Article pubs.acs.org/Langmuir
Detection of Nanosized Ordered Domains in DOPC/DPPC and DOPC/ Ch Binary Lipid Mixture Systems of Large Unilamellar Vesicles Using a TEMPO Quenching Method Keishi Suga and Hiroshi Umakoshi* Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan S Supporting Information *
ABSTRACT: Nanosized ordered domains formed in 1,2-dioleoyl-sn-glycero-3phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DOPC/DPPC) and DOPC/cholesterol (Ch) liposomes were characterized using a newly developed (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) quenching method. The membrane fluidity of the DOPC/DPPC liposomes, evaluated by the use of 1,6-diphenyl-1,3,5hexatriene (DPH), increased significantly above their phase-transition temperature. The fluorescence spectra of 6-lauroyl-2-dimethylamino naphthalene (Laurdan) indicated the formation of an immiscible ordered phase in the DOPC/DPPC (50/50) liposomal membrane at 30 °C. The analysis of the membrane polarity indicated that the surface of the liquid-disordered phase was hydrated whereas that of the ordered phase was dehydrated. DOPC/DPPC and DOPC/Ch (70/30) liposomes exhibited heterogeneous membranes, indicating that nanosized ordered domains formed on the surface of the DOPC/DPPC liposomes. The size of these nanosized ordered domains was estimated using the TEMPO quenching method. Because TEMPO can quench DPH distributed in the disordered phases, the remaining fluorescence from DPH is proportional to the size of the ordered domain. The domain sizes calculated for DOPC/DPPC (50/50), DOPC/DPPC (25/75), DOPC/Ch (70/30), and DOPC/DPPC/Ch (40/40/20) were 13.9, 36.2, 13.2, and 35.5 Å, respectively.
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INTRODUCTION Insights into the possible effect of the surrounding environment on the conformation and function of enzymes and nucleic acids are important to clarifying the in vivo mechanisms of biomacromolecules. The formation of membrane lipid domains (rafts) in cells has attracted much attention because of its implications in membrane-related processes, including bacterial and viral infections and signal transduction.1 Recent studies have also revealed that lipid membranes can activate biomacromolecules and regulate their functions.2−4 Liposomes, which are self-assemblies of various kinds of phospholipids, have been intensely studied as biomembrane mimics. In our previous reports, we showed that large unilamellar vesicles (LUVs) with a diameter of 100 nm regulate the in vitro expression of green fluorescent protein through the interaction between single-stranded RNAs and the unfolded polypeptide.3,5,6 Other studies have also found that nucleic acids can accumulate on the microdomains of lipid membranes.7,8 From the aspect of membrane−protein (or enzyme) interaction, LUVs modified with a positively charged cholesterol derivative show enhanced accumulation and enzymatic activity for hexokinase.4 The heterogeneous liposomes, including cholesterol (Ch), have been reported to induce the remarkable behaviors that can affect various biochemical reactions. The oxidation of cholesterol in the liposomes was found to modulate the catalytic functions of the amyloid β-Cu complex.9 © 2013 American Chemical Society
In addition, the refolding of carbonic anhydrase (CAB) was effectively enhanced in the presence of 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC)/Ch (2/1) whereas that was not very significant with POPC only.10 These previous findings suggest that the Ch-modified liposome can recognize the unstable polypeptides, showing that the microdomain can act as a platform of the recognition, folding, and functionalization of the biomacromolecules. Recently, it has also been reported that heterogeneous cationic liposomes can induce partial conformational changes in mRNA and regulate translation in an Escherichia coli cell-free translation system.11 It is therefore important to understand the physicochemical properties of LUVs, such as their phase state, microphase separation, and microdomain formation on the membrane. Lipid membranes composed of natural lipid molecules exhibit several important dynamic behaviors such as thermal undulation, the morphological change of the entire lipid membrane (e.g., fusion/fission), (transient) pore formation, and the formation of heterogeneous structures (i.e., phase separation/domain formation).12,13 Sphingomyelin (SM) and cholesterol (Ch) are typical components present in the raft domains of liquid-disordered (ld) membranes.14 Because the Received: December 1, 2012 Revised: February 12, 2013 Published: March 18, 2013 4830
dx.doi.org/10.1021/la304768f | Langmuir 2013, 29, 4830−4838
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similar to that in previous reports.29,30 Fluorescent probe DPH was added to a liposome suspension in a molar ratio of 250/1 lipid/DPH; the final concentrations of lipid and DPH were 100 and 0.4 μM, respectively. The fluorescence polarization of DPH (Ex = 360 nm, Em = 430 nm) was measured using a fluorescence spectrophotometer (FP-6500 or FP-8500; Jasco, Tokyo, Japan) after incubation at 30 °C for 30 min. The sample was excited with vertically polarized light (360 nm), and emission intensities both perpendicular (I⊥) and parallel (I∥) to the excitation light were recorded at 430 nm. The polarization (P) of DPH was then calculated from the following equations:
formation of membrane domains depends on the lipid composition and surrounding environment (e.g., temperature), the phase diagrams of membranes in binary and tertiary lipid mixtures have been systematically studied.15−18 Fluorescence microscopy and fluorescence resonance energy transfer (FRET) assays have been developed to detect domains in living membranes. However, despite advances in far-field optical microscopy, subwavelength lipid domains have never been directly visualized, although their sizes are predicted to be 75−100 nm (microscopy and FRET),
