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Toxicon 67 (2013) 1–11

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Toxicon

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Purification, characterization and gene cloning of Da-36, anovel serine protease from Deinagkistrodon acutus venom

Ying Zheng a,b,1, Feng-Ping Ye b,c,1, Jie Wang b, Guo-Yang Liao a, Yun Zhang c,Quan-Shui Fan b,**, Wen-Hui Lee c,*

a Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, Chinab Institute of Military Medical, Chengdu Military Region’s Center for Disease Control & Prevention, Kunming 650032, ChinacKey Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province,Kunming Institute of Zoology, The Chinese Academy of Sciences, Kunming, Yunnan 650223, China

a r t i c l e i n f o

Article history:Received 24 September 2012Received in revised form 9 January 2013Accepted 16 January 2013Available online 24 February 2013

Keywords:Deinagkistrodon acutusGeneSerine proteasePurification

* Corresponding author. Tel.: þ86 871 5192476; fa** Corresponding author.

E-mail addresses: fqs168@126.com (Q.-S. Fan),(W.-H. Lee).

1 These authors contributed equally to this work.

0041-0101/$ – see front matter � 2013 Elsevier Ltdhttp://dx.doi.org/10.1016/j.toxicon.2013.01.021

a b s t r a c t

A serine protease termed Da-36 was isolated from crude venom of Deinagkistrodon acutus.The enzyme was a single chain protein with an apparent molecular weight of 36,000 onSDS–PAGE with an isoelectric point of 6.59. Da-36 could clot human plasma by cleaving theAa, Bb and g chains of fibrinogen and also exhibited arginine esterase activity. The pro-teolytic activity of Da-36 toward TAME was strongly inhibited by PMSF and moderatelyaffected by benzamidine and aprotinin, indicating that it was a serine protease. Mean-while, Da-36 showed stability with wide temperature (20–50 �C) and pH value ranges (pH6–10). Divalent metal ions of Ca2þ, Mg2þ, and Mn2þ had no effects but Zn2þ and Cu2þ

inhibited the arginine esterase activity of Da-36. Total DNA was extracted directly from thelyophilized crude venom and the gene (5.5 kbp) coding for Da-36 had been successfullycloned. Sequence analysis revealed that the Da-36 gene contained five exons and fourintrons. The mature Da-36 was encoded by four separate exons. The deduced matureamino acid sequence of Da-36 was in good agreement with the determined N-terminalsequence of the purified protein and shared high homology with other serine proteasesisolated from different snake venoms. Blast search using amino acid sequence of Da-36against public database revealed that Da-36 showed a maximal identity of 90% withboth Dav-X (Swiss-Prot: Q9I8W9.1) and thrombin-like protein 1 (GenBank: AAW56608.1)from the same snake species, indicating that Da-36 is a novel serine protease.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Snake venom contains a mixture of proteins and poly-peptides which exhibit various biochemical and pharma-cological functions. These venom components functiontogether to immobilize, kill and initiate digestion of theprey (Aird, 2002; Dufton, 1993). Based on their structural

x: þ86 871 5191823.

leewh@mail.kiz.ac.cn

. All rights reserved.

similarities, snake venom proteolytic enzymes are gener-ally classified into two major groups: serine proteases andmetalloproteases. Both groups affect the blood coagulationprocess in various ways (Braud et al., 2000; White, 2005).

Snake venom serine proteases (SVSPs) are a majorcomponent of venoms and have been identified in thefamilies of Viperidae, Crotalidae, Elapidae and Colubridae(Serrano and Maroun, 2005). Although SVSPs have similarprimary sequences (50–70% identity) and adopt similarthree-dimensional structures, these venom proteasesexhibit different substrate specificity and showed variousphysiological functions (Serrano and Maroun, 2005;Vaiyapuri et al., 2011). SVSPs generally effect hemostatic

Y. Zheng et al. / Toxicon 67 (2013) 1–112

system by acting on specific targets of the coagulationcascade, such as induction of platelet aggregation, fibrin(-ogen)olysis, factor V activation, plasminogen activation orprotein C activation (Markland, 1998; Kini et al., 2002; Kini,2005).

Thrombocytin from Bothrops atrox (Niewiarowski et al.,1977) causes platelet aggregation, release of platelet se-rotonin, and activation of factor XIII (Kirby et al., 1979).Both Batroxobin [Defibrase� (Pentapharm, Switzerland)]from B. atrox moojeni venom (Stocker and Barlow, 1976)and Ancrod (American Knoll Pharmaceutical Company)from Calloselasma rhodostoma venom (Nolan et al., 1976)induce the clotting of fibrinogen through fibrinopeptiderelease. Batroxobin and Ancrod can cause blood clottingin vitro, while acting in vivo, they apparently deplete thecirculating fibrinogen and render the blood uncoagulation.Batroxobin and Ancrod have been used as defibrinoge-nating agents for the treatment and prevention ofthromboembolic diseases in clinic (Pirkle, 1998; Matsuiet al., 2000). RVV-V from Vipera russelli (Kisiel, 1979) ac-tivates factor V by cleavage of a single peptide bond andgenerates active form of factor Va (Tokunaga et al., 1988).VLFVA from Vipera lebetina (Siigur et al., 1998) and con-tortrixobin from Agkistrodon contortrix contortrix (Amiconiet al., 2000) are also reported to activate factor V. TSV-PAfrom Trimeresurus stejnegeri activates plasminogen togenerate plasmin by cleavage plasminogen at the peptidebond Arg561–Val562, which does not activate nor degradeprothrombin, factor X, or protein C and does not clotfibrinogen nor show fibrino(geno)lytic activity in theabsence of plasminogen (Zhang et al., 1995). ACC-C from A.contortrix contortrix (Stocker et al., 1986; Kisiel et al., 1987),commercially available under the trade name Protac, hy-drolyzes the Arg169–Leu170 bond of the heavy chain ofprotein C, leading to the formation of activated protein C(Kisiel et al., 1987). As the activation of protein C can easilybe determined by means of coagulation or chromogenicsubstrate techniques, ACC-C has found a broad applicationin diagnostic practice for the determination of disorders inthe protein C pathway. For example, in assays of functionalprotein C determination, total protein S content and otherprotein S functional assays in plasma (Gempeler-Messinaet al., 2001).

