Schwegman J Jeff, Carpenter John F, Nail Steven L
Department of Industrial and Physical Pharmacy, School of Pharmacy, Purdue University, 575 W. Stadium Mall Dr., West Lafayette, Indiana 47907, USA.
J Pharm Sci. 2007 Jan;96(1):179-95. doi: 10.1002/jps.20630.
A commercially available freeze-dry microscopy stage interfaced with an IR microscope is described as a method of in situ measurement of protein secondary structure in the liquid, frozen and freeze-dried states. Studies using solutions of model proteins demonstrated that spectra collected using the IR microscope have resolution and sensitivity that is comparable to techniques using a conventional infrared spectrometer. Additionally, spectra collected in triplicate on the microscope in the solution, frozen, and freeze-dried states and after reconstitution were shown to be reproducible. The limiting factor when collecting spectra on the infrared microscope appears to be the higher level of water vapor inherently present within the optical path of the microscope used in this study. Results demonstrate that the native secondary structure is perturbed in both the frozen and freeze-dried states, and bands characteristic of structural changes associated with freezing and drying stresses were observed in the Amide I region. Freeze-drying studies conducted in the presence of mannitol and sucrose demonstrated that perturbation to the native state secondary structure after freeze-drying was considerably reduced in the presence of these excipients.
一种与红外显微镜相连的市售冻干显微镜载物台被描述为一种在液体、冷冻和冻干状态下原位测量蛋白质二级结构的方法。使用模型蛋白溶液进行的研究表明，使用红外显微镜收集的光谱具有与使用传统红外光谱仪的技术相当的分辨率和灵敏度。此外，在溶液、冷冻、冻干状态下以及复溶后在显微镜上一式三份收集的光谱显示具有可重复性。在本研究中使用的红外显微镜上收集光谱时的限制因素似乎是显微镜光路中固有存在的较高水平的水蒸气。结果表明，天然二级结构在冷冻和冻干状态下均受到扰动，并且在酰胺I区域观察到与冷冻和干燥应力相关的结构变化特征带。在甘露醇和蔗糖存在下进行的冻干研究表明，在这些赋形剂存在下，冻干后对天然状态二级结构的扰动大大降低。
