Identification and partial purification of an anticoagulant factor from the venom of the Iranian snake Agkistrodon halys

An anticoagulant factor was purified from the venom of the Iranian snake Agkistrodon halys by gel filtration on Sephadex G-50 and ion-exchange chromatography on DEAE-Sepharose. In the final stage of purification, the percentage recovery of purified anticoagulant factor was found to be 83%. The purified anticoagulant factor revealed a single protein band in SDS-polyacrylamide electrophoresis under reducing conditions and its molecular weight was about 22 kDa. The purified peptide did not show any effect on casein, BApNA or plasma.


INTRODUCTION
Snake venoms are rich sources of pharmacologically active proteins and peptides.
They play an important role in incapacitating, immobilizing and digesting prey.Thus toxins have evolved to specifically target various critical points in the physiological systems of prey.Over the years, a number of toxins that affect blood circulation have been isolated and characterized from various snake venoms (1,2).Within each family of snakes, the venom components seem to be fairly common and similar to one another.
Nerve toxins are generally found in the Hydrophidae and Elapidae venoms whereas hemorrhagic and myonecrotic toxins are generally found in the venoms of the Viperidae and Crotalidae families of snakes (3).Snake venom toxins affecting hemostasis have been classified by virtue of their overall effect into procoagulant and anticoagulant ones (2)(3)(4).Snake venoms have different types of anticoagulant proteins, some of which have enzymatic activity, represented by phospholipase A 2 , metalloproteinases like α-fibrinogenase, serine proteinases and L-Amino acid oxidase.But others (C-type lectin-related proteins and three-finger toxins) do not show any enzymatic activity (1,(3)(4)(5).Phospholipases A 2 are esterolytic enzymes that hydrolyze acyl-ester bonds of 1,2-diacyl-3-snphosphoglycerides and release fatty acids (3,6,7).Snake venoms contain a number of serine and metalloproteinases including fibrino(geno)lytic enzymes (8,9).Thrombin-like enzymes and fibrinolytic enzymes, which act on fibrinogen, lead to defibrinogenation of blood and a consequent decrease in blood viscosity.Fibrinolytic enzymes have been reported in species of the Crotalidae and Viperidae families including Agkistrodon contortrix contotrix and Agkistrodon acutus (10)(11)(12).L-amino acid oxidases are flavoenzymes that catalyze the stereospecific oxidative deamination of an L-amino acid substrate to a corresponding a-ketoacid by the production of ammonia and hydrogen peroxide (13).
In this study, we report the purification and characterization of an anticoagulation protein from the venom of the Iranian snake species Agkistrodon halys that exhibits anticoagulant activity on human blood.

Material
Fresh crude venom of A. halys was obtained directly from a local snake in Iran, lyophilized and preserved at -20ºC.DEAE-Sepharose, Sephadex G-50 and C18 columns were purchased from Pharmacia Biotech Company (Sweden).Bovine serum albumin, BApNA (N2-benzoyl-dl-arginine-p-nitroanilide), the kit of standard protein markers and other reagents for enzymatic and biochemical assays were purchased from Sigma (USA).All other chemicals were of analytical reagent grade.

Isolation of the Thrombin-Like Enzyme
Lyophilized crude venom of A. halys (200 mg) was dissolved in 8 mL of 50 mM ammonium acetate buffer (pH 7.4) and centrifuged at 5,000 rpm for 15 minutes at 4ºC and was filtered by 0.45 microfilter to remove the insoluble materials.
The clear supernatant was applied on a molecular exclusion chromatographic column of Sephadex G-50 (150 x 3 cm), previously equilibrated with the ammonium acetate buffer (pH 7.4) and then eluted with the same buffer.Fractions of 9 mL/tube were collected at a flow rate of 60 mL/hour at 4ºC.The obtained fractions were denominated AH1 to AH5, indicating A. halys fractions 1 to 5.
The fraction (AH2), which showed clotting activity from the gel chromatography step, was pooled and dialyzed overnight at 4°C against distilled water and applied on DEAE-Sepharose CL-6B (2.5 x 20 cm) column, equilibrated with 20 mM Tris buffer, at pH 8.2.In this step, proteins were eluted with a linear gradient of NaCl from 0.0 to 0.5 mM.The flow rate was 17 mL/hour and 5 mL fractions were collected at 4ºC.The peaks were monitored at A 280 .

Blood Collection
Normal pooled plasma was obtained from ten individual healthy donors, without history of bleeding or thrombosis.Blood was centrifuged for 20 minutes at 2,400 g, and the plasma was fresh when used.

Determination of Molecular Weight
12% SDS-PAGE was performed and utilized a low molecular weight standard ranging from 6.5 to 66 kDa (11).

Purity Analysis
Sample aliquots (100 μL) applied on an HPLC C18 column, were equilibrated with solvent A (H 2 O, 0.1% trifluoroacetic acid), and eluted with a concentration gradient of solvent B (acetonitrile, 0.1% trifluoroacetic acid) from 0 to 30%, at a flow rate of 0.5 mL/minute for 30 minutes.

Prothrombin Time (PT) Assay
Prothrombin time reagent (200 μL) and sample aliquots (200 μL) were pre-incubated for ten minutes at 37ºC and mixed for five seconds and then 100 μL of plasma was added and clotting time was recorded (13).One unit of anticoagulant activity corresponds to an increase of 20 seconds in normal plasma coagulation.

Amidolytic Activity
Proteolytic activity in the samples was assayed using BApNA as substrate.The substrate solution was prepared by dissolving BApNA in 5 mL of dimethyl methyl sulfoxide (DMSO) and adding 95 mL of 0.05 M Tris-HCl buffer (pH 8.2).Proteolytic activity was monitored as BApNA hydrolysis by mixing 50 μL of sample with 100 μL of the substrate solution.After ten minutes of incubation at 37ºC, the absorbance at 410 nm was measured.One unit of protease activity corresponds to an increase of A 410 = 0.001/minute (9).

Coagulant Activity
Plasma (200 μL) and different amounts of purified factor were pre-incubated briefly at 37ºC, then mixed and shaken, and the clotting times were recorded (3,8,14).

Proteolytic Activity on Casein
Proteolytic activity was determined by addition of various amounts of the enzyme to a buffer solution of 0.1 M Tris-HCl, pH 9.0, and the final volume was adjusted to 250 μL, followed by the addition of 750 μL of 1% (m/v) casein and incubated for 15 minutes at 37ºC.The reaction was stopped by adding 1.5 mL of 30% TCA.The resulting proteolysis products in the supernatant solution were evaluated spectrophotometrically at λ = 280 nm after centrifugation at 5000 rpm for ten minutes.
One unit of caseinolytic activity corresponds to an increase of A 280 = 0.001/minute (9).

Protein Determination
Protein concentration was measured by the method of Lowry et al. (15), using bovine serum albumin (BSA) as standard.

Selection of Snake Species
Crude venom from the Iranian snake species E. carinatus and A. halys were selected and assayed with PT test.The E. carinatus venom coagulated plasma very rapidly, which leads to the conclusion that E. carinatus venom contains procoagulant factors.
Crude venom of Agkistrodon halys was selected next: this venom delayed plasma coagulation.Thus it was concluded that the A. halys snake venom contains one or more anticoagulant factors.

Isolation of Anticoagulant Factor
In the initial Sephadex G-50 fractionation of the crude Agkistrodon halys venom, five peaks at 280 nm were obtained (AH1 to AH5) as shown in Figure 1.When all the fractions were tested for anticoagulation, it was found that fraction AH2 showed anticoagulation effect.Further purification was carried out by ion exchange chromatography on DEAE-Sepharose resin (Figure 2).In this purification step six fractions were obtained (AH21-AH26) out of which fraction AH21 showed anticoagulant activity.By this purification procedure, about 16.75 mg of purified enzyme was obtained from 182.5 mg of the venom (Table 1).

Purity and Determination of Molecular Weight
The homogeneity of purified enzyme was confirmed by SDS-PAGE and HPLC as shown in Figures 3 and 4, respectively.Isolated anticoagulant factor (AH21) showed high purity as analyzed by C18 reverse phase HPLC.This factor also showed a single band in SDS-PAGE.The molecular weight of this anticoagulant factor was estimated to be about 22 kDa under reduced conditions.Caseinolytic Clotting Assay and Amidolytic Activity AH21 did not exert any proteolytic activity on casein, coagulant activity on BAPNA or clotting activity upon human plasma.
Ghorbanpur M et al.Identification an partial purification of an anticoagulant factor from the venom of the Iranian 99 snake Agkistrodon halys.J Venom Anim Toxins incl Trop Dis.2010;16(1): Ghorbanpur M et al.Identification an partial purification of an anticoagulant factor from the venom of the Iranian 100 snake Agkistrodon halys.J Venom Anim Toxins incl Trop Dis.2010;16(1): Ghorbanpur M et al.Identification an partial of an anticoagulant factor from the venom of the Iranian 101 snake Agkistrodon halys.J Venom Anim Toxins incl Trop Dis.2010;16(1):

Figure 2 .
Figure 2. DEAE-Sepharose chromatography of AH2 obtained from Sephadex G-50.The pooled AH2 anticoagulant fraction was applied to DEAE-Sepharose column (2.5 x 20 cm) equilibrated with 0.05 M Tris-HCl buffer (pH 8.2).Proteins were eluted with a linear concentration gradient of NaCl from 0.0 to 0.5 M, and 4.5 mL fractions were collected from each tube.Series 1: NaCl concentration (mol/L) and series 2: absorbance at 280 nm.
Ghorbanpur M et al.Identification an partial purification of an anticoagulant factor from the venom of the Iranian 98 snake Agkistrodon halys.J Venom Anim Toxins incl Trop Dis.2010;16(1):

Table 1 .
Purification of an anticoagulant from A. halys venom