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Quantitative Chirality Measures Applied to Domain Formation in Langmuir Monolayers Suzanne Amador Kane* Physics Department, Haverford College, Haverford Pennsylvania 19041 Received July 19, 2002. In Final Form: September 9, 2002 A quantitative chirality measure previously developed by Zabrodsky et al. (Zabrodsky, H.; Peleg, S.; Avnir, D. J. Am. Chem. Soc. 1992, 114, 7843-7851) has been applied to the analysis of liquid-condensed domain shapes in Langmuir monolayers of the phospholipid D-R-phosphatidylcholine, dipalmitoyl (dipalmitoylphosphatidylcholine or DPPC). We present data on the variation of the quantitative chirality measure as a function of in-plane molecular area and temperature, as well as different subphase electrolyte concentration. Upon compression, the chirality is shown to grow monotonically with decreasing molecular area (increasing surface pressure) and to correlate linearly with the domain shape factor (a measure of noncompact growth). The chirality was similar for temperatures e25 °C and decreased substantially by 30 °C. For a subphase electrolyte concentration high enough to screen out electric dipole interactions, the dependence of chirality measure on molecular area is the same as that measured for low-electrolyte concentration data taken at 30 °C. Sample chirality calculations are also presented for a variety of other amphiphiles using previously published data.
Introduction In a series of papers, Zabrodsky et al. have shown that chirality, as well as other symmetries, can be quantitatively measured for any object.1-5 Previously, other chirality measures had been shown to distinguish deviations from mirror symmetry, but they were less sensitive to distinctions between merely random or labyrinthine shapes and visibly chiral ones.6 This technique has been applied to a variety of problems in chemistry7-11 and other fields, including applications to systems as diverse as molecular wave functions,1 supramolecular aggregates,12,13 and archaeological artifacts.14 Meanwhile, a diverse array of notably chiral domain growth has been documented for Langmuir monolayers made from a variety of amphiphiles, including naturally occurring phospholipids with and without cholesterol,15-20 and amphiphiles of nonbiological * To whom correspondence should be addressed. (1) Zabrodsky, H.; Peleg, S.; Avnir, D. J. Am. Chem. Soc. 1992, 114, 7843-7851. (2) Zabrodsky, H.; Avnir, D. J. Am. Chem. Soc. 1995, 117, 462-473. (3) Zabrodsky, H.; Peleg, S.; Avnir, D. J. Am. Chem. Soc. 1993, 115, 8278-8289 (erratum p 11656). (4) Zabrodsky, H.; Avnir, D. Adv. Mol. Struct. Res. 1995, 1, 1-31. (5) Avnir, D.; Katzenelson, O.; Keinan, S.; Pinsky, M.; Pinto, Y.; Salomon, Y.; Zabrodsky Hel-Or, H. In Concepts in Chemistry; Rouvray, D. H., Ed.; Research Studies Press: Somerset, U.K., 1996; pp 283-324. (6) Gilat, G. J. Phys. A: Math. Gen. 1989, 22, L545-550. (7) Pinto, Y.; Avnir, D. Enantiomer 2001, 6, 211-217. (8) Buch, V.; Greshgoren, E.; Zabrodsky, Hel-Or, H.; Avnir, D. Chem. Phys. Lett. 1995, 247, 149-153. (9) Kanis, D. R.; Wong, J. S.; Marks, T. J.; Ratner, M. A.; Zabrodsky, H.; Keinan, S.; Avnir, D. J. Phys. Chem. 1995, 99, 11061-11066. (10) Keinan, S.; Zabrodsky Hel-Or, H.; Avnir, D. Enantiomer 1996, 1, 351-357. (11) Pinto, Y.; Zabrodsky Hel-Or, H.; Avnir, D. J. Chem. Soc., Faraday Trans. 1996, 92, 2523-2527. (12) Katzenelson, O.; Avnir, D. Chemistry 2000, 6 (8), 1346-1354. (13) Katzenelson, O.; Zabrodsky Hel-Or, H.; Avnir, D. Chem. Eur. J. 1996, 2, 174-181. Katzenelson, O.; Avnir, D. Chem. Eur. J. 2000, 6, 1346-1354. (14) Saragusti, I.; Sharon, I.; Katzenelson, O.; Avnir, D. J. Archaeological Sci. 1998, 25, 817-825. (15) Weis, R. M. Chem. Phys. Lipids 1991, 57, 227-239. (16) Weis, R. M.; McConnell, H. M. Nature 1984, 310, 47-49. (17) Gaub, H. E.; Moy, V. T.; McConnell, H. M. J. Phys. Chem. 1986, 90, 1721-1725. (18) Weis, R. M.; McConnell, H. M. J. Phys. Chem. 1985, 89, 44534459.
