Langmuir 1996,11,281-285
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Interaction of Phosphotungstate Ions with Phospholipid Monolayers: A Synchrotron X-ray Study G. T. Barnes,* I. R. Gentle, C. H. L. Kennard, and J. B. Peng Department of Chemistry, The University of Queensland, Brisbane, Australia
I. McL. Jamie Department of Physics, University College, University of New South Wales, Canberra, Australia Received August 10, 1994. In Final Form: October 17, 1994@ The surface pressure-area isotherms of monolayers of several phospholipids are markedly altered by the presence of phosphotungstate ions in the aqueous subphase at low values of pH (Gorwyn,D.; Barnes, G. T. Langmuir 1990,6,222). These changes have now been studied by grazing incidence synchrotron X-ray diffraction. Where the monolayerwas in a condensed monolayer state the 28 scans revealed a single peak consistent with hexagonal packing of the acyl chains despite the diacyl structure of the phospholipids. Addition of phosphotungstate to the subphase at pH 2 caused this peak to disappear, suggesting a disruption of the regular chain packing, and examination of the concomitantsurface pressure-area isotherms showed that there had been a transition from a condensed to an expanded phase.
Introduction
Experimental Section
Phospholipids are major components of the bilayer structures that form the matrix of most biological membranes.l The integrity of such structures during preparation for and examination under the electron microscope is therefore ofconcern in a great deal ofbiological research. The use of electron-dense materials as stains to fix specimens and to enhance contrast has been a common practice, but in recent years there has been an accumulation of e v i d e n ~ e ,particularly ~'~ by Talmon and his associates,4s5 indicating that such stains may alter the structures under examination, sometimes substantially. A phospholipid monolayer at the airlwater interface is similarto one side of a membrane bilayer and thus provides a useful and accessible model for certain interactions that may occur at membranes. Recently, Gorwyn and Barnes6z7 showed that the surface areas of several phospholipid monolayers were markedly changed by the addition to the subphase of phosphotungstic acid or uranyl acetate, both of which are used as stains in electron microscopy. There appeared to be profound changes in monolayer structure induced by these large ions, and it was therefore considered that a n examination of these structures by synchroton X-ray techniques could assist in explaining the changes. Because of operating practice it was not possible to use uranyl acetate in these experiments so this report is concerned solely with the substantial expansions in monolayer area that occur with phosphotungstate ion at low pH values. Grazing incidence X-ray diffraction measurements in the plane of the monolayer (20 or Qx scans) and out of the plane (Braggrods or Qz scans) were made on three phospholipid monolayers at various surface pressures and temperatures both without and with phosphotungstate in the subphase.
Materials. The phospholipids used in this study were 1,2dimyristoyl-sn-glycero-3-phosphoethanolamine (puriss grade) from Fluka and DL-a-phosphatidylcholine dipalmitoyl (purity
Abstract published in Advance A C S Abstracts, December 15, 1994. (1)Yeagle, P., Ed. The Structure of Biological Membranes, CRC Press: Boca Raton, 1991. (2) Lukac, S.; Perovic, A. J . Colloid Interface Sci. 1985,103,586. (3) Dreher, K. D.; Schulman, J. H.; Anderson, 0. R.; b e l s , 0. A. J. Ultrastruct. Res. 1967,19,586. (4) Talmon, Y. J . Colloid Interface Sci. 1983,93,366. (5) Kilpatrick, P. K.; Miller, W. G.; Talmon, Y. J. Colloid Interface Sci. 1986,107,146. ( 6 ) Gorwyn, D.; Barnes, G. T. Ultramicroscopy 1989,27,113. (7) Gorwyn, D.; Barnes, G. T. Langmuir 1990,6, 222. @
99%) from Sigma. Behenic acid from the Hormel Foundation was used to verify the experimental system. Chloroform from Wako Pure Chemical Ltd. was used as the spreading solvent in all cases. Subphasebuffer solutionswere prepared with doubly distilled water (Photon Factory) or MilliQ water (University of Queensland) and contained 0.10 mol L-' of NaCl (BDH, Analar) and 0.01 mol L-' of Tris buffer (tris(hydroxymethyl)aminomethane, Merck). The pH of these solutions was adjusted with HC1 (Katayama Chemical) to approximately 2.0. One series of subphase solutions also contained 1.0 mmol L-I of phosphotungstic acid, sodium salt (PTA) (Sigma). Apparatus and Procedures. The surface pressure-area isotherms were measured on a film balance at The University of Queensland that has been described in earlier publications.8 X-ray diffraction experiments were performed on wiggler beamline 16A at the Photon Factory, Tsukuba, Japan. The wavelength, A, was 1.488 A. The film balance consisted of a PTFE-coated trough of surface area 133 cm2fitted with a paper Wilhelmy plate and force transducerto measure surface pressure and mounted on the sample stage of the diffractometer. The geometry of this diffractometer has been described previously.9 A Soller collimator in front of the vertical position-sensitive detector gave a resolution in the plane of the surface of 0.009 A-l. The moving barrier could be controlled remotely by the operator or could be set to maintain a predetermined, constant surface pressure. The trough temperature was controlled by water circulated from a thermostated bath through channels in the base of the trough.
