Langmuir 2000, 16, 7315-7317
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A Comparative Monolayer Film Behavior Study of Monoglucosyl Diacylglycerols Containing Linear, Methyl Iso-, and ω-Cyclohexyl Fatty Acids B. Asgharian and D. A. Cadenhead* Department of Chemistry, 410 Natural Sciences Complex, State University of New York at Buffalo, Buffalo, New York 14260-3000
D. Mannock and R. N. McElhaney Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 Received March 11, 2000. In Final Form: June 12, 2000 A comparative monomolecular film study of a series of 1,2-di-O-(R-D-glucopyranosyl)-sn-glycerols in which the acyl chains were 16:0, 18:0, 19:iso, and 15:ω-cyclohexyl, was carried out at the air/water interface. Area/temperature isobars were obtained in the 20° through 60 °C range with the lipids going from fully condensed to fully expanded. It was found that the area/molecule shift between the expanded and condensed states decreased in the order 16:0 and 18:0 > 19:iso > 15-ω-cyclohexyl, with the expanded state of the ω-cyclohexyl glycolipid being the most condensed. Reduction of the area/molecule shift arose through an expansion of the condensed state and a condensation of the expanded state. Isotherms of the straight-chain glycolipids showed only a small expansion on changing the pH from 5.6 to 2.0, but the ω-cyclohexyl glycolipid showed no effect. All the glycolipids studied showed thermal stability up to the 60 °C range, in contrast with previously obtained results on similar phosphatidylcholines, which were unstable above 40 °C. These results help explain the greater than 70% abundance of ω-cyclohexyl glycolipids in several strains of Bacillus acidocaldarius, whose natural environment is in hot springs with a pH as low as 3 and a temperature of up to 65 °C. It is postulated that the ability to overcome the acidity arises through the insensitivity of the polar headgroup and the highly impermeable bilayer, whereas a glycolipid headgroup appears essential for survival at high temperatures.
Introduction Both branched and cyclic chain substituted fatty acids are common constituents of membrane lipids in bacterial microorganisms. The major fatty acids found in such organisms include methyl iso- and methyl anteiso-,1 cyclopropyl-,2,3 and ω-cyclohexyl-substituted4,5,6,7 compounds. Although it is generally agreed that bacteria utilize branched and cyclic chains to regulate their fluidity and the phase state of their membrane lipids, the precise way in which such lipids achieve this is not fully understood. It is clear, however, that branched and cyclic lipids show a substantially reduced area/molecule range, from a fully condensed state to a fully expanded state, in monomolecular films.8,9,10 This is achieved by an expansion of the condensed state and a condensation of the expanded state, resulting in a greatly reduced shift in the area per molecule for the transition. Calorimetric and 31P NMR studies with model membrane bilayers of phosphatidyl* Corresponding author. (1) Kaneda, T. Microbiol. Rev. 1977, 41, 391. (2) Hoffmann K.; O’Leary, W. M.; Yoho, C. W.; Liu, T. Y. J. Biol. Chem. 1959, 234, 1672. (3) Christie, W. W. Top. Lipid Chem. 1969, 1, 1. (4) DeRosa, M.; Gambacorta, A.; Minale, L.; Bu´Lock, J. D. J. Chem. Soc. 1971, 1334. (5) DeRosa, M.; Gambacorta, A.; Minale, L.; Bu´Lock, J. D. Biochem. J. 1972, 128, 751. (6) Blume, A.; Dreher, R.; Porolla, K. Biochim. Biophys. Acta 1978, 512, 489. (7) Oshima, M.; Ariga, T. J. Biol. Chem. 1975, 250, 6963. (8) Rice, D. K.; Cadenhead, D. A.; Lewis, R. N. A.; McElhaney, R. N. Biochemistry 1987, 26, 3205. (9) Balthasar, D. M.; Cadenhead, D. A.; Lewis, R. N. A.; McElhaney, R. N. Langmuir 1988, 4, 180. (10) Asgharian, B.; Rice, D. K.; Cadenhead, D. A.; Lewis, R. N. A. H.; McElhaney, R. N. Langmuir 1989, 5, 30.
