Relationship Between Cryoprotectant Potential and Protein Hydration in Aqueous Zwitterionic Solutions

The cryoprotectant potential of 3-(1-(2-(2-methoxyethoxy)ethyl)imidazol-3-io)butane-1-carboxylate (OE₂imC₃C) for proteins necessitates assessment to elucidate its relationship with protein hydration. To reveal this relationship, we assessed the protein stability (pre-freezing and post-thawing) and melting behavior in dilute aqueous protein–OE₂imC₃C solutions containing varying mole fractions (x) of OE₂imC₃C (x = 0, 7.7 × 10⁻³, and 1.7 × 10⁻²) using Fourier-transform infrared (FTIR) and near-UV circular dichroism (near-UV CD) spectroscopy and differential scanning calorimetry (DSC) techniques. Following freezing/thawing using a deep freezer, protein stability in aqueous OE₂imC₃C solutions (x = 1.7 × 10⁻²) preserved the folded state owing to the protein–OE₂imC₃C interaction, whereas stability at x = 7.7 × 10⁻³ was reduced. These results indicate that the protein cryoprotectant potential in aqueous OE₂imC₃C solutions at x = 1.7 × 10⁻² is higher than that at x = 7.7 × 10⁻³, owing to the preferential binding of OE₂imC₃C with proteins.