Langmuir 1991, 7, 272-276
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Mixