Isolation, properties and N-terminal amino acid sequence of a factor V activator from Vipera lebetina (Levantine viper) snake venom

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Isolation, properties and N-terminal amino acid sequence of a factor V activator from Vipera lebetina (Levantine viper) snake venom Ene Siigur, Mari Samel, Ku«lli To¬nisma«gi, Juhan Subbi, To¬nu Reintamm, Ju«ri Siigur * Institute of Chemical Physics and Biophysics, Akadeemia tee 23, EE-0026 Tallinn, Estonia Received 8 September 1998; received in revised form 7 October 1998; accepted 8 October 1998

Abstract A factor V activator (VLFVA) was separated from Vipera lebetina venom by gel filtration on Sephadex G-100 superfine, followed by chromatography on CM-cellulose and on heparin-agarose. This enzyme (VLFVA) with a molecular mass of 28.4 kDa, as determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry, is a single-chain glycoprotein containing seven residues of neutral sugars, seven residues of hexosamines and three residues of neuraminic acid per molecule. The treatment with N-glycosidase F lowered the molecular mass approximately 6%. The N-terminal sequencing of VLFVA up to the 30th residue evidenced a high homology with Vipera russelli factor V activator RVV-VQ (90% identity). Aside from factor V, no other protein substrate for VLFVA has yet been identified. VLFVA hydrolyzes several synthetic arginine ester substrates, such as benzoylarginine ethyl ester (BAEE), tosylarginine methyl ester (TAME) and amide substrates such as Pro-Phe-Arg-MCA. The arginine ester hydrolase activity of the enzyme is markedly lower than that of the crude venom. The ability of VLFVA to activate factor V and its activity to BAEE and TAME were inhibited by the serine proteinase inhibitor, diisopropylfluorophosphate. VLFVA is thermostable protein, heating for 20 min at 70³C does not alter the arginine esterase activity of the enzyme. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Factor V activator; Glycoprotein; Snake venom; (Vipera lebetina)

1. Introduction Snake venoms, particularly those belonging to the Crotalidae and Viperidae families, are known to con-

Abbreviations: VLFVA, Vipera lebetina factor V activator; BAEE, benzoylarginine ethyl ester; TAME, tosylarginine methyl ester; DFP, diisopropyl£uorophosphate; DHB, 2,5-dihydroxybenzoic acid; PMSF, phenylmethylsulfonyl £uoride; MCA, 4methylcoumarinyl-7-amide ; MALDI-TOF, matrix assisted laser desorption ionization time-of-£ight; HPLC, high performance liquid chromatography; PRP, platelet-rich plasma; RP-HPLC, reverse phase HPLC * Corresponding author. Fax: +372 (6) 398 313; E-mail: siigur@kb¢.ee

tain a number of components a¡ecting blood coagulation. These venoms have been found to have potent e¡ects on coagulation through both pro- and anticoagulant mechanisms (reviews: [1^8]). These highly speci¢c proteinases which cleave limited bond(s) in the blood coagulation factors are usually divided into two groups: (1) serine proteinases^arginine esterases (factor V activator, protein C activator, kinin-releasing and thrombin-like enzymes, L-¢brinogenases); (2) metalloproteinases which need Ca2‡ or Zn2‡ (or both) for their hydrolytic activity and are inhibited by metal chelating agents (factor X activator, prothrombin activator, K(L)-¢brinogenases). We showed that the venom of Vipera lebetina con-

