CcMP-II, a new hemorrhagic metalloproteinase from Cerastes cerastes snake venom: Purification, biochemical characterization and amino acid sequence analysis Hinda Boukhalfa-Abib, Fatima Laraba-Djebari PII: DOI: Reference:

S1532-0456(14)00126-4 doi: 10.1016/j.cbpc.2014.09.004 CBC 8060

To appear in:

Comparative Biochemistry and Physiology Part C

Received date: Revised date: Accepted date:

6 July 2014 3 September 2014 12 September 2014

Please cite this article as: Boukhalfa-Abib, Hinda, Laraba-Djebari, Fatima, CcMP-II, a new hemorrhagic metalloproteinase from Cerastes cerastes snake venom: Purification, biochemical characterization and amino acid sequence analysis, Comparative Biochemistry and Physiology Part C (2014), doi: 10.1016/j.cbpc.2014.09.004

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ACCEPTED MANUSCRIPT CcMP-II, a new hemorrhagic metalloproteinase from Cerastes cerastes snake venom: Purification, biochemical characterization and amino acid sequence analysis

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Boukhalfa-Abib Hinda and Laraba-Djebari Fatima USTHB, Faculty of Biological Sciences, Laboratory of Cellular and Molecular Biology, BP 32, El-Alia Bab Ezzouar, 16111, Algiers, Algeria.

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E-mail: [email protected]/[email protected] Fax: 00213 21 33 60 77

Abstract

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Snake Venom Metalloproteinases (SVMPs) are the most abundant components in snake venoms. They are important in the induction of systemic alterations and local tissue damage after envenomation. CcMP-II, which is a metalloproteinase purified from Cerastes cerastes snake venom, was obtained by a combination of gel filtration, ion-exchange and affinity chromatographies. It was homogeneous on SDS-PAGE, with a molecular mass estimated to 35 kDa and presents a pI of 5.6. CcMP-II has an N-terminal sequence of EDRHINLVSVADHRMXTKY, with high levels of homology with those of the members of class P-II of SVMPs, which comprises metalloproteinase and disintegrin-like domains together. This proteinase displayed a fibrinogenolytic and hemorrhagic activities. The proteolytic and hemorrhagic activities of CcMP-II were inhibited by EDTA and 1,10-phenanthroline. However, these activities were not affected by aprotinine and PMSF, suggesting that CcMP-II is a zinc-dependent hemorrhagic metalloproteinase with an α-fibrinogenase activity. The hemorrhagic metalloproteinase CcMP-II was also able to hydrolyze extracellular matrix components, such as type IV collagen and laminin. These results indicate that CcMP-II is implicated in the local and systemic bleeding, contributing thus in the toxicity of Cerastes cerastes venom.

1. Introduction

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Keywords : Cerastes cerastes ; Snake venom ; Metalloproteinase ; Hemorrhage.

Viperidae venoms produce cutaneous or subcutaneous bleeding at the bite site, including systemic bleeding, contributing to the severity of envenomation. Venom-induced

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hemorrhage affects multiple organs, giving rise to complications such as cardiovascular shock, pulmonary bleeding and hemorrhage in the central nervous system (Otero et al., 2002). Local and systemic complications following Viperidae snakebites are due to protease components of the venom, especially hemorrhagic metalloproteinases. The metalloproteinases from snake venoms (SVMPs) are synthesized by the glands as pro-enzymes with a preserved zinc-proteinase domain (HEBxHxBGBxHD). In its catalytic site the zinc ion is coordinated by three histidine residues and it is essential for the action SVMPs (Stocker et al., 1995; Swenson and Markland, 2005; Moraes and Selistre-de-Araujo, 2006 ; Cintra et al., 2012 ; Camacho et al., 2014). The SVMPs shared structural and functional motifs with other metalloproteinases, such

as

MMPs

(Matrix

Metalloproteinases)

and

ADAMs

(A

Disintegrin

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Metalloproteinase) (Stocker et al., 1995). They are classified from PI to PIII according to their domains (Fox and Serrano, 2008 ; Markland and Swenson, 2013). The mature form of the PI

ACCEPTED MANUSCRIPT class is only composed by the metalloproteinase domain, which can show weak or nonhemorrhagic activity, whereas P-II and P-III SVMPs exhibit additional non-catalytic domains, such as disintegrin, disintegrin-like and cysteine-rich domains. These classes of SVMPs can

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cause potent hemorrhage, suggesting the important role of disintegrin-like and cysteine-rich

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domains in inducing hemorrhage (You et al., 2006 ; Chen et al., 2008 ; Pinyachat et al., 2011 ; Suntravat et al., 2013).

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The action of SVMPs is related to the proteolysis of extracellular matrix components (type IV collagen, laminin, fibronectin and nidogen) (Gutiérrez and Rucavado, 2000; Sanchez et al., 2010; Escalante et al., 2011; Bernardes et al., 2013), and endothelial cell surface

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proteins (integrins and cadherins) involved in cell-matrix and cell-cell adhesion (Wu and Huang, 2003 ; Gutiérrez et al., 2005 ; Pinyachat et al., 2011). Some metalloproteases affect

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also platelet function and hyrolyzed blood clotting factors, leading to hemorrhage (Wang et al., 2005; Girón et al., 2011; Suntravat et al., 2013). Many hemorrhagic SVMPs have been isolated from the Crotalidae or Viperidae

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snakes. However, only one hemorrhagic protein of SVMPs was reported from Cerastes

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cerastes (Boukhalfa-Abib et al., 2009). In the present study, we describe the purification and biochemical characterization of hemorrhagic metalloptroteinase, named CcMP-II, isolated

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from the venom of Cerastes cerastes. 2. Materials and methods 2.1. Venom and Animals

