Phase Diagram of Androsterol

J. Phys. Chem. B 2008, 112, 8375–8382

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Phase Diagram of Androsterol-Dipalmitoylphosphatidylcholine Mixtures Dispersed in Excess Water Wenying Gao,† Lin Chen,† Ruiguang Wu,† Zhiwu Yu,*,† and Peter J. Quinn‡ Key Laboratory of Bioorganic Phosphorous Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua UniVersity, Beijing 100084, China, and Department of Biochemistry, King’s College London, 150 Stamford Street, London SE1 9NH, U.K. ReceiVed: December 22, 2007; ReVised Manuscript ReceiVed: April 17, 2008

The effect of androsterol, whose structure resembles that of cholesterol but without the alkyl side chain, on the phase behavior of aqueous dispersions of dipalmitoylphosphatidylcholine has been studied to understand the role of the side chain played in the formation of ordered phases of the type observed in membrane rafts. Thermotropic changes in the structure of mixed dispersions and transition enthalpies have been examined by synchrotron X-ray diffraction, Fourier transform infrared spectroscopy, and differential scanning calorimetry. From these results a partial phase diagram of the binary system has been constructed. The three-phase line is determined to be 34.5 °C, which is 3-5 °C lower than that observed in binary mixtures of cholesterol, ergosterol, or stigmasterol with dipalmitoylphosphatidylcholine. The proportions of androsterol in mixtures representing the “left end point” and “right end point” of the three-phase line are 11.1 and 30.9 mol %, respectively. These proportions are greater than that seen in phase diagrams of other sterols codispersed with dipalmitoylphosphatidylcholine. We conclude that androsterol is less effective in promoting the formation of an ordered phase, and furthermore, this ordered phase is less compact than the normal lamellar liquid-ordered phase. 1. Introduction Sterols are omnipresent lipids in eukaryotes and play indispensable roles in regulating the physical properties of biomembranes.1–3 One putative structural role of cholesterol is to act as a key and integral component of membrane microdomains such as caveolae and rafts. Such domains are rich in cholesterol and saturated lipids and are found to coexist with domains where unsaturated molecular species of phospholipids predominate.4–8 There have also been reports that some other naturally occurring sterols may also play a role in the formation of ordered lipid domains.9–12 The incorporation of sterols into membranes induces a more ordered packing of the fluid acyl chains of phospholipids in the hydrophobic section of the bilayer, increases mechanical strength of the structure, and reduces permeability to solutes.13 To get a better understanding to the essential structural features of sterols that are required to support mammalian cell growth, mutant strains of Chinese hamster ovary cells defective in sterol biosynthesis have been cultured with different sterols.1 It was found that sterols with minor modifications to the side chain such as campesterol, β-sitosterol, and desmosterol, supported long-term growth of the mutant cells, but sterols with more complex modifications to the side chain or the sterol nucleus itself (androsterol) did not. Although androsterol has precisely the same structure and stereochemistry of the rigid planar fused ring system as the parent cholesterol molecule except lacking the C17 alkyl side chain, the above observations demonstrate the importance of the side chain of the sterols. * Corresponding author. Tel.: (+86) 10 6279 2492. Fax: (+86) 10 6277 1149. E-mail: [email protected]. † Tsinghua University. ‡ King’s College London.

Phosphatidylcholines are among the major phospholipids of plasma membranes.14 Saturated molecular species of phosphatidylcholines, such as the dipalmitoyl derivative of phosphatidylcholine (dipalmitoylphosphatidylcholine, DPPC), are known to have very similar physical properties to that of sphingolipids with saturated, long-chain amide-linked fatty acids commonly found in the plasma membranes of mammalian cells.3,15 Thus, sterol/DPPC mixtures have been selected by many research groups as simple models of biomembrane microdomains or the basic structure of rafts. A partial phase diagram of cholesterol/ DPPC has been constructed by Vist and Davis16 and has gotten both experimental and theoretical support.17–19 Recently, Hsueh’s group and our group have constructed partial phase diagrams of ergosterol/DPPC (the predominant sterol in fungi) and stigmasterol/DPPC (the predominant phytosterol), respectively.20,21 These diagrams are characterized by three two-phase regions and a three-phase line. Three distinct phases have been identified: (1) lamellar liquid-crystal (Lr) or liquid-disordered phase (Ld), (2) lamellar-gel (Lβ) or solid-ordered phase (so), and (3) liquid-ordered phase (Lo).16,20 Nonetheless, no phase diagrams of androsterol-containing lipid mixtures have been published so far. The aim of this work is to construct a partial phase diagram of aqueous dispersions of androsterol, as well as to compare this phase diagram with those of ergosterol/DPPC, cholesterol/DPPC, and stigmasterol/DPPC binary mixtures. 2. Materials and Methods 2.1. Materials. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was purchased from Sigma Chemicals (St. Louis, MO) and androsterol (5-androsten-3β-ol) was obtained from Steraloids Inc. (99%, Rhode Island). Both lipids were used without further purification. Androsterol/DPPC mixtures with designated mole ratios were dissolved in chloroform, dried under a stream of oxygen-free dry nitrogen, and stored in vacuum overnight