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tains: bradykinin releasing enzymes [9], L- and K(L)¢brin(ogen)olytic enzymes [10^12] and factor X activator [13]. Yukelson et al. [14] demonstrated the presence of factor V activator in V. lebetina venom. Factor V is a protein cofactor that accelerates the conversion of prothrombin to thrombin by activated factor X in the presence of phospholipid and calcium ions. In plasma its activation was shown to occur by limited proteolysis by thrombin to give a multiple subunit protein [15]. In the present work we describe the puri¢cation, characterize properties and the N-terminal amino acid sequence of a previously unidenti¢ed factor V activator from V. lebetina venom. 2. Materials and methods 2.1. Materials The venom of V. lebetina was a commercial preparation from Tashkent Integrated Zoo Plant (Uzbekistan). Sephadex G-100 (super¢ne) and markers for isoelectric focusing were the products of Pharmacia (Uppsala, Sweden). Ampholytes for isoelectric focusing were from LKB (Bromma, Sweden). Heparinagarose was the product of Kemotex Bio AE (Tallinn, Estonia). BAEE and TAME were from Reanal (Budapest, Hungary). Neuraminidase, Pro-Phe-ArgMCA and DFP were obtained from Serva (Heidelberg, Germany), PMSF, DHB, factor V de¢cient plasma and thromboplastin were from Sigma (St. Louis, MO, USA). Ultra¢ltration membranes PM10 were obtained from Amicon (Oosterhout, Holland). All other reagents used were of analytical grade. 2.2. Isolation of VLFVA All fractionation steps were performed at 4³C in a cold room. Step 1. Gel ¢ltration. Crude V. lebetina venom (1.5 g) was dissolved in 15 ml of 0.2 M ammonium acetate, pH 6.7. Insoluble material was removed by centrifugation (5000Ug for 15 min) and the supernatant was applied to the column (2.8U128 cm) of Sephadex G-100 super¢ne equilibrated with 0.2 M

ammonium acetate. The elution was carried out with the same solution at a £ow rate of 6.8 ml/h and fractions were collected at 1 h intervals. Step 2. Ion exchange chromatography on CM-52 cellulose. The column (1.5U9 cm) was equilibrated with 0.2 M ammonium acetate, pH 6.7. Combined and concentrated by lyophilization or by ultra¢ltration, fraction IV from gel ¢ltration (0.3 g in 10 ml of 0.2 M ammonium acetate, pH 6.7) was applied onto the column. Non-adsorbed material was washed with the equilibration solution. The fraction containing factor V activating protein was eluted with a gradient of 0.2^0.6 M ammonium acetate, pH 6.7. The £ow rate was 12 ml/h; fractions of 5 ml were collected. Step 3. The column of heparin-agarose (1.7U 10 cm) was equilibrated with 0.2 M ammonium bicarbonate. 35 mg of lyophilized material (fractions 33^48, Fig. 1B) from ion exchange chromatography was dissolved in 5 ml of 0.2 M ammonium bicarbonate and applied to the column. Non-adsorbed material was washed with the equilibration solution. The column was eluted with 0.75 M ammonium bicarbonate, with a gradient of 0.75^1.3 M ammonium bicarbonate and with 2 M ammonium bicarbonate. The £ow rate was 12 ml/h; fractions of 3 ml were collected. 2.3. Measurement of activities 2.3.1. Factor V activator assay The reaction mixture, consisting of 0.05 ml of diluted VLFVA and 0.05 ml of barium sulfate-adsorbed human plasma, was incubated at 37³C for 1 min in a plastic culture tube. An aliquot of the incubation mixture was diluted 100-fold in 50 mM Tris-HCl (pH 7.5), containing 100 mM NaCl and 0.1% of bovine serum albumin, and 0.05 ml of the diluted sample transferred to a glass tube. The following reagents were added sequentially to the sample: 0.05 ml of factor V-de¢cient plasma, 0.05 ml of thromboplastin, and 0.05 ml of 25 mM CaCl2 . A timer was started with the addition of the calcium chloride, and the clotting time was determined. A straight line was identi¢ed when the log of the clotting time was plotted against the log of VLFVA concentration.

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2.3.2. Arginine esterase and amidase activities Arginine esterase activity was determined by the method of Schwert and Takenaka [16] using BAEE and TAME as substrates. One unit of arginine esterase activity is de¢ned as the amount of protein that hydrolyzes 1 Wmol of substrate per min. MCA-substrate hydrolytic activity was assayed by the method of Iwanaga et al. [17]. Pro-Phe-Arg-MCA was dissolved in dimethylsulfoxide and the solution was diluted to ¢nal concentration of 0.1 mM, using 50 mM Tris-HCl at pH 8.0, containing 100 mM NaCl and 10 mM CaCl2 . The reaction was started by addition of 10 Wl of enzyme in a total volume of 1.0 ml. The £uorescence of 7-amino-4-methylcoumarin produced was monitored using a Hitachi £uorescent spectrophotometer, model 850 (Tokyo, Japan). Measurements were carried out with excitation at 380 nm and emission at 460 nm. Hydrolytic activity is expressed as nmol 7-amino-4-methylcoumarin per min per mg protein. 2.3.3. Proteolytic activity Caseinolytic activity was assayed by the method of Kunitz as modi¢ed by Mebs [18]. Azocaseinolytic activity was estimated according to the method of Charney and Tomarelli [19]. Fibrinolytic activity was estimated by the ¢brin plate method of Astrup and Mu«llertz [20]. 2.3.4. Blood coagulation and platelet aggregation Plasma recalci¢cation time was studied according to Dimitrov and Kankonkar [21]. Plasma prothrombin time was measured by the method of Furie and Furie [22]. Platelet aggregation was measured photometrically in a Chrono-Log aggregometer according to the method of Born [23]. Platelet aggregation assays were performed with human platelet-rich plasma (PRP). Blood was collected from healthy volunteers, who had not taken any medication for at least 2 weeks prior to sampling. It was stabilized by 0.129 M sodium citrate (9 vol. blood/1 vol. citrate) and centrifuged at room temperature for 10 min at 180Ug to obtain PRP. The extent of platelet aggregation was quantitated by measuring the total amplitude at a predetermined time interval following addition of the platelet stimulant (ADP).

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2.4. Protein determination Protein concentrations were determined using the Pierce micro BCA kit. Bovine serum albumin was used as standard for detecting the protein concentration. The method is based upon the reaction of protein with Cu2‡ to form Cu‡ , which complexes with bicinchoninic acid (BCA), forming a colored product. The intensity of this product at 562 nm is related to protein concentration. 2.5. Molecular mass of VLFVA 2.5.1. SDS-PAGE SDS-PAGE was carried out in 10% and 12.5% gels at pH 8.3 by the method of Laemmli [24]. The following molecular mass indicators were used: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), bovine carbonic anhydrase (29 kDa), soybean trypsin inhibitor (20.1 kDa), cytochrome c (12.3 kDa). Staining was performed with Coomassie brilliant blue R250. 2.5.2. MALDI-TOF mass spectrometry The MALDI mass spectra were measured with a home-built gridless time-of-£ight MALDI mass spectrometer designed for maximum £exibility in use (Institute of Chemical Physics and Biophysics). It can be used in linear and re£ection modes with both static extraction ¢eld and delayed extraction without any change in ion source con¢guration. For this work it was used in linear delayed extraction mode with 3 kV pulsed extraction and 19 kV total acceleration voltage. A 1.6 m £ight tube was used. A double multichannel plate detector with conversion dynode was used for ion detection and Tectronics TDS 520 digitizing oscilloscope for data accumulation. An excimer laser pumped dye laser working on 340 nm was used for desorption/ionization, and the delay between laser pulse was 700 ns. The mass calibration was done with bovine carbonic anhydrase (29.023 kDa). DHB was used as matrix. 2.6. Carbohydrate test. Treatment with N-glycosidase F and with neuraminidase Neutral sugars were determined by the phenol-sul-

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furic acid method [25], using a standard solution containing D-glucose. Hexosamines were assayed by the procedure of Winzler [26] after acid hydrolysis in 3 N HCl for 4 h at 100³C. Glucosamine was used as a standard. To demonstrate the presence of neuraminic acid the VLFVA (0.42 mg) was incubated at 37³C for 40 h with neuraminidase (0.21 units) in 0.21 ml of 0.05 M sodium acetate bu¡er, pH 5.6. Neuraminic acid content was measured with HPLC by isocratic elution (0.6 ml/min) of a Bio-Rad HPX-87H column (300U7.8 mm) equipped with UV206 and refractive index detectors with 0.009 N H2 SO4 using N-acetylneuraminic acid as a standard. Deglycosylation with N-glycosidase F was performed according to the manufacturer's advice (Boehringer Mannheim Biochemica). Denatured VLFVA (0.5 mg in 100 Wl of 50 mM phosphate bu¡er, pH 7.5, 2% SDS, 5 min at 95³C) was deglycosylated with 2 units of N-glycosidase F in 900 Wl of 50 mM phosphate bu¡er, pH 7.5, containing 40 mM EDTA and 2% of n-octylglucoside, by overnight incubation at 37³C. The mixture was desalted on Sephadex G-25 and deglycosylated protein was analyzed on SDS-PAGE. 2.7. Isoelectric focusing Analytical isoelectric focusing was carried out using an LKB £at-bed electrofocusing apparatus Multiphor 2117 in 5% polyacrylamide gels according to standard procedure [27] in the pH range 3^10 at 10³C. 2.8. HPLC chromatography Reverse-phase chromatography was performed on a DuPont HPLC system with a variable wavelength detector at 280 nm and room temperature on WBon-

dapak C18 columns (3.9U300 mm) from Waters (Milford, MA). Activator fraction (50 Wl, 2 mg/ml in 0.1 M Tris-HCl, pH 8.1) was injected to the C18 column. The £ow rate was kept constant at 1 ml/min. The elution protocol was as follows: a linear gradient 0.1% TFA to 80% acetonitrile in 0.1% TFA was run over 60 min, the run was completed by isocratic elution with 80% acetonitrile in 0.1% TFA for 10 min. 2.9. Heat treatment The enzyme (1 mg/ml in 0.1 M ammonium acetate, pH 6.7) was incubated for 20 min at 37, 50, 70 and 95³C, and then quickly cooled to room temperature. Remaining activity was determined with BAEE as substrate. 2.10. Partial amino acid sequence of VLFVA Partial protein sequence of VLFVA was obtained after SDS-PAGE (as described above) and subsequent electrotransfer to PVDF membrane. The protein band was excised and N-terminal sequence was determined directly in an Applied Biosystems 494 Protein Sequencer. 3. Results and discussion 3.1. Isolation of V. lebetina venom factor V activator V. lebetina venom contains several proteolytic enzymes, among them coagulants and anticoagulants, as has been established in our previous studies [9^13]. The distribution and quantity of coagulant and anticoagulant components varies in di¡ering venom batches. Puri¢cation of the factor V activator (VLFVA) was achieved by a three-step procedure

Table 1 Summary of puri¢cation of VLFVA Step

Total protein (mg)

Speci¢c activity substrate BAEE (U/mg)

Speci¢c activity ProPheArg-MCA (U/mg)

Crude venom Sephadex G-100 fourth fraction CM-cellulose second fraction Heparin-agarose third fraction

1500 300 42 23

3.2 8.1 1.0 1.2

1444 79 8.0 4.9

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(Fig. 1 and Table 1). The gel ¢ltration of crude venom on Sephadex G-100 super¢ne gave nine fractions. VLFVA eluted with the fourth fraction, containing approximately one-¢fth of proteins loaded to the column. The fourth fraction contains bradykinin-releasing enzyme and other arginine esterases, serine proteinase and several other proteins [9]. VLFVA from the fourth gel ¢ltration fraction was separated from the caseinolytic activity and from other arginine esterases such as bradykinin-releasing enzyme, on a CM-cellulose column indicating that factor V activating activity and caseinolytic activity are associated with di¡erent enzymes. The enzymes eluting in the ¢rst peak have pIs in the acid region and also have higher BAEE hydrolyzing activity [9] than VLFVA. VLFVA elutes in the second peak (Fig. 1B). After the second step puri¢cation the preparation contained impurities (Fig. 2, lane 3; Fig. 3A). Final puri¢cation was achieved by heparin-agarose chromatography (Fig. 1C). After elution with 0.75^1.3 M ammonium bicarbonate gradient the homogeneous activator was obtained as judged by SDS, HPLC and MALDI-TOF analysis (Fig. 2, lane 4, Figs. 3B

6

Fig. 1. (A) Gel ¢ltration of V. lebetina venom. Crude V. lebetina venom (1.5 g) was dissolved in 15 ml of 0.2 M ammonium acetate, pH 6.7. Insoluble material was removed by centrifugation (5000Ug for 15 min) and the supernatant was applied to the column (2.8U128 cm) of Sephadex G-100 super¢ne equilibrated with 0.2 M ammonium acetate. The elution was carried out with the same solution at a £ow rate of 6.8 ml/h and fractions were collected at 1 h intervals. (B) Ion exchange chromatography on CM-52 cellulose. The column (1.5U9 cm) was equilibrated with 0.2 M ammonium acetate, pH 6.7. Combined and concentrated by lyophilization, fraction IV from gel ¢ltration (0.3 g in 10 ml of 0.2 M ammonium acetate, pH 6.7) was applied onto the column. Non-adsorbed material was washed with the equilibration solution. The fraction containing factor V activating protein was eluted with a gradient of 0.2^0.6 M ammonium acetate, pH 6.7. The £ow rate was 12 ml/h; fractions of 5 ml were collected. (C) The column of heparin-agarose (1.7U10 cm) was equilibrated with 0.2 M ammonium bicarbonate. 0.035 g of lyophilized material (fractions 33^48, B, peak II) from ion exchange chromatography was dissolved in 5 ml of 0.2 M ammonium bicarbonate and applied to the column. Non-adsorbed material was washed with the equilibration solution. The column was eluted with 0.75 M ammonium bicarbonate, with a gradient of 0.75^1.3 M ammonium bicarbonate and with 2 M ammonium bicarbonate. The £ow rate was 12 ml/h; fractions of 3 ml were collected.

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28 000^29 000 [29]. Although thrombin-catalyzed activation of factor V occurs in three cleavages, RVVV cleaves only Arg1545 -Ser1546 in factor V [15]. The molecular mass and pI of VLFVA are close to Vipera russelli activator RVV-V. 3.2.2. Carbohydrate content VLFVA contains seven residues of neutral sugars, seven residues of hexosamine and three residues of

Fig. 2. (A) SDS-PAGE of VLFVA in 12.5% gel. 1: V. lebetina venom, 2: fraction IV from Sephadex G-100 sf., 3: fraction II from CM-52 cellulose, 4: fraction III from heparin-agarose, 5: standard proteins (molecular weight standard proteins were bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), soybean trypsin inhibitor (20.1 kDa), and cytochrome c (12.3 kDa)). (B) SDS electrophoresis of VLFVA in 10% gel. 1: VLFVA, 2: VLFVA treated with N-glycosidase F, 3: molecular weight markers: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), soybean trypsin inhibitor (20.1 kDa).

and 4). However, we cannot rule out the existence of very close isoforms of VLFVA due to a shoulder of BAEE esterase activity in the elution curve. The single step puri¢cation of VLFVA from crude venom on heparin-agarose column was unsuccessful. 3.2. Characterization of VLFVA 3.2.1. Molecular mass and isoelectric point The puri¢ed activator was homogeneous by several criteria. In SDS electrophoresis it moved according to a molecular mass of 30 þ 2 kDa (Fig. 2A). The molecular mass of native VLFVA estimated by MALDI-TOF mass spectrometry was 28.4 þ 0.2 kDa (Fig. 4). The broad band produced by VLFVA following SDS-PAGE and a broad ion peak following mass analysis refer to the probable glycoprotein nature of the enzyme which is also con¢rmed by carbohydrate analysis. Analytical isoelectric focusing studies revealed a protein band near the cathode (pI higher than 9.3 for the enzyme). Factor V can also be activated by a component of Russell's viper venom, RVV-V [28]. RVV-V is a glycoprotein with Mr of

Fig. 3. (A) HPLC chromatography of the second fraction from CM-52 cellulose on C18 column. (B) HPLC chromatography of VLFVA on C18 column. Reverse-phase chromatography was performed on a DuPont HPLC system with a variable wavelength detector at 280 nm on WBondapak C18 columns (3.9U300 mm) from Waters (Milford, MA). Activator fraction (50 Wl, 2 mg/ml in 0.1 M Tris-HCl, pH 8.1) was injected to the C18 column. The £ow rate was kept constant at 1 ml/min. The elution protocol was as follows: a linear gradient 0.1% TFA to 80% acetonitrile in 0.1% TFA was run over 60 min, the run was completed by isocratic elution with 80% acetonitrile in 0.1% TFA for 10 min.

