The Studies Of The Synthetic Mutant K27A Of Huwentoxin-â…£ And The Structures And Functions Of Two Neurotoxins From The Spider Chilobrachys Jingzhao

The Solid-phase chemical synthesis of K27A-HWTX-IV, a mutant of neurotoxic peptide huwnetoxin-IV(HWTX-IV), in which Lys-27 was substituted with Ala, was carried out using the Fmoc chemistry on the Pioneer Peptide Synthesizer System. The synthetic mutant was identified by MALDI-TOF-MS(matrix-assisted laser desorption/ionization-time of flight mass spectrometry). Reverse-phase HPLC was used to monitor the oxidative folding of the mutant under defferent reaction conditions in order to find the optimal condition forrenaturation of the mutant. The best renaturatioh yield wasreceived in 1mmol/L GSH and 0.1mmol/L GSSG at pH 8.0 in 0.1mol/L Tris-HCl buffer, and the concentration of the sample was 0.1mg/mL. The renaturated mutant was identified by MALDI-TOF-MS. The exiperiments of isolated mouse phrenic nerve-diaphragm preparation showed that the mutant K27A-HWTX-IV lost 86% of the biological activity of the native toxin, which indicates that Lys was the key residue to HWTX-IV.