Lyophilized Cerastes cerastes venom was provided from Laboratory of Cellular and

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Molecular Biology of Biological Sciences Faculty. Male NMRI mice (20 ± 2 g of body mass (BW) were obtained from animal breeding of Biological Sciences Faculty of the University of Sciences and Technology Houari Boumediène of Algeria. They were housed in temperaturecontrolled rooms and received water and food until use. Experimental protocol was carried out according to the European Community rules of the Ethical Committee for Animals Welfare. Animals were used in compliance with the guidelines from the European Community Council Directive (86/609/EEC). 2.2. Reagents DEAE-Sephadex A-50, Sephadex G-75 and Benzamidine Sepharose 6B were purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. Phenylmethylsulphonyl fluoride (PMSF), aprotinin, ethylenediaminetetraacetic acid (EDTA), N-benzoyl-L-arginine ethyl ester (BAEE), Nα-CBZL-arginine-p-nitroanilide hydrochloride (CBZ) and 1,10phenanthroline were from Sigma Chemicals Co., St Louis, USA. All of reagents used were of

ACCEPTED MANUSCRIPT analytical grade. 2.3. Purification of CcMP-II from Cerastes cerastes venom 2.3.1. Fractionation of Cerastes cerastes venom by gel filtration chromatography on

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Sephadex G-75

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Cerastes cerastes venom (500 mg) was dissolved in distilled water, and centrifuged at 10 000 g for 5 min. The supernatant was applied to a Sephadex G-75 column (2.5 x 100 cm)

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equilibrated and eluted with 0.1 M ammonium acetate, pH 8.5 (AcNH4 0.1 M, pH 8.5) at 4°C. The venom was eluted at a flow rate of 18 mL/h (3 ml/fraction) and the elution profile was monitored at 280 nm. The hemorrhagic fractions were pooled and lyophilized prior to the next

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step.

2.3.2. Ion exchange chromatography on DEAE-Sephadex A-50

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Active fraction obtained from the previous step was applied to a DEAE-Sephadex A50 column (1.5 x 27 cm) equilibrated with 0.1 M ammonium acetate, pH 8.5 (AcNH4 0.1 M, pH 8.5) at 4°C. Samples were eluted with an ionic strength (0.1-2 M) linear gradient in

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ammonium acetate, pH 8.5, at a flow rate of 18 ml/h, and were monitored at 280 nm.

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Fractions (3 ml) were collected and screened for hemorrhagic activity. Active fraction was pooled for the next chromatographic step.

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2.3.3. Affinity chromatography on Benzamidine Sepharose 6B The pooled active fraction from the previous step was applied to an Benzamidine Sepharose 6B gel (1.5 x 6 cm) equilibrated with 0.05 M Tris-HCl, pH 7.5, at 4°C. The column was washed with the same buffer to remove unbound material. Bound proteins were then

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eluted with a linear NaCl gradient (0-2 M) at a flow rate of 18 mL/h. The elution profile was monitored at 280 nm. Fractions (3 mL) were collected and analyzed for hemorrhagic activity. Active fraction was pooled and dialysed prior to the next step. 2.3.4. Reverse-phase HPLC of active fraction Lyophilized sample was applied onto RP-HPLC C8 column (4.6 x 150 mm). The column was eluted with a linear acetonitrile gradient of 0-60 % (v/v) in 0.1 % trifluoroacetic over 60 min. TFA at a flow rate of 1ml/min was used to elute the hemorrhagic molecule. Homogeneity of the hemorrhagic fraction was tested by SDS-PAGE. 2.4. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was carried out according to the method of Laemmli (1970). Samples were pretreated in reducing conditions at 100°C for 5 min. Gels were stained with Coomassie blue R-250. The molecular mass was estimated by interpolation from a linear logarithmic plot of relative molecular mass versus distance of migration. Standard molecular weight markers

ACCEPTED MANUSCRIPT (Merck) were phosphorylase b (94,000), bovine serum albumin (BSA) (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,100) and α-lactalbumin (14,400).

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2.5. N-terminal amino acid sequencing

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Bands of purified CcMP-II obtained by SDS gels were transferred to a ProBlott membrane (Applied Biosystems, Foster City, CA, USA), and N-terminal sequence analysis

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was performed by automated Edman’s degradation using an Applied Biosystem Model 473A gas-phase sequencer (Hewick et al., 1981). The sequence was compared to those in the UniProtKB/Swiss-Prot or GenBank databases using the BLAST homology search program

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www.ncbi.nlm.nih.gov/blast. 2.6. Isoelectric focusing (IEF)

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Isoelectric point (pI) of CcMP-II was determined by performing isoelectric focusing in a PhastSystem Apparatus (GE Healthcare, Sweden) with precast polyacrylamide gels (PhastGel IEF 3-10). Calibration standards (GE Healthcare, Sweden) were between pH 3.5

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and 9.5. Gels were silver stained (Blum et al., 1987).

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2.7. Hemorrhagic activity

Hemorrhagic activity of the venom and purified hemorrhagin were tested using the

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method described by Kondo et al. (1960), and modified by Gutiérrez et al. (1985). Different doses of purified hemorrhagin (1, 2, 5, 10, 20 and 40 µg/20 g BW dissolved in 100 µL of 0.9 % NaCl) were injected into the back skin of three mice. Control mice received 0.9 % NaCl. After two hours, mice were humanely euthanized, the back skin was removed and the

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diameter of the hemorrhagic spot in the inner side of the back skin was measured. Hemorrhagic activity was expressed as mean ± S.E. The minimum hemorrhagic dose (MHD) was defined as the amount of venom or hemorrhagin that produced a hemorrhagic lesion of 10 mm in diameter. 2.8. Caseinolytic activity Proteolytic activity was tested using casein as substrate and following the method described by Kunitz (1947), modified by Van Der Watt and Joubert (1971). Samples of purified hemorhagin (10 µg) were incubated with 1% casein in 0.2 M Tris-HCl, pH 7.2, for 30 min at 37°C. Trichloro-acetic acid (5 %) was then added for protein precipitation. Samples were centrifuged for 20 min at 1000 g, and supernatant absorbance was recorded at 280 nm.

