title: Interfacial Recognition of Acetylcholine by an Amphiphilic p-Sulfonatocalix[8]arene Derivative Incorporated into Dimyristoyl Phosphatidylcholine Vesicles.
abstract: Dodecyl ether derivatives 1-3 of p-sulfonatocalix[n]arene were incorporated into dimyristoyl phosphatidylcholine (DMPC) vesicles, and their binding abilities for acetylcholine (ACh) were examined by using steady-state fluorescence/fluorescence anisotropy and fluorescence correlation spectroscopy (FCS). For the detection of ACh binding to the DMPC vesicles containing 5 mol % of 1-3, competitive fluorophore displacement experiments were performed, where rhodamine 6G (Rh6G) was used as a fluorescent guest. The addition of Rh6G to the DMPC vesicles containing 3 resulted in a decrease in the fluorescence intensity of Rh6G with an increase of its fluorescence anisotropy, indicating that Rh6G binds to the DMPC-3 vesicles. In the case of DMPC-1 and DMPC-2 vesicles, significant changes in the fluorescence spectra of Rh6G were not observed. When ACh was added to the DMPC-3 vesicles in the presence of Rh6G ([3]/[Rh6G]=100), the fluorescence intensity of Rh6G increased with a decrease in its fluorescence anisotropy. From the analysis of fluorescence titration data, the association constants were determined to be 7.1×10⁵ M-1for Rh6G-3 complex and 1.1×10² M-1for ACh-3 complex at the DMPC-3 vesicles. To get a direct evidence for the binding of Rh6G and its displacement by ACh at the DMPC-3 vesicles, diffusion times of the Rh6G were measured by using FCS. Binding selectivity of the DMPC-3 vesicles for ACh, choline, GABA, L-aspartic acid, L-glutamic acid, L-arginine, L-lysine, L-histamine and ammonium chloride was also evaluated using FCS.
authors.affiliation: Nano-bio Materials Laboratory, Immunology Frontier Research Center, Osaka University, Suita, Osaka, 565-0871, Japan. jin@fbs.osaka-u.ac.jp.|Graduate School of Frontier Biosciences, High Performance Bioimaging Facility, Suita, Osaka University, Osaka 565-0871, Japan. jin@fbs.osaka-u.ac.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Jin
authors.affiliation: Nano-bio Materials Laboratory, Immunology Frontier Research Center, Osaka University, Suita, Osaka, 565-0871, Japan.
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Fujii
authors.affiliation: Division of Pathogenesis and Control of Oral Disease, Graduate School of Dentistry, Osaka University, Suita, Osaka, 565-0781, Japan. ymgtooi@fbs.osaka-u.ac.jp.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Ooi
keywords: Interfacial recognition; acetylcholine; fluorescence correlation spectroscopy; lipid bilayer; sulfonatocalixarene; vesicle