Heptakis (2,3,6-tri-O-methyl)-b-cyclodextrin (TOM-b-CyD) has recently been used to alter the membrane cholesterol content. For example, using TOM-b-CyD, cholesterol of the myometrial plasma was selectively depleted from the myometrial plasma membrane.1) TOM-b-CyD has also been used to clarify whether membrane proteins exist at an association with specialized microdomains called lipid rafts by depleting cholesterol contained in them.2) It is clear that cholesterol forms a soluble complex with TOM-b-CyD in aqueous solution. Although the interactions of cholesterol with TOM-b-CyD are particularly important, to the best of our knowledge, no studies on the thermodynamic parameters of the interaction in aqueous solution have been reported.We therefore investigated the interactions of cholesterol with TOM-b-CyD in aqueous solution quantitatively using the solubility measurement method now common in the pharmaceutical field, measurement of proton nuclear magnetic resonance ( 1 H-NMR), and Corey-Pauling-Koltum (CPK) atomic models. As cholesterol exists in a buried form in the phospholipid bilayer in biomembranes 3,4) the interactions of cholesterol with phospholipids are suggested to be based on the hydrophobic interaction. On the other hand, TOM-b-CyD has a deeper and more hydrophobic cavity than the parent cyclodextrin.5) Therefore, it is suggested that the interaction of cholesterol with TOM-b-CyD is based on the hydrophobic interaction in aqueous solution. In a previous paper, 6) we reported that heptakis (2,6-di-O-methyl)-b-cyclodextrin (DOM-b-CyD), which like TOM-b-CyD has a deeper and more hydrophobic cavity than the parent cyclodextrin, forms soluble complexes with cholesterol in aqueous solution.However, the inclusion behaviors of these hydrophobic cyclodextrins differ in general. For example, DOM-b-CyD penetrates the matrix of liposomes and extracts phospholipid from liposomes to form a soluble complex, whereas only a small amount of TOM-b-CyD penetrates the matrix of liposomes to remain there and therefore, TOM-b-CyD has very week ability to form a soluble complex with phospholipids. 7,8) For these reasons, TOM-b-CyD is used to alter membrane cholesterol content as mentioned above, although DOM-b-CyD is not used for this purpose. As another example, the formation constant of the complex between TOMb-CyD 9) and 8-anilino-1-naphthalenesulfonate (ANS) is smaller than that between DOM-b-CyD 10) and ANS. Furthermore, each incluson mode is different in the TOM-b-CyD-ANS complex and DOM-b-CyD-ANS complex. Therefore the comparison of the interaction between TOM-b-CyD and cholesterol with that between DOM-b-CyD and cholesterol in present paper would be informative and pertinent.
ExperimentalMaterials TOM-b-CyD purchased from Nacalai Tesque Co. (Kyoto, Japan) was used after recrystallization from water and dried for 12 h at 110°C in a vacuum before use. Cholesterol purchased from Sigma (St. Louis, MO, U.S.A.) was used without further purification. Water purified with Milli-Q Labo (Ͼ18 MW · cm) was used throughout the ex...