Development of high pressure cells for circular dichroism and high pressure effects on protein secondary structure.

为了在生物科学和生物技术领域建立高压光谱的原位观测方法，研制了一种方便、实用的圆二色性（CD）测定池，并研究了聚谷氨酸和聚赖氨酸的α、β和随机结构随压力的变化。这些研究扩展到测量核糖核酸酶A和羧肽酶Y及其遗传突变形式的压力效应。研究结果如下：1.以石英、蓝宝石和人造钻石作为高压CD盒的窗口，测试了其耐压性和紫外光吸收率。结果表明，金刚石窗适用于230 ~可见光区CD的测量.测量了不同pH值下压力对聚谷氨酸和聚赖氨酸的α、β和无规结构的影响。不幸的是，由于金刚石窗强烈吸收220 nm的紫外光，光谱很难清晰描述.测定了不同pH和温度下牛胰核糖核酸酶A和羧肽酶Y的压力效应，并测定了它们的Pm。用突变酶测定了Pm.用核糖核酸酶A测量了CD在280 nm处的压力效应，证明了其三级结构随压力的变化与随温度的变化一样是两态跃迁。

To establish in situ observation of high pressure spectroscopy in the field of bioscience and biotechnology, convenient and routine cells for circular dichroism (CD) were developed and pressure dependent changes of α, β and random structures in poly-glutamic acid and poly-lysine were studied. These studies were extended to measure the pressure effects on ribonuclease A and carboxypeptidase Y and their genetically mutated forms. The results are as follows:1. Pressure tolerance and absorbance of ultra violet light were tested with quartz, sapphires, and artificial diamonds as windows of high pressure CD cells. As the results, the diamond window was found to be applicable to measure CD from 230 to visible light region.2. Pressure effects on α, β and random structures of poly-glutamic acid and poly-lysine were measured under various pH. Unfortunately, since diamond windows absorbed strongly ultra violet light at 220 nm, clear description of spectrum was difficult.3. Pressure effects on bovine pancreatic ribonuclease A and carboxypeptidase Y were measured under various pH and temperature and their Pm were determined. Pm was also determined with mutant enzymes.4. Pressure effects of CD at 280 nm were measured with ribonuclease A and the pressure dependent change of the tertiary structure was proved to be the two state transition as the temperature dependent change.