ACCEPTED MANUSCRIPT 2.9. Amidase activity Protease activity on the chromogenic substrate Nα-CBZL-arginine-p-nitroanilide hydrochloride (CBZ) was assayed at a wavelength of 405 nm, by monitoring the rate of

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hydrolysis of CBZ (1.08 x 10-4 M) by CcMP-II (10 µg) at 37°C in a 0.01 M Tris HCl pH 8.7,

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0.1 M NaCl 0.1% (w/w) PEG buffer according to the method of Stocker et al. (1986). 2.10. Esterolytic activity

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Esterolytic activity was evaluated according to the Nishikata method (1984). Samples of purified hemorhagin (10 µg) dissolved in 0.04 M Tris-HCl buffer, pH 8, containing 0.01 M CaCl2 were incubated with N-benzoyl-L-arginine ethyl ester (BAEE) (6.6 x 10-4 M) for 30 min

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at 37°C. Absorbance was measured at 253 nm. 2.11. Fibrinogenolytic activity

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Fibrinogenolytic activity was examined using 10 mg/mL of human fibrinogen solution in 50 mM Tris–HCl buffer (pH 7.4). Samples (10 µg in 100 µL of crude venom or CcMP-II) or seven units of thrombin (used as control) were mixed with fibrinogen solution (10 mg/mL).

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The mixtures were incubated for 24 h at 37°C, centrifuged at 3,000 g for 20 min, supernatant

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containing the fibrinogen degradation products, was loaded onto a C18 RP-column. The products were eluted using a linear gradient of acetonitrile (ACN) (5–75% in presence of

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0.05% TFA) in 30 min at flow of 1 mL/min. Specific cleavage of fibrinogen by CcMP-II was also analyzed on 10% SDS-polyacrylamide gels as described by Peichoto et al. (2007). Fibrinogen solution (2 mg/mL) and isolated protein CcMP-II (10 µg) were mixed and incubated at 37° C. Aliquots were collected from the reactionnel mixture after 0, 3 and 24 h of

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incubation. Samples were then denatured and reduced before loading on 10% SDS polyacrylamide gel.

2.12. Fibrinolytic activity Fibrinolytic activity was assayed as described by Astrup and Mûllertz (1952). Thrombin (50 mL, 10 U NIH) were added to fibrinogen solution (1.5 mg/mL in Tris-HCl buffer, pH 7.4) and incubated in order to obtain a fibrin clot. Aliquots of 20 µL containing different amounts of CcMP-II (1, 2, 5, 10, 20 and 40 µg) were added to the fibrin gel and incubated at 37°C for 24 h. The fibrinolytic activity was evaluated visually and quantified according to the halo diameter, which was compared to a negative control (PBS only). 2.13. Proteolytic activity upon extracellular matrix components Proteolytic activity of CcMP-II towards purified extracellular matrix components was investigated according to Moraes and Selistre-de-Araujo (2006). Laminin solutions (2 mg/ml) (from Engelberth Holm Swarm murine sarcoma, Sigma) were mixed with CcMP-II (10 µg)

ACCEPTED MANUSCRIPT for 5, 15, and 30 min at 37°C. Aliquots were collected, denatured and loaded onto SDSPAGE (10%) under reduced conditions. Collagenase activity was also tested using type IV collagen (from human placenta,

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Sigma) according to the method described by Martinez et al. (1990). Samples of CcMP-II (1,

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2, 5, 10, 20 and 40 µg in 0.025 M CaCl2) were incubated with type IV collagen in 0.05 M Tris–HCl, pH 7.8, for 24 h at 37°C. Hydroxyproline released during an incubation of 24 h

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incubation was detected by ninhydrin reagent after boiling for 20 min. N-propanol 50% was added after cooling. Collagenase activity was expressed as absorbance increase at 600 nm. 2.14. Local damage induced by CcMP-II in mice

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Groups of three male NMRI mice (18-20 g BW) were injected in the right gastrocnemius muscle with CcMP-II (10 µg). The animal controls received only saline

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solution (0.9% NaCl). After 2 h the animals were sacrificed by deep anesthesia with ethyl ether and a small section of the central region of the muscle was excised and soaked in fixing solution (10% formaldehyde). Thereafter, the tissue sample was dehydrated in a graded

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alcohol series and embedded in paraffin. Sections (7 µm) were cut and stained with

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hematoxylin and eosin to be examined under a light microscope. 2.15. Enzymatic properties of the hemorrhagin

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2.15.1. pH optimum

The pH optimum for the caseinolytic activity of CcMP-II was determined over the pH range of 5.0-10.0 using the caseinolytic assay in phosphate (50 mM ; pH 5.0-6.0), Tris (50 mM ; pH 7.0-8.0), and glycine (50 mM ; pH 9.0-10.0).

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2.15.2. Effect of inhibitors

The effects of 1,10-phenanthroline, EDTA, Aprotinin and PMSF were examined by pre-incubating the hemorrhagin CcMP-II with these compounds (final concentration, 5 mM in all cases) for 30 min at 37°C. After incubation, the residual proteolytic and hemorrhagic activities were evaluated. 2.16. Statistical analysis Obtained results were expressed as the mean ± Standard Deviation (S.D.). The significance of the differences between the mean values of two experimental groups was assessed by the Student’s t-test. Values of p lower than 0.01 were considered significant. 3. Results 3.1. Purification and biochemical characterization Hemorrhagic protein (CcMP-II) was purified using several chromatographic steps. Fractionation of the crude venom on Sephadex G-75 yielded three peaks (GF1, GF2 and GF3).

ACCEPTED MANUSCRIPT Hemorrhagic activity was found in peaks 1 and 2 (GF1 and GF2) (Fig. 1A). The pooled active fraction GF2 was applied onto DEAE-Sephadex A-50 column. This step yielded four peaks (D1, D2, D3 and D4), first and second peaks (D1 and D2) showed hemorrhagic activity when

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tested in mice (Fig. 1B). The active fraction D2 was further submitted to Benzamidine

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Sepharose 6 B column. Hemorrhagic activity was eluted in the fraction B1 (Fig. 1C). Reversed-phase HPLC analysis of the active fraction showed only one peak which was

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homogeneous on SDS-PAGE (CcMP-II) (Fig. 1D). The molecular mass of CcMP-II was estimated at 35 kDa (Fig. 2). Isoelectric focusing revealed a pI of 5.6 for this hemorrhagic protein (data not shown).

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3.2. N-terminal amino acid sequence of CcMP-II

The amino acid sequence of CcMP-II was determined for the 19 N-terminal residues

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(EDRHINLVSVADHRMXTKY). This sequence was compared with existing protein sequences in the GenBank non-redundant nucleotide database, using the BLASTP and tBlastn search programs, and the BLASTP software available at the Swiss-Prot database

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(http://www.expasy.ch/sprot/).

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As illustrated in Table 1, a multiple alignment analysis revealed a strong identity of the CcMP-II sequence with those found in other class P-II of SVMPs, reaching 85% identity

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with ZnMc adamalysin II like from Protobothrops flavoviridis (accession no. AN89389), 76% with Stejnitin from Trimeresurus stejnegeri (accession no. P0DM87) and 71% with CaVMP-II from Crotalus adamanteus (accession no. C9E1R7). 3.3. Hemorrhagic activity

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CcMP-II induced hemorrhage after subcutaneous injection in mice, with a MHD of 5.5 μg/20 g BW (Fig. 3) which was nearly 2-fold more low than the venom (MDH, 3 µg/20 g BW). Similar results were obtained when Cerastes cerastes crude venom or purified CcMP-II was injected by i.m. route. Indeed, Microscopic and histological observations of the right gastrocnemius muscle removed two hours after injection of crude venom (Figs. 4C and D) revealed a more extensive hemorrhage than that observed after injection of CcMP-II (Figs. 4E and F). Whereas, the control muscle retained normal appearance (Figs. 4A and B). 3.4. Caseinolytic activity CcMP-II demonstrated caseinolytic activity, but no amidolytic and esterase activities towards CBZ and BAEE were detected. The effect of pH on caseinolytic activity was also assayed, and the optimum activity for CcMP-II was noticed at pH 8.0. Incubation of CcMP-II at pH values below 6.0 or above 9.0 resulted in an abrupt decrease in activity (Fig. 5).

ACCEPTED MANUSCRIPT 3.5. Effect of protease inhibitors on hemorrhagic and proteolytic activity of CcMP-II The inhibition of CcMP-II hemorrhagic and proteolytic activity by metal chelating agents (EDTA and 1,10-phenanthroline), but not by serine proteinase inhibitors (aprotonin

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and PMSF), suggested that this protein is a zinc-metalloproteinase (Figs. 6 and 7).

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3.6. Fibrinogen and fibrin degradation

In order to determine whether CcMP-II was potentially able to interfere with the

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coagulation system, proteolytic effect of CcMP-II upon fibrinogen and fibrin was examined. Fibrinogen was incubated with CcMP-II and RP-HPLC chromatography fractionation was used to analyze the fibrinogen degradation by CcMP-II. The purified protein hydrolyzed only

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the Aα-chain of fibrinogen (Fig. 8C). Similar result was observed with the analysis of electrophoretic migration patterns of the Aα-chain, which showed a progressive

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disappearance of this band, whereas the Bβ-chain was unaffected. The γ-chain was resistant, even when the incubation time was prolonged to 24 h (Fig. 9, Lanes 2 and 3). Based on these characteristics, CcMP-II was classified as an α-fibrinogenase. Fibrinolytic activity tested on

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gel containing fibrin indicated that CcMP-II was able to degrade the fibrin gel at all tested

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doses. The fibrin hydrolysis occurred in a dose-dependent manner (Fig. 10). 3.7. Proteolytic activity of CcMP-II upon extracellular matrix components

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To determine the mechanism underlying the induction of hemorrhage by CcMP-II, its capacity to hydrolyze extracellular matrix components was assessed. Obtained results showed that CcMP-II was able to hydrolyze type IV collagen in a dose-dependent manner (Fig. 11A). Furthermore, CcMP-II was also able to cleave laminin after 15 min of incubation, presenting

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a complete digestion after 30 min (Fig. 11B, Lanes 3 and 4). This proteolytic activity of CcMP-II towards the laminin was time-dependent. The ability of CcMP-II to hydrolyze these components is consistent with its ability to induce hemorrhage by degrading extracellular matrix components of the blood vessel basement membranes. 4. Discussion Snake venom metalloproteinases are widely distributed in Viperidae and Crotalidae snake venoms and are involved in several pathological and biological effects, such as hemorrhage, inflammation, necrosis, hypotension, pro-coagulant, anticoagulant, and antiplatelet activities (Bjarnason and Fox, 1994; Fox and Serrano, 2005 ; Calvete, 2011). They are multifunctional-domain proteins with variability in snake venoms and are divided into several subclasses depending on the organization of their domains (Fox and Serrano, 2008 ; Markland and Swenson, 2013). The P-II and P-III metalloproteinases are divided into P-IIa to P-IIe and P-IIIa to P-IIId subclasses, respectively, based on their variable post-translation modifications

ACCEPTED MANUSCRIPT (Fox and Serrano, 2009). In this study, we describe the purification and biochemical characterization of a hemorrhagic metalloproteinase from Cerastes cerastes snake venom. The proteinase was isolated by the combination of three steps chromatographic: a gel-

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filtration, an ion-exchange and affinity, which provided a high level of homogeneity as

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confirmed by reverse phase chromatography, SDS-PAGE and N-terminal amino acid sequencing.

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The purified protein had a molecular mass of 35 kDa, which is similar to other previously identified metalloproteinases such as: Ac4-proteinase from Agkistrodon acutus at 33 kDa (Mori et al., 1984), the 36 kDa hemorrhagic metalloproteinase jerdonitin for

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Trimeresurus jerdonii (Chen et al., 2003), Stejnitin from Trimeresurus stejnegeri at 35 kDa (Han et al., 2007), the 35 kDa hemorrhagic metalloproteinase albolamin for Cryptelytrops

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albolabris (Jangprassert and Rojnuckarin, 2014), among others. CcMP-II showed relatively strong hemorrhagic activity, with an MHD of 5.5 µg/ 20 g BW, which was comparable with other SVMPs, especially when compared to P-II and P-III

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class SVMPs, usually showing MHD between 0.2 and 5 μg (Mazzi et al., 2004 ; Cintra et al.,

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2012 ; Bernardes et al., 2013), such as, BaH4 from Bothrops asper has an MHD of 2 µg (Franceschi et al., 2000), alternagin, a hemorrhagic factor isolated from Bothrops alternatus

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has an MHD of 1 μg (Souza et al., 2000) and B-JussuMP-I from Bothrops jararacussu has an MHD of 4 µg (Mazzi et al., 2006). Based on the molecular mass, hemorrhagic activity, and sequence similarity to other class P-II SVMPs, we can include it in the class P-II of SVMPs. The strong activity of hemorrhagic CcMP-II can be explained by its structure, since all

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SVMPs belonging to the class P-II are characterized by a metalloproteinase and disintegrinlike domains. Therefore, the hemorrhage caused by this toxin must be due to its proteolytic action on components of the basement membrane of capillary vessels. Furthermore, the similar inhibition profiles of the proteolytic and hemorrhagic activities of CcMP-II in the presence of EDTA and 1,10-phenanthroline, suggested that these two activities may be related and interdependent. This is in accordance with the widely accepted concept about the proteolytic degradation of basement membrane components of capillary vessels by hemorrhagic metalloproteinases is a key step in their pathologic effect (Bjarnason and Fox, 1994 ; Gutièrrez et Rucavado, 2000 ; Bello et al., 2006 ; Bernardes et al., 2013). The ability of this SVMP to degrade fibrinogen might potentiate the hemorrhagic activity. CcMP-II hydrolyzes only the Aα-chain of fibrinogen throughout the incubation period examined, while the Bβ- and γ-chains remained resistants to proteolysis even when the incubation time was increased to 24 h. Venom fibrinogenases are either α- or β-type enzymes.

ACCEPTED MANUSCRIPT Alpha fibrinogenases have been isolated from the venoms of Trimeresurus gramineus (Ouyang and Huang, 1979), Agkistrodon contortrix mokasen (Moran and Geren, 1981) and Vipera lebetina (Siigur and Siigur, 1991). The α-fibrinogenases enzymes preferentially attack

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the Aα-chains are metalloproteinases, can possess hemorrhagic activity and they also cleave

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the Bβ-chain but at a slow rate (Peichoto et al., 2007), whereas β-fibrinogenases are serine proteases and show arginine-esterase activity (Shimokawa and Takahashi, 1995). According

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to Markland (1991 ; 1998), the Aα-chain specificity is typical of the majority of fibrinogenolytic snake venom metalloproteinases (SVMPs). While some of them degrade preferentially, although not exclusively, the Aα- chain of fibrinogen, such as BjussuMP-I

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from Bothrops jararacussu venom, which degrades the Aα-chain within 15 min and the Bβchain within 6 h (Mazzi et al., 2004), others degrade exclusively the Aα-chain of fibrinogen,

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such as jararhagin from Bothrops jararaca venom (Kamiguti et al., 1994), Patagonfibrase from Philodryas patagoniensis (Peichoto et al., 2007) and the enzyme isolated in this work. Based on the degradation of fibrinogen chains, the metalloproteinase CcMP-II can be

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classified as an α-fibrinogenase.

