Measuring changes in protein secondary structure in-situ during freezing and freeze-drying using infrared microscopy.

摘要

The goal of this research was twofold, (1) determine the feasibility of combining a freeze-dry microscopy stage with an infrared microscope as a method of in-situ measurement of protein secondary structure in the liquid, frozen and freeze-dried states, and (2) use this instrumentation to gain a better understanding at the molecular level of the mechanism of loss of integrity of proteins during freezing. Studies using solutions of model proteins demonstrated that spectra collected using the infrared microscope had resolution and sensitivity that was better or comparable to techniques using a conventional infrared spectrophotometer. Additionally, spectra collected in triplicate on the microscope in the solution state, the frozen state, the freeze-dried state 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 this microscope. Results demonstrated 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. Annealing in the presence of mannitol prior to freeze-drying resulted in an increase in the loss of native state structure which is consistent with loss of the protective effect due to crystallization of mannitol. Annealing in the presence of both mannitol and sucrose prior to freeze-drying did not however show a significant loss of native state structure due to the fact that sucrose is remaining amorphous. Spectra of model proteins collected both in the interstitial space and on the surface of ice crystals in a partially frozen system revealed that significant structural changes were occurring on the surface of ice crystals relative to the interstitial space These results demonstrate evidence of ice-induced protein denaturation at the ice/freeze concentrate interface. The addition of surfactants significantly reduced the amount of structural perturbation at this interface.