色谱

色谱

• 研究论文 • 上一篇    下一篇

海南捕鸟蛛毒素-Ⅲ的固相化学合成和氧化复性

皮建辉1,2,全妙华1,2,曾雄智1

  

  1. 1.Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education,
    Hunan Normal University, Changsha 410081, China; 2.Department of Biological Engineering of Huaihua College, Huaihua 418008, China
  • 收稿日期:2006-09-14 修回日期:2006-11-28 出版日期:2007-05-30 发布日期:1985-09-25
  • 通讯作者: 曾雄智

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

PI Jianhui1,2, QUAN Miaohua1,2, ZENG Xiongzhi1   

  1. 1.Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education,
    Hunan Normal University, Changsha 410081, China; 2.Department of Biological Engineering of Huaihua College, Huaihua 418008, China
  • Received:2006-09-14 Revised:2006-11-28 Online:2007-05-30 Published:1985-09-25

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摘要：

应用芴甲氧羰基(Fmoc)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pH 7.5的重蒸水溶液体系，样品质量浓度为0.1 g/L，还原型谷胱甘肽和氧化型谷胱甘肽的浓度分别为 1.0 mmol/L和0.1 mmol/L、L-Arg浓度为1.0 mol/L。复性产物经质谱测定其相对分子质量为3607.68;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。

关键词: 反相高效液相色谱, 固相化学合成, 海南捕鸟蛛毒素-Ⅲ, 基质辅助激光解吸电离飞行时间质谱 , 结构与功能, 氧化复性

Abstract:

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 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 words: Hainantoxin-Ⅲ, martix-assisted laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS) , oxidative refolding, reversed-phase high performance liquid chromatography (RP-HPLC), structure and function, solid-phase chemical synthesis