[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 3 607.68 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 ratio of 1 : 1, and the effect of refolded HNTX-III on neuromuscular transmission in isolated mouse phrenic nerve-diaphragm preparations was almost 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.
Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, Hunan Normal University, Changsha 410081, China. pijh817@yahoo.com.cn
Jianhui
J
Pi
Miaohua
M
Quan
Xiongzhi
X
Zeng
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003964:Diaphragm / Q000187:drug effects / Q000294:innervation; D066298:In Vitro Techniques; D051379:Mice; D010084:Oxidation-Reduction; D010791:Phrenic Nerve / Q000187:drug effects; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013111:Spider Venoms / Q000138:chemical synthesis* / Q000737:chemistry* / Q000633:toxicity
D013111:Spider Venoms; C488683:hainantoxin III, Seleconosmia hainana