Purification and Characterization of a Nonenzymatic Neurotoxin from Hippasa partita (Lycosidae) Spider Venom Gland Extract
Figure 1
Isolation of neurotoxin from H. partita venom. (a) Elution profile from Sephadex G-100 column chromatography. The column (1.5 × 75 cm) was eluted with 0.1 M NaCl at a flow rate of 22 mL/hr, and 2.2 mL fractions were collected. Protein elution was monitored at 280 nm (—). Neurotoxin fractions (bar line) were pooled, concentrated, desalted, and applied onto CM-Sephadex C-25 column for further fractionation. (b) Elution profile from CM-Sephadex C-25 column chromatography. The column (1.6 × 25 cm) was equilibrated with 0.05 M sodium phosphate buffer (pH 7.0). Subfractions were eluted stepwise using sodium phosphate buffer of different molarities (0.05 M–0.1 M) and pH (7.0–7.5). Neurotoxin fractions (bar line) were pooled, concentrated, and desalted. (c) RP-HPLC profile of neurotoxin on a Vydac C4 column (5 μm, 0.21 × 25 cm), that had been equilibrated with 0.1% TFA in water. Protein was eluted using linear gradient from solution A (0.1% TFA in water) to 100% solution B (0.1% TFA in acetonitrile) over 40 min. Protein was eluted at a flow rate of 1 mL/min and monitored at 280 nm. (d) SDS-PAGE pattern of purified neurotoxin. Purification of neurotoxin as shown in SDS-PAGE (10%). Samples containing 80 μg H. partita venom (1), 75 μg Sephadex G-100 fraction (2), and 20 μg of neurotoxin under nonreduced (3) and reduced (4) conditions. M represents the molecular weight markers in kDa (from top to bottom: phosphorylase b (97.4), bovine serum albumin (66.2), ovalbumin (45.0), carbonic anhydrase (31.0), soya bean trypsin inhibitor (21.5), and lysozyme (14.4)). (e) Mass spectrometry of neurotoxin using Voyager DE-PRO (PerSeptive Biosystems) MALDI-TOF machine in positive ionization mode. 3,5-Dimethoxy-4-cinnamic acid is used as matrix.
