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 (OE₂imC₃C) [10 wt %, mole fraction of solute (x) = 7.75 × 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 OE₂imC₃C solutions [x = 0-2.96 × 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 OE₂imC₃C with two general cryoprotectants (CPAs), glycerol and dimethyl sulfoxide. DSC results indicated that aqueous OE₂imC₃C solutions can be melted at lower temperatures with less energy than the control CPA solution, with increasing x, primarily due to OE₂imC₃C having a higher content of unfrozen water molecules. Moreover, Raman and FTIR results showed that the high content of unfrozen water molecules in aqueous OE₂imC₃C 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 OE₂imC₃C 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.