Emiko Okamura

Fourier transform infrared-attenuated total reflection spectroscopy of hydration of dimyristoylphosphatidylcholine multibilayers

The effect of hydration on the structure and molecular orientation of multibilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cast on a germanium plate, was studied by means of polarized Fourier transform infrared (FT-IR)-attenuated total reflection spectroscopy. Compared with the dry state, the antisymmetric and symmetric CH2 stretching bands of fully hydrated DMPC in the liquid-crystalline state were shifted to the higher frequency side, indicating the increase in the number of the gauche conformers. However, the dichroism of these bands revealed that the hydrocarbon chains of DMPC were still ordered and titled. The absorption bands of the glycerol ester, phosphoryl, and choline groups were broadened upon hydration, suggesting the activation of the librational or torsional motion. Furthermore, the dichroism of the polar head group bands of DMPC indicated that these groups retained a slight orientation even in the fully hydrated and fluid multibilayers.

ORIENTATION STUDIES OF HYDRATED DIPALMITOYLPHOSPHATIDYLCHOLINE MULTIBILAYERS BY POLARIZED FTIR-ATR SPECTROSCOPY

Polarized Fourier-transform infrared-attenuated total reflection spectroscopy has been applied to explore the temperature-dependence of molecular orientations in multibilayers of 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC) of various degrees of hydration. The order parameter of the hydrocarbon chain, evaluated from the dichroic ratios of the antisymmetric and symmetric CH2 stretching bands, was drastically decreased at the main (or gel to liquid-crystalline phase) transition temperature (Tm) irrespective of the water content, suggesting that the hydrocarbon chain is in a disordered state as a result of chain-melting associated with an increase in the number of the gauche conformers. On the other hand, the dichroic ratios of the polar bands of hydrated DPPC assignable to the symmetric PO2- stretching and asymmetric N+ (CH3)3 stretching modes were increased mainly at the pretransition temperature (Tp), except for less hydrated case. The dichroic ratios of both the OH stretching and OH2 bending bands of water showed the same temperature-dependence as those of the polar bands. These results indicate that the pretransition is ascribable mainly to the reorientation of the polar groups of the DPPC and bound water, while the main transition is due to the orientational disorder of the hydrocarbon chains. For less hydrated DPPC, the reorientation of the polar groups and water did not occur around Tp, but in the higher temperature region around Tm. This is in accord with the previously reported observation that the pretransition disappears for less hydrated DPPC. In this case, the polar groups and water may reorient following the reorientation of the hydrocarbon chains near Tm.

Fourier transform infrared-attenuated total reflection spectra of dipalmitoylphosphatidylcholine monomolecular films

Abstract Single monomolecular films of 1,2- dipalmitoyl -3-sn- phosphatidylcholine (DPPC) were transferred onto a germanium plate by the Langmuir-Blodgett technique at various surface pressures encompassing a plateau region in a surface pressure(π)-area( A ) isotherm. Molecular orientation and structure of the monomolecular films were investigated by the Fourier transform infrared-attenuated total reflection spectroscopy. Appreciable changes in frequency and halfbandwidth of the antisymmetric and symmetric CH2 stretching bands of the palmitoyl chains were found at the onset of the plateau region in the π-A isotherm . By analyzing polarized infrared-attenuated total reflection spectra, the palmitoyl chains of DPPC films were proved to be oriented vertically to the germanium surface with all -trans conformation, irrespective of the surface pressure on the film transfer. This finding suggests the existence of islands or surface micelles on the surface throughout the surface pressure examined. The changes in the spectral parameters mentioned above were explained by changes in the state of packing of these islands. The plateau is ascribable to a transition process from a close-packed island phase to a molecularly homogeneous phase.

