© 2006 The Japanese Biochemical Society.
Regular Paper |
Methanol-Induced Tertiary and Secondary Structure Changes of Granulocyte-Colony Stimulating Factor
1 Product Development Section, CMC R&D Laboratories, Pharmaceutical Division, Kirin Brewery Co., Ltd, Takasaki, Gunma 370-0013; and 2 Department of Chemistry, School of Science and Technology, Kwansei-Gakuin University, Sanda 669-1337
* To whom correspondence should be addressed. Tel: +81-79-565-8349, Fax: +81-79-565-9077, E-mail: ozaki{at}kwansei.ac.jp
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 
-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.