Methanol-induced tertiary and secondary structure changes of granulocyte-colony stimulating factor.
We have studied methanol-induced conformational changes in rmethuG-CSF at pH 2.5 by means of circular dichroism (CD), fluorescence and infrared (IR) spectroscopy, and 8-anilino-1-naphthalene sulfonic acid (ANS) binding. Methanol has little effect on the secondary and tertiary structures of rmethuG-CSF when its concentration is in the range of 0 to 20% (v/v). At 30% (v/v) methanol, rmethuG-CSF has ANS binding ability. In the methanol concentration range of 30 to 70% (v/v) the amount of alpha-helix decreases a little, and the tertiary structure decreases significantly. At methanol concentrations above 70% (v/v), a transition to a more helical state occurs, while there is little change in the tertiary structure, and no ANS binding ability. Thermal denaturation studies involving CD have demonstrated that as the methanol concentration increases the melting temperature and the cooperativity of transition decrease, and the transition covers a much wider range of temperature. It seems that the decreased cooperativity means an increase in the concentration of partially folded intermediate states during the unfolding of rmethuG-CSF.
Yamazaki
Katsuyoshi
K
Product Development Section, CMC R&D Laboratories, Pharmaceutical Division, Kirin Brewery Co., Ltd, Takasaki, Gunma 370-0013.
Iwura
Takafumi
T
Ishikawa
Rika
R
Ozaki
Yukihiro
Y
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D000069585:Filgrastim; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry*; D006358:Hot Temperature; D000432:Methanol / Q000008:administration & dosage / Q000494:pharmacology*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D013055:Spectrophotometry, Infrared
D000817:Anilino Naphthalenesulfonates; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; C027132:1-anilino-8-naphthalenesulfonate; D000069585:Filgrastim; D000432:Methanol