[Reversed-phase high performance liquid chromatographic behavior of unfolding procedure of Huwentoxin-V]

Huwentoxin-V (HWTX-V) is an insecticidal peptide purified from the venom of spider Selnocosmia huwena. HWTX-V contains 33 amino acid residues, including six cysteine residues that form three pairs of disulfide bond. A method for measuring unfolding intermediates of HWTX-V by reversed-phase high performance liquid chromatography (HPLC) has been established. HWTX-V was first denatured over 30 min in the guanidine hydrochloride, and then reduced by using tris-(2-carboxyethyl)-phosphine at pH 3.0 over 12 min. All intermediates were separated on a C18 column (4.6 mm i. d. x 390 mm) with a linear gradient elution of acetonitrile containing 0.1% (v/v) trifluoroacetic acid, and identified by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Finally, these intermediates were carboxamidomethylated by iodoacetamide at the concentration of 0.5 mol/L and verified by MALDI-TOF MS. The diverse chromatographic retention behaviors for intermediates of Huwentoxin-V are discussed. This method is helpful to reveal the conformation changes in the procedures of proteins/peptides unfolding.