Protein Cryoprotectant Ability of the Aqueous Zwitterionic Solution

Area of Science:

• Biochemistry and Molecular Biology
• Materials Science
• Physical Chemistry

Background:

• Protein cryopreservation is crucial for long-term storage of unstable proteins, but requires specific solvent properties to prevent damage.
• Previous work demonstrated cryopreservation of N-acetylglucosaminyltransferase-V (GnT-V) using a dilute aqueous solution of the artificial zwitterion 3-(1-(2-(2-methoxyethoxy)ethyl)imidazol-3-io)butane-1-carboxylate (OE2imC3C).
• The specific solvent properties enabling cryoprotection by OE2imC3C remained unclear.

Purpose of the Study:

• To investigate the melting phenomena and solution structure of dilute binary aqueous OE2imC3C solutions.
• To compare the cryoprotectant ability of OE2imC3C with conventional cryoprotective agents (CPAs) like glycerol and dimethyl sulfoxide.
• To elucidate the solvent properties required for effective protein cryopreservation.

Main Methods:

• Differential scanning calorimetry (DSC) was used to analyze melting phenomena.
• Raman and Fourier transform infrared (FTIR) spectroscopies were employed to study solution structure.
• Molecular dynamics (MD) simulations were conducted to complement experimental findings and analyze water molecule behavior.

Main Results:

• Aqueous OE2imC3C solutions exhibited lower melting temperatures and required less energy for melting compared to control CPA solutions, attributed to a higher content of unfrozen water.
• Raman and FTIR data revealed that the high unfrozen water content resulted from hydration around the ionic groups (COO- and imidazolium ring) and the OCH2CH2O segment of OE2imC3C.
• MD simulations indicated fewer structured water molecules around the OCH2CH2O segment compared to the ionic hydration sites.

Conclusions:

• Dilute aqueous OE2imC3C solutions possess unique solvent properties, including a high content of unfrozen water, making them effective in preventing freezing even under deep freezer conditions.
• The specific hydration patterns around the zwitterionic molecule contribute to its cryoprotective capabilities.
• OE2imC3C demonstrates potential as a novel cryoprotectant for proteins in biotechnology and biomedical applications.