摘要
应用芴甲氧羰基(Fm o c)固相化学合成了海南捕鸟蛛毒素-Ⅲ,并优化了其最佳氧化复性条件。最佳的氧化复性条件为pH7.5的重蒸水溶液体系,样品质量浓度为0.1g/L,还原型谷胱甘肽和氧化型谷胱甘肽的浓度分别为1.0mm o l/L和0.1mm o l/L、L-A rg浓度为1.0m o l/L。复性产物经质谱测定其相对分子质量为3 607.68;与天然毒素等量混合后用高效液相色谱分析得到单一峰;膈神经-膈肌标本生理实验结果表明,合成的毒素具有与天然毒素相同的生物学活性,从而可确定二者在结构与功能上具有一致性。
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-argiirine, 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 l: 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.
出处
《色谱》
CAS
CSCD
北大核心
2007年第3期399-403,共5页
Chinese Journal of Chromatography
基金
国家自然科学基金重点项目(No.30430170)
关键词
固相化学合成
海南捕鸟蛛毒素-Ⅲ
氧化复性
结构与功能
反相高效液相色谱
基质辅助激光解吸电离飞行时间质谱
solid-phase chemical synthesis
Hainantoxin-Ⅲ
oxidative refOlding
structure and function
reversed-phase high performance liquid chromatography (RP-HPLC)
martix-assisted laser desorption/ionization time-of-flight mass spectrometry ( MALDI-TOF MS)