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sialic acid per molecule. The treatment with N-glycosidase F lowered the molecular weight about 6% (Fig. 2B), showing that the enzyme is N-glycosylated. In this respect VLFVA is also rather close to RVV-V which contains 6% of carbohydrate [15]. 3.3. Activation of factor V The e¡ect of gel ¢ltration fractions and of the puri¢ed component, VLFVA, on blood coagulation

Fig. 5. Coagulation time dependence from the concentration of VLFVA. 50 Wl of barium sulfate-adsorbed human plasma was added to various amounts of VLFVA (1^10 000 ng) in 50 Wl of 50 mM Tris-HCl (pH 7.5), containing 100 mM NaCl and 0.1% bovine serum albumin. After incubation at 37³C for 1 min, aliquots of the reaction mixtures were diluted 100-fold in the same bu¡er. 50 Wl each of diluted sample, factor V-de¢cient plasma, thromboplastin and 25 mM CaCl2 were pipetted into a glass tube. A timer was started with the addition of the calcium chloride.

was studied by measuring the factor V activating activity. Factor V activating activity was found in the fourth gel ¢ltration fraction, in the second CMcellulose fraction and in the third heparin-agarose fraction. VLFVA converts factor V to the active form Va in the presence of Ca2‡ ions and phospholipid (Fig. 5). 3.4. Esterase and amidolytic activities

Fig. 4. MALDI-TOF mass spectrum of VLFVA. The MALDI mass spectra were measured with a home-built gridless time-of£ight MALDI mass spectrometer designed for maximum £exibility in use (Institute of Chemical Physics and Biophysics). The mass calibration was done with bovine carbonic anhydrase (29.023 kDa). DHB was used as matrix. 10 mg of DHB was dissolved in 1 ml of a 1:1 mixture of 0.1% tri£uoroacetic acid and acetonitrile for sample preparation. 1 Wl of this mixture was deposited on a stainless steel probe tip, mixed there with 1 Wl of VLFVA (1 mg/ml in a 1:1 mixture of 0.1% tri£uoroacetic acid and acetonitrile), and allowed to dry at room temperature.

VLFVA is active towards low molecular weight substrates such as BAEE and TAME. BAEE hydrolytic activity of VLFVA is relatively low (1.2 U/mg), about 37% of the activity of crude venom. It also has weak amidase activity towards Pro-Phe-Arg-MCA: 4.9 U/mg (Table 1). No other protein substrate besides factor V has yet been found. Enzyme does not hydrolyze casein or asocasein, ¢brinogen and ¢brin. Inhibition of esterase activity of enzyme by DFP and PMSF demonstrates the serine proteinase nature of the protein. The factor V activating enzyme is thermostable.

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Fig. 6. N-terminal sequence of VLFVA and comparison with other related serine proteinases from snake venoms. The dots signify identical amino acids for the various species reported.

The arginine esterase activity of VLFVA after heating at 70³C for 20 min was retained. 3.5. E¡ect on blood coagulation and platelet aggregation The puri¢ed enzyme showed coagulant activity, as judged by a decrease in normal human plasma recalci¢cation time and prothrombin time. VLFVA did not act on ¢brinogen or on prothrombin. This enzyme had no aggregatory e¡ect on human PRP and did not inhibit platelet aggregation induced by ADP. 3.6. Sequencing of VLFVA and comparison with other serine proteinases The ¢rst 30 amino acid residues of VLFVA were determined by Edman degradation and compared for homologies with other serine proteinases from snake venoms [30^36] (Fig. 6). The sequence of the novel VLFVA revealed that (1) V. russelli factor V activator (RVV-VQ) shows 90% sequence similarity with the ¢rst 30 amino acids analyzed and (2) the N-terminus of these snake venom components is strongly conserved (Fig. 6). Factor V activator from V. russelli venom has several molecular forms: RVV-VK,

RVV-VL and RVV-VQ. The complete amino acid sequences of RVV-VK and RVV-VQ have been determined [30]. Both enzymes consist of 236 residues with six disul¢de bridges and di¡er in only six amino acid residues in the molecule. All these puri¢ed activators are glycoproteins. Factor V activators from Viperidae venoms [15] are quite di¡erent from the Naja naja oxiana venom activator [37]. In conclusion, the procedure used in this work enabled us to isolate a novel factor V activating enzyme from V. lebetina venom. The enzyme was characterized as a serine proteinase because of its substrate speci¢city, inhibitor action and N-terminal sequence homology. Acknowledgements We are grateful to Dr. Nisse Kalkkinen (Institute of Biotechnology, University of Helsinki) for performing the N-terminal amino acid sequencing for VLFVA. We are indebted to Tiiu-Mai Laht for detection of neuraminic acid. The work was ¢nancially supported by Estonian Science Foundation Grant 3441.

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