An infrared spectroscopy study of acid stability and thermal unfolding process of granulocyte-colony stimulating factor.
Temperature-dependent (25-80 degrees C) infrared (IR) spectra were obtained for recombinant methionyl human granulocyte-colony stimulating factor (rmethuG-CSF) in aqueous solutions over the pD range of 5.5-2.1 to investigate its thermal stability at various pDs. Second derivative, Fourier self-deconvolution, and curve-fitting analyses were performed to analyze the obtained spectra. These spectral analyses demonstrated that in the thermal unfolding process the alpha-helix structure of rmethuG-CSF partially changes to an unordered structure and then the unordered structure forms aggregates. The temperature-dependent IR spectra revealed that the structure of rmethuG-CSF is the most stable at pD 2.5 in the pD range of 5.5-2.1. It has been suggested that the unordered structure formed before the marked structural change in the whole molecule is a perturbed form of the native structure of rmethuG-CSF and plays a role as a precursor for the aggregation. This alteration to the perturbed form is likely to be the first secondary structure change that occurs along the aggregation pathway. Of particular note is that the stability at pD 2.1 is slightly lower than that at pD 2.5, but that aggregates are formed at higher temperature at pD 2.1 than at pD 2.5, probably because the repulsive interaction between the unordered structure is stronger at pD 2.1.
Yamazaki
Katsuyoshi
K
Product Development Section, CMC R&D Laboratories, Pharmaceutical Division, Kirin Brewery Co., LTD., Takasaki, Gunma 370-0013.
Murayama
Koichi
K
Ishikawa
Rika
R
Ozaki
Yukihiro
Y
D004355:Drug Stability; D005583:Fourier Analysis; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation / Q000502:physiology*; D017510:Protein Folding; D011994:Recombinant Proteins; D013055:Spectrophotometry, Infrared
D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor