中文摘要
利用悬滴法在微液滴中培养溶菌酶和胰岛素酶微晶，获得不同培养条件下多种的蛋白质微晶（单晶）。结合原位显微拉曼技术，改变环境湿度，实现了微晶中各类水分子脱水过程中，蛋白质微晶的原位拉曼观测。发现棒状的溶菌酶单晶具有较高的水含量，当相对湿度由95%下降到86%时，晶体首先失去大量的体相水（占总量的80%），溶菌酶的疏水腔结构没有变化，继续降低湿度由86%到65%，虽然仅有有少量溶剂化水继续失去（15%），但疏水腔结构发生突变。对于胰岛素酶，也发现结构不同的两种微晶（R6和T6），在失水过程中，蛋白分子结构的细微变化。围绕微液滴中的一些物理化学过程，如晶体的形成、相分离、离子对结构、晶体表面结构、相变等基本问题，利用微区拉曼，结合高速摄像，FTIR-ATR等技术，开展了系统研究。
英文摘要
Micro crystals of lysozyme and Insulin were prepared in droplets with the hanging drop vapor diffusion technique. It was found that the Lysozyme and Insulin crystals with different structures can be obtained when changing the conditions of mother solutions and the reservoirs. Con-focal Raman technique was set up to monitor structure change of the microcrystals of the proteins with changing the relative humidity. For the rod-like lysozyme crystal with highest water content, there was no change for the hydrophobic clusters of lysozyme when 80% total water （bulk water）in the crystal envaporated with dcreasing relative humidity (RH) to 86%. Further decreasing RH to 65%, the destruction of hydrophobic clusters of lysozyme was induced by the expulsion of the hydration water (only 15% in total) from the crystal.In contrast to the rod-like lysozyme crystal, tetragonal crystal and floor-like precipitate with a smaller amount of water showed no change of the structures of the hydrophobic clusters when the relative humidity was decreasing. The presence of bulk water in the rod-like crystal is believed a necessary factor for the function of the hydration water which promotes the hydrophobicity of hydrophobic clusters. For the Insulin crystal (R6 and T6), structure change were also observed when the RH decreasd. In order to understand the physical and chemical process of the protein crystal in the droplets, foundmental problems such as the formation of crystal, phase separation, structure of ion pairs, phase transformation, were also studied by FTIR-ATR, micro-Raman, high speed imagimg techniques.
结题摘要
利用悬滴法在微液滴中培养溶菌酶和胰岛素酶微晶，获得不同培养条件下多种的蛋白质微晶（单晶）。结合原位显微拉曼技术，改变环境湿度，实现了微晶中各类水分子脱水过程中，蛋白质微晶的原位拉曼观测。发现棒状的溶菌酶单晶具有较高的水含量，当相对湿度由95%下降到86%时，晶体首先失去大量的体相水（占总量的80%），溶菌酶的疏水腔结构没有变化，继续降低湿度由86%到65%，虽然仅有有少量溶剂化水继续失去（15%），但疏水腔结构发生突变。对于胰岛素酶，也发现结构不同的两种微晶（R6和T6），在失水过程中，蛋白分子结构的细微变化。围绕微液滴中的一些物理化学过程，如晶体的形成、相分离、离子对结构、晶体表面结构、相变等基本问题，利用微区拉曼，结合高速摄像，FTIR-ATR等技术，开展了系统研究。