cholines (iso-, anteiso-, and ω-cyclohexyl PCs) also demonstrated significantly lower chain-melting phase transition temperatures than those of straight-chain PCs.11,12,13 Similar results have been obtained in membranes of Acholeplasma laidlawii B for both chain branching and ω-cyclohexyl fatty acids.14,15 One consequence of such a lowered transition temperature is that a membrane will remain fluid to lower temperatures. However, the fact that mesophilic16 and thermophilic bacteria utilize branched-chain and cyclic lipids to a high extent suggests that lower transition temperatures by themselves are not the crucial factor in understanding the advantages of branched and cyclic chains versus straight chains. In this article, we will concentrate on the comparative monomolecular film behavior of the lipids 1,2-di-O-acyl3-O-(R-D-glucopyranosyl)-sn-glycerol, (di- acyl-R-GlcDG), where the acyl chains selected were hexadecanoyl (16:0), octadecanoyl (18:0), iso-methyl-nonadecanoyl (19:iso) and ω-cyclohexyl pentadecanoyl (15:ch) fatty acids. With the exception of 16:0, the equivalent chain length for the other three fatty acids was 18 carbon atoms. The results will be compared with previous data from the corresponding (11) Lewis, R. N. A. H.; McElhaney, R. N. Biochemistry 1985, 24, 2431. (12) Macdonald, P. M.; Sykes, B. D.; McElhaney, R. N. Biochemistry 1985, 24, 2412. (13) Lewis, R. N. A. H.; McElhaney, R. N. Biochemistry 1985, 24, 4903. (14) Macdonald, P. M.; McDonough, B.; Sykes, B. D.; McElhaney, R. N. Biochemistry 1983, 22, 5103. (15) Macdonald, P. M.; Sykes, B. D.; McElhaney, R. N. Biochemistry 1985, 24, 4651. (16) Suzuki, K.; Saito, K.; Kawaguchi, A.; Okuda, S.; Komagata, K. J. Gen. Appl. Microbiol. 1981, 27, 261.
10.1021/la0003645 CCC: $19.00 © 2000 American Chemical Society Published on Web 08/09/2000
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Langmuir, Vol. 16, No. 18, 2000
Figure 1. Isobars of 1,2-di-O-acyl-3-O-(R-D-glucopyranosyl)sn-glycerol having the acyl chains as indicated in the figure. All isobars were carried out at 10 dyn/cm.
PCs,8,9,10 so that the role of both the acyl chains and the polar headgroup can be established. In this way, we hope to understand why several strains of Bacillus acidocaldarius select in excess of 70% of their lipids with ω-cyclohexl chains and an R-GlcDG or similar headgroup when grown at high temperatures or low pH. B. acidocaldarius is a bacterium characterized by its tolerance to high temperatures (65 °C) and low pH (pH 3).7 Experimental Section The approach taken here was to determine molecular area/ temperature isobars that permit the physical states of the lipid film (condensed and expanded) to be examined independently. The isobars were obtained at 10 dyn/cm with a film balance system that is described elsewhere17 and that was operated with a servomechanism in order to maintain a fixed pressure with increasing temperature. A pressure of 10 dyn/cm was selected for all of the isobars reported here. This selection was made in order to avoid the required higher compensating temperature (70° -80 °C), where considerable experimental difficulties arise. The basic film balance has also been described previously17 and was used to determine the isotherms of the straight-chain glycolipids at 36.5 °C. The glycolipids used in this study were synthesized by Drs. Ronald N. McElhaney and David A. Mannock of the Biochemistry Department of the University of Alberta, Edmonton, Canada, and were used as supplied. Details of the synthesis of the ω-cyclohexyl fatty acids were described previously18 as were those of the 1,2-di-O-acyl-3-O-(R-D-glucpyranosyl)-sn-glycerols,19 except that ω-cyclohexyl fatty acids were also incorporated in this case. Purification of these lipids has also been described,19 and the final lipids were chromatographically pure and of high anomeric and chiral purity. The lipids were spread over deionized, quadruply distilled water at pH 5.6 using a 9:1 volume ratio of n-hexane/ethanol as a spreading solvent.
Results and Discussion The isobars of 1,2-di-O-acyl-3-O-(R-D-glucopyranosyl)sn-glycerols having straight- (16:0 and 18:0), methyl isobranched- (19:iso), and ω-cyclohexyl- (15:ch) chains, obtained at 10 dyn/cm, are shown in Figure 1. All isobars show a relatively abrupt increase in the area/molecule as the temperature is raised from 20 to 60 °C. Comparison of the 16:0 and the 18:0 isobars show similar area/molecule shifts but with an approximately 15 °C temperature shift, that is, a 7.5 °C/2 carbon atom increase/chain. This temperature shift is consistent with that expected for the increased chain length.20 (17) Asgharian, B.; Cadenhead, D. A. J. Colloid Interface Sci. 1990, 134, 522. (18) Lewis, R. N. A. H.; McElhaney, R. N. Biochemistry 1985, 24, 2431. (19) Mannock, D. A.; Lewis, R. N. A. H.; McElhaney, R. N. Chem. Phys. Lipids 1990, 55, 309.