origin.21-24 During this same time, distinctive changes in domain structure have been shown to occur upon the addition of pulmonary surfactants25,26 and local anesthetics,27 among other additives. According to X-ray diffraction results, in Langmuir monolayers the phase referred to as the LE (liquid-expanded) phase corresponds to a twodimensional fluid, with disordered hydrocarbon chains, while the LC (liquid-condensed) phase has longer distance positional correlations and long-range bond orientational order possibly corresponding to a two-dimensional hexatic phase.28,29 The chiral domain growth of interest results when LC domains nucleate and grow in the LE-LC coexistence region of the isotherm near room temperature. These systems differ notably from the labyrinthine domain growth found in many systems, including ferrofluids,30 and because of this chirality, the handedness is determined by the enantiomeric chirality of the constituent molecules. For example, the stereoisomers of DPPC form LC domains that are exact mirror images. To model such effects, several authors have added chiral terms to a Landau free energy by including terms that model interactions between the (19) Heckl, W. M.; Losche, M.; Cadenhead, D. A.; Mohwald, H. Eur. Biophys. J. 1986, 14, 11-17. (20) Kane, S. M. A.; Compton, M. A.; Wilder, N. Langmuir 2000, 16, 8447-8455. (21) Stine, K. J.; Uang, J. Y.-J.; Dingsman, S. D. Langmuir 1993, 9, 2112-2118. (22) Parazak, D. P.; Uang, J. Y.-J.; Terner, B.; Stine, K. J. Langmuir 1994, 10, 3787-3793. (23) Stine, K. J.; Whitt, S. A.; Uang, J. Y.-J.Chem. Phys. Lipids 1994, 69, 41-50. (24) Stine, K. J.; Whitt, S. A.; Parazak, D. P.; Uang, J. Y.-J. Chem. Phys. Lipids 1995, 75, 155-161. (25) Taneva, S. G.; Keough, K. M. Biophys J. 2000, 79, 2010-23. Ruano, M. L.; Nag, K.; Casals, C.; Perez-Gil, J.; Keough, K. M. Biophys. J. 1999, 77, 1469-76. (26) Lee, K. Y. C.; Majewski, J.; Kuhl, T.; Howes, P. B.; Kjaer, K.; Lipp, M. M.; Waring, A. J.; Zasadzinski, J. A.; Smith, G. S. Biophys. J. 2001, 81, 572-585. Lipp, M. M.; Lee, K. Y. C.; Zasadzinski, J. A. Biophys. J. 1997, 72, 2783-2804. (27) Kane, S. M. A.; Floyd, S. D. Phys. Rev. E 2000, 62, 8400-8408. (28) Kjaer, K.; Als-Nielsen, J.; Helm, C. A.; Laxhuber, L. A.; Mohwald, H. Phys. Rev. Lett. 1987, 58, 2224-2227. (29) Helm, C. A.; Mohwald, H.; Kjaer, K.; Als-Nielsen, J. Biophys. J. 1987, 52, 381-390. (30) Seul, M.; Monar, L. R.; O’Gorman, L.; Wolfe, R. Science 1991, 254, 1616-1618.
