Purification and characterization of a novel type of neurotoxic peptides from the venom of the Iranian scorpion Hemiscorpius lepturus

Abstract

Objectives: Scorpion venom has toxic effects on mammals, insects and crustaceans. Toxicogenic peptides are major contributors to the scorpion venom, which make it toxic. The Hemiscorpius lepturus (H. lepturus) is one of the most common scorpion bites agent, and responsible for 95% of scorpion bite deaths cases in Iran.

Materials and methods: In this project, we fractionated the H. lepturus scorpion venom and analyzed toxic fractions of the venom. The crude venom of H. lepturus was dialyzed against distilled water and then the soluble part of the venom was isolated from the non-soluble (mucoproteins) part of the venom and loaded onto the Sephadex G-50 gel filtration column, then after determining the toxicity of the obtained fractions (fractions toxicity were detected in mice by IV injection), the resulting toxic fraction was purified with three stages of ion-exchange chromatography (anion and cationic) and RP-HPLC. The purity of the fractions was verified by SDS-PAGE electrophoreses.

Results: The LD₅₀ of H. lepturus venom was 177.01 µg/mouse. The crude venom had 7 detectable bands with molecular weights of 10-100 KDa and one band less than 10 KDa. Finally, after the different stages of chromatography, two HL2153 and HL2155 peaks were obtained from the RP-HPLC, which were depicted single bands and high purity. The electrophoretic analysis showed molecular weight 4874 Da for HL2153 and 5107 Da for HL2155 toxins.

Conclusion: It is concluded that H. lepturus venom contains two HL2153 and HL2155 toxins with a relatively similar molecular weight and similar electrical charge 4874 and 5107 Da, respectively.

Keywords: Hemiscorpius lepturus; Purification; Scorpion venom; Toxic peptides; Toxin.

Figure 1

Gel filtration chromatogram of Hemiscorpius lepturus crude venom: The crude venom amount 500 mg, stationary phase Sephadex G50, column 150×2.3 cm, mobile phase ammonium acetate buffer, absorbance wave number 280 nm, and collection tubes 10 ml/tube

Figure 2

Anion-exchange chromatogram of HL2 peak obtained from gel filtration: stationary phase DEAE-Sepharose, column 50×1.6 cm, elution flow rate with a linear gradient of 300 ml of 20 mM tris base (pH 8.3) and 300 ml of the same buffer containing 0.5 M NaCl, and absorbance wave number 280 nm, and collection tubes 5 ml/tube

Figure 3.

Cation-exchange chromatogram of HL21 peak obtained from anion-exchange chromatography: stationary phase CM- Sepharose, column 20×1.6 cm, elution flow rate 30ml/hr with a linear gradient of 250 ml of 20 mM sodium acetate and 250 ml of the same buffer containing 0.5 M NaCl, and absorbance wave number 280 nm, and collection tubes 5 ml/tube

Figure 4

RP-HPLC chromatogram of HL215 obtained from Cation-exchange chromatography: C8 column with a linear gradient of solution A (0.1% trifluoroacetic acid in water) and solution B (0.1% TFA in acetonitrile). The protein fractions were detected at 215 and 280 nm. Totally 10 protein peaks were obtained and pooled

Figure 5

SDS-PAGE of crude Hemiscorpius lepturus venom and toxic protein peaks from chromatographic columns: Crude venom (Line 2), HL2 (Line 3) from Sephadex G50 column, HL21 (Line 4) from DEAE-Sepharose column, HL215 (Line 5) from CM- Sepharose column, HL2153 (Line 6) and HL2155 (Line 7) from RF-HPLC system were run on 20% SDS-PAGE under non-reducing condition. Line 1 shows the protein molecular weight marker: (A) 100 kDa, (B) 75 kDa, (C) 50 kDa, (D) 37 kDa, (E) 25 kDa (F) 20 kDa,(G) 15 kDa, (H) 10 kDa