Results The operation of the equipment was first checked with a monolayer of behenic acid, and results are summarized in Figures 1and 2 and Table 1. An investigation of this material has been reported by Kenn et aZ.,1° and Table 1 also shows a comparison of their results with ours. Qualitatively there is good agreement between the two sets of data: there is a single peak at low surface pressures (8) Barnes, G . T.; Hunter, D. S. J . Colloid Interface Sci. 1990,136, 198. Lawrie, G.A.; Barnes, G. T. J . Colloid Interface Sci. 1994,162, 36. (9) Matsushita, T.; Iida, A,; Takeshita, K.; Saito, K.; Kuroda, S.; Oyanagi, H.; Sugi,M.; Furukawa,Y.Jpn. J.Appl.Phys. 1991,30, L1674. (10)Kenn, R. M.; Bohm, C.; Bibo, A. M.; Peterson, I. R.; Mohwald, H. J . Phys. Chem. 1991,95,2092.
0743-7463/95/2411-0281$09.00/00 1995 American Chemical Society
Barnes et al.
282 Langmuir, Vol. 11, No. I, 1995
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and two sharp peaks a t higher surface pressures. Quantitatively, the differences in the temperatures and surface pressures of these two sets make exact comparisons difficult, but nevertheless the Qx values show excellent agreement. The peak observed a t low surface pressure in the Qz scan (Figure l a ) was slightly asymmetric, and the &,-resolved contour plot (Figure lb) shows indications of two maxima, a t Q2= 0 and a small peak a t Q, = 0.6 A-l. This observation is consistent with a n overlap of the (1,l) and (2,O)peaks in the LZphase, where the maximum at Q2 = 0 is contributed by the (2,O) peak and the smaller maximum by the (1,l)peak. While no Bragg rod scans are given by Kenn et al. for 5 mN m-l and 7 "C, they noted a similar asymmetry in the Lz phase region and obtained similar Bragg rod profiles for the better-resolved peaks at 6 mN m-l and 18.5 "C. Both peaks a t the higher surface pressure (Figure 2) showed a maximum a t Q2= 0, which is also consistent with the results of Kenn et a1.l0 for behenic acid in the CS phase. Diffraction peaks were observed for monolayers of DMPE and DPPC, but no peaks were found for DMPC. Figures 3 and 4 show the results for DMPE, and Table 2 summarizes the useful data for DMPE and DPPC. Solid lines in Figures 3a and 4a are Lorentzian profiles, which fitted significantly better in all cases than Gaussian profiles. Bragg rod scans were performed for these monolayers under various conditions of temperature and pressure, and a n example is shown for the sharp single
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peak of DMPE a t 10 "C and 25 mN m-l (Figure 3a) in Figures 3b and 3c. Figure 4a shows the shift in peak position as a DMPE monolayer was compressed from 25 mN m-l to 45 mN m-l, and Figure 4b shows curves fitted to the corresponding Bragg rod profiles. I t is clear from Table 2 that sharp diffraction peaks are only observed under certain conditions: low temperature (10 "C) and high surface pressure ('25 mN m-l) being the most favorable combination. The surface pressure-area isotherms in Figure 5 show that DMPE monlayers at 10 and 15 "C are condensed a t surface pressures higher than 2 mN m-l, whereas a t 25 "C the transition to an expanded phase occurs at about 10 mNm-l. Comparison with Table 2 shows that sharp peaks were observed only when the monolayer was in a condensed phase and a t a surface pressure somewhat above the condensed to expanded phase transition and clear of the upward curving region a t the low area end of the transition. This suggests that the transition is not complete until the surface pressure has risen above this curved region, a concept supported
Phosphotungstate Ion Interactions
Langmuir, Vol. 11, No. 1, 1995 283
