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College Park, Maryland      June 6 - 10 , 2004

MP50: Role of solvent in protein preservation

G. H. Caliskan (The University of Akron, Polymer Science Department), M. T. Cicerone (NIST Polymer Division), M. Gangoda, R. B. Gregory (Kent State University, Department of Chemistry), A. Kisliuk (The University of Akron, Polymer Science Department), I. Peral (University of Maryland, College Park), J. H. Roh, A. P. Sokolov (The University of Akron, Polymer Science Department)

Protein dynamics span over a broad time range from picoseconds, where vibrations take place, up to years, the target of pharmaceutical industry for biopreservation. Understanding the dynamics is vital in controlling the activity of biological macromolecules, and improving the designs for biopreservation formulations. High TG sugars are usual choice for biopreservation at ambient temperatures. However, glycerol (TG ~ 190 K) is the most effective solvent for long-term (years) cryo-preservation. The reason for this difference in effectiveness of the solvents remains unclear.

We measured dynamics of hen egg white lysozyme in various solvent conditions, (glycerol, water, trehalose and dry state) in a broad temperature (150 K to 320 K) range. Time-of-Flight Spectrometer at the National Institute of Standards and Technology Center for Neutron Research offered a broad range of energy from ~ 40 μeV up to ~ 65 meV where the fast relaxation (local conformational fluctuations) and slow vibrations of proteins become accessible. Our results show that the fast relaxation depends strongly on the solvent. It is strongly suppressed in liquid glycerol compared to solid trehalose (TG ~390 K) at low temperatures. At high temperatures (> 270 K), it becomes enhanced in glycerol and in water environments, as opposed to that in trehalose and in dry conditions. This observation quantitatively agrees with bioactivity measurements on Myoglobin in glycerol and trehalose at different temperatures. The results suggest that the viscosity of the solvent is not the only parameter affecting the protein dynamics. Fast picosecond relaxations should also be taken into consideration. We propose a mechanism that might explain extreme biopreservation efficiency of glycerol at low temperatures. Possible implications for development of better formulations are discussed.

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