Protein Cryoprotectant Ability of the Aqueous Zwitterionic Solution

Protein cryopreservation is important for the long-term storage of unstable proteins. Recently, we found that N-acetylglucosaminyltransferase-V (GnT-V) can be cryopreserved in a deep freezer without temperature control using a dilute binary aqueous solution of 3-(1-(2-(2-methoxyethoxy)ethyl)imidazol-3-io)butane-1-carboxylate (OE(2)imC(3)C) [10 wt %, mole fraction of solute (x) = 7.75 x 10(-3)], an artificial zwitterion. However, it is unclear which solvent properties are required in these media to preserve unstable proteins, such as GnT-V. In this study, we investigated the melting phenomena and solution structure of dilute binary aqueous OE(2)imC(3)C solutions [x = 0-2.96 x 10(-2) (0-30 wt %)] using differential scanning calorimetry (DSC) and Raman and Fourier transform infrared (FTIR) spectroscopies combined with molecular dynamics (MD) simulation to compare the cryoprotectant ability of OE(2)imC(3)C with two general cryoprotectants (CPAs), glycerol and dimethyl sulfoxide. DSC results indicated that aqueous OE(2)imC(3)C solutions can be melted at lower temperatures with less energy than the control CPA solution, with increasing x, primarily due to OE(2)imC(3)C having a higher content of unfrozen water molecules. Moreover, Raman and FTIR results showed that the high content of unfrozen water molecules in aqueous OE(2)imC(3)C solutions was due to the hydration around the ionic parts (the COO- group and imidazolium ring) and the OCH2CH2O segment. In addition, the MD simulation results showed that there were fewer structured water molecules around the OCH2CH2O segment than the hydration water molecules around the ionic parts. These solvent properties suggest that dilute aqueous OE(2)imC(3)C solutions are effective in preventing freezing, even in a deep freezer. Therefore, this medium has the potential to act as a novel cryoprotectant for proteins in biotechnology and biomedical fields.