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Figure 4. (a) &,-resolved scans for DMPE at 15 "C and three differentsurfacepressures. (b)&,-resolved(Braggrod)profiles for the three peaks shown in Figure 4a. The curves are least squares fits of sin2(1/2Q~)/(1/2Q~)2, at the Q, maxima, representing an approximation t o the variation of the intensity due to the longitudinal part of the structure factor.14 L is a length parameter, and the peak maximum at Q, = 0 was excluded from the fitting procedure. and 25 "C (Figure 5). The X-ray peaks reproduced in Figure 4a span this transition pressure, but while these peaks do not given any indication of a structural change, examination of the Bragg rod profiles (Figure 4b) reveals a contraction of the peak profile toward Qz= 0 when the surface pressure is raised above IT,, indicating that the molecules are in a more ordered and upright orientation above this transition. Superficially, the IT-A isotherms for DPPC (Figure 6) are very similar to those of DMPE, but closer examination reveals that the areas are slightly larger with DPPC, a n (12) Helm, C. A.;Mohwald, H.; Kjaer, K.; As-Nielsen, J. Biophys. J. 1987,52, 381.
Barnes et al.
284 Langmuir, Vol. 11,No. 1, 1995 Table 2. Synchrotron X-ray Results for Phospholipid Monolayers on NaCl + Buffer at pH = 2 lipid
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effect that can reasonably be attributed to the bulkier phosphatidyl choline group compared with the phosphatidyl ethanolamine group. This difference is clearly seen in theX-ray diffraction patterns: in the condensed phases there are sharp peaks with DMPE but broad and weak peaks with DPPC. With DMPC the condensed phase is only reached a t comparatively high surface pressures: about 35-40 mN m-l a t 10 "C (isotherms not shown). No peaks were observed a t surface pressures lower than this value, and no measurements were made a t higher surface pressures. The addition of phosphotungstate to the subphase clearly caused substantial changes in the structures of DMPE and DPPC monolayers (Figures 5 and 6), and the X-ray diffraction data (Figure 7) demonstrate the nearly total loss of order in the monolayer upon addition of PTA. Apart from a weak broad peak for DMPE at 10 "C and 25 mN m-l (Figure 7a1, no peaks were observed in the presence of PTA.
Discussion The single sharp X-ray peak found with DMPE monolayers is similar to results obtained by others with DMPE
are given in Table 3. The shift with surface pressure (Figure 4a) is appreciable and clearly shows the compression of the monolayer, more precisely demonstrated by areas per molecule calculated from d-spacings. It is worth noting that these area values arise only from the ordered domains in the film and show slightly lower areas per molecule than the geometrical film area, which averages the areas per molecule over the entire film. The width of the diffraction peak (at half maximum) yields, after correction for the instrumental line width, the positional correlation length, 5. The experimental peaks ranged in width from only marginally greater than the resolution-limited line width for behenic acid a t low temperatures to approximately 2.5 times the direct beam width for DMPE (Figure 3a) of 0.022 A-l. This leads to a n uncorrected correlation length of 91A, or approximately 22 d-spacings, which is a n estimate of the lower limit of the correlation length. Thus, the crystallites are quite small. Similar small values of 6 were found for DMPA monolayers at high surface pressures by Helm et aZ.13and, when considered in conjunction with electron diffraction data,15 were interpreted as indicative of a hexatic phase structure. In a hexatic phase there is long range bondorientational order but only short range positional order, corresponding to small crystallites with the axes of the crystallites oriented in the same directions. Peterson et a1.I6have suggested that various monolayer phases are mesophases with structures analogous to the smectic categories. Accordingto this approach, the structures that yield a single peak in the 28 scans with Qz of zero correspond to the smectic BH category: a hexatic structure with hydrocarbon chains normal to the interface. (13)Helm, C. A.; Mohwald, H.; Kjaer, K.; Als-Nielsen, Europhys. Lett. 1987,4, 697. (14) Helm, C. A.; Tippmann-Krayer, P.; Mohwald, H.; Als-Nielsen, J.;Kjaer, K. Biophys. J. 1991,60, 1457. (15) Fischer, A.; Sackmann, E. J.Phys. (Paris) 1984,45,517. (16) Bibo, A. M.; Knobler, C. M.; Peterson, I. R. Makromol. Chem. Makromol. Symp. 1991,46,55.
