Lamellar neutron diffraction data from a DLPC/d62-DPPC mixture are shown in . Two series of lamellar diffraction peaks, which index on two different d-spacings, are present and indicate two lamellar phases. This requires separate stacking of d62-DPPC-rich and DLPC-rich 2-dimensional (2D) domains into 3D-domains. Such stacking is also readily observed by diffraction for mixtures of DOPC/DPPC/cholesterol, but not for all lipid mixtures14
and may depend on 2D-domain size, lipid molecular area, and hydration in addition to such conditions as composition and temperature.15
Intensities of the two series of neutron diffraction peaks result from the different deuterium compositions of the two domains. For the H2
O hydration here, the intensities from the d62-DPPC-rich domains are strong, due to high contrast between H2
O and CD2
, whereas diffraction from DLPC-rich domains is much less intense since H2
O and CH2
have about the same neutron scattering length density and so contrast is very small. Thus the larger d-spacing is identified as a d62-DPPC rich phase and the smaller d-spacing is the DLPC rich phase. With increased temperature, the d62-DPPC-rich (“solid”) phase intensities decrease dramatically, while the disordered DLPC-rich (“liquid”) phase intensities increase, demonstrating transfer of d62-DPPC to the DLPC-rich liquid phase and changing the contrast by mixing of the hydrogen and deuterium-containing chains. Lateral diffusion constants for DPPC and DLPC in multi-layers (>103
are sufficient to mix sub-micron sized domains in a few minutes. At 15° C there are only small liquid phase peaks, whereas at 35° C, only the liquid phase peaks remain and have become much more intense. Addition of halothane at 25° C shifts the intensities of the solid and liquid peaks so that the intensities of the smaller d-spacing predominate.
Figure 1 Neutron diffraction of 1:1 d62-DPPC/DLPC oriented multi-layers on glass at different temperatures and after addition of halothane. Q = 4πsin(θ)/λ is the momentum transfer directed perpendicular to the membrane plane. θ (more ...)
We also measured in-plane neutron diffraction on oriented d62-DPPC/DLPC multilayer membranes (). In-plane neutron diffraction by non-crystalline phospholipids in the region of the chain diffraction (Q=1.4 Å−1
to 1.5 Å−1
) depends strongly on the deuterium content of the fatty acid chains. The negative neutron scattering length of hydrogen makes the net scattering length of the CH2
group small and negative, whereas CD2
is large and positive. As a result, in-plane neutron diffraction from the chains of non-crystalline phospholipids is only observed with chain-perdeuterated phospholipids. For a mixture of H and D-chains, such as in a d62-DPPC/DLPC mixture, the intensity of the chain diffraction depends on the size and number of domains consisting of primarily d62-chains and on the order of d62-chains within these domains. Mixing of the H and D lipids decreases the intensity, while separation of d62-DPPC into 2D domains increases the intensity. This in-plane neutron diffraction method, with H/D chain mixing, is especially effective for observing changes in composition of lateral domains17
since it does not require vertical alignment of the lipid domains into 3D domains as lamellar diffraction does. We found the mixing transition for d62-DPPC/DLPC is broad, beginning at about 22° C and extending to 31° C. Other techniques have found similarly broad mixing transitions in other lipid mixtures 5a
. The breadth of these transitions is not apparent in phase diagrams for these mixtures18
. Halothane at 1.5 mol %, about twice the minimum alveolar concentration for anesthesia, produces a marked shift of about 5°C in the mixing transition towards lower temperatures (an order of magnitude larger than the shift in the main melting transition of pure DPPC induced by anesthetic concentrations of octanol 19
), while 7.5 mol % F6 produces a shift of about half this magnitude. We obtained similar in-plane data for binary lipid bilayers supported in pores of anodisc filters, where the geometry is very different from that of planar multilayers (Supporting Figure 1a
). In-plane neutron diffraction measurements were also performed for a 1/1 mixture of dimyristoylphosphatidylcholine (DMPC) and d70-distearoylphosphatidylcholine (DSPC) as planar multilayers, with a midpoint transition temperature of 51°C (Supporting Figure 1b
Figure 2 Neutron diffraction of 1:1 d62-DPPC/DLPC oriented multi-layers on glass. Q [1.4 Å−1 −1.5 Å−1] is directed parallel to the plane of the membrane. Neutron diffraction in this plane is generated by the d62-lipid chains (more ...)
Because the domains of d62-DPPC/DLPC mixtures stack separately as 3-D structures with different d-spacings, we were able to study the co-existence of the two domains at a range of concentrations of halothane and F6 using X-ray diffraction. Sample preparation and measurement procedures were as for neutron diffraction. illustrates separation of phases measured by X-ray diffraction. Two series of peaks, corresponding to the solid and liquid phases, are seen, as in the neutron diffraction studies. Introduction of halothane increases the intensity of the liquid phase peaks and decreases the intensity of the solid phase peaks. This change was evident within 5 minutes, remained stable at fixed halothane concentrations, and was reversible on halothane removal. In contrast, F6 produced only minor effects on the relative intensities. Note that the diffraction pattern is qualitatively different from that obtained using neutron scattering and d62-DPPC/DLPC because both lipid phases scatter X-rays in proportion to their similar electron densities, whereas for neutrons, the phase containing mainly d62-DPPC has a very different neutron scattering length density than the mainly DLPC phase.
Figure 3 Change in Bragg X-ray diffraction for a 1:1 mixture of DPPL/DLPC multi-layers on a glass before (solid line) and after (dotted line) addition of halothane 2.9 mol %. The first peak corresponds to the DPPC-rich “solid” phase, and the second (more ...)
To quantify the difference between halothane and F6, we measured the ratio of the intensities of the first order diffraction peaks for each phase as an estimate of the ratio of the lipid mass in the two phases. Halothane and F6 both produce nonspecific changes in X-ray Bragg diffraction. The intensity of a diffraction peak depends upon the structure factor, and the geometrical and mosaic spread factors. We did not observe any change in θ scans after addition of halothane or F6, suggesting that the mosaic spreads remained constant. Thus, both halothane and F6 appear to affect the level of disorder in the lipid multilayers, but only halothane produces the large shift in phase transition temperature. While the initial ratio of solid to liquid lipid phases varied between samples, the relative content of solid phase always decreased upon halothane addition. illustrates the change in the ratio of solid/liquid phases plotted against concentrations of halothane and F6. There is a marked difference between the slopes of the linear regression lines for halothane, −17.6±3.5, and for F6, −0.6±0.75 (s.d.). Even corrected by the relative solubility of halothane and F6 (halothane has a 5-fold higher oil/gas partition coefficient than F6—see Supporting Information Note 5
) the average slope for the halothane effect is about 5-fold higher than that for F6, significantly different at p < 0.05.
Relative change in the intensity of 1st order solid/liquid peaks in 1:1 mixtures of DPPC/DLPC at 28±1°C (s.d.) as a function of halothane (squares) and F6 (circles) concentrations.
We obtained very similar data for the effects of halothane on the phase mixing of ternary mixtures of DOPC/DPPC (1:1) and DOPC/sphingomyelin (1:1) with 20% cholesterol. demonstrates the effect of 4 mol % halothane on the ratio of 1st order peak areas in a mixture of DOPC/sphingomyelin (1:1) with 20% cholesterol. Note that the ratio declines by about 20% and quickly reverses after the halothane is withdrawn. Further description of the phase behavior of these mixtures and their responses to different anesthetics will be presented elsewhere.
Figure 5 Ratio of first order X-ray diffraction peak areas for a DOPC/sphingomyelin (1:1) with 20% cholesterol mixture as a function of time, at 27°C. (Porcine brain sphingomyelin obtained from Avanti.) Humidity maintained at 99% throughout. This mixture (more ...)
To compare our methods with those of previous investigations2
we recorded X-ray diffraction (Supporting Figure 2
) from multilayer samples of DMPC with 40 molar % cholesterol, in the absence and presence of 7.3 mol % halothane (about ten times the minimum alveolar concentration). In accord with the earlier findings, the headgroup to headgroup distance in the DMPC/cholesterol membrane () does not shift significantly (< 0.03 Å) in the presence of this large anesthetic concentration. However, repeating the measurements for membranes composed of dioleoylphosphatidylcholine (DOPC), an unsaturated lipid (Supporting Figure 3
) we found that 3.4 mol % halothane produces a small, but measurable change (−0.65 Å) in the thickness of the bilayer between headgroups with a −0.23 Å change in d-spacing (). We have scaled the plots to match the electron density at the center of the water layers. The change in electron density in the interfacial area is consistent with the preferential partitioning of halothane into this region of the membrane, as described in simulations. 20
(See Supporting Information Note 4
for further details on the relationship between the diffraction plots and electron density profiles). Concentrations of F6 up to 7.5 mol % produced no significant change (< 0.03 Å) in headgroup to headgroup distance (data not shown).
Figure 6 Electron density profiles for DMPC+40% cholesterol mulitlayers at 28° C before (solid) and after (dashed) the addition of halothane at 7.3 mol %. No significant shift in location of headgroups is observed, only the slight changes towards the center (more ...)
Figure 7 Electron density profiles for the DOPC multilayers at 28° C before (solid) and after (dashed) the addition of 3.4 mol % halothane. Small perturbations in the headgroup to headgroup distance (−0.65 Å) across the bilayer are evident (more ...)