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In conclusion, a new hemorrhagic metalloproteinase, named CcMP-II, was characterized for first time from Cerastes cerastes venom. This hemorrhagin was

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homogeneous on SDS-PAGE, with a molecular mass of 35 kDa. The molecular mass, the inhibition assays and N-terminal amino acid sequence data, suggest that metalloproteinase CcMP-II belongs to P-II class of SVMPs. CcMP-II exhibited a strong hemorrhagic activity, with a MHD of 5.5 µg/20 g BW, and was able to hydrolyze extracellular matrix components,

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such as type IV collagen and laminin. This metalloproteinase may contribute to the hemorrhage, local tissue damage and hemostatic disorders seen in patients envenomed by Cerastes cerastes venom. However, there are additional metalloproteinases and likely a number of other enzymes and toxins present in the venom that could damage structural proteins in prey tissue, leading to "predigestion"of prey, and CcMP-II is only one of these. References Astrup, T., Mûllertz, S., 1952. The fibrin plate method for estimating fibrinolytic activity. Archs. Biochem. Biophys. 40, 346-351. Bello, C. A., Hermogenes, A. L. N., Magalhàes, A., Veiga, S. S., Gremski, L. H., Richardson, M., Sanchez, E. F., 2006. Isolation and biochemical characterization of a fibrinolytic proteinase from Bothrops leucurus (white-tailed jararaca) snake venom. Biochimie 88, 189-200. Bernardes, C.P., Menaldo, D.L., Camacho, E., Rosa, J.C., Escalante, T., Rucavado, A., Lomonte, B., Gutiérrez, J.M., Sampaio, S.V., 2013. Proteomic analysis of Bothrops pirajai snake venom and characterization of BpirMP, a new P-I metalloproteinase. J. of Proteomics 80, 250-267.

ACCEPTED MANUSCRIPT Bjarnason, J. B., Fox, J.W., 1994. Hemorrhagic metalloproteinases from snake venoms. Pharmac. Ther. 62, 325-372. Blum, H., Beier, H., Gross,H.J., 1987. Improved silver staining of plant proteins. RNA and DNA in polyacrylamide gels. Electrophoresis 8, 93-99.

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Boukhalfa-Abib, H., Meksem, A., Laraba-Djebari, F., 2009. Purification and biochemical characterization of a novel hemorrhagic metalloproteinase from horned viper (Cerastes cerastes) venom. Comparative Biochemistry and Physiology, Part C 150, 285-290.

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Calvete, J.J., 2011. Proteomic tools against the neglected pathology of snake bite envenoming. Expert Rev Proteomics 8,739-58.

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Camacho, E., Villalobos, E., Sanz, L., Pérez, A., Escalante, T., Lomonte, B., Calvete J.J., Gutiérrez, J.M., Rucavado A., 2014. Understanding structural and functional aspects of PII snake venom metalloproteinases: Characterization of BlatH1, a hemorrhagic dimeric enzyme from the venom of Bothriechis lateralis. Biochimie 4398_proof.

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Chen, R.Q., Jin, Y., Wu, J.B., Zhou, X.D., Lu, Q.M., Wang, W.Y., Xiong, Y.L., 2003. A new protein structure of P-II class snake venom metalloproteinases: it comprises metalloproteinase and disintegrin domains, Biochem. Biophys. Res. Commun. 310, 182-187.

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Chen, H.S., Tsai, H.Y., Wang, Y.M., Tsai, I.H., 2008. P-III hemorrhagic metalloproteinases from Russell's viper venom: cloning, characterization, phylogenetic and functional site analyses. Biochimie 90, 1486-1498.

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Cintra, A.C., De Toni, L.G., Sartim, M.A., Franco, J.J., Caetano, R.C., Murakami, M.T., Sampaio, S.V., 2012. Batroxase, a new metalloproteinase from B. atrox snake venom with strong fibrinolytic activity. Toxicon 60, 70–82. Escalante, T., Rucavado, A., Fox, J. W., Gutiérrez, J. M., 2011. Key events in microvascular damage induced by snake venom hemorrhagic metalloproteinases. Journal of proteomics 74, 1781-1794.

AC

Fox, J.W., Serrano, S.M., 2005. Structural considerations of the snake venom metalloproteinases, key members of the M12 reprolysin family of metalloproteinases. Toxicon 45, 969–85. Fox, J.W., Serrano, S.M., 2008. Insights into and speculations about snake venom metalloproteinases (SVMP) synthesis, folding and disulfide bond formation and their contribution to venom complexity. FEBS J. 275, 3016-3030. Fox, J.W., Serrano, S.M., 2009. Timeline of key events in snake venom metalloproteinase. Research. J. Proteomics 72, 200-209. Franceschi, A., Rucavado, A., Mora, N., Gutiérrez, J. M., 2000. Purification and characterization of BaH4, a hemorrhagic metalloproteinase from the venom of snake Bothrops asper. Toxicon 38, 6377. Gutiérrez, J. M., Geni, J. A., Rojas, G., and Cerdas, L., 1985. Neutralization of proteolytic and hemorrhagic activities of Costa Rican snake venom by a polyvalent antivenom. Toxicon 23, 887893. Gutiérrez, J. M., Rucavado, A., 2000. Snake venom metalloproteinases : Their role in the pathogenesis of local tissue damage. Biochimie 82, 841-850.

ACCEPTED MANUSCRIPT Gutiérrez, J.M., Rucavado, A., Escalante, T., Diaz, C., 2005. Hemorrhage induced by snake venom metalloproteinases: biochemical and biophysical mechanisms involved in microvessel damage. Toxicon 45, 997-1011.

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T

Girón, M. E., Estrella, A., Sanchez, E. E., Galan, J., Andy Tao, W., Guerrero, B., Salazar, A. M., Rodriguez-Acosta, A., 2011. Purification and characterization of a metalloproteinase Porthidin-1, from the venom of lansberg’s hog-nased piyvipers (Porthidium lansbergii hutmanni). Toxicon 57, 608-618.

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Han, Y.P., Lu, X.Y., Wang, X.F., Xu, J., 2007. Isolation and characterization of a novel P-II class snake venom metalloproteinase from Trimeresurus stejnegeri. Toxicon 49, 889-898. Hewick, R.M., Hunkapiller, M.W., Hood, L.E., Dreyer, W.J., 1981. A gas-liquid solid phase peptide and protein sequenator, J. Biol. Chem. 256, 7990-7997.