10.1021/la0262708 CCC: $22.00 © 2002 American Chemical Society Published on Web 11/07/2002
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director and the dipole orientation.31,32 These efforts have resulted recently in the successful computation of domain shapes very similar to those seen experimentally by including in the free energy an effective pair potential between the chiral molecules making up the monolayers, in addition to the usual terms modeling the effect of inplane line tension and dipole-dipole interactions.33 However, these two areas of researchsthe quantitative shape analysis of Langmuir monolayers and quantitative chirality measurementshave not been brought together to allow a quantitative determination of how domain chirality varies with relevant experimental variables. In this paper, we compute the chirality measure of LC domains in Langmuir monolayers of the chiral phospholipid L-DPPC and determine its dependence on in-plane molecular area (Amol), temperature, and electrolyte concentration. We also compare these findings with chirality computations on previously published work on other amphiphiles for comparison. Since earlier studies have established that domain twinning can occur via chiral segregation in monolayers formed from racemic mixtures of DPPC, we did not pursue the analysis of mixed chirality monolayers.34,35 Materials and Methods 1. Sample Preparation. All phospholipid samples were purchased from Avanti Polar Lipids (Alabaster, Alabama) and used without further purification. The fluorescent probe used was the acyl-chain labeled 2-(12(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1hexadecanoyl-sn-gl ycero-3-phosphocholine (NBD-PC). (Avanti Polar Lipids, Alabaster, Alabama). The NBD-PC probe is labeled on the acyl chain and partitions unequally between the LC and LE phases, accumulating preferentially in the latter phase. The phase behaviors and domain shapes described here were independent of the specific probe used. Probe concentrations of 0.5 mol % were used to label the lipid samples. Studies of isotherms for various probe concentrations were performed to establish that this probe concentration has negligible effect on the monolayer behavior. All solvents used in the experiment were HPLC grade, purchased from either Aldrich or Fisher Scientific. Ultrapure Milli-Q water (Millipore Corporation, Bedford, Massachusetts) was used to prepare the subphase. Subphases used consisted of either ultrapure water at pH 6.0 or 0.15 M NaCl titrated to 7.0 pH with HCl and NaOH. Setting the pH in this way allowed us to maintain the desired pH and electrolyte concentration without the complications of introducing other ionic species via a buffer; pH values were measured using a low-volume probe and were stable during the measurement. All chemicals used to prepare the subphase were highest-grade ACS reagents. All samples were spread from a 2:1 v/v chloroform/methanol solution at lipid concentrations of 1 mg/ mL. All phospholipid and lipid compositions are given as w/w ratios. 2. Langmuir Monolayer Isotherm Measurements. The Langmuir trough used was a KSV Instruments (Riverside, CT) Minitrough, modified to mount on a Nikon Labophot 2A epifluorescence microscope. The trough itself was Teflon, and area compression was achieved using two hydrophilic Delrin barriers. The trough area was 7.5 cm (31) Pikin, S. A. Physica A 1992, 191, 139-142. (32) Kam, R.; Levine, H. Phys. Rev. E 1996, 54, 2797-2801. (33) Kruger, P.; Losche, M. Phys. Rev. E 2000, 62, 7031-7043. (34) Moy, V. T.; Keller, D. J.; McConnell, H. M. J. Phys. Chem. 1986, 90, 3198-3202. (35) Moy, V. T.; Keller, D. J.; McConnell, H. M. J. Phys. Chem. 1988, 92, 5233-5238.
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by 25.5 cm, giving a surface area of 190 cm2. The subphase volume was 0.10 L. Surface pressure measurements were made using a KSV Wilhelmy electrobalance with a platinum plate. To eliminate air convection and dust, the entire microscope was enclosed in a sealed plexiglass box. The trough itself was enclosed in another box covered with thermofoil heaters, which enabled the regulation of the temperature of the air above the monolayers. The temperature of the trough and its subphase was controlled using a water bath, which circulated water through a heating block on the trough’s base. To avoid condensation, the microscope objective was also heated to just below the ambient temperature of the trough; this had the added advantage of minimizing domain drift. Calibrated Teflon coated thermistors were used to measure the temperature of the subphase and air. Temperatures were controlled to (0.1 °C over the course of an isotherm, and subphase and air temperatures were carefully equilibrated before each data-taking run. Clean water isotherms were performed before the monolayers studies began, yielding reproducibly lowsurface-pressure increases of 0.1 dyn/cm or less on a 10:1 area compression. Both the trough and Wilhelmy plate were cleaned and extensively rinsed with ultrapure water between sample runs with differing compositions. After spreading, films were allowed to equilibrate for 10 min before compression began. Compression rates corresponded to 0.13 Å2/molecule/s. Isotherms corresponding to stepwise compression during fluorescence microscopy runs were in good agreement with those taken in one shorter, continuous compression. Imaging was performed both during compression and while the barriers were paused at fixed area; little evolution of domain shapes was seen during the pauses. Further details about the experimental technique have been published previously.20 3. Epifluorescence Microscopy. For the epifluorescence measurements, samples were illuminated with a 100 W mercury lamp source filtered through a dichroic mirror/filter combination (Omega Optical). No photobleaching occurred because of a slight drift in domain position during a typical measurement. A long working distance 40X objective and a 5X projection lens combination was used to give a field of view of 105 micrometers by 135 micrometers. Images were collected using a Quantex QC-100 image-intensified camera and stored on VHS videotape. For quantitative analysis, individual images were digitized using a PCVisionPlus framegrabber from Imaging Technologies in a Pentium personal computer. 