蜂毒肽的分离纯化及结构鉴定

Isolation, purification and structural identification of melittin

中文关键词:  蜂毒肽  蜂毒  结构鉴定  分离纯化

英文关键词:melittin  bee venom  structural identification  isolation and purification

中文摘要:

      目的 对蜂毒肽进行分离纯化, 并进行结构鉴定。方法 以意大利蜜蜂蜂毒为原料, 经溶解、超声、浸提、过滤等, 并制备C18液相色谱柱, 对一次纯化采用的流动相、洗脱程序进行优化, 对二次纯化时进样量、进样浓度进行选择, 冻干后得到高纯度蜂毒肽。利用基质辅助激光解析电离飞行时间质谱法(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF MS)和液相色谱-串联质谱法(liquid chromatography-tandem mass spectrometry, LC-MS/MS)进行分子量测定等定性研究, 利用傅里叶变换红外光谱(Fourier transform infrared spectroscopy, FT-IR)进行二级结构表征, 尝试利用低场核磁共振(nuclear magnetic resonance, NMR)技术对其氢谱定性解析, 基于高效液相色谱法(high performance liquid chromatography, HPLC)的面积归一化法测定蜂毒肽纯度。结果 蜂毒第一次采用三氟乙酸水(2/998, V/V)和乙腈洗脱, 第二次采用三氟乙酸水(2/998, V/V)和甲醇洗脱, 经过冻干后成功得到相对分子质量为2845.9 Da的蜂毒肽, 纯化过程中未发生结构修饰, 红外光谱显示蜂毒肽的二级结构呈α-构象并有部分弯曲, 液相色谱面积归一化法测定蜂毒肽纯度达99.67%。结论 采用制备液相法分离纯化蜂毒肽操作简单、方便易行, 对设备要求低, 纯化后不会改变蜂毒肽的结构, 可用于蜂毒肽标准品原料积累, 为蜂毒肽纯度标准物质的研制和市场上相关产品中蜂毒肽含量准确测定奠定基础。

英文摘要:

      Objective To isolate and purify melittin and identify its structure. Methods The venom of Apis mellifera was used as raw material, which was pretreated by lysis, sonication, extraction and filtration, etc., combined with the preparation of liquid chromatography with C18 column, the mobile phase and elution procedure used for primary purification were optimized, the injection volume and concentration were selected for secondary purification, and high purity melittin was obtained after lyophilization. Qualitative studies such as molecular weight determination were carried out by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fourier transform infrared spectroscopy (FT-IR) was used to characterise the secondary structure of melittin, and low field nuclear magnetic resonance (NMR) was used to analyze the hydrogen spectrum of melittin, and the purity of melittin was determined by area normalization method based on high performance liquid chromatography (HPLC). Results The melittin was firstly purified and lyophilized by trifluoroacetic acid in water (2/998, V/V) and acetonitrile. Melittin was then lyophilised with trifluoroacetic in water (2/998, V/V) and methanol, giving a liquid phase melittin purity of 99.67%. The molecular weight of melittin was determined by MALDI-TOF MS to be 2845.9 Da, and no other impurities were found after the full scan by LC-MS/MS. The secondary structure of melittin showed α-helix with partial bending. Conclusion The preparation liquid phase method is simple, convenient and easy to separate and purify melittin. It has low requirements on equipment and does not change the structure of melittin after purification. It can be used for the accumulation of standard materials of melittin, which lays a foundation for the development of standard materials of melittin purity and the accurate determination of content of melittin in related products on the market.