Orientation of gramicidin D incorporated into phospholipid multibilayers: a Fourier transform infrared-attenuated total reflection spectroscopic study

Polarized Fourier transform infrared (FTIR)-attenuated total reflection (ATR) spectroscopy was applied to study the orientation of the linear pentadecapeptide antibiotic gramicidin D incorporated into phospholipid multibilayers, which were cast on a germanium ATR plate from chloroform solution. In DMPC and DPPC multibilayers, the CH2 stretching bands of lipid hydrocarbon chains were slightly shifted to the higher frequency side and bandwidth was increased in the presence of gramicidin. However, in DPPE multibilayers, frequencies and bandwidths of these bands were unaltered. In each case, gramicidin produced little effect on the orientation of lipid hydrocarbon chains, suggesting that gramicidin penetrates into lipid layers without noticeable perturbations. Upon incubation of cast films in contact with water above the gel-liquid-crystalline transition temperature (Tc) of lipids, the reorientation of gramicidin in lipid multibilayers occurred, the degree thereof depending upon the fluidity of the lipid hydrocarbon chains and the amount of surrounding water. In DMPC multibilayers, the helix axis of gramicidin was oriented almost parallel to the lipid hydrocarbon chains after incubation. In DPPC multibilayers, on the other hand, the helix axis of gramicidin was tilted on average about 15 degrees from the lipid hydrocarbon chains after incubation. However, in DPPE multibilayers, which are known to have the most rigid bilayer structures, the reorientation of gramicidin could not be seen.

Drug binding and mobility relating to the thermal fluctuation in fluid lipid membranes

Drug binding and mobility in fluid lipid bilayer membranes are quantified in situ by using the multinuclear solution NMR combined with the pulsed-field-gradient technique. One-dimensional and pulsed-field-gradient (19)F and (1)H NMR signals of an anticancer drug, 5-fluorouracil (5FU) are analyzed at 283-313 K in the presence of large unilamellar vesicles (LUVs) of egg phosphatidylcholine (EPC) as model cell membranes. The simultaneous observation of the membrane-bound and free 5FU signals enables to quantify in what amount of 5FU is bound to the membrane and how fast 5FU is moving within the membrane in relation to the thermal fluctuation of the soft, fluid environment. It is shown that the mobility of membrane-bound 5FU is slowed down by almost two orders of magnitude and similar to the lipid movement in the membrane, the movement closely related to the intramembrane fluidity. The mobility of 5FU and EPC is, however, not similar at 313 K; the 5FU movement is enhanced in the membrane as a result of the loose binding of 5FU in the lipid matrices. The membrane-bound fraction of 5FU is approximately 0.1 and almost unaltered over the temperature range examined. It is also independent of the 5FU concentration from 2 to 30 mM with respect to the 40-50 mM LUV. The free energy of the 5FU binding is estimated at -4 to -2 kJ/mol, the magnitude always close to the thermal fluctuation, 2.4-2.6 kJ/mol.

Cholesterol location and orientation in aqueous suspension of large unilamellar vesicles of phospholipid revealed by intermolecular nuclear overhauser effect.

The locational and orientational structure and the dynamics of cholesterol in the bilayer membrane were studied by using the solution-state NMR. The intermolecular nuclear Overhauser effect (NOE) was analyzed for large unilamellar vesicles (100 nm in diameter) composed of dimyristoylphosphatidylcholine (DMPC) and cholesterol at cholesterol concentrations of 9-33 mol %. The DMPC headgroups show (1)H-{(1)H}-NOEs with the methyl groups at the hydrophobic terminals of both cholesterol and DMPC, illustrating the significant fluctuation of the bilayer membrane in the vertical (bilayer normal) direction. Cholesterol was found to keep the hydroxyl (OH) group toward the outer water pool on the basis of the following observations: (1) the cross correlation between the DMPC headgroup and the cholesterol terminal methyl group is weaker than those between the DMPC headgroups and (2) the methyl group at the hydrophobic terminal of cholesterol shows strong correlation with the terminal group of the DMPC chain portion. The OH group plays a crucial role in orienting cholesterol with its OH group outward, since cholestane, which has a molecular structure similar to that of cholesterol except for the absence of the OH group, was found to have no orientational preference in the bilayer membrane. The dynamic slowdown at high cholesterol concentrations is demonstrated on the basis of the correlation times for NOE as well as the broadening of the proton linewidths.