4. Image Analysis. The 8 bit gray scale 480 by 640 pixel images obtained from epifluorescence microscopy were analyzed using the MOCHA image processing package (Jandel Scientific, California) and ScionImage (Scion Corporation, Frederick, MD). Images were stored in pixels calibrated to represent the actual spatial dimensions, although this did not influence the computation of the chirality measure. The simpler calculations, such as edge tracing and computation of the domain area and perimeter, were all performed within MOCHA and ScionImage. To obtain edge tracings, the images were first corrected for nonuniform illumination by using a 5 × 5 pseudoclearfield correction in MOCHA, then smoothed once using a 3 × 3 averaging filter. The resulting images were further smoothed using an erosion filter followed by a dilation filter, then thresholding was used to separate LC domains from the surrounding LE regions. Edge tracings were then obtained from the thresholded images. More complex calculations were performed in the spreadsheet and data analysis program Origin (Microcal Soft-
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ware, Inc., Northampton, MA) using spreadsheets into which the edge-tracking data had been saved as x-y coordinates. Edge tracings were smoothed to reduce noise due to pixel noise in the original image, which leads to pixel-scale roughness along the perimeter after thresholding. Origin was used in particular to compute the arc length as a function of position along the edge-tracing perimeter. Files containing the x-y coordinates and corresponding arc length were then analyzed to determine the local curvature at each point on the edge tracing, using a defined region of neighboring points centered on the point of interest. (The region of perimeter used to compute the local curvature was fixed at approximately 20 points, which corresponds to a spatial distance twice the computed optical resolution, 2 µm, for our imaging optics.) The local curvature was obtained by fitting this local neighborhood of edge-tracing points to a quadratic polynomial using a nonlinear curve-fitting program written in C++ and adapted from Press et al.36 This fitted curvature was then used to compute a smoothed version of the edge tracing which eliminated noise on length scales small compared to the optical resolution. Chirality measures were computed before and after smoothing to determine the effect of this procedure on the chirality measure. In general, smoothing reduced the chirality measure by an amount either comparable to (for low-chirality images) or onethird of (for higher chirality images) the error bars quoted below; the effect of smoothing was to consistently decrease the chirality measure, since random noise in general increases the chirality measure. 5. Computation of the Continuous Chiral Symmetry Measure. The chirality measure, C, was computed using the method of Avnir et al.2 The actual program which implemented this technique was made available to us by David Avnir (Hebrew University) and was run on a Silicon Graphics Indigo II workstation running the operating system IRIX 6.5.37 This approach computes a continuous measure of deviations from perfect mirror symmetry, C, where C ranges from 0 (for a perfectly mirror-symmetric shape) to greater values for progressively more chiral shapes. This method amounts to taking the original shape, described by a series of n vertices, P Bi (where i ) 1, ..., n), and finding the set of vertices, P B′i, that define the nearest mirror-symmetric shape. (This method can be extended to treat other symmetry groups or elements, as described in the original papers.) The continuous chirality measure, C, is then defined as
C)
100 n
n
|(P Bi - P B′i)|2 ∑ i)1
(1)
The program controls for the effect of image size by a normalization procedure that involves scaling the image dimensions so the distance from the image center of mass to the farthest vertex is equal to 1 exactly. All values of B′i are then expressed in these normalized coorP Bi and P dinates. The factor of 100 is used merely to set the range of possible C values to 0 e C e 100. Thus, C can be thought of as an average percent deviation from perfect mirror symmetry. Reference 2 contains a more detailed explanation of why C is bounded; in brief, the upper limit on C arises because the worst case disagreement between (36) Press: W. H.; Flannery, B. P.; Teukolsky, S. A.; Vetterling, W. T. Numerical Recipes in C++: The Art of Scientific Computing; Cambridge University Press: Cambridge, U.K., 2002. (37) Readers interested in applying these programs for their own research are encouraged to contact Prof. David Avnir at david@ chem.ch.huji.ac.il.
