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A Microfluidic Approach for the Study of Ice and Clathrate Hydrate Crystallization
Published on: August 18, 2022
Freezing-induced phase separation and spatial microheterogeneity in protein solutions
Jinping Dong1, Allison Hubel, John C Bischof
1Characterization Facility, Institute of Technology, Mechanical Engineering Department, 111 Church Street Southeast, University of Minnesota, Minneapolis, Minnesota 55455, USA.
The Journal of Physical Chemistry. B
|July 8, 2009
Summary
Understanding cryoprotectant mechanisms is key for stabilizing biomolecules. Confocal Raman microspectroscopy reveals how trehalose and supercooling impact protein structure and aggregation during freezing, offering insights into lyophilization and cryopreservation.
Area of Science:
- Biophysics
- Materials Science
- Biochemistry
Background:
- Lyophilization and cryopreservation are vital for stabilizing biomolecules but their molecular mechanisms remain unclear.
- Molecular interactions significantly influence preservation outcomes, yet direct probing remains challenging.
Purpose of the Study:
- To quantify freezing-induced microheterogeneity and phase separation in protein-trehalose solutions.
- To investigate the impact of supercooling and trehalose concentration on molecular interactions and protein structure during freezing.
Main Methods:
- Confocal Raman microspectroscopy was employed to analyze frozen solutions of lysozyme (model protein) and trehalose (cryoprotectant).
- Quantitative spectral analysis was performed across ice, freeze-concentrated liquid (FCL), and interface regions.
- Samples were subjected to varying degrees of supercooling before freezing.
Main Results:
- Freezing microstructure depended on both trehalose concentration and the degree of supercooling.
- High supercooling led to occlusion of lysozyme and trehalose within the ice phase.
- Lysozyme retained native structure in FCL but denatured in ice; high trehalose and supercooling induced protein-sugar aggregation.
Conclusions:
- The degree of supercooling and cryoprotectant concentration critically influence phase separation and molecular stability during freezing.
- Confocal Raman microspectroscopy provides quantitative insights into molecular behavior during cryopreservation.
- Understanding these interactions is crucial for optimizing lyophilization and cryopreservation protocols for proteins and complex organisms.
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