MA

NU

Jangprasert, P., Rojnuckarin, P., 2014. Molecular cloning, expression and characterization of albolamin: A type P-IIa snake venom metalloproteinase from green pit viper (Cryptelytrops albolabris). Toxicon 79, 19-27.

Kamiguti, A.S., Desmond, H.P., Theakston, R.D.G., Hay, C.R., Zuzel, M., 1994. Ineffectiveness of the inhibition of the main haemorrhagic metalloproteinase from Bothrops jararaca venom by its only plasma inhibitor, α2-macroglobulin. Biochim. Biophys. Acta 1200, 307-314.

TE

D

Kondo, H., Kondo, S., Ikezawa, H., Murata, R., and Ohsaka, A., 1960. Studies on the quantitative method for determination of hemorrhagic activity of habu snake venom. Jap. J. Med. Sci. Biol. 13, 43-51.

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Kunitz, M., 1947. Cristalline soybean trypsin inhibitor : General properties. J. Gen. Physiol. 30, 291310. Laemmli, U. K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.

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Markland Jr, F. S., 1991. Inventory of α- and β-fibrinogenases from snake venoms, Thromb. Haemost. 65, 438-443. Markland Jr, F. S., 1998. Snake venoms and hemostatic system. Toxicon 36, 1749-1800. Markland Jr, F.S., Swenson, S., 2013. Snake venom metalloproteinases. Toxicon 62, 3–18. Martinez, M., Rael, E. D., Naddux, N. L., 1990. Isolation of a hemorrhagic toxin from Mojave rattlesnake (Crotalus scutulatus scutulatus) venom. Toxicon 28, 685-694. Mazzi, M.V., Marcussi, S., Carlos, G.B., Stábeli, R.G., Franco, J.J., Ticli, F.K., 2004. A new hemorrhagic metalloprotease from Bothrops jararacussu snake venom: isolation and biochemical characterization. Toxicon 44, 215–23. Mazzi, M.V., Magro, A.J., Amui, S.F., Oliveira, C.Z., Ticli, F.K., Stábeli, R.G., Fuly, A.L., Rosa, J.C., Braz, A.S., Fontes, M.R., Sampaio, S.V., Soares, A.M., 2006. Molecular characterization and phylogenetic analysis of BjussuMP-I: a RGD-P-III class hemorrhagic metalloprotease from Bothrops jararacussu snake venom. J. Mol. Graph Model. 26, 69–85. Moran, J.B., Geren, C.R., 1981. Characterization of a fibrinogenase from northern copperhead (Agkistrodon contortrix mokasen) venom. Biochim. Biophys. Acta 659, 161-168.

ACCEPTED MANUSCRIPT Moraes, C.K., Selistre-de-Araujo, H., 2006. Effect of rACLF, a recombinant snake venom metallopeptidase on cell viability, chemokine expression and degradation of extracellular matrix proteins. Toxicon 48, 641-648.

IP

T

Mori, N., Nikai, T., Sugihara, H., 1984. Purification of a proteinase (Ac5-proteinase) and characterization of hemorrhagic toxins from the venom of the Humdred-PACE snake (Agkistrodon acutus). Toxicon 22, 451-461.

SC R

Nishikata, M., 1984. Trypsin-like protease soybean seeds: Purification and some properties. J. Biochem 95, 1169-1177.

NU

Otero, R., Gutiérrez, J., Mesa, J.M.B., Duque, E., Rodriguez, O., Arango, J.L., Gomez, F., Toro, A., Cano, F., Rodriguez, L.M., Caro, E., Martinez, J., Cornejo, W., Gomez, L.M., Uribe, F.L., Cardenas, S., Nunez, V., Diaz, A., 2002. Complications of Bothrops, Porthidium, and Bothriechis snakebites in Colombia. A clinical and epidemiological study of 39 cases attended in a university hospital. Toxicon 40, 1107-1114.

MA

Ouyang, C., Huang, T.F., 1979. Alpha and beta-fibrinogenase from Trimeresurus gramineus snake venom. Biochim. Biophys. Acta 571, 270-283.

TE

D

Peichoto, M.E., Teibler, P., Mackessy, S.P., Leiva, L., Acosta, O., Gonçalves, L.R.C., TanakaAzevedo, A.M., Santoro, M.L., 2007. Purification and characterization of patagonfibrase, a metalloproteinase showing a-fibrinogenolytic and hemorrhagic activities, from Philodryas patagoniensis snake venom. Biochim. Biophys. Acta 1770, 810-819.

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Pinyachat,A., Rojnuckarin,P., Muanpasitporn, C., Singhamatr,P., Nuchprayoon, S., 2011. Albocollagenase, a novel recombinant P-III snake venom metalloproteinase from green pit viper (Cryptelytrops albolabris), digests collagen and inhibits platelet aggregation. Toxicon 57, 772-780.