Asgharian et al.
Figure 2. Isobars of 1,2-di-O-acyl-3-O-(R-D-glucopyranosyl)sn-glycerol having the acyl chains indicated in the figure. The ordinate is plotted as a reduced surface pressure with all isobars initiating with the same condensed reduced area (that of 16:0). All isobars were carried out at 10 dyn/cm.
In comparing the area/molecule shift for the 16:0, the 19:iso, and the 15:ch, it is clear that the shift diminishes in that order. This is more clearly seen in Figure 2, where the ordinate is placed on a reduced area basis with the isobar for 18:0 removed. All isobars are adjusted to the same condensed state. Clearly, the expanded areas decrease in the order 16:0 or 18:0 > 19:iso > 15:ch. Of these three lipids, 15:ch has the most condensed expanded state either on an absolute or reduced area basis. Moreover, based on the slopes of the expanded states, 15:ch has the lowest coefficient of thermal expansion. Since the monolayer expanded and condensed states correspond to liquid crystalline and crystalline states, respectively, it is the expanded state that relates to a fluidity essential in lipid bilayers for cell viability. Thus, of the three lipids, 15:ch has the most condensed packing, which is consistent with the enhanced barrier properties required to produce a viable cell membrane at pH 2 and 65 °C. While branched and ω-cyclohexyl-chain substitution shows very similar effects in free fatty acids, phospholipids,10 and glucolipids, the magnitude of the effect is significantly different. The larger the size of the chain substitution, and the smaller the polar headgroup, the smaller the difference between the condensed and expanded state areas. For fatty acids, ω-cyclohexyl substitution results in featureless isotherms with only a gradual change in slope at the transition (unpublished results). In contrast, ω-cyclohexyl glycolipids show a reduced gap between the expanded and condensed states, even more so than ω-cyclohexyl PCs of similar chain length.10 Therefore, whereas a similar effect is obtained with ω-cyclohexyl chains in the various lipids studied, the magnitude of the effect appears to be linked to the size and nature of the polar headgroups. It has been postulated21 that the large size and the intrinsic rigidity of the ω-cyclohexyl group reduces the conformational order of the liquid-crystalline state by “dampening” all acyl chain motions. Thus, compared to straight-chain saturated fatty acyl groups, the increased orientational order and reduced rates of motion of the ω-cyclohexyl chains in the center of the bilayer would create a less permeable membrane. The 16:0- R-GlcDG shows a slight expansion of the expanded and intermediate states when the pH is lowered to pH 2 (see Figure 3); however, the isotherms of the ω-cyclohexyl glycolipid were identical at pH 2 and 5.6. One might have expected ester-linked glycolipid chains to show some pH sensitivity, but it is not clear why these (20) Kellner, B. M. J.; Muller-Landau, F.; Cadenhead, D. A. J. Colloid Interface Sci. 1978, 66, 597. (21) Mantsch, H. H.; Madec, C.; Ruthven, L. N. A. H.; McElhaney, R. N. Biochim. Biophys. Acta 1989, 980, 42.
Monolayer Film Behavior Study of Diacylglycerols
Figure 3. The effect of substrate pH on the surface pressure (π)/area per molecule isotherms of di-16:0-R-GlcDG. The temperature was 36.5 °C.
show little or none. One possible reason is that the sugar headgroup protects the ester linkage from hydrolysis. In addition, such an uncharged polar headgroup should be
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less sensitive to pH than those having zwitterionic or acidic headgroups. This ability to resist pH changes could be critical in the tolerance of the harsh, low pH, hot springs environment in which the bacteria exist. Finally, the isobars of the glycolipids (Figures 1 and 2) show that the films are stable up to the 60 °C range, something that we have not found for ω-cyclohexyl PCs,10 which became unstable at about 40 °C. It is clear, therefore, that the high-temperature stability of this lipid is very much dependent on the glycolipid polar headgroup. To some extent, the ability to resist low pH media also seems to depend on the headgroup, although the very low permeability of an ω-cyclohexyl bilayer clearly also plays a role. In none of this are we suggesting that other chains or other headgroups may not perform similar functions, only that the chain and headgroup of di-15ch-R-GlcDG do indeed confer both the ability to tolerate low pH media and to survive at high temperatures. These results help explain the greater than 70% abundance of ω-cyclohexyl glycolipids in several strains of Bacillus acidocaldarius whose natural environment is in hot springs with a pH as low as 3 and a temperature of up to 65 °C. LA0003645
