2.8. Thermal Stability Determination by Differential Scanning Calorimetry

Differential scanning calorimetry (DSC) was performed using a CSC-6300 NanoDSC III calorimeter, and data were acquired and interpreted using the DSCRun 4.4.9 and NanoAnalyze 3.7.5 software, respectively (TA Instruments, New Castle, DE, USA). Purified, dialysed RBDmfc protein samples were adjusted to 0.605 mg/mL with PBS. The instrument sample cell was filled with the protein sample (exactly 299 µL), and the reference cell was filled with PBS (the same PBS batch as used for sample dialysis and concentration adjustment). A PBS blank was also performed for further baseline correction.

Data were collected using a heating rate of 1 °C/min from 20 °C to 100 °C under 3 atmospheric pressure and after a pre-equilibration step of 600 sec at 20 °C. The sample’s raw thermogram (µJ/s vs. temperature) was baseline subtracted versus the PBS thermogram, then converted to molar heat capacity (defined as the amount of energy in the form of heat needed to raise the temperature of one mole of purified RBDmfc protein by one Kelvin) using their respective molecular weight to obtain kJ/mol.K vs. temperature thermograms. The integration of transition peaks was then achieved using a sigmoidal baseline method before calculating thermodynamic parameters.