10.1021/jp712032v CCC: $40.75  2008 American Chemical Society Published on Web 06/21/2008

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to remove any remaining traces of solvent. The lipid films were hydrated with Tris-HCl buffer consisting of 50 mM Tris-HCl, 150 mM NaCl, and 0.1 mM CaCl2 (pH ) 7.2). The lipid to solvent ratio is 1/4 (w/w). The dispersions were mixed with repeated vortex and thermal cycling between 60 and -20 °C to ensure homogeneous dispersion. Samples with mole percentage of androsterol of 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, and 40% were prepared for differential scanning calorimetry (DSC) and X-ray diffraction (XRD) examination. More detailed DPPC samples with mole percentage of androsterol 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 17%, 19%, 21%, 23%, 25%, 27%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, and 38% were hydrated with D2O for Fourier transform infrared spectroscopy (FTIR) experiments in order to do image analysis accurately. 2.2. Experimental Methods. 2.2.1. X-ray Diffraction Experiments. Static synchrotron XRD experiments were performed at Station BL40B2 of SPring-8, Japan. The XRD data were recorded with an image plate detector. The wavelength was 0.12 nm, and camera length was 400 mm. X-ray scattering intensity patterns were recorded during 30 s exposure time of the sample to the synchrotron beam. A standard silver behenate sample was used for calibration of diffraction spacings. Each sample was sealed in a copper sample holder with thin mica windows about 1 mm apart. The sample holder, with a volume of about 20 µL, was mounted on a programmable Linkam thermal stage (Linkam Scientific Instruments, U.K., ( 0.1 °C). Samples were equilibrated at desired temperatures for several minutes before recording the diffraction patterns with a scanning rate 0.5 °C/ min. Static X-ray powder diffraction intensity data were analyzed and integrated by a program Fit2D. Electron density distributions across the unit cell of the lamellar repeat of DPPC or its mixtures with androsterol dispersed in water were calculated as follows. Integrated intensities I(h) for a range of diffraction orders (h) were obtained from small-angle scattering intensity profiles. Electron density profiles in a one-dimension space x, in arbitrary units, were calculated from Fourier reconstructions using the X-ray structure factors:22,23

F(x) ) Σ[g(h)|F(h)| cos(2πhx/d)] h

(1)

where F(h) is the structure factor, equal to the root of [h2I(h)], g(h) is the phase of F(h) of the hth order diffraction, and d is the repeat spacing of the multibilayers. To calculate a true electron density profile, it is first necessary to assign phase angles to each of the diffraction orders. One way to solve this problem is to adopt a model-building approach.24 In the camera configuration used in this study, five orders of diffraction were collected from the dispersions of DPPC containing different fractions of androsterol. Assuming that the multilamellar structures of the dispersions are centrosymmetric, the phase angles of the structure factor F(h) are restricted to either π or 0. This implies that g could only be -1 or 1.25 Thus, there are 32 combinations of phases for one dispersion sample in the electron density analysis. After examination of all possible phase sets, the only plausible combination of phases consistent with a bilayer repeat is (- - + - -), which is in agreement with previous results of DPPC systems.26 A direct way to solve the phase problem is to obtain data from a swelling series,27,28 in which lipids are dispersed in different amounts of solvent, assuming the electron density profile of the lipid structure remains constant over the range of solvent/lipid ratios examined. Mixtures of DPPC containing 30%

androsterol with different hydration were examined at several temperatures. The swelling series indicated phase combinations of either (- - + - -) or (+ + - + +). Because the electron density in the center of the hydrocarbon region is lower than that in the solvent layer, the former is correct. Therefore the electron density profile of androsterol/DPPC was calculated using the phase combination (- - + - -). 2.2.2. Differential Scanning Calorimetry. Calorimetry was performed using a Mettler-Toledo DSC821e differential scanning calorimeter. Samples (20 µL) were examined at a scan rate of 0.5 °C/min, and at least three scans were performed to verify the reproducibility. Other scanning rates such as 0.2 °C/min and 0.15 °C/min have been evaluated in DSC measurements. The general features of the thermal peaks are similar to those at 0.5 °C/min. The selection of 0.5 °C/min in the DSC measurement, as well as in the XRD and IR studies, was the result of balanced judgment between equilibration requirement and good signal-to-noise ratio. Deconvolution of the experimental curves was conducted by the aid of PeakFit software (Aisn Software Inc.). The peak type was chosen Gauss+Lorentz for all deconvolution treatments, and baseline was created by the two-point linear method. 2.2.3. Fourier Transform Infrared Spectroscopy. FTIR spectra were recorded using a Nicolet 5700 spectrometer. Androsterol/DPPC mixed lipid samples with mole percentages of androsterol from 8% to 38% were used in the FTIR study. Samples were coated onto the inner surfaces of a pair of CaF2 windows, which were mounted on a temperature-controlled sample holder. The lipid dispersions were sealed with silicone grease between the two CaF2 windows to prevent evaporation of the water. Samples were heated from 20 to 70 °C at 0.5 °C/ min. Spectra were recorded in the range of 4000-900 cm-1, with a resolution of 2 cm-1. The spectral image analysis method has been employed to evaluate spectral changes over a range of wavenumbers.29 For two data sets X and Y such as two FTIR spectra, a parameter, correlation coefficient C(X, Y), is expressed with the following equation:

C(X, Y) )

SXY

√SXXSYY

(2)

where SXY is the covariance between X and Y and SXX and SYY are the covariances of X and Y themselves. The correlation coefficient reflects the similarity between two selected spectra. It is zero if X and Y are statistically independent and 1 when X and Y are the most significantly correlated. The parameter can remove some system errors caused by the inconsistencies of the Fourier transform and solvent suppression.29–31 In this work, the spectrum parameter correlation coefficient C has been evaluated using two neighboring spectra. The spectral region used for this analysis is 2800-3000 cm-1, which covers the characteristic bands of the symmetrical and asymmetrical stretching vibrations of CH2 and CH3. The parameter was then used to identify phase transitions triggered by either temperature or concentration. 3. Results and Discussion A partial phase diagram of androsterol/DPPC, as shown in Figure 1, has been established based on the experimental data of DSC (solid circles), XRD (solid triangles), and FTIR (open circles). The boundaries of Pβ′/(Pβ′ + Lr) and (Pβ′ + Lr)/Lr, where Pβ′ is the rippled gel phase, were determined from the onset temperature and end point temperature of the main

Phase Diagram of Androsterol-DPPC Mixtures

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Figure 1. Partial phase diagram of the androsterol/DPPC mixtures in excess water. The boundaries of the Pβ′ + Lr region and the threephase line of Pβ′ + Lo + Lr are delineated from DSC data (b); the left and right boundaries of Pβ′ + Lo are based on FTIR (O) and XRD results (2); The boundary of Lβ′/Pβ′ is determined by both DSC and XRD results. The boundary of Lo/(Lr + Lo) and (Lr + Lo)/Lr are determined by both FTIR and XRD information. See text for details.

transitions detected by DSC; the three-phase line was gained from the deconvolution results of DSC traces. The boundary of Lβ′/Pβ′ was defined from pretransition peak temperatures of DSC thermograms of DPPC and DPPC with 2.5 mol % androsterol and the midpoints of the phase transitions based on XRD information of DPPC with 5 mol % androsterol and DPPC with 10 mol % androsterol. The boundaries of Pβ′/(Pβ′ + Lo) and (Pβ′ + Lo)/Lo were determined based on the XRD information and FTIR image analysis of the lipid mixtures at fixed temperatures. The boundary of (Lr + Lo)/Lr is determined jointly from the end points of transition peaks in the FTIR image analysis curves and the end points of the broad peaks of the deconvolution results of DSC traces. The boundary of Lo/(Lr + Lo) at higher androsterol concentration range was estimated based on the onset temperatures of image analysis of FTIR spectra of DPPC with 34, 35, 36, and 38 mol % androsterol and the onset temperatures of DSC thermograms of DPPC with 35 and 40 mol % androsterol. The ripple phase was identified by both DSC and XRD techniques.32 Details are explained below. 3.1. Phase Analysis of DSC Results. 3.1.1. Analysis of the Main Transition and Pretransition. DSC thermograms of mixed aqueous dispersions of androsterol/DPPC recorded during a heating scan at 0.5 °C/min are presented in Figure 2. In the absence of the sterol, DPPC dispersions display a low enthalpic pretransition (Lβ′ to Pβ′) at 35.1 °C and a sharp main transition (Pβ′ to Lr) at 41.6 °C, which are identified as the peak temperatures of the DSC curves according to McMullen and McElhaney.33 The phase transition temperatures are in good agreement with the published data.34,35 In the presence of androsterol, the endothermic peak of the main phase transition of the phospholipid becomes broad, and the transition temperature decreases markedly. The onset and end point temperatures of the transition in mixed dispersions containing 2.5, 5, and 10 mol % androsterol are used to delimit the coexisting region of Pβ′ and Lr phases. It is noteworthy that the end point temperature observed during heating and the onset temperature during cooling through the main phase transition shows a hysteresis of less than 0.5 °C. With further increase in the proportion of androsterol, the endothermic peaks of the main transition become broader, and an asymmetric feature appears when the molar ratio of androsterol is greater than 10%. This suggests that the dispersions may be comprised of more than one phase. We have, therefore, deconvoluted these

Figure 2. DSC thermograms of androsterol/DPPC mixtures containing different molar percentage of androsterol. Samples equilibrated at 20 °C were heated to 60 °C at a scanning rate of 0.5 °C/min. A faster scanning rate (5 °C/min) was also employed in 2.5 mol % androsterol to achieve larger differential signals.