Phosphotungstate Zon Interactions
Langmuir, Vol. 11, No. 1, 1995 285 a change in the monolayer state from condensed to expanded arising from a substantial increase in the surface pressure of the condensed-to-expandedphase transition. The absence of diffraction peaks in the presence of PTA is consistent with a liquid-like structure for the expanded monolayer state. A small broad peak was observed for the DMPE monolayers in the presence of PTA at low temperature (Figure 7a), the only instance in which there was any evidence of structure with the larger PTA ions present. Inspection of the isotherm (Figure 5) reveals that under these conditions the monolayer is only marginally below the surface pressure of the expandedcondensed phase transition, n,. Unfortunately, due to the broadness of the peak, it is difficult to determine whether the center has shifted relative to the corresponding peak in the absence of PTA, as could have been expected from the indirect evidence of the isotherms. The effects of bulk phase pH on the interaction of phosphotungstate ion (PW120403-)with phospholipids, as revealed by surface pressure-area data, led Gorwyn and Barnes' to conclude that interaction occurred almost exclusivelywith the protonated forms of the phospholipids. They also pointed out that the expansion induced by PTA corresponds to approximately one-third of the crosssectional area of the PTA anion, suggesting a ratio of one adsorbed PTA anion to three phospholipid molecules. Such an aggregate of three protonated phospholipid molecules and one PTA anion would thus have zero net charge. Two models for the interaction were proposed: in one, the PTA anion lies between the nearly horizontal dipoles which are oriented with their positive poles toward the anion; in the other, the dipoles have been changed to a vertical orientation with the PTA anion between the positively charged ends. Both structures would probably lead to a disordering of the monolayer packing which is in accord with the present X-ray diffraction data. However, the X-ray data do not provide information which would enable a distinction to be made between the proposed models.
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The addition of PTA to the subphase caused the complete elimination of diffraction peaks for those phospholipid monolayers that otherwise exhibited such peaks, with the exceptionof DMPE a t 10 "C and 25 mNm-l. Examination of the corresponding surface pressure-area isotherms (Figures 5 and 6) shows that the addition of PTA causes
Sharp synchrotron X-ray diffraction peaks were observed for DMPE only when the monolayers were in a condensed phase. These data are consistent with an hexatic phase structure corresponding to the smectic BH category. For DPPC, peaks were found in similar situations but they were broader and weaker in conformity with the slightly larger areas per molecule observed for this substance. With DMPE, only single peaks were found showing that the packing was determined by the individual acyl chains rather than by the double-chained phospholipid molecules. Addition of phosphotungstate ions to the subphase caused major increases in the surface pressure of the condensed-to-expanded phase transition, and X-ray examination of the expanded structures clearly revealed a loss of structural regularity.
Acknowledgment. Financial support of the Australian Nuclear Science and Technology Organization and the Australian National Beamline Facility is acknowledged. The ANBF is funded by a consortium comprising the ARC, DITARD, Ansto, CSIRO, ANU,and UNSW. We are also indebted to Prof. T. Matsushita, Photon Factory, Tsukuba, Japan, for the invitation to use his equipment, to Dr. K. Takeshita and Mr. E. Arakawa for their assistance with the experiments,and Dr. R. Steitz (Visiting Scholar,University of Queensland) for useful discussions. LA940628L
