purified protein pool using several purification steps leads to a loss of approximately 30% of the material in each step performed.Therefore, it is important that an efficient method is deployed, standardized, and validated for the chromatographic purification of other thrombin-like enzymes such as those found in B. alternatus and B. moojeni snake venoms.
Aiming at a higher yield this work aimed to search a new fast and optimized process combining only two chromatographic techniques that allow the isolation and consequently the characterization of these thrombin-like enzymes.In the end, structural analysis and biological activities were compared with other SVTLEs that have already been deposited in databases.

Venom
The venoms were obtained from a venom-pool had been milked from Bothrops snakes (B.alternatus and B. moojeni) adults, of both sexes, individually microchipped, created and maintained in the CEVAP´s Serpentarium, located at CEVAP-UNESP, Botucatu, São Paulo Brazil, according to the methodology developed by this Translational Center (https://youtu.be/CPcs4ity-Uw).

Purification Method
Five hundred miligrans of whole venom were obtained from each pool of venoms of two Bothrops genus studied were applied to a Benzamidine-Seharose 6B affinity column in a ÄKTA Explorer 100 liquid chromatography system pH 7.4 (GE Healthcare, Diegem, Belgium), previously equilibrated with 0.05M Tris-HCl buffer, pH 7.4 (buffer 1).Elution was performed with 30 mL of 0.05M Tris-HCl pH 7.4 + 0.5M NaCl (buffer 2) and 20 mL of 0.02M glycine pH 3.2 (buffer 3) with fractions eluted in glycine were neutralized directly in the collection tube with 400 µL of 1.0 M Tris-HCl pH 9.0.The collection flow was 30 mL/hour, with 3.0 mL/tube collected at a temperature of 25°C.
The fractions isolated by affinity chromatography were diluted in 250µL of 0.1% (v/v) trifluoroacetic acid (TFA) and subjected to a High Performance Liquid Chromatography system (Shimadzu, Kyoto, Japan) on a reverse phase column (RP-HPLC) C18 (20 x 250 mm).The sample was initially eluted in 0.1% (v / v) trifluoroacetic acid (TCA), followed by a linear gradient of 70% acetonitrile in a flow of 1mL/min at room temperature.The peak of each serine proteinase in question was separated and lyophilized for further characterization.

In Vitro Coagulant Activity Assay
The process of purifying TLE serine proteinases was monitored by coagulant activity on blood.Blood samples were collected from healthy donors in the presence of 3.8% sodium citrate, in the proportion of 9:1 and centrifuged at 2500 xg at 4°C for 15 minutes to obtain plasma.Coagulant activity was performed using 200 µL of citrated human plasma incubated with 25 µg of each of the serine proteinases isolated from the snake venoms, diluted in milliQ water at a concentration of 1mg/mL.This mixture was incubated in a water bath at 37°C and the time of formation of the fibrin network was timed.Each sample assay was performed in triplicate.The maximum observation period for the formation of the fibrin network was five minutes, and after that it was considered as no coagulant activity (Theakston & Reid, 1983).

Mass Spectrometry
After running SDS-PAGE gels, the protein bands were excised and subjected to an in-gel trypsin digestion followed by ESI-QUAD-TOF Mass Spectrometry and Mascot (Matrix Science, CO, UK) search engine against the NCBI NR database restricted to the taxa Snakes.For the mass spectrometry analysis, it was used the quadrupole electrospray (ESI) mass spectrometry equipment, model micrOTOF-Q III (Bruker Daltonics, Bremen, Germany), coupled to an LC-20AT liquid chromatograph (Shimadzu, Kyoto, Japan).The liquid chromatography was coupled to the mass spectrometry equipment, composed of a binary pump system and an automatic sample applicator.The mobile phase consisted of water (A) and acetonitrile (B), containing 0.1% (v / v) formic acid or 0.1% (v / v) TFA.In addition, chromatographic separation was performed using a C18 reverse phase column (4.5 mm x 100 mm, 1.8 µm).The elution conditions were optimized in a linear gradient from 0 to 85% of solvent B for 60 minutes, in a flow of 0.2 mL / min.The column and the automatic sample applicator were maintained at 25°C and 10°C, respectively.The injection volume of the reference compounds and samples obtained by digestion in-gel was 2 µL.
MASCOT v.2.1 server (www.matrixscience.com)was used as a bioinformatic tool to assist identification of the isolated protein samples.The protein and fragmentation spectra obtained experimentally by mass spectrometry were compared with the set of peptide sequences allocated in the SwissProt database.To increase the precision of the identification processsof proteins, the following parameters were used: Enzyme: Trypsin as a cleavage agent; Taxonomy: Snakes; Carbamidomethylation (cysteine) as a fixed modification; Oxidation (methionine and tryptophan) as a variable modification; Number of cleavages lost by the enzyme: 1; Molecular weight of type: Monoisotopic; Molecular weight: without restrictions; Peptide tolerance error ± 0.5 Da and MS / MS tolerance error ± 0.8 Da for MS / MS analysis; Protonation: +1, +2, +3 for the state of the peptide charge; Instrument type: ESI-Q-ToF.