Figure 3. Representative deconvoluted endotherms of the main transition of DPPC with different molar percentage of androsterol in DPPC as indicated. Either one peak (a) or two peaks can be deconvolved (b). Solid lines represent experimental curves, dash-dotted lines represent the deconvoluted peaks, and the red dotted line is the overall fitted curve.

endotherms and found that thermograms of mixtures containing androsterol from 15 and 30 mol % could be well fitted by two components as depicted in Figure 3. The deconvolution gives rise to one sharp (the left peak) and one broad component (the right peak), as previously described, representing the melting of sterol-poor and sterol-rich domains, respectively.35–38 All the left peaks appear at almost the same temperature, 34.5 °C, with a variation of (0.5 °C. By contrast, the thermograms of the samples containing less than 10% or more than 35% androsterol are symmetric and are likely to originate from a single phase transition. In addition, faster scanning rate (5 °C/min) was also employed in 2.5 and 5 mol % androsterol to achieve larger differential signals in Figure 2 as insertion. Clearly, the pretransition

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temperature decreases with adding androsterol into DPPC. The pretransition cannot be detected by DSC in mixtures containing more than 5 mol % androsterol, in agreement with the report by McMullen et al.38 The partial Lβ′/Pβ′ boundary was evaluated by the peak temperatures of pretransition. 3.1.2. Determination of the Three-Phase Coexistence Line by Calorimetry. On the basis of the results shown in Figure 3, the following conclusions can be drawn: (1) the temperature, where Pβ′ + Lr + Lo three phases coexist, is 34.5 °C; (2) the minimum sterol concentration of the three-phase line, or the “left end point” (φ), is between 10 and 15 mol %; (3) the maximum sterol concentration of the three-phase line, or the “right end point” (θ), is between 30 and 35 mol %. Evaluation of the precise values of φ and θ has been undertaken by means of the method developed by Wu et al.21 Briefly, we assume that, for the two deconvoluted thermal events shown in Figure 3b, the sharp component is attributed to the phase transition enthalpy from Pβ′ to Lr phase containing φ mol % androsterol, and the broad component represents the phase transition enthalpy from Lo to Lr phase containing θ mol % androsterol. ARp and ARl are defined as the areas of the two components, np and nl are the moles of molecules of each phase involved, ∆Rp Hm and ∆Rl Hm are the phase transition enthalpy per mole of DPPC from Pβ′ to Lr and from Lo to Lr, respectively. Accordingly, the following equations can be expressed:

ARp ) np(100 - φ)∆Rp Hm

(3)

AlR ) nl(100 - θ)∆lRHm ARp /ARl

(4) ∆Rp Hm/∆Rl Hm.

We further define λ ) and h ) The former can be determined by deconvolving endothermic peaks. Then for a given sterol molar percentage x in a sterol/DPPC mixture, the following equation can be obtained according to the lever rule:

λ)

(θ - x) (100 - φ) h (x - φ) (100 - θ)

(5)

The three parameters in the equation, h, φ, and θ, can be determined by nonlinear fitting of Microcal Origin software. The results obtained are that φ ) 11.1 and θ ) 30.9. These values fall precisely into the range predicted before and are consistent with the extrapolation from other boundaries. 3.2. Phase Analysis from X-ray Diffraction Results. X-ray diffraction patterns were recorded over the temperature range from 20 to 50 °C at intervals of 5 °C. Representative smallangle (SAXS) and wide-angle X-ray scattering (WAXS) intensity patterns for different phases are presented in Figure 4, parts a and b (solid lines). Simultaneously shown in the figure are selected diffraction patterns of stigmasterol/DPPC dispersions for comparison (dash-dotted lines). In the small-angle region, the reciprocal spacings of the first four diffraction orders show a ratio of 1:2:3:4, revealing all the structures to be lamellar structure. Especially, the lamellar-gel phase (Lβ′) of pure DPPC dispersion in the wide-angle region is characterized by an asymmetric scattering centered at 0.42 nm. The ripple phase (Pβ′) of DPPC is characterized by a diffuse SAXS patterns with a “distinct” peak at S-1 ) 7.1 nm corresponding to the lamellar repeat distance of the ripple phase and a near-symmetric WAXS scattering centered at 0.42 nm. The WAXS pattern for the lamellar liquid-crystal phase (Lr) is a diffuse scattering band centered at 0.46-0.47 nm. However, the WAXS pattern of the androsterol/DPPC dispersion (x ) 35%) shows a maximum around 0.43 nm, similar to that of the stigmasterol/DPPC dispersion (the Loβ phase, dash-dotted line), but the diffraction

Figure 4. Comparison of the small-angle (a and c) and wide-angle (b and d) X-ray diffraction patterns of different phases. Solid lines: Lβ′, pure DPPC at 30 °C; Pβ′, pure DPPC at 38 °C; Lo, DPPC with 35 mol % androsterol at 35 °C; Lr, DPPC with 25 mol % androsterol at 50 °C. Dash-dotted line: Loβ, DPPC with 35 mol % stigmasterol at 35 °C; Lr, DPPC with 25 mol % stigmasterol at 50 °C. Lower panel curves are representative diffraction patterns of (Pβ′ + Lr), DPPC with 5 mol % androsterol at 40 °C; (Lo + Lr), DPPC with 20 mol % androsterol at 40 °C; (Pβ′ + Lo), DPPC with 25 mol % androsterol at 25 °C.