Figure 1. (a) Sample edge tracing of a liquid-condensed domain taken from Langmuir monolayers of L-DPPC with 0.5 mol % NBD-PC probe at 25 °C on ultrapure water at Amol ) 54 Å2. (Compare Figure 3e.) (b) Closest mirror-symmetric shape computed for a, corresponding to a value of chirality measure C ) 0.42.
each pair of P Bi and P B′i is constrained to be at most 1 for this choice of normalization. Reference 5 gives illustrations of examples of closed-two-dimensional curves having both high and low values of C. The computer program used to compute C works by B′i, that describe the nearest finding the set of vertices, P mirror-symmetric shape, defined as the choice of the axis B′i that of mirror symmetry and the consequent set of P require the smallest amount of displacement to overlap with the original set of P Bi. The resulting computation of C thus also yields the nearest mirror-symmetric shape and mirror symmetry axis for inspection. This program first creates a dense set of vertices evenly spaced around the original shape defined by experimental x-y coordinates. In our case, 2000 vertex points were sampled for each image; since the largest perimeters observed were at most 500 µm, with most images having smaller perimeters, this corresponded to a spacing between sampled vertex points quite small compared to the optical resolution of the images. It then uses a genetic algorithm employing a procedure called the folding/unfolding method to find the set of vertices that minimize C. The folding/ unfolding algorithm can be understood as a process that averages the original shape and its mirror-image (for a specific choice of mirror symmetry axis passing through the image center of mass) point-by-point to create a mirrorsymmetric shape. A genetic algorithm is then used to perform a search for all orientations of mirror symmetry axis to yield the orientation with the lowest resulting value of C. The result for a typical LC domain shape is shown in Figure 1, along with its symmetrized form. Experimental Results Figure 2 shows representative isotherms and sample LC domain images for DPPC monolayers on ultrapure water at pH 6.0 with no additional buffer or salt at 18, 21, 25, and 30 °C. We also examined L-DPPC Langmuir monolayers formed at 25 °C on a 0.15 M NaCl, unbuffered subphase titrated to pH 7.0 with NaOH and HCl (Figure 3). The epifluorescence images used for quantitative image analysis were collected across the LC-LE coexistence region at fixed temperature. For each data set, a uniformly bright background, corresponding to uniformly distributed fluorescent probe, was observed before the beginning of the LC/LE coexistence region. The exclusion of probes from the LC phases made their growing domain shapes visible throughout the coexistence region. For each temperature, small, circular nuclei were observed soon after the onset of the LC-LE coexistence region. These initially approximately circular domains evolve to progressively more chiral and less compact domain shapes. In general, the LC domains formed on the 0.15 M NaCl subphase were notably more compact than those formed on ultrapure water with no added electrolyte. This is presumably due
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Figure 2. Surface pressure vs molecular area isotherm for Langmuir monolayers of L-DPPC with 0.5 mol % NBD-PC probe on ultrapure water subphase, pH 6.0 at (a) 18 °C, (b) 21 °C, (c) 25 °C, and (d) 30 °C. Sample edge tracings of epifluorescence images of characteristic liquid-condensed domain shapes are shown, with corresponding values of molecular area, Amol, and surface pressure indicated by arrows. Scale bars are in microns.
to electrostatic screening at high-salt concentrations of the repulsive dipole-dipole interactions between the zwitterionic DPPC headgroups, which favor ramified, less compact structure in the low electrolyte case.20 For each monolayer, images of multiple domains were collected at a variety of points along the LC/LE coexistence region. Images were selected either by time or molecular area and used without further editing to avoid selection bias. The one exception was images that clearly appeared to have twinning of domains. At most, three or four images in total were eliminated because of this effect. These images were digitized and analyzed as explained in the Materials and Methods section to compute the chirality measure (C), domain perimeter (P), and domain area (A). These last two quantities were then used to compute the shape factor, S, defined as
S)
P2 4πA
(2)
The values of this geometrical measure of compactness range from a low of 1 for a perfectly circular shape to higher values for less compact shapes. Thus, the shape factor provides a way to characterize the evolution of domains from more to less compact geometries.38 It does not, however, distinguish between chiral and achiral shapes. We have previously reported experimental results for the variation of S with molecular area and electrolyte concentration for these same systems.20 (38) Stine, K. J.; Stratmann, D. T. Langmuir 1992, 8, 2509-2514.
Our results for the dependence of C on both molecular area, Amol, and temperature are displayed in Figure 4. We also investigated how the chirality measure, C, varied with shape factor, S, for various temperatures and subphase conditions. These data are displayed in Figure 5. Sources of error in C include the natural hetereogeneity of the domain shapes themselves and also variation in the choice of the mirror symmetry axis by the program. Small variations in the actual domain shape, in the amount of residual roughness in the edge tracing, or in the choice of axis will influence the shape of the final closest symmetrical shape and hence will introduce significant variations in the chirality of shapes that appear similar to an observer. To estimate the uncertainty in the chirality measure, multiple images were sampled for the 0.15 M NaCl subphase, 25 °C L-DPPC monolayers. All images obtained at fixed molecular area for these samples appeared very similar to the eye; for comparison, the scatter in the shape factor has previously been computed and shown to vary smoothly, within much smaller percentage uncertainties, for these systems.20 Seven to 10 domains were used to compute the standard deviation for each chirality measurement. These results were used to compute the error bars displayed in the values reported for these samples. The more ramified and more chiral LE domains formed on ultrapure water-only subphases exhibited greater heterogeneity of detailed shapes, so we chose to represent these data by using individual data points. These same samples exhibited a monotonic, smooth, and reproducible evolution of shape factor upon
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Figure 5. (a) Chirality measure, C, vs shape factor, S, for L-DPPC Langmuir monolayers on ultrapure water subphase for 18 °C (3), 21 °C (0), 25 °C (O), 30 °C (]), and for 25 °C, 0.15 M NaCl subphase (b).
Figure 3. Isotherm and samples images for L-DPPC at 25 °C on 0.15 M NaCl subphase, with molecular area, Amol, and surface pressure equal to (a) 71 Å2, 8.88 dyn/cm, (b) 66 Å2, 9.98 dyn/cm, (c) 61 Å2, 11.1 dyn/cm, (d) 58 Å2, 11.8 dyn/cm, (e) 54 Å2, 14.0 dyn/cm, (f) 51 Å2, 16.6 dyn/cm, (g) isotherm.
Figure 4. (a) Chirality measure, C, vs molecular area for L-DPPC Langmuir monolayers on ultrapure water subphase for 18 °C (3), 21 °C (0), 25 °C (O), 30 °C (]), and for 25 °C, 0.15 M NaCl subphase (b).
compression, with a much lower percent noise than was measured for the chirality.20 To compare these DPPC samples with Langmuir monolayers formed from other chiral amphiphiles, we obtained edge tracings and analyzed domains from publications by several other research groups. These
Figure 6. Liquid-condensed domain edge tracings and chirality values for sample images from previously published work on liquid-condensed domains in Langmuir monolayers made from (a) R-DPPC with 2 mol % cholesterol, 23 °C, Amol ) 69 Å2 (solid fraction of 0.38), C ) 4.0, S ) 5.7 (data from Figure 5 of reference 17); (b) N-stearoylvaline, pH 2 HCl subphase at 22 °C, Amol) 37 Å2, C ) 0.37, S ) 3.3 (data from Figure 6a of reference 22); (c) N-stearoylserine methyl ester (SSME), respectively, C ) 2.0, S ) 3.5 (24 °C, Amol) 35 Å2, data from Figure 4a of reference 21); and (d) C ) 0.24, S ) 2.8 (37 °C, Amol) 36 Å2, data from Figure 6b of reference 21) both on ultrapure water subphase.