N-terminal Amino Acids Sequence
The analysis of the N-terminal sequence of each purified serine proteinase was performed in an automatic protein sequencer Shimadzu model PPSQ-23A (Shimadzu, Kyoto, Japan).A solution with approximately 1mg/mL of the enzyme was applied to the sequencer and the amino acids sequence was determined by the Edman degradation method [16].After determining the sequence of each serine proteinase, their amino acid homology was compared with other serine proteinases, by alignment using the BLAST protein program [2].N-terminal sequence alignments of purified serine proteinases with others thrombin-like serine proteinases were made using ClustalW in Edit Seq 5.01 © DNASTAR (Madison, WI., USA).

Fibrinogenolytic Activity
Fibrinogenolytic activity was determined according to the methodology described by Rodrigues et al [30].Samples containing 30µg of bovine fibrinogen were incubated with 10µg of the purified proteins at 37°C for 2h.The reaction was stopped by adding 10µL of sample buffer 0.05M Tris-HCl, pH 8.0 containing glycerol 10% (v / v), βmercaptoethanol 10% (v / v), SDS 2% (v / v) and 0.05% bromophenol blue (w / v), followed by boiling at 100ºC for 5 minutes.Then the samples were analyzed by 13% SDS-PAGE.

Purification of Thrombin Like Enzymes from B. Alternatus and B. moojeni Venoms
The elution profiles of B. alternatus and B. moojeni venoms subjected to an affinity chromatography (benzamidine-sepharose 6B column) showed 3 fractions each (BaI-III andBmI-III, respectively) (Figure 1A, B).A coagulant activity was observed in the fraction III of both venoms.After asecond chromatographic step was performed (reverse phase HPLC system, using an analytical column C18), the fractionation of BaIII resulted in 8 peaks (BaIII-1 to 8).Peaks BaIII-4 to BaIII-8 showed coagulant activity (Figure 1B).On the other hand, the BmIII fraction revealed 5 peaks (BmIII-1 to 5) and a coagulant activity was observed for peaks BmIII-2 to BmIII-5 (Figure 1D).These peaks were then selected for the analysis of mass spectrometry, determination of the N-terminal sequence and fibrinogenolytic activity.
The inserts in figures 1B-D show the presence of a single electrophoretic band of all proteins purified under denaturing conditions, with relative molecular mass around 35 kDa in relation to the molecular mass markers (lane 1).These results indicate a high degree of homogeneity of each fraction.Table 1 shows a summary of the steps involved in the purification process and protein recovery, with BaIII-6, BmII-2 and BmIII-4 having the best performance (36.4%, 31.2% and 51.5% respectively).

Identification of Peptides by Mass Spectrometry of Thrombin Like Enzymes from B. alternatus and B. moojeni Venoms
Proteins bands were excised from SDS-PAGE gels and ingel trypsin digestion was performed followed by ESI-QUAD-TOF Mass Spectrometry and Mascot search engine against the NCBI NR database restricted to the taxa Snakes.
Table 2 shows serine proteinases identified from the peptide fragments analyzed by mass spectrometry.
Crotalus adamanteus, Trimeresurus albolabris, Agkistrodon bilineatus, B. jararaca and B. atrox were the species of snakes to which the obtained serine proteinases were compared.The identified peptides have an ion score greater than 34, which indicates identity or a high degree of similarity.Analysis of N-terminal amino acid sequence of the serine proteinases obtained from B. alternatus and B. moojeni was carried out using the Edman degradation method.The results showed that between 22 to 25 amino acids from the Nterminal region were sequenced, as shown in table 3.

Alignment of the N-terminal Sequences of the Thrombin Like Enzymes Isolated from B. alternatus and B. moojeni Venoms
Figure 2 shows the alignment of the N-terminal sequences of the serine proteinases isolated from the B. alternatus venom, BaIII-4 -8 with other TLEs from snake venoms deposited in the NCBI Database (Figure 2A -E, respectively), as well as the sequence comparison of these 5 isolated serine proteinases (Figure 2F).The figure 2 also shows the conserved amino acid residues compared to each isolated protein.In the same way, the N-terminal sequence of serine proteinases isolated from B. moojeni venom were aligned with sequences of other toxins from the same snake family (Figure 3A -E

Discussion
Purification of snake venoms requires standard handling criteria and techniques.The purified sample must have a high qualitywhich means a certain degree of purity, in addition to maintaining its original biological activity.Therefore, methods of purification should provide the venom sample with the minimum of changes in its composition and, consequently, in its biological activities [5].
The isolation and purification of SVTLEs usually use conventional purification systems, which consist of three chromatographic techniques that combine gel filtration, affinity and ion exchange, such as DEAE-Sephacel, Sephadex G-75 and Benzamidine-Sepharose [11]; Benzamidine-Sepharose, Sephadex G-25 and reversed phase high performance chromatography [46]; Sephacryl S-100, Enzyme from Two Bothrops-genus Snake Venoms Benzamidine-Sepharose and cation-exchange column [17]; Sephacryl S-200, sepharose-agmatine and ion-exchange chromatography on DEAE-Sepharose CL-6B [24].In this work, SVTLEs from B. moojeni and B. alternatus were isolated using an optimized method that is faster than traditional protocols.This new procedure involved two chromatographic steps, combining affinity chromatography (Benzamidine-Seharose 6B) and reverse phase HPLC with high purity degree.Five SVTLEs (designated BaIII-4 -8) from B. alternatus and four SVTLEs (designated BmIII-2 -5) from B. moojeni were purified.All these SVTLEs seem to be homogenous on SDS-PAGE since a single protein band was detected for all these fractions.The relative molecular mass of the purified SVTLEs was 30-40 kDa.Most of SVTLEs isolated from snake venoms were single chain with molecular mass of 26 to 67 kDa, depending on their carbohydrate content [15,22,27].
One of the major consequences of using several steps during a protein purification is the loss of material due to freeze-drying intermediate steps.The purification of Leucuribin from B. leucurus, for instance, was described as 36.7%yield after three chromatographic steps [24]; Agkihpin from G. halys Pallas had a yield of 51% and 5.5 mg of protein were obtained from a gram of venom subjected to four stages of purification [39]; after three chromatographic steps the yield of VLCII from 500 mg of Vipera lebetina venom was about 4.35 mg [3].The protocol presented in this work allowed us to process half a gram of each venom in a few days with fairly reproducible results.Interestingly, a yield of 51.5% was reached for BmIII-4 and 16.5 mg of protein was obtained with specific activity increased 30-fold.
The single bands of SVTLEs were excised from SDS-PAGE gels and digested by trypsin digestion followed by ESI-QUAD-TOF Mass Spectrometry and the sequencing of the N-terminal amino acids was performed.The partial sequences of SVTLEs BaIII-5 shared high identity with serine proteinases from other Bothrops snake venoms such as Bhalternin, Asperase, BpirSP27 from B. alternatus, B. asper and B. pirajai, respectively [11,26,29].The N-terminal amino acids BaIII-4, BaIII-7 and BaIII-8 showed high identity with SVTLEs of B. jararaca (TL-BJ2 and SVSP HS114) [31,35] and non-bothropic venoms.In addition to Bhalternin other studies have reported thrombin-like enzymes purified from B. alternatus venom in three chromatographic steps.Balterobin and SPBA were reported with apparent molecular weight of 30 and 32 kDa, respectively, and presence of coagulant and fibrinogenolytic activities [17,36].
Results obtained in the analysis of triptych ions by mass spectrometry, indicated that the BmIII-4 and BmIII-5 proteins of B. moojeni correspond to SVTLE Batroxobin [21], with a Mascot / Ion relationship Score of 106/47 and 142/53 respectively.Therefore, these results indicate either identity or high structural similarity.Analysis of the alignment of the amino acid residue sequences of the N-terminal of the isolated proteins with other SVTLEs suggest a high identity of BmIII-4 with Leucurobin, Bothrombin and Batroxobin.Proteins BmIII-2 and 3 with coagulant activity showed high similarity with the enzyme PA-BJ isolated from B. jararaca venom which induces platelet aggregation (Serrano, Mentele, Sampaio & Fink, 1995 Finally, this new purification alternative approach developed using only two steps was faster and more economical than the traditional process.Faster purification and better-improved extraction yield can provide new insights into these enzymes including the use as a candidate molecule in the production of new drugs.

Conclusion
We have presented an optimized purification protocol to obtain SVTLEs from B. alternatus and B. moojeni venoms only in two steps with a high degree of purity.The enzymes were characterized using SDS-PAGE and in-gel protein digestion coupled to LC-MS/MS and Mascot protein identification, and their coagulant activity was assessed.All SVTLEs had molecular masses of 30-40 kDa and their partial sequences shared high identity with venom plasminogen activator, plateletaggregating enzyme, SVSPs and batroxobin from Bothrops venoms.The SVTLEs purification process developed in this study is faster and more economical compared to other traditional methods.The faster purification and high yield can provide new insights about these enzymes, including the use as a candidate molecule in the production of new drugs.formal analysis, writing-original draft preparation, review and funding acquisition.Edda Evnet Newball-Noriega and Salomon Huancahuire-Vega developed the formal analysis, investigation, original draft preparation and writing-editing.Benedito Barraviera and Rui Seabra Ferreira Junior developed the conceptualization, formal analysis, supervision, project administration and review & editing.All authors approved the submitted version.
Figure 2 shows the alignment of the N-terminal sequences of the serine proteinases isolated from the B. alternatus venom, BaIII-4 -8 with other TLEs from snake venoms deposited in the NCBI Database (Figure 2A -E, respectively), as well as the sequence comparison of these 5 isolated serine proteinases (Figure 2F).The figure 2 also shows the conserved amino acid residues compared to each isolated protein.In the same way, the N-terminal sequence of serine proteinases isolated from B. moojeni venom were aligned with sequences of other toxins from the same snake family (Figure 3A -E).BmIII-2 showed a great identity matching with PA-BJ of B. jararaca, BmIII-3 with the serine proteinase 3 of Protobothrops jerdonii, BmIII-4 with Leucurobin of B. leucurus and BmIII-5 with Cerastotin of C. cerastes.

Table 3 .
Sequencing of the amino acids N-terminal portion of TLEs purified from B. alternatus and B. moojeni venoms.
). BmIII-5 did not show structural similarity with any of the thrombin-like enzymes from botropic venoms deposited in the Mascot and BLASTp / NCBI databases.Other studies have reported the isolation and amino acid sequence of SVTLEs from the B. moojeni venom, BMII32, BMII35 and BmooSP with coagulant / fibrinogenolytic activity and molecular masses of 32, 35 and 36 kDa, respectively.Additionally, amino acid sequences of those SVTLEs showed high identity with batroxobin (de Oliveira et al., 2016; Fernandes de Oliveira et al., 2013).According to their specificity for cleaving fibrinogen chains, the serine proteinases have been classified as α, β and γ-fibrinogenases.SVSPs preferentially cleave the βb-chain (Menaldo et al., 2012), however, some of them such as Batroxobin from B. atrox, BjussuSP-I from B. jararacussu and Bhalternin from B. alternatus (Costa Jde et al., 2010; Sant' Ana et al., 2008; Stocker & Barlow, 1976) cleave the Aα-chain.The latter are considered α-fibrinogenases, classical enzymes studied in basic and clinical research.The fibrinogenolitic activity test performed with samples of thrombin-like serine proteinases obtained from B. alternatus and B. moojeni venoms in this work demonstrated that these enzymes can be considered α-fibrinogenases, mainly due to the fact they hydrolyze the Aα chain.Curiously, SVTLEs from B. moojeni showed greater fibrinogenolitic activity than those from B. alternatus.