peak slightly broader. We assign this phase as Lo, which is clearly different from Lβ′, Pβ, and Lr. Detailed analyses of the ripple and Lo phases are as follows. The SAXS (Figure 5a) and WAXS (Figure 5b) intensity patterns of various androsterol/DPPC dispersions at 30 °C, as well as those of pure DPPC at the same temperature are presented in Figure 5. By comparison with the representative Pβ′ diffraction pattern of the DPPC dispersion (in Figure 4), the phase state of the mixed lipid dispersions containing 5 and 10 mol % androsterol have the feature of ripple phase, and the mixture containing 10 mol % androsterol can be assigned as ripple phase (with diffuse SAXS patterns). Particularly, the (01) plane of the undulation structure present in a ripple phase was detected in this work, which can be used to identify the Pβ′ phase directly. As regard to pure DPPC, the periodicity lies within the range of 12-16 nm according to literature reports.39–41 In the case of androsterol/DPPC dispersions containing up to 30 mol % of the androsterol, as shown in Figure 5c, the unique scattering peak can also be seen unambiguously with a periodicity from 14.7 to 16.8 nm. The WAXS pattern in Figure 4 indicates that the Lo phase of androsterol/DPPC is similar to the ordered phase observed in the stigmasterol/DPPC dispersion. Electron density was calculated to compare the thickness of the bilayer. In Figure 6, the bilayer thickness dp-p of DPPC with 33 mol % cholesterol

Phase Diagram of Androsterol-DPPC Mixtures

Figure 5. Small-angle (a) and wide-angle (b) X-ray diffraction patterns recorded from androsterol/DPPC codispersions at 30 °C. The WAXS intensities have been enlarged to show the patterns more clearly. The (01) peak profiles of the ripple period of the Pβ′ phase in the SAXS region are shown in (c); the inserted illustration is an enlarged local part with baseline adjustment.

Figure 6. Electron density profiles of DPPC dispersions in the presence of sterol (33 mol % cholesterol, 35 mol % androsterol) and absence of androsterol at the indicated temperatures.

is about 4.69 nm, whereas dp-p of DPPC with 35 mol % androsterol is about 4.52 nm, suggesting that cholesterol makes the hydrocarbon chains of DPPC more extended than the case

J. Phys. Chem. B, Vol. 112, No. 28, 2008 8379 with androsterol. On the other hand, in the WAXS region, the periodicity of DPPC with 35 mol % stigmasterol is 0.430 nm, whereas it becomes 0.435 nm with 35 mol % androsterol, implying that androsterol does not have as great a condensing effect as sterols with an alkyl chain on the DPPC multibilayer structures. This can be attributed to the shorter length of androsterol unlike cholesterol or stigmasterol, which can restrict the motion of the hydrocarbon chains of DPPC more effectively. The representative diffraction patterns from the two-phase regions in the phase diagram have also been shown in Figure 4, parts c and d, for comparison. Only the first- and secondorder peaks are shown to demonstrate the details of the peak overlap feature of the mixed phases. Clearly we can see shoulder peaks in the SAXS patterns of (Pβ′ + Lr) and (Lo + Lr). What is problematic is the near symmetric feature of the peaks of (Pβ′ + Lo). Similar problems exist in the SAXS patterns of the mixtures containing 15-30 mol % androsterol (Figure 5a). Interestingly, by performing the second-derivative treatment of the SAXS curve, the overlapped feature can be displayed unambiguously (see the inserted curve of the second-order SAXS, linear plot). To further address the problem, we propose that the SAXS intensity of the ripple phase in the two-phase region is much lower than the Lo phase. The same situations have been reported in other similar systems. In the study of the effect of cholesterol on the ripple phase of a series of phosphatidylcholines, Hicks et al.39 reported that the introduction of increasing concentrations of cholesterol led to the increase in repeat distance and decrease in amplitude. With the use of 2HNMR technique, Vist and Davis16 found that, for concentration from 7 to 22 mol % cholesterol, at temperatures between about 25 and 37 °C, the spectra do not appear to be obvious superpositions of two distinguishable end point spectra. The concerned region is actually the (gel-Lo) two-phase region. In another study of the ripple phase induced by R-tocopherol in DMPC, Wang et al.42 reported that, although only symmetric SAXS peaks were seen in the two-phase region and no apparent ripple phase reflections were observed in SAXS, ripples could be identified by the freeze-fracture electron microscopy (FFEM) technique. It should be noted that the effect of R-tocopherol on the phase behavior of phospholipids is frequently linked to that of cholesterol. We would like to stress that, although assignment of different phases was mainly based on XRD results, other techniques (FTIR and DSC) also provided important information. For the two-phase region, for example, the (Pβ′ + Lo), the two individual phases on both sides of the region (Pβ′ and Lo) were first assigned. Then we used the FTIR-based image analysis, as described in the following section, to determine the phase boundaries. Finally we used the (01) diffraction peak of the undulation structure to check the existence of the ripple phase in the region. 3.3. Image Analysis Based on FTIR Spectra. 3.3.1. Determination of the (Lr + Lo)/Lr Boundary. FTIR spectra were recorded over the temperature range of 20-70 °C at intervals of 0.25 °C at a heating rate of 0.5°C/min. A wavenumber range from 2800 to 3000 cm-1 has been selected to calculate correlation coefficients of mixtures of DPPC and androsterol. On the basis of the left end point of the three-phase line, the samples with mole percentage of androsterol from 13 to 38 mol % were used to determine the (Lr + Lo)/Lr boundary. Presented in Figure 7a are the representative original FTIR spectra of DPPC with 25 mol % androsterol. Shown in Figure 7b are the representative correlation coefficient curves from image analysis of two neighboring spectra upon heating.