AC

Sanchez, E.F., Schneider, F. S., Yarleque, A., Borges, M. H., Richardson, M., Figueiredo, S. G., Evangelista, K. S., Eble, J. A., 2010. The novel metalloproteinase atroxlysin-I from Peruvian Bothrops atrox (Jergón) snake venom acts both on blood vessel ECM and platelets. Arch. Biochem. Biophys. 496, 9–20. Shimokawa, K.I., Takahashi, H., 1995. Comparative study of fibrinogen degradation by four arginine ester hydrolase from the venom of Agkistrodon caliginosus (Kankoku mamushi). Toxicon 33, 179186. Siigur, E., Siigur, J., 1991. Purification and characterization of lebetase, a fibrinolytic enzyme from Vipera lebetina (Snake) venom. Biochim. Biophys. Acta 1074, 223-229. Souza, D.H., Iemma, M.R., Ferreira, L.L., Faria, J.P., Oliva, M.L., Zingali, R.B., Niewiarowski, S., Selistre-de-Araujo, H.S., 2000. The disintegrin-like domain of the snake venom metalloprotease alternagin inhibits α2β1integrin-mediated cell adhesion, Arch. Biochem. Biophys. 384,341–350. Suntravat, M., Jia, Y., Lucena, S.E., Sánchez, E.E., Pérez, J.C., 2013. cDNA cloning of a snake venom metalloproteinase from the eastern diamondback rattlesnake (Crotalus adamanteus), and the expression of its disintegrin domain with anti-platelet effects. Toxicon 64, 43-54. Stocker, K., Fisher, H., Brogli, M., 1986. Chromogenic assay for the prothrombin activator ecarin from the venom of the saw-scaled viper (Echis carinatus). Toxicon 24, 81-89. Stocker, W., Grams, F., Baumann, U., Reinermer, P., Gomis-Ruth, F.X., McKay, D.B., Bode, W., 1995. The metzincins-topological and sequential relations between the astacins, adamalysins,

ACCEPTED MANUSCRIPT serralysins, and matrixins (collagenases) define a superfamily of zinc-peptidases. Protein. Sci. 4, 823–840. Swenson, S., Markland, F.S., 2005. Snake venom fibrin(ogen)olytic enzymes. Toxicon 45, 1021– 1039.

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T

Van Der Watt, S. J., and Joubert, F. J., 1971. Studies on puff adder (Bitis arietans) venom. Purification and properties of protease A. Toxicon 9, 153-161.

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Wang, W.J., Shih, C., Huang, T., 2005. Primary structure and antiplatelet mechanism of snake venom metalloproteinase, acurhagin, from Agkistrodon acutus venom. Biochimie 87, 1065-1077. Wu, W.B., Huang, T.F., 2003. Activation of MMP-2, cleavage of matrix proteins, and adherens junctions during a snake venom metalloproteinase induced endothelial cell apoptosis. Exp. Cell Res. 288, 143-157.

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You, W.K., Jang, Y.J., Chung, K.H., Jeon, O.H., Kim, D.S., 2006. Functional roles of the two distinct domains of halysase, a snake venom metalloprotease, to inhibit human platelet aggregation. Biochemical and Biophysical Research Communications 339, 964-970.

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Fig. 1. Purification of hemorrhagic protein (CcMP-II) from Cerastes cerastes venom. (A)

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Fractionation of Cerastes cerastes venom by gel filtration on Sephadex G-75. Venom (1 g) was dissolved in distilled water, centrifuged and the supernatant was applied to the column (2.5 x 100 cm). The fractions corresponding to peak 1 (GF2) showed hemorrhagic activity and were pooled for further purification. (B) Fractionation of GF2 by ion-exchange chromatography on DEAE-Sephadex A-50 (1.5 x 27 cm). The active fraction corresponding to peak D2 was pooled for the next chromatographic step. (C) Affinity chromatography of D1 on Benzamidine Sepharose 6B (1.5 x 6 cm). Only peak B1 displayed hemorrhagic activity. (D) The active fraction B1 was further purified on a RP-HPLC C8 column (4.6 x 150 mm). The only peak corresponding to CcMP-II and displaying hemorrhagic activity. Fractions with hemorrhagic activity are indicated by a bar above the corresponding peak.

Fig. 2. SDS-PAGE of CcMP-II. Molecular mass markers (lane 1) and CcMP-II (lane 2) were run on

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a 15 % gel. The gel was stained with Coomassie blue R250.

Fig. 3. Determination of the minimal hemorrhage dose (MHD) of CcMP-II. Different

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concentrations of CcMP-II (1, 2, 5, 10, 20 and 40 µg in 100 µL of 0.9 % NaCl) were injected subcutaneous into the dorsal skin of mice and the hemorrhage halo formation was measured after two hours. The results were expressed as the mean halo diameter (mm) ± S.D. Each concentration was tested in a group of 3 mice (n = 3).

Fig. 4. Macroscopic and histological changes induced by CcMP-II in mice gastrocnemius. (A and B) Injection of saline solution (50 µL) as a control. (C and D) Injection of Cerastes

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cerastes venom (10 µg/50 µL). (E and F) Injection of CcMP-II (10 µg/50 µL) by i.m. route. Tissue samples were collected 2 h after injection. Notice abundant erythrocytes in the interstitium of muscle tissue injected with venom or CcMP-II, whereas normal histological features are observed in control muscle injected with saline solution. Hematoxylin and eosin stain, magnification x 400. H: hemorrhage.

Fig. 5. pH-dependence of the proteolytic activity of CcMP-II. The caseinolytic activity of

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CcMP-II was determined over the pH range of 5–10 using the standard proteolytic assay. The points are the mean ± S.D. of three determinations.

Fig. 6. Inhibition of the hemorrhagic activity of CcMP-II. (A) Control 0.9 % NaCl; (B) CcMP-II

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(10 μg/ 20 g BW) alone; (C) CcMP-II incubated with 5 mM EDTA; (D) CcMP-II incubated with 5 mM 1,10phenanthroline; (E) CcMP-II incubated with 5 mM aprotinin; (F) CcMP-II incubated with 5 mM PMSF.

Fig. 7. Effect of some reagents on proteolytic activity of CcMP-II. The chelating agents EDTA and 1,10-phenanthroline, the specific inhibitors aprotinin (competitive inhibitor of serine protease) and PMSF (inhibitor of serine and cysteine proteases). Bars represent the mean ± S.D. of three individual experiments. Asterisks indicate statistically significant differences between treatments and CcMP-II alone (positive control), (p