Effects of the concentration and heating rate on the thermal denaturation and reversibility of granulocyte-colony stimulating factor studied by circular dichroism and infrared spectroscopy

Temperature-dependent (25-90 °C) circular dichroism (CD) spectra were measured for recombinant methionyl human granulocyte-colony stimulating factor (rmethuG-CSF) in aqueous solutions over a pH range of 2.0-5.0 to investigate its thermal stability and reversibility. The effects of the protein concentration and heating rate on the thermal denaturation and reversibility were studied. We also compared the melting temperature (Tm) of rmethuG-CSF obtained from the CD spectra with that from the infrared (IR) spectra. The CD study of rmethuG-CSF with the concentration of 0.5 mg/mL has demonstrated that rmethuG-CSF is the most stable at pH 2.8 in the pH range of 2.0-5.0 when the heating rate is 1 °C/min. This result does not always agree with the IR result, obtained using the concentration of 10 mg/mL, that the secondary structure of rmethuG-CSF is the most stable at pD 2.5 in the pD range of 2.5-5.5. However, when the CD measurements are curried out for the protein concentration of 5.0 mg/mL and the heating rate of 0.2 °C/min, which are similar measurement conditions to those for the IR study, Tm obtained from the CD data is almost identical with that from the IR data. The reversibility of rmethuG-CSF has been found to be the highest at pH 2.0 by the CD analysis. It seems that the Tm of rmethuG-CSF is easily affected by the measurement conditions such as protein concentration and heating rate as its reversibility is low. The CD study have also revealed that as pH is increased β-structure is increased after heating.