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Figure 7. (a) Representative infrared spectra of DPPC with 25 mol % androsterol, (b) correlation coefficient curves of various androsterol/DPPC mixtures during a heating process, (c) comparison of the WAXS patterns of mixed lipids with 10 and 15 mol % androsterol, and (d) the WAXS patterns and the concentration-dependent reciprocal spacing of lipid mixtures with 25, 30, 35, and 40 mol % androsterol at 40 °C.

With increasing temperature, all the correlation coefficient curves undergo an identical process: slight change, rapid decrease, marked increase, and steady state, forming a negative or bottom-up peak. Because smaller correlation coefficients indicate dissimilarity between neighboring spectra, the bottomup peaks are evidence of phase transition processes. Not surprisingly, the onset temperatures of all the peaks are between 32 and 35 °C, which is consistent with the temperature where three phases coexist. The end points of the peaks, on the other hand, can be used to determine the (Lr + Lo)/Lr phase boundary (open circles in Figure 1). DSC and XRD results have also been used to delineate the boundary. For the former, the end points of the deconvoluted broad DSC peaks of DPPC with 15, 20, 25, and 30 mol %, and the end point temperature of DSC peaks of DPPC with 35 and 40 mol % androsterol are depicted in Figure 1 as solid circles. For the latter, there are two significant changes which provide information on the boundary: one is the change of position and shape of WAXS intensity peaks at 40 °C in mixtures containing between 10 and 15 mol % sterol (Figure 7c); the other is the abrupt change of the reciprocal spacing of WAXS maxima between 30 and 35 mol % sterol at 40 °C (Figure 7d). Therefore, the average values 12.5 and 32.5 mol % androsterol were taken as the boundary compositions, depicted in Figure 1 as solid triangles. 3.3.2. Determination of Left and Right Boundaries of (Pβ′ + Lo). In Figure 8a, on the basis of the comparison of the firstorder and the second-order diffraction peaks in the SAXS region and the shape of the WAXS intensity peaks, it is clear that the patterns of DPPC with 10 mol % androsterol have markedly

diffuse characteristics at temperatures of 20, 25, and 30 °C. There is a manifest sharpening of the peaks when the mole percentage of androsterol content reaches 15 mol % inferring that there is a transition between 10 and 15 mol % at the temperature range of 20-30 °C. Figure 8b shows relative electron density distributions through the bilayer normal to compare the thicknesses of the bilayers. The comparison shows that the thickness of the lipid bilayer and the water layer of DPPC containing 25, 30, 35, and 40 mol % androsterol undergoes the greatest change of thickness between 30 and 35 mol % at 20, 25, and 30 °C. This suggests that there should be a transition between 30 and 35 mol % sterol. Accordingly, the average value 12.5 and 32.5 mol % were plotted in Figure 1 with solid triangles. In order to determined the precise boundaries, image analysis of FTIR spectra were conducted by calculating correlation coefficients of the neighboring two spectra of the lipid mixtures containing androsterol over the range of 8-38 mol % at various fixed temperatures. The correlation coefficient curves at representative temperatures, 25, 27, 30, and 33 °C, respectively, are shown in Figure 9. They have been treated with three-point smoothing to reduce experimental error. The left and right boundaries of the (Pβ′ + Lo) region are determined by the two peak positions, which are around 12 and 31 mol % at 25 °C. The peak width of half-height is taken as the error bar. The phase boundaries at other temperatures can also be obtained in this way. Finally, the boundary of Lβ′/Pβ′ was determined with both DSC (pretransition, solid circles in Figure 1) and XRD methods (comparing XRD patterns, solid triangles in Figure 1). And the

Phase Diagram of Androsterol-DPPC Mixtures

Figure 8. (a) Comparison of the XRD patterns of two mixed lipids with 10 and 15 mol % androsterol at 20, 25, and 30 °C; (b) the electron density profiles of mixed lipids with 25, 30, 35, and 40 mol % androsterol.

