Solid-Phase Chemical Synthesis and Oxidative Refolding of Hainantoxin-III

Hainantoxin-III (HNTX-III) was synthesized using solid-phase chemical method with Fmoc-protected amino acids. Reversed-phase high performance liquid chromatography (RP-HPLC) was used to monitor the oxidative refolding of linear HNTX-III. It is found that the best refolding yield for 0.1 g/L linear HNTX-III could be obtained in double-distilled aqueous solution at pH 7.5 containing 1.0 mol/L L-arginine, 1.0 mmol/L reduced glutathione (GSH), and 0.1 mmol/L oxidized glutathione (GSSG). The relative molecular mass of the refolded HNTX-III was determined to be 3607.68 by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Only one peak was found in the RP-HPLC chromatogram for the mixed sample of native HNTX-III and refolded HNTX-III at a ratio of 1:1, and the effect of refolded HNTX-III on neuromuscular transmission in isolated mouse phrenic nerve-diaphragm preparations was almost the same as that of native HNTX-III. The results indicate that refolded HNTX-III had the same structure and biological activities as the native HNTX-III.