results are summarized in Figure 6. One of the most striking cases of chiral domain growth is the case of Langmuir monolayers made of phospholipids containing several mol % cholesterol. For example, Figure 5 of reference 17 shows a clear example of the “triskelion” LC domains which resulted for Langmuir monolayers made from R-DPPC with 2 mol % cholesterol at Amol ) 69 Å2 and 23 °C. These structures result when the characteristically spiral domains form 3-fold rotationally symmetric twinned structures. Our analysis of one typical single domain (Figure 6a) gave a chirality measure of 4.0. These techniques also can be applied to the study of nonbiological amphiphiles. For example, Langmuir monolayers made from N-stearoylvaline form upon slow compression whiskerlike domains with chirality measure 0.37, as determined from an analysis of Figure 6a of reference 22 (see Figure 6b). By contrast, the nonbiological amphiphile N-stearoylserine methyl ester (SSME) has been determined to form spiral-shaped LC domains after undergoing chiral segregation upon repeated compression at 24 °C; an analysis of typical domains from Figure 4a of reference 21 resulted in the edge tracing shown in Figure 6c, with a chirality measure of C ) 2.0 and S ) 3.5. When the temperature was raised to 37 °C, these systems
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instead formed LC domains with lower chirality measure C ) 0. 24 and S ) 2.8. Discussion An analysis of our data for DPPC monolayers showed a monotonic increase of chirality measure upon compression (Figure 4). In general, C increased significantly upon compression for all temperatures and subphase conditions investigated. The greatest increases were seen for the ultrapure water subphase data at 25 °C, although there was little difference on average between that temperature and the corresponding data taken at 18 °C and 21 °C. The dependence of C on molecular area was similar for the 30 °C, ultrapure water subphase and the 25 °C, 0.15 NaCl subphase data. Although there was a clear correlation and an approximately linear relationship between C and S for all samples investigated, we were unable to discern any clear temperature dependence of the slope for the ultrapure water-only subphase data (Figure 5). The slope of the C versus S data taken at 25 °C for both the ultrapure water and 0.15 M NaCl data agreed, indicating that the main effect of the salt is to limit the growth of more ramified, less compact structures, presumably by screening out dipole-dipole interactions. This linear dependence of C on S for these samples indicates that the shape factor is an adequate characterization of these samples for fixed phospholipid composition. However, racemic domains with equal D- and L-compositions would give low C values, but larger values of S, so these measures are not interchangeable. An analysis of DPPC containing 1 mol % cholesterol gave a value of C ) 4.0 (Figure 6a), significantly higher than the values of C ) 0.05 ( 0.02 for pure L-DPPC measured for our systems at a similar temperature and molecular area. The falloff of chirality with temperature was seen to be a phenomenon which also occurred in the nonbiological amphiphile SSME, which undergoes a decrease in domain chirality from C ) 2.0 (S ) 3.5) at 24 °C to C ) 0.24 (S ) 2.8) at 37 °C, in agreement with the temperature trend seen for our results for DPPC; the chirality measure was more sensitive than shape factor to the visible change in chirality between these two cases.21 (Figure 6c and d) Similar domain shapes for SSME and N-stearoylvaline gave similar values of chirality measure (C ) 0.24 and 0.37, respectively)22 (see Figure 6b and d). We have shown that quantitative chirality measures that previously have lent insight into diverse problems in
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stereochemistry and supramolecular aggregation can be successfully used to quantify the evolution of twodimensional chiral domain growth in Langmuir monolayers. These results show that chirality can indeed be quantified and usefully tracked for these samples, despite the relatively small values of the chirality measure obtained and the experimental uncertainty inherent in the relatively heterogeneous samples encountered. Since the domains studied here are compact compared to many of the cases of interest, we expected that this method should prove useful for studying the effects of impurities such as cholesterol, local anesthetics, other surfactants such as proteins important to pulmonary surfactant function, and for studying other factors in monolayer ordering. For example, this technique could be used in analyzing the growth of chiral domains upon compression in systems such as those found in Figure 1 of reference 19 for dimyristoylphosphatidylcholine (D-R-phosphatidylcholine, dimyristoyl or DMPA) Langmuir monolayers containing 1 mol % of cholesterol, on pH 11.4, T ) 10 °C subphases; these images consist of single, untwinned spiral domains, thus eliminating the uncertainties involved in extracting a presumed single domain from the triskelions of reference 17. Other interesting outstanding questions include correlating the stereochemical chirality of the phospholipid (or other amphiphile) headgroup with the macroscopic chirality of the domains formed from different species and the effect of mixtures of various phospholipids on chirality. Acknowledgment. This work was supported in part by National Science Foundation Grant No. NSF-DMB9109460 and by a William and Floral Hewlett Foundation Award from the Research Corporation. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research. We are grateful to David Avnir for his willingness to share his computer programs for chirality analysis and for his assistance in carrying out this research. We also would like to express our appreciation to Idit Saragusti and Dina Yogev for their assistance in obtaining the chirality analysis software, including their help in running test and control cases to verify that our computations were in agreement with their laboratory’s results. We have also benefited greatly from conversations with Robert Manning. LA0262708