J. Phys. Chem. B, Vol. 112, No. 28, 2008 8381 androsterol. In this high androsterol concentration region, both DSC and IR image analysis show very broad phase transition peaks. It is for this reason that dotted lines are used to show the trends of respective phase boundaries. 3.4. Comparison of the Phase Behavior of Sterols Codispersed with DPPC. At present, phase diagrams of three sterol-DPPC binary systems, namely, cholesterol-DPPC,16 ergosterol-DPPC,20 and stigmasterol-DPPC,21 have been reported. It is thus worthwhile to compare them and to examine the effects of the sterol nucleus or androsterol on the phase behavior of DPPC. First, androsterol reduces the main transition temperature of DPPC more effectively than the other three sterols, and the threephase line in the phase diagram of androsterol/DPPC (34.5 °C) is 3-5 °C lower than that of the other three binary lipid mixtures. The results indicate less close packing of the androsterol/DPPC self-assembled structure than is the case with the other three sterols. Second, the two end points of the threephase line are 6-8 and 20-25 mol %, respectively, in the phase diagrams of DPPC with cholesterol, ergosterol, or stigmasterol, whereas they turn to 11.1 and 30.9 mol % in androsterol/DPPC mixtures. This implies that a greater proportion of androsterol in DPPC is required to achieve a single ordered phase. The presence of the alkyl side chain would serve to extend the overall length of the sterol molecules to match that of the phospholipid between the hydrocarbon-water interface and the central plane of the bilayer where the ends of the acyl chains reside. In this configuration, naturally occurring sterols tend to stabilize the membrane structure and exert their influence on membrane permeability. Desmosterol and lanosterol are two precursors in the biosynthesis of cholesterol. There have been comparative studies on the molecular interactions of these sterols with DPPC or other lipid species.43–46 It was demonstrated that the properties of phospholipid membranes such as lipid packing in the presence of cholesterol or desmosterol are similar. However, for lanosterol, a more distant precursor of cholesterol synthesis, more significant differences were observed. It has been proposed that desmosterol is able to replace cholesterol in lipid membranes.45 Phase diagrams of two binary mixtures have been constructed. The lipid used was 1-palmitoyl-2-petroselinoyl-sn-glycero-3phosphatidylcholine (PPetPC), and the sterols were cholesterol and lanosterol.43 The general feature of the phase diagram of cholesterol/PPetPC is similar to that of cholesterol/DPPC mixtures. However, in the case of lanosterol, the phase diagram is significantly different; no three-phase line and eutectic point were seen. All these studies support the notion that sterols with structures similar to that of cholesterol are more effective in forming ordered structures with phosphatidylcholine molecules. 4. Conclusions

Figure 9. Correlation coefficient curves of androsterol/DPPC mixtures obtained from FTIR image analysis of neighboring concentration samples, smoothed with a three-point average.

Lo/(Lr + Lo) boundary was determined by the onset temperatures of thermal curves of DPPC with 35 and 40 mol % androsterol by DSC and the onset temperatures of correlation coefficient curves of DPPC with 34, 35 36, and 38 mol %

The phase behavior of androsterol/DPPC binary mixtures has been examined by DSC, synchrotron XRD, and FTIR methods. A partial phase diagram of the system has been constructed. The overall profile of the phase diagram resembles that of other sterol/DPPC binary systems, including cholesterol, stigmasterol, and ergosterol, but with lower three-phase line temperature and higher sterol concentrations at the end points of the three-phase line. We found that androsterol is less efficient in promoting the formation of an ordered phase rich in sterol, which is less compact than the normal lamellar liquid-ordered phase. It can be inferred from the structure and phase behavior of DPPC and androsterol that the alkyl chain on the sterol functions to position the sterol nucleus within the bilayer so as to interact with the

8382 J. Phys. Chem. B, Vol. 112, No. 28, 2008 hydrocarbon chains of the phospholipid and induce a liquidordered phase. Acknowledgment. This work was supported by Grants from the Natural Science Foundation of China (NSFC: 20633080 and 20675046) and the Human Frontier Science Programme (RPG0016/2005C). Assistance of Dr. Takahashi from Gunma University and Dr. Inoue from SPring-8 in the synchrotron station facility setup is gratefully acknowledged. References and Notes (1) Xu, F.; Rychnovsky, S. D.; Belani, J. D.; Hobbs, H. H.; Cohen, J. C.; Rawson, R. B. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 14551. (2) Simons, K.; Ikonen, E. Science 2000, 290, 1721. (3) Ohvo-Rekila¨, H.; Ramstedt, B.; Leppimaki, P.; Slotte, J. P. Prog. Lipid Res. 2002, 41, 66. (4) Brown, D. A.; Rose, J. K. Cell 1992, 68, 533. (5) Brown, D. A.; London, E. Annu. ReV. Cell DeV. Biol. 1998, 14, 111. (6) Brown, D. A.; London, E. J. Membr. Biol. 1998, 164, 103. (7) Brown, D. A.; London, E. J. Biol. Chem. 2000, 275, 17221. (8) Simons, K.; Toomre, D. Nat. ReV. Mol. Cell Biol. 2000, 1, 31. (9) Peskan, T.; Westermann, M.; Oelmuller, R. Eur. J. Biochem. 2000, 267, 6989. (10) Mongrand, S.; Morel, J.; Laroche, J.; Claverol, S.; Carde, J. P.; Hartmann, M. A.; Bonneu, M.; Simon-Plas, F.; Lessire, R.; Bessoule, J. J. J. Biol. Chem. 2004, 279, 36277. (11) Borner, G. H. H.; Sherrier, D. J.; Weimar, T.; Michaelson, L. V.; Hawkins, N. D.; MacAskill, A.; Napier, J. A.; Beale, M. H.; Lilley, K. S.; Dupree, P. Plant Physiol. 2005, 137, 104. (12) Martin, S. W.; Glover, B. J.; Davies, J. M. Trends Plant Sci. 2005, 10, 263. (13) Bloch, K. E. CRC Crit. ReV. Biochem. 1983, 14, 47. (14) Karmakar, S.; Raghunathan, V. A. Phys. ReV. E 2005, 71, 061924. (15) Stottrup, B. L.; Keller, S. L. Biophys. J. 2006, 90, 3176. (16) Vist, M. R.; Davis, J. H. Biochemistry 1990, 29, 451. (17) Linseisen, F. M.; Thewalt, J. L.; Bloom, M.; Bayerl, T. M. Chem. Phys. Lipids 1993, 65, 141. (18) Murtola, T.; Falck, E.; Patra, M.; Karttunen, M.; Vattulainen, I. J. Chem. Phys. 2004, 121, 9156. (19) Ipsen, J. H.; Karlstroem, G.; Mouritsen, O. G.; Wennerstroem, H.; Zuckermann, M. J. Biochim. Biophys. Acta 1987, 905, 162. (20) Hsueh, Y. W.; Gilbert, K.; Trandum, C.; Zuchermann, M.; Thewalt, J. Biophys. J. 2005, 88, 1799.