Deinagkistrodon acutus is a specific snake and widelydistribute in Southern China. Previous work suggested thatserine proteases distributed divergently in the venom of D.acutus. A serine protease with determined mass of 34 kDawas purified from D. acutus venom and the crude venomwas originated from Anhui Province of China (Xin et al.,2007). The isolated enzyme was designated as acutobin IIby the same research group and revealed that acutobin IIcomposed of a 28 kDa peptide chain plus 6 kDa of O-linkedglycan chains (Xin et al., 2009). As many as 7 completecDNAs coding for different thrombin-like enzymes werecloned from a venom gland of this species. The living snakeused for the experiment was fromHunan Province of China(Zha et al., 2006). However, only 2 venom serine proteaseswere identified by transcriptome analysis of 8696expressed sequence tags of a constructed venom glandcDNA library. The snake used in the experiments was fromFujian Province of China (Zhang et al., 2006).

In the present work, a new serine protease was purifiedand characterized from the snake venom of D. acutus. Inaddition, the gene coding for this enzyme was also clonedfrom the total DNA which was extracted directly from thelyophilized dry venom.

2. Materials and methods

2.1. Materials

Lyophilized crude venom ofD. acutuswas obtained froma serpentarium from Jiangxi Province, China. DEAE-Sepharose Fast Flow, Superdex-75 Prep grade, DEAE-Sephadex A-50, Sephacryl S-100 and Sephadex G-25 werepurchased from GE Healthcare Life Science Inc. Humanthrombin and human fibrinogen were purchased fromTianjin Biochemical Technology Co. (Tianjin, China). Phe-nylmethyl sulfonylfluoride (PMSF), aprotinin, benzamidine,p-toluenesulfonyl arginine methyl ester (TAME) and a-N-benzoyl-L-arginine amide ethyl ester (BAEE) were pur-chased from the Sigma–Aldrich Co. (USA). Ex Taq poly-merase, pMD19-T vector, and DH5a competent cells werefrom TaKaRa Biotechnology Co. Ltd. (Dalian, China). Allother reagents used for chemical and biological character-ization were of analytical grade.

2.2. Isolation of the thrombin-like enzyme

Crude venom (20 g) was dissolved in 300 mL of 50 mMTris–HCl (pH 8.0), and centrifuged at 3500 g for 30 min. Theclear supernatant was filtered through a 0.22 mM filter andloaded onto a DEAE-Sepharose Fast Flow column(5� 60 cm) equilibratedwith 50mMTris–HCl (pH 8.0). Thecolumnwas washed with a linear gradient of 1 M NaCl at aflow rate of 10 mL/min. Peak III showing clotting activitywas collected and lyophilized then desalted by a SephadexG-25 column with 50 mM Tris–HCl, 10 mM KCl (pH 7.5)buffer. Lyophilized fraction was dissolved in water andloaded onto a Superdex-75 PG column (5 � 100 cm) equil-ibrated with 50 mM Tris–HCl, 100 mM KCl (pH 7.5) andeluted with the same buffer. Peak IV of Superdex-75showing clotting activity was pooled and lyophilized thendesalted by a Sephadex G-25 columnwith 10 mM Tris–HCl(pH 7.5) buffer, collected protein peak was lyophilized andredissolved inwater and then loadedonto aDEAE-SephadexA-50 column (5 � 100 cm) pre-equilibrated with 50 mMTris–HCl (pH 7.5) buffer. The column was eluted with alinear NaCl gradient (0–0.5 M) in equilibration buffer andpeak II was pooled and lyophilized. After dissolved in 10mLwater, the fractionwasfinally loaded onto a Sephacryl S-100column (5�100 cm) equilibratedwith 50mMTris–HCl (pH7.5) buffer. The columnwas elutedwith the same buffer andpeak I having clotting activity was pooled and lyophilized.The purified enzyme was named as Da-36. Protein con-centration was determined according to the method ofLowry et al. (1951) using BSA as standard.

2.3. Clotting assay

The clotting activity was assayed at 37 �C by mixing200 mL of preheated citrated human plasma with 20 mL of

Y. Zheng et al. / Toxicon 67 (2013) 1–11 3

the protease. Then the time from the enzyme addition tothe clot formation was recorded. One unit of clotting ac-tivity was defined as the amount of enzyme that could clot200 mL of preheated citrated human plasma within60 � 20 s. Accordingly, specific clotting activity wasexpressed as units/mg protein. The resistance of the clotwas tested by adding 1 mL of 5 M urea and stirring for5 min.

2.4. Arginine-esterase activity

The method previously described by Hahn et al. (1996)was used with some modifications. Briefly, 0.25 mg of Da-36 were incubated with 1 mL of 1 mM TAME or BAEE in10 mM Tris–HCl, pH 7.5. The activity was carried out byspectrophotometric analysis at 247 nm during the first10min of reaction. One unit of esterase activity was definedas the increase of 0.01 units in the absorbance, resulting inhydrolytic activity of the enzyme upon substrates. Thespecific activity was expressed by the units of TAME orBAEE/mg of protein. The stability study of Da-36 was per-formed by preincubating the enzyme for 30 min indifferent conditions as follows: pH (2.0–11.0), temperature(10–90 �C), divalent ions (10 mM Ca2þ, Mg2þ, Cu2þ, Zn2þ

andMn2þ) and inhibitors (5 mM PMSF, b-mercaptoethanol,EDTA, 2.0 mM aprotinin and benzamidine). Then thearginine-esterase activities of the enzyme on TAME wereassayed as mentioned above.

2.5. Fibrinogenolytic activity

Fibrinogenolytic activity was examined according to themethods reported by Edgar and Prentice (1973) with somemodifications. Samples containing 10 mL of human fibrin-ogen (2 mg/mL in 10 mM Tris buffer, pH 7.4) were incu-bated with 10 mL of different concentrations of enzyme(0.005 mg/mL to 0.1 mg/mL) at 37 �C for 1 h. The reaction wasterminated by the addition of 5 mL of 250 mM Tris–HClbuffer, in the pH value of 6.8, containing 50% (v/v) glycerol,5% (v/v) b-mercaptoethanol, 10% (m/v) SDS, and 0.05% (m/v) bromophenol blue and boiled at 100 �C for 5 min. Thesamples were analyzed by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The sameprocedure was performed incubating 0.3 mg of Da-36 with15 mg of human fibrinogen at different time intervals (1–27 h) at 37 �C.

2.6. N-terminal amino acid sequence determination

Da-36 was run on a 12% SDS–PAGE gel and the proteinband of Da-36 was transferred into a PVDF membraneusing a Trans-blot SD (Bio-Rad, USA) in accordancewith themanufacturer’s instructions. The N-terminal sequence ofDa-36 was determined by automated Edman degradationusing an Applied Biosystems 494 Procise ProteinSequencing System by PVDF protocol.

2.7. Determination of molecular weight and isoelectric point

The apparent molecular weight of Da-36 was deter-mined by conventional 12.5% SDS–PAGE under reducing

and non-reducing conditions. The gel was stained withCoomassie brilliant blue R-250. The standard proteins usedwere rabbit phosphorylase b (97,400), bovine serum albu-min (66,200), rabbit actin (43,000), bovine carbonic anhy-drase (31,000), trypin inhibitor (20,100) and hen egg whitelysozyme (14,400).

Isoelectric point (pI) of Da-36 was determined bycapillary electrophoresis on a Beckman P/ACE 5500 in-strument. The absorbance of the focused proteins wasdetected at 280 nm. Samples were applied by filling thewhole capillary with a mixture of ampholyte, Da-36 and pImarkers. The pI markers used were 5.10, 5.90 and 9.45,respectively. The catholyte consisted of 20 mmol/L NaOHand the anolyte of 20 mmol/L H3PO4. The capillary wasthermostated at 25 �C and focused at 25kV.

2.8. DNA cloning of Da-36

Total DNA was extracted directly from lyophilizedvenom. Approximately 100 mg of venom sample wasresolved in 1 mL distilled water and placed at room tem-perature for a fewminutes until the venomwas completelydissolved. It was then centrifuged at 12 000 g for 10 minand the supernatant was discarded. The pellet was resus-pended in 1 mL distilled water and centrifuged at 12 000 gfor 2 min. DNA was extracted from the pellet using theTIANamp genomic DNA kit (Tiangen Biotech. Co. Ltd., Bei-jing, China) according to the manufacturer providedprotocols.

The extracted total DNA served as the template for theamplification ofDa-36 gene. In order to clone the 30-terminalsequence of Da-36, primers CD-f (50-GGAGGTAATGAATGT-GACAC-30) and CD-r (50-TGGTTAGGGATATAGAAGAG-30)were used. The primer CD-f was a sense primer that wasdesigned based on the N-terminal amino acid sequence‘GGNECDT’ of Da-36, and the primer CD-r was an antisenseprimer designed based on the alignment of the conserved 30

Non-coding region of other serine proteases from differentspecies of snakes. In the case of molecular cloning of the 50-terminal sequence of Da-36, primers ND-f (50-GCA-GAGTTGAAGCTATGGT-30) and ND-r (50-ACTGAAAAGTC-CAAGGCCG-30) were used. The sense primer ND-f wasdesigned based on the alignment of the conserved 50 Non-coding region of serine proteases in different snake venom,and the gene-specific antisense primer ND-r was designedbased on the determined nucleotide sequence of CD-f andCD-rproduct. PCRswereperformedusingExTaqpolymerase(Takara, Dalian, China) under the following conditions: pre-incubation at 94 �C for 3 min; 35 cycles consisting of dena-turation at 94 �C for 0.5 min, annealing at 60 �C for 0.5 minandextension at 72 �C for 4minor 2min; andfinal extensionat 72 �C for 10 min.

Amplified products were subjected to 1% agarose gelelectrophoresis, visualized by ethidium bromide stainingand purified using a gel extraction kit. Purified PCR prod-ucts were ligated into the pMD19-T vector (Takara, Dalian,China). Ligated vectors were transformed to DH5acompetent cells by heat shock. Ampicillin (100 mg/mL) wasused for antibiotic resistance selection. Positive colonieswere selected and further proved by colony PCR. DNA

Y. Zheng et al. / Toxicon 67 (2013) 1–114

sequencing was done by a commercial company (SangonBiotech Co., Ltd., Shanghai, China).

Analyses of the cloned DNA sequence and the deducedamino acid sequence of Da-36 were carried out usingDNAStar and blast search programs. The multiple align-ments of amino acid sequences were performed using theClustal Wmultiple alignment program. Potential sites of N-glycosylation were predicted in Da-36 by the programNetNGlyc v.1.0, available at the Center for BiologicalSequence Analysis server (http://www.cbs.dtu.dk/).

2.9. Statistical analysis

The results regarding biological activity were presentedas means and standard deviation. Statistical significance ofresults was evaluated using Student’s t-test. The value ofp < 0.05 was considered significant.

3. Results

3.1. Purification and biochemical characterization

Da-36 was purified from D. acutus venom by DEAE-Sepharose ion-exchange chromatography, Superdex-75PG gel filtration, DEAE-Sephadex A-50 ion-exchange chro-matography and Sephacryl S-100 gel filtration. Fraction-ation by DEAE-Sepharose produced nine major proteinpeaks (Fig. 1A). Peak III showed the highest clotting activityupon human plasma and was further isolated on Superdex-75 PG (Fig. 1B). Peak IV of Superdex-75 showed the clottingactivity and was submitted to DEAE-Sephadex A-50 isola-tion step, resulting into two fractions (Fig. 1C). Fraction IIwas finally loaded onto a Sephacryl S-100 column, threeisolation peaks were obtained (Fig. 1D). Fraction I wascharacterized to be a new serine protease and designated asDa-36. Da-36 was a minor component of the crude venom,85 mg final product was produced from 20 g crude venomwith a yield of 0.43%. Meanwhile, high purification factorwas reached using present reported purification pro-cedures (Table 1).

Da-36 was a single chain protein showing molecularweight of approximately 36 kDa in SDS-PAGE under bothreducing and non-reducing conditions (Fig. 1D insert). ThepI value of the protein was determined to be 6.59 bycapillary electrophoresis (Fig. 1E). The N-terminal 25 aminoacid residues of Da-36 were determined to beVIGGNECDTNEHRFLAAFFTSRPWT.

3.2. Functional properties

Da-36 showed higher clotting activity on human plasmawith a clotting activity of 2416 U/mg. The clot formed bythis reaction was resolved when urea was added into theclot.

The purified enzyme showed esterase activities on bothTAME and BAEE. The esterase activities were determined tobe 158,435 U/mg and 332,518 U/mg toward TAME andBAEE, respectively. Da-36 showed thermal and pH stabilityafter pre-incubation the enzyme in different conditions andthen assay its esterase activities on TAME. At the temper-atures ranging from 20 to 50 �C (Fig. 2A) and in different pH

between 6.0 and 10.0 (Fig. 2B), the enzymatic activity of Da-36 toward TAME had no obvious changes. Esterase activityof Da-36 was inhibited by Zn2þ and Cu2þ (Fig. 2C) and alsoby PMSF, b-mercaptoethanol, aprotinin and benzamidine(Fig. 2D). PMSF and Zn2þ inhibited the esterase activity ofthe enzyme by 94% and 37%, respectively. However, theenzymatic activity was not affected by EDTA.

The proteolytic activity of Da-36 on human fibrinogenwas investigated. Da-36 could cleave human fibrinogen ina dose and time dependent manner (Fig. 3). The Aa-, theBb- and the g-chain of human fibrinogen were highlydegraded within 1 h when 0.6 mg enzyme was added into20 mg fibrinogen (Fig. 3A). Moreover, the Aa-, the Bb- andthe g-chain of human fibrinogen could be fully digestedwhen the incubation time was prolonged to 27 h at thecondition of 0.3 mg enzyme added into 15 mg fibrinogen(Fig. 3B). Thus, Da-36 is the first reported land snakeserine protease that could cleave Aa, Bb and g chains ofhuman fibrinogen.

3.3. DNA cloning of Da-36

The gene encoding for Da-36 (Fig. 4A) was successfullycloned from total DNA, which was extracted from 100 mglyophilized venom sample. Molecular cloning strategy ofpresent work is summarized in Fig. 4B. Amplification of thetotal DNA by primers CD-f and CD-r resulted in a fragmentof 3854 bp. The fragment was cloned and sequenced. Thesequence covered the mature protein coding region exceptfor the first 2 amino acids of the N-terminal sequence. Toobtain the 50-terminal nucleotide sequence of Da-36, theforward primer ND-f, corresponding to 50-terminal non-coding region of snake venom serine proteases, and thereverse primer ND-r, designed according to the sequence ofthe CD-f and CD-r amplification product, were used. As aresult, a 1701 bp product was amplified from the total DNAby PCR with the pair of ND-f and ND-r, and the fragmentwas cloned and sequenced. An identical overlapping 76 bpsequence was found for the mentioned 2 PCR fragmentsand gave a final nucleotide sequence of 5479 bp. These twofragments covered the entire Da-36 gene. Finally, the genecoding for Da-36 was constructed based on the two roundsof PCR results and deposited to GenBank with accessionnumber of JX220981.

To analyze the exon/intron organization of Da-36 gene,the Da-36 and Batroxobin gene (Itoh et al., 1988) werecompared. The results showed that Da-36 gene containedfive exons (exon 1 ¼ 66 bp, exon 2 ¼ 157 bp, exon3 ¼ 260 bp, exon 4¼ 140 bp, and exon 5 ¼ 241 bp) and fourintrons (intron 1 ¼ 1.5 kbp, intron 2 ¼ 0.9 kbp, intron3¼ 1.8 kbp, and intron 4¼ 0.3 kbp) (Fig. 4A), and the Da-36genewas estimated to span approximately 5.5 kbp (Fig. 4B).Batroxobin gene spanned 8 kbp and shared similar exon/intron organization with that of Da-36. However, intron 1(2.5 kbp) and intron 2 (1.7 kbp) of Batroxobin gene weremuch longer that those of Da-36 gene, respectively (Itohet al., 1988).

The sequences of the exon-intron boundaries of Da-36gene are shown in Fig. 4A. All the 50 and 30 ends of theintron sequences adhered to the “GT/AG rule”which beganat the 50 end with the dinucleotide G T and terminated with

Fig. 1. Purification of Da-36 from the crude venom of D. acutus. (A) Chromatograph of 20 g crude venom by DEAE-Sepharose Fast flow column. The column waswashed with a linear gradient of 0–1 M NaCl in 50 mM Tris–HCl (pH 8.0) buffer at a flow rate of 10 mL/min. Peak III having plasma-clotting activity was indicatedby an arrow. (B) Chromatograph of peak III from DEAE-Sepharose on a Superdex-75 PG column in 50 mM, Tris–HCl buffer (pH 7.5), containing 100 mM KCl. PeakIV having plasma-clotting activity was indicated by an arrow. (C) Chromatograph of Peak IV from Superdex-75 gel filtration on a DEAE-Sephadex A-50 column.The sample was loaded and washed with a linear gradient of NaCl (0–0.5 M) in 50 mM, Tris–HCl (pH 7.5) buffer. Peak II showing plasma-clotting activity wasindicated by an arrow. (D) Chromatograph of Peak II from DEAE-Sephadex A-50 on a Sephacryl S-100 column. The column was equilibrated with 50 mM Tris–HCl(pH 7.5) and eluted with the same buffer. Final product (Da-36) was indicated by an arrow. Insert: SDS–PAGE pattern of Da-36. Molecular weight standardproteins are indicated at left (kDa). Lanes 2 and 3, Da-36 under reducing and non-reducing conditions, respectively. (E) Isoelectric point determination of Da-36by capillary electrophoresis. The pI markers were 5.10, 5.90 and 9.45, respectively.

Y. Zheng et al. / Toxicon 67 (2013) 1–11 5

the dinucleotide A G (Breathnach and Charnbon, 1981; Itohet al., 1988). The intron sequences flanking the 50 and 30

boundaries were purine and pyrimidine-rich, respectively,and in agreement with the consensus sequences ofeukaryotic splice junctions (Breathnach and Charnbon,1981).

The exon nucleotide sequence of the Da-36 gene wastranslated into protein sequence (Fig. 4A). Da-36 geneencoded an open reading frame of 260 amino acids as fol-lows: 18 amino acids – a signal peptide, six amino acids –

an activation peptide, and 236 amino acids – the matureprotein. The calculated molecular mass for Da-36 was

Table 1Summary of the Da-36 purification.

Step Protein (mg) Specific clottingactivity (units/mg)

Total clottingactivity (units)

Yield (%) Purificationfactor

Crude venom 20,000 78 1,560,000 100 1DEAE-Sepharose 2057 460 946,220 10.29 5.90Superdex-75 303 953 288,759 1.52 12.21DEAE-Sephadex A-50 123 1951 239,973 0.62 25.01Sephacryl S-100 85 2416 205,360 0.43 30.97

Y. Zheng et al. / Toxicon 67 (2013) 1–116

26,359 Da. Exon 1 of Da-36 gene encoded the 50-noncodingregion and a large part of the signal peptide. Matureenzyme was encoded separately by exons 2–5. Exon 2encoded the rest of the signal peptide, the propeptide, andthe N-terminal region of mature Da-36. Exon 5 encoded theC-terminal region of Da-36 and the 30-noncoding region.The putative catalytic residues of Da-36, His43, Asp88, andSer182, were encoded by separate exons, exons 2, 3, and 5,respectively (Fig. 4A).

The deduced N-terminal mature amino acid sequence ofDa-36 (VIGGNECDTNEHRFLAAFFTSRPWT) was consistentwith the determined N-terminal sequence of the purifiedenzyme, indicating that the cloned gene do encode for Da-36. The primary structure of Da-36 was deduced from the

Fig. 2. The stability study of Da-36. Da-36 (0.25 mg) was used as control and the arginof temperature; (B) Effect of pH; (C) Effect of different ions (10 mM Ca2þ, Mg2þ, Cu2

EDTA, 2.0 mM aprotinin and benzamidine). Bars represent the mean of % esterase acto the control.

cloned gene and aligned with the amino acid sequences ofother homologous SVSPs. Da-36 shared 64% identity withsalmobin from Agkistrodon halys (Jeong et al., 2001), 63%with acutobin from D. acutus (Wang et al., 2001), 60% withcalobin from Agkistrodon caliginosus (Hahn et al., 1996), 58%with halystase from Agkistrodon halys blomhoffii (Matsuiet al., 1998), 57% with KN-BJ from Bothrops jararaca(Serrano et al., 1998), 56% with Batroxobin from B. atrox,moojeni (Itoh et al., 1988) and Aav-Sp-I from D. acutus (Zhuet al., 2005), 55% with ancrod from C. rhodostoma (Au et al.,1993) and flavoxobin from Trimeresurus flavoviridis (Shiehet al., 1988), 53% with TSV-PA from T. stejnegeri (Zhanget al., 1995), respectively (Fig. 5). It should be noted thatDa-36 only showed 63% and 56% sequence identities with

ine-esterase activity was examined using 1 mM TAME as substrate. (A) Effectþ, Zn2þ and Mn2þ); (D) Effect of inhibitors (5 mM PMSF, b-mercaptoethanol,tivity � SD (n ¼ 3), asterisks (*) represent significance level (p < 0.05) related

Fig. 3. Fibrinogenolysis patterns of Da-36 judged by SDS–PAGE. (A) Con-centration dependent effects. Different concentrations of Da-36 incubated at37 �C for 1 h with 20 mg human fibrinogen. Lane 1: control (fibrinogen), Lane2: 0.05 mg, Lane 3: 0.1 mg, Lane 4: 0.3 mg, Lane 5: 0.6 mg and Lane 6: 1.0 mg; (B)Time dependent effects. Da-36 (0.3 mg) incubated at 37 �C with 15 mgfibrinogen. At indicated interval times, samples were taken and assayed;Lane 1: control (fibrinogen), Lane 2: 1 h, Lane 3: 2 h, Lane 4: 6 h, Lane 5: 12 hand Lane 6: 27 h.

Y. Zheng et al. / Toxicon 67 (2013) 1–11 7

acutobin and Aav-Sp-I, which were characterized fromsame snake species, respectively.

4. Discussion

Da-36, a novel serine protease, was purified by four-stepchromatographic procedures from the crude venom of D.acutus and its chemical and biological properties werecharacterized. This enzyme is a single chain proteinwith anapparent molecular weight of 36 kDa and a pI of 6.59 asanalyzed by SDS–PAGE and capillary electrophoresis,respectively. Most SVSPs isolated from different snakevenoms were single-chain proteins with molecular massvarying between 26 and 67 kDa (Serrano and Maroun,2005). One exception was brevinase, which was a two-chain enzyme isolated from the Agkistrodon blomhoffiibrevicaudus venom (Lee et al., 1999). Nearly all SVSPs wereglycoproteins having a few N- or O-glycosylation sites intheir amino acid residues that differed greatly in variousenzymes (Serrano and Maroun, 2005; Castro et al., 2004).Interestingly, SVSPs characterized from the same D. acutusspecies but originated from different geographic areasshowed difference in their primary structures. For example,acutobin II isolated from D. acutus crude venom originatedfrom Anhui Province of China was not identified from thesame species distributed in Hunan Province of China by

powerful specific thrombin-like enzyme cDNA clonemethods (Xin et al., 2009; Zha et al., 2006). Blast searchusing amino acid sequence of Da-36 against public data-base revealed that Da-36 showed amaximal identity of 90%with both Dav-X (Swiss-Prot: Q9I8W9.1) and thrombin-likeprotein 1 (GenBank: AAW56608.1) from the same snakespecies, the rest of the best hit 100 serine proteinases hadan identity around 60–65% (data not shown). Blast resultsrevealed that Da-36 is a novel serine protease.

SVSPs could act on different natural and synthetic sub-strates. Da-36 clotted human plasma, displayed esteraseactivity upon TAME and BAEE, and digested fibrinogen. Theclotted humanplasma induced in vitro by Da-36was fragileand easily dissolved in urea, suggesting that blood coagu-lation FXIII was not activated by Da-36. Normally, clotsformed by SVSPs were not cross-linked in vivo and could beremoved from the circulatory system either by the fibri-nolytic reaction or via the reticuloendothelial system(Mahir et al., 1987). Therefore, some SVSPs acted in vitro asprocoagulants by converting fibrinogen to fibrin, whilein vivo they cause benign defibrination and were used inclinic (Marsh, 1994). The esterase activity of Da-36 uponTAME was stable at 20–50 �C and in the pH values of 6.0–10.0. Other homologous enzymes, e.g., Batroxobin from B.atrox (Stocker and Barlow, 1976), TLBm from Bothropsmarajoensis (Vilca-Quispe et al., 2010), BjussuSP-I fromBothrops jararacussu (Sant’ Ana et al., 2008) were alsothermally stable. The enzymatic activity of SVSPs wasgenerally inhibited by PMSF, DPF, benzamidine and p-aminobenzamidine (Serrano and Maroun, 2005). UsingTAME as substrate, the activity of Da-36 was almostcompletely inhibited by PMSF, and moderately inhibited byb-mercaptoethanol, aprotinin and benzamidine.

Studies of the fibrinogenolytic action of Da-36 showedthat it preferentially degraded both Aa- and Bb-chains offibrinogen. Interestingly, all the Aa-, Bb- and g-chains ofhuman fibrinogen could be degraded after 27 h incubationwith fibrinogen. Most SVSPs had both fibrinogenolytic andfibrinolytic activities, but many of them specifically cleavedonly fibrinogen releasing preferentially either fibrinopep-tide A (FPA) or B (FPB), or both Aa and Bb chains offibrinogen, promoting coagulation (Serrano and Maroun,2005). Digestion of g-chain of fibrinogen by SVSP hadbeen seldom reported except Harobin, a serine proteaseisolated from a sea snake Lapemis hardwickii, which coulddigest the Aa-, Bb- and g-chains of fibrinogen (He et al.,2007). To our knowledge, Da-36 represents the first landsnake serine protease that could cleave Aa-, Bb- and g-chains of human fibrinogen. This unique acting property onfibrinogen characterized Da-36 as a novel serineproteinase.