Quantitative analysis of molecular orientation in chlorophyll a Langmuir monolayer: a polarized visible reflection spectroscopic study

Polarized visible reflection spectra of a chlorophyll a (Chl.a) Langmuir monolayer have been measured in situ at various surface pressures. By applying Hansens optics to the three-phase plane-bounded system (air/Chl.a monolayer/water), the negative reflection absorbances observed were reproduced satisfactorily by the theoretical calculation. Molecular orientation of Chl.a in the monolayer was evaluated quantitatively as a function of surface pressure, from the reflection absorbance of p- and s-polarized spectra of the red (Qy) band. It has been proven that Chl.a molecules in the monolayer form aggregates (islands) even in the low surface pressure region and that during the monolayer compression the molecules are gradually reorganized from inhomogeneous islands to ordered structures, with the chromophores oriented on the average vertically to the water surface.

Binding of hydrophobic fluorinated bisphenol A to large unilamellar vesicles of egg phosphatidylcholine.

The kinetics of membrane binding and dissociation of fluorinated bisphenol A (FBPA, (CF(3))(2)C(C(6)H(4)OH)(2)) is quantified by 1D (19)F NMR spectra in situ. Although the bound and free components are in fast exchange, the rate constants and bound fraction is nonetheless determined from an analysis of the spectra. The analysis relies on the expression of 1D NMR signal intensity by a set of Bloch equations with exchange terms. The time span of the binding and dissociation of hydrophobic FBPA to large unilamellar vesicles of egg phosphatidylcholine (EPC) is 10(-3) to 10(-2) s. The rates of FBPA binding and dissociation are kept constant per EPC molecule even at different concentrations of the vesicle. The free energy of FBPA transfer is -20 ± 2 kJ/mol at 303 K. The process is entropy-driven. The efficiency of FBPA transfer is enhanced by a factor of 7 × 10(4), as compared with the hydrophilic 5-fluorouracil.

Fourier transform infrared / attenuated total reflection study on subtransition of hydrated dipalmitoylphosphatidylcholine multibilayers

Abstract Changes of the infrared bands of water occurring at the subtransition of a 1,2-dipalmitoyl- sn -glycero-3-phosphocholine (DPPC)-water system were studied by Fourier transform infrared/attenuated total reflection spectroscopy. When hydrated DPPC multibilayers (with 85 wt% of water) were annealed at 2°C for 115 h, the intensities of the water bands were substantially decreased below the subtransition temperature ( T s ) around 20°C, whereas those of the hydrocarbon and polar group bands of DPPC were increased. These results suggest that the annealed DPPC multibilayers are in a poorly-hydrated state; water is squeezed out of the bilayer, the phase separation between bulk water and the multibilayer occurs and consequently only bound (unfreezable) water remains in the bilayer region. On heating the sample above T s , the intensity of the water bands was increased and that of the DPPC bands was decreased. This means that the sample acquires a more hydrated state above T s owing to the penetration of bulk water into the interbilayer region. The dichroic ratios of the polar group and water bands also changed at T s . It is concluded that the subtransition is a process in which poorly hydrated DPPC is fully hydrated, being accompanied by the reorientation of the polar groups and water.

Microstructure of thin Langmuir-Blodgett films of dipalmitoylphosphatidylcholine: electron microscopic images replicated with plasma polymerized film by glow discharge

Abstract Electron-microscopic images of 1- and 5-monolayer LB films of dipalmitoylphosphatidylcholine, transferred to a glass substrate at various surface pressures encompassing the plateau region of the surface pressure-area isotherm, were observed by using a plasma polymerization replica method. One-monolayer films are flat and there is no substantial evidence of structural defects up to the high surface pressures. Crystalline domain structures appear in the 5-monolayer films even at the low surface pressures below the plateau, which accords well with our previous spectroscopic results. The domains decrease their size with increases in surface pressure and finally disappear at the high surface pressures above the plateau. These are explained by the formation of a closely packed homogeneous structure around the plateau region. Further compression of the monolayer before the collapse pressure leads to the reappearance of the inhomogeneous crystalline domain structures, due to the local collapse of the monolayer just before the main collapse pressure.