Gao et al. (21) Wu, R. G.; Chen, L.; Yu, Z. W.; Quinn, P. J. Biochim. Biophys. Acta 2006, 1758, 764. (22) McIntosh, T. J.; Magid, A. D.; Simon, S. A. Biochemistry 1989, 28, 17. (23) McIntosh, T. J.; Magid, A. D.; Simon, S. A. Biochemistry 1989, 28, 7904. (24) Warren, R. C. Physics and Achitecture of Cell Membranes; Adam Hilger: Bristol, U.K., 1987. (25) McDaniel, R. V.; McIntosh, T. J.; Simon, S. A. Biochim. Biophys. Acta 1983, 731, 97. (26) Yu, Z. W.; Quinn, P. J. Biophys. J. 1995, 69, 1456. (27) Torbet, J.; Wilkins, M. H. F. J. Theor. Biol. 1976, 62, 447. (28) Franks, N. P.; Lieb, W. R. J. Mol. Biol. 1979, 133, 460. (29) Yan, Y. B.; Jiang, B.; Zhang, R. Q.; Zhou, H. M. Protein Sci. 2001, 10, 321. (30) Yan, Y. B.; Zhang, R. Q.; Zhou, H. M. Eur. J. Biochem. 2002, 269, 5314. (31) Yan, Y. B.; Wang, Q.; He, H. W.; Hu, X. Y.; Zhang, R. Q.; Zhou, H. M. Biophys. J. 2003, 85, 1959. (32) Matuoka, S.; Kato, S.; Hatta, I. Biophys. J. 1994, 67, 728. (33) McMullen, T. P. W.; McElhaney, R. N. Biochim. Biophys. Acta 1995, 1234, 90. (34) Koynova, R.; Caffrey, M. Biochim. Biophys. Acta 1998, 1376, 91. (35) Halling, K. K.; Slotte, J. P. Biochim. Biophys. Acta 2004, 1664, 161. (36) McMullen, T. P. W.; Lewis, A. H.; McElhaney, R. N. Biochemistry 1993, 32, 516. (37) Ghosh, Y. K.; Indi, S. S.; Bhattacharya, S. J. Phys. Chem. B 2001, 105, 10257. (38) McMullen, T. P. W.; Lewis, R. N. A. H.; McElhaney, R. N. Biophys. J. 1994, 66, 741. (39) Hicks, A.; Dinda, M.; Singer, M. A. Biochim. Biophys. Acta 1987, 903, 177. (40) Copeland, B. R.; McConnell, H. M. Biochim. Biophys. Acta 1980, 599, 95. (41) Hatta, I. J. Therm. Anal. Calorim. 2005, 82, 189. (42) Wang, X. Y.; Semmler, K.; Richter, W.; Quinn, P. J. Arch. Biochem. Biophys. 2000, 377, 304. (43) Miao, L.; Nielsen, M.; Thewalt, J.; Ipsen, J. H.; Bloom, M.; Zuckermann, M. J.; Mouritsen, O. G. Biophys. J. 2002, 82, 1429. (44) Bernsdorff, C.; Winter, R. J. Phys. Chem. B 2003, 107, 10658. (45) Huster, D.; Scheidt, H. A.; Arnold, K.; Herrmann, A.; Muller, P. Biophys. J. 2005, 88, 1838. (46) Scheidt, H. A.; Huster, D.; Gawrisch, K. Biophys. J. 2005, 89, 2504.

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