DNA could be recovered from the crude snake venombecause small quantity of cellular debris might retain in thesample (Pook and McEwing, 2005; Chen and Zhao, 2009).Da-36 gene was successfully amplified using total DNA asthe template, which was extracted from the lyophilizedvenom. The determined N-terminal amino acid sequence ofpurified Da-36 matched well with the nucleotide sequencededuced protein sequence, indicating that the cloned genereally coding for Da-36. The result presented here justifiedthe application of using the crude snake venom as the

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Fig. 4. Gene structure and cloning strategy of the Da-36. (A) Nucleotide and the deduced amino acid sequence of Da-36. Exon sequences are denoted by capitalletters. Intron sequences are denoted by lowercase letters. The numbering of the amino acid sequence starts at the amino-terminal amino acid of Da-36.Overlapping sequence between primers ND-f w ND-r and CD-f w CD-r PCR products is denoted by dashed line. Direct N-terminal sequence of Da-36 (under-lined) confirmed the DNA sequence. The catalytic amino acids are boxed. (B) Cloning strategy of the Da-36. PCR amplification of total DNA with two pairs ofprimers: primers CD-f and CD-r gave a PCR product of 3854 bp, covering exons 2–5 and introns 2–4; primer ND-f and ND-r, gave a PCR product of 1701 bp,covering exon 1 and the rest of exon 2, together with intron 1. Da-36 gene was composed of 5479 bp with identical 76 bp overlapping region in 2 PCR products.

Fig. 5. Amino acid sequence alignment of Da-36 with other SVSPs. Multiple alignment was made by Clustal W 2.1 program. Asterisk (*) indicates positions whichhave a single, fully conserved residue; Colon (:) indicates conservation between groups of strongly similar properties – scoring > 0.5 in the Gonnet PAM 250matrix; Period (.) indicates conservation between groups of weakly similar properties – scoring � 0.5 in the Gonnet PAM 250 matrix. The numbering starts fromthe amino terminal amino acid of Da-36. Gaps have been inserted to maximize similarities. Residues composing the catalytic site are in red. Cys residues areboxed. Residues composing the binding pocket S1 (D176), S2 (G199) and S3 (G210) are in green, blue and yellow, respectively. Enzymes used were as follows:Acutobin from D. acutus (gi: 13959650), Ancrod from C. rhodostoma (gi: 403078), Salmobin from A. halys (gi: 3668352), Aav-Sp-I from D. acutus (gi: 49258366),TSV-PA from T. stejnegeri (gi: 13959636), Flavoxobin from T. flavoviridis (gi: 3915685), Calobin from A. caliginosus (gi: 13959630), Halystase from A. halys blomhoffii(gi: 3122187), KN-BJ from B. jararaca (gi: 13959622) and Batroxobin from B. atrox,moojeni (gi: 211031). (For interpretation of the references to colour in this figurelegend, the reader is referred to the web version of this article.)

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origin source to clone the gene of a specific protein, whichavoided the sacrifice of living snakes.

The difference between the apparent molecular weight(36 kDa) and the calculated molecular mass (26,359 Da) ofDa-36 indicated that Da-36 was also a glycoprotein. Twoputative N-glycosylation sites, Asn-X-Thr/Ser, were locatedat the amino acid residues of 81–83 and 124–126 of Da-36by NetNGlyc 1.0 program. The role of the carbohydratemoiety in SVSPs structure has not yet been fully elucidated.However, evidences indicated that the carbohydrate moi-ety of some SVSPs interfered with the catalytic activity ofthese enzymes and was important to protein stabilization(Costa et al., 2009; Zhu et al., 2005). The role of carbohy-drate in Da-36 needs to be further investigated.

Up to now, only one snake venom serine protease geneorganization has been reported. The enzyme was Batrox-obin and the gene coding for Batroxobinwas obtained fromB. atrox,moojeni (Itoh et al., 1988). Batroxobin gene spanned8 kbp pairs and comprised five exons and four introns. Themature enzyme was encoded by exons 2–5 while the pu-tative catalytic triad residues were encoded by separateexons (2, 3, and 5). Gene comparison of the Da-36 and theBatroxobin revealed that the exon/intron organization ofthem was the same, which suggested that the Da-36 genewas also a member of the trypsin/kallikrein gene family(Itoh et al., 1988). In addition, the intron 1 (1.5 kbp) andintron 2 (0.9 kbp) of Da-36 gene was significantly shorterthan the intron 1 (2.5 kbp) and intron 2 (1.7 kbp) ofBatroxobin gene, respectively. More information on SVSPgenes should be desired for better understanding of theevolutionary development of these genes.

Da-36 contained 12 cysteine residues at conserved po-sitions, which formed six putative disulfide bridges in po-sitions of Cys7–Cys141, Cys28–Cys44, Cys78–Cys234, Cys120–Cys188, Cys152–Cys167 and Cys178–Cys203 (Fig. 5). These di-sulfide bridge pairings were confirmed experimentally onbilineobin from Agkistrodon bilineatus (Nikai et al., 1995)and contortrixobin from A. contortrix contortrix (Amiconiet al., 2000), and the same as those found in the crystal-lographic structure of TSV-PA (Parry et al., 1998). Thesedisulfide bonds were very important in the stabilization ofprotein structure, e.g. the Cys120–Cys188 bond is moreessential for the maintenance of the SVSP native structure(Castro et al., 2004). Based on the homology with othermature serine proteases, the catalytic amino acid residuesof Da-36 were deduced to be His43, Asp88, and Ser182, whichare encoded by separate exons and corresponding to thoseof His40, Asp84 and Ser176 in trypsin. The sequences flankingthe catalytic sitewere found to be highly conserved in theseenzymes. The primary (S1) and secondary (S2) specificitypositions (Asp176 and Gly199, respectively) were alsoconserved in SVSPs, while the tertiary specificity site (S3)was occupied by Gly210 on most SVSPs and replaced byalanine on the rest enzymes analyzed (Fig. 5). The bottomof the S1 pocket was highly conserved with an Asp, whichguaranteed the interaction with basic P1 residues of sub-strates such as TAME and a-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) (Wang et al., 2001; Guo et al., 2001). Itis of great interesting to resolve the crystal structure of Da-36 to help us understand the unique mechanism of Da-36on fibrinogen.

In summary, a novel serine protease termed Da-36 wasisolated and characterized from crude venom of D. acutus.Da-36 gene was successfully cloned using total DNA extrac-ted from the lyophilized venom. Da-36 gene composed of 5exons and 4 introns with a span of 5479 bp. Good thermaland pH stability, combined with cleavage Aa-, Bb- and g-chains of humanfibrinogen,makeDa-36 becoming a leadingstructure to explore novel defibrinogenating agents for thetreatment and prevention of thromboembolic diseases inclinic.

Ethical statement

All procedures are agreed upon standards of expectedethical behavior.

Acknowledgments

This work was supported by The National Natural Sci-ence Foundation of China (31071926), 2011CI139 fromYunnan Province and China Postdoctoral Science Founda-tion (20080440214, 200902656).

Conflict of interest

None declared.

References

Aird, S.D., 2002. Ophidian envenomation strategies and the role of pu-rines. Toxicon 40, 335–393.

Amiconi, G., Amoresano, A., Boumis, G., Brancaccio, A., De Cristofaro, R.,De Pascalis, A., Di Girolamo, S., Maras, B., Scaloni, A., 2000. A novelvenombin B from Agkistrodon contortrix contortrix: evidence forrecognition properties in the surface around the primary specificitypocket different from thrombin. Biochemistry 39 (33), 10294–10308.

Au, L.C., Lin, S.B., Chou, J.S., Teh, G.W., Chang, K.J., Shih, C.M., 1993. Mo-lecular cloning and sequence analysis of the cDNA for ancrod, athrombin-like enzyme from the venom of Calloselasma rhodostoma.Biochem. J. 294 (Pt 2), 387–390.

Braud, S., Bon, C., Wisner, A., 2000. Snake venom proteins acting on he-mostasis. Biochimie 82, 851–859.

Breathnach, R., Charnbon, P., 1981. Organization and expression ofeucaryotic split genes coding for proteins. Annu. Reu. Biochem. 50,349–383.

Castro, H.C., Zingali, R.B., Albuquerque, M.G., Pujol-Luz, M.,Rodrigues, C.R., 2004. Snake venom thrombin-like enzymes: fromreptilase to now. Cell. Mol. Life Sci. 61 (7–8), 843–856.

Chen, N., Zhao, S.J., 2009. Forensic identification of snake crude venom bymtDNA analysis. Lishizhen Med. Materia Med. Res. 20 (8), 2001–2003.

Costa, F.L., Rodrigues, R.S., Izidoro, L.F., Menaldo, D.L., Hamaguchi, A., Homsi-Brandeburgo, M.I., Fuly, A.L., Soares, S.G., Selistre-de-Araújo, H.S.,Barraviera, B., Soares, A.M., Rodrigues, V.M., 2009. Biochemical andfunctional properties of a thrombin-like enzyme isolated from Bothropspauloensis snake venom. Toxicon 54 (6), 725–735.

Dufton, M.J., 1993. Kill and cure: the promising future for venom research.Endeavour 17 (3), 138–140.

Edgar, W., Prentice, C.R.M., 1973. The proteolytic action of ancrod onhuman fibrinogen and its polypeptide chains. Thromb. Res. 2, 85–95.

Gempeler-Messina, P.M., Volz, K., Bühler, B., Müller, C., 2001. Protein Cactivators from snake venoms and their diagnostic use. Haemostasis31 (3–6), 266–272.

Guo, Y.W., Chang, T.Y., Lin, K.T., Liu, H.W., Shih, K.C., Cheng, S.H., 2001.Cloning and functional expression of the mucrosobin protein, a beta-fibrinogenase of Trimeresurus mucrosquamatus (Taiwan Habu). Pro-tein Expr. Purif. 23, 483–490.

Hahn, B.S., Yang, K.Y., Park, E.M., Chang, I.M., Kim, Y.S., 1996. Purificationand molecular cloning of calobin, a thrombin-like enzyme fromAgkistrodon caliginosus (Korean viper). J. Biochem. 119 (5), 835–843.

Y. Zheng et al. / Toxicon 67 (2013) 1–11 11

He, J., Chen, S., Gu, J., 2007. Identification and characterization of Harobin,a novel fibrino(geno)lytic serine protease from a sea snake (Lapemishardwickii). FEBS Lett. 581 (16), 2965–2973.

Itoh, N., Tanaka, N., Funakoshi, I., Kawasaki, T., Mihashi, S., Yamashina, I.,1988. Organization of the gene for batroxobin, a thrombin-like snakevenom enzyme. J. Biol. Chem. 263, 7628–7631.

Jeong, H., Yoo, S., Kim, E., 2001. Cell surface display of salmobin, athrombin-like enzyme from Agkistrodon halys venom on Escherichiacoli using ice nucleation protein. Enzym. Microb. Technol. 28 (2–3),155–160.

Kini, R.M., Rao, V.S., Joseph, J.S., 2002. Procoagulant proteins from snakevenoms. Haemostasis 31, 218–224.

Kini, R.M., 2005. Serine proteases affecting blood coagulation and fibrino-lysis from snake venoms. Pathophysiol. Haemost. Thromb. 34, 200–204.

Kirby, E.P., Niewiarowski, S., Stocker, K., Kettner, C., Shaw, E.,Brudzynski, T.M., 1979. Thrombocytin, a serine protease fromBothrops atrox venom. 1. Purification and characterization of theenzyme. Biochemistry 18, 3564–3570.

Kisiel, W., 1979. Molecular properties of the factor V-activating enzymefrom Russell’s viper venom. J. Biol. Chem. 254, 12230–12234.

Kisiel, W., Kondo, S., Smith, K.J., McMullen, B.A., Smith, L.F., 1987. Char-acterization of a protein C activator from Agkistrodon contortrix con-tortrix venom. J. Biol. Chem. 262, 12607–12613.

Lee, J.W., Seu, J.H., Rhee, I.K., Jin, I., Kawamura, Y., Park,W.,1999. Purificationand characterization of brevinase, a heterogeneous two-chain fibri-nolytic enzyme from the venom of Korean snake, Agkistrodon blom-hoffii brevicaudus. Biochem. Biophys. Res. Commun. 260, 665–670.

Lowry, O.H., Rosebrough, N.J., Farr, A.J., Randall, R.J., 1951. Protein mea-surement with the folin phenol reagent. J. Biol. Chem. 193, 265–275.

Mahir, M.S., Hynd, J.W., Flute, P.T., Dormandy, J.A., 1987. Effect of defib-rinogenation on the early patency rate of experimental small calibrearterial grafts. Br. J. Surg. 74 (6), 508–510.

Markland, F.S., 1998. Snake venoms and the hemostatic system. Toxicon36, 1749–1800.

Marsh, N.A., 1994. Snake venoms affecting the haemostatic mechanism –

a consideration of their mechanisms, practical applications and bio-logical significance. Blood Coagul. Fibrinolysis 5 (3), 399–410.

Matsui, T., Sakurai, Y., Fujimura, Y., Hayashi, I., Oh-Ishi, S., Suzuki, M.,Hamako, J., Yamamoto, Y., Yamazaki, J., Kinoshita, M., Titani, K., 1998.Purification and amino acid sequence of halystase from snake venomof Agkistrodon halys blomhoffii, a serine protease that cleaves specif-ically fibrinogen and kininogen. Eur. J. Biochem. 252 (3), 569–575.

Matsui, T., Fujimura, Y., Titani, K., 2000. Snake venom proteases affectinghemostasis and thrombosis. Biochim. Biophys. Acta 1477, 146–156.

Niewiarowski, S., Kirby, E.P., Stocker, K., 1977. Thrombocytin – a novelplatelet activating enzyme from Bothrops atrox venom. Thromb. Res.10, 863–869.

Nikai, T., Ohara, A., Komori, Y., Fox, J.W., Sugihara, H., 1995. Primarystructure of a coagulant enzyme, bilineobin, from Agkistrodon bili-neatus venom. Arch. Biochem. Biophys. 318, 89–96.

Nolan, C., Hall, L.S., Barlow, G.H., 1976. Ancrod, the coagulating enzymefrom Malayan pit viper (Agkistrodon rhodostoma) venom. MethodsEnzymol. 45, 205–213.

Parry, M.A., Jacob, U., Huber, R., Wisner, A., Bon, C., Bode, W., 1998. Thecrystal structure of the novel snake venom plasminogen activatorTSV-PA: a prototype structure for snake venom serine proteinases.Structure 6, 1195–1206.

Pirkle, H., 1998. Thrombin-like enzymes from snake venoms: an updatedinventory. Scientific and Standardization Committee’s Registry ofExogenous Hemostatic Factors. Thromb. Haemost. 79, 675–683.

Pook, C.E., McEwing, R., 2005. Mitochondrial DNA sequences from driedsnake venom: a DNA barcoding approach to the identification ofvenom samples. Toxicon 46, 711–715.

Sant’ Ana, C.D., Ticli, F.K., Oliveira, L.L., Giglio, J.R., Rechia, C.G., Fuly, A.L.,Selistre de Araújo, H.S., Franco, J.J., Stabeli, R.G., Soares, A.M.,Sampaio, S.V., 2008. BjussuSP-I: a new thrombin-like enzyme isolatedfrom Bothrops jararacussu snake venom. Comp. Biochem. Physiol. AMol. Integr. Physiol. 151 (3), 443–454.

Serrano, S.M., Hagiwara, Y., Murayama, N., Higuchi, S., Mentele, R.,Sampaio, C.A., Camargo, A.C., Fink, E., 1998. Purification and charac-terization of a kinin-releasing and fibrinogen-clotting serine pro-teinase (KN-BJ) from the venom of Bothrops jararaca, and molecularcloning and sequence analysis of its cDNA. Eur. J. Biochem. 251 (3),845–853.

Serrano, S.M.T., Maroun, R.C., 2005. Snake venom serine proteinases:sequence homology vs. substrate specificity, a paradox to be solved.Toxicon 45, 1115–1132.

Shieh, T.C., Kawabata, S., Kihara, H., Ohno, M., Iwanaga, S., 1988. Aminoacid sequence of a coagulant enzyme, flavoxobin, from Trimeresurusflavoviridis venom. J. Biochem. 103 (4), 596–605.

Siigur, E., Samel, M., Tõnismägi, K., Subbi, J., Reintamm, T., Siigur, J., 1998.Isolation, properties and N-terminal amino acid sequence of a factorV activator from Vipera lebetina (Levantine viper) snake venom. Bio-chim. Biophys. Acta 1429 (1), 239–248.

Stocker, K., Barlow, G.H., 1976. The coagulant enzyme from Bothrops atroxvenom (batroxobin). Methods Enzymol. 45, 214–223.

Stocker, K., Fischer, H., Meier, J., Brogli, M., Svendsen, L., 1986. Protein Cactivators in snake venoms. Behring Inst. Mitt. 79, 37–47.

Tokunaga, F., Nagasawa, K., Tamura, S., Miyata, T., Iwanaga, S., Kisiel, W.,1988. The factor V-activating enzyme (RVV-V) from Russell’s vipervenom. Identification of isoproteins RVV-V alpha, -V beta, and -Vgamma and their complete amino acid sequences. J. Biol. Chem. 263,17471–17481.

Vaiyapuri, S., Wagstaff, S.C., Harrison, R.A., Gibbins, J.M., Hutchinson, E.G.,2011. Evolutionary analysis of novel serine proteases in the venomgland transcriptome of Bitis gabonica rhinoceros. PLoS ONE 6 (6),e21532.

Vilca-Quispe, A., Ponce-Soto, L.A., Winck, F.V., Marangoni, S., 2010.Isolation and characterization of a new serine protease withthrombin-like activity (TLBm) from the venom of the snake Bothropsmarajoensis. Toxicon 55 (4), 745–753.

Wang, Y.M., Wang, S.R., Tsai, I.H., 2001. Serine protease isoforms ofDeinagkistrodon acutus venom: cloning, sequencing and phylogeneticanalysis. Biochem. J. 354 (Pt 1), 161–168.

White, J., 2005. Snake venoms and coagulopathy. Toxicon 45, 951–967.Xin, Y., Dong, D., Chen, D., Li, R., 2009. Structural and biological charac-

terization of a novel acutobin-like enzyme isolated from the venom ofthe sharp-nosed pit viper (Deinagkistrodon acutus). Biotechnol. Appl.Biochem. 53 (Pt 2), 123–131.

Xin, Y., Dong, D., Wang, T., Li, R., 2007. Affinity purification of serineproteinase from Deinagkistrodon acutus venom. J. Chromatogr. BAnalyt. Technol. Biomed. Life Sci. 859 (1), 111–118.

Zha, X.D., Huang, H.S., Zhou, L.Z., Liu, J., Xu, K.S., 2006. Thrombin-likeenzymes from venom gland of Deinagkistrodon acutus: cDNA cloning,mechanism of diversity and phylogenetic tree construction. ActaPharmacol. Sin. 27 (2), 184–192.

Zhang, B., Liu, Q., Yin, W., Zhang, X., Huang, Y., Luo, Y., Qiu, P., Su, X., Yu, J.,Hu, S., Yan, G., 2006. Transcriptome analysis of Deinagkistrodon acutusvenomous gland focusing on cellular structure and functional aspectsusing expressed sequence tags. BMC Genom. 7, 152.

Zhang, Y., Wisner, A., Xiong, Y., Bon, C., 1995. A novel plasminogen acti-vator from snake venom. Purification, characterization, and molecularcloning. J. Biol. Chem. 270 (17), 10246–10255.

Zhu, Z., Liang, Z., Zhang, T., Zhu, Z., Xu, W., Teng, M., Niu, L., 2005. Crystalstructures and amidolytic activities of two glycosylated snake venomserine proteinases. J. Biol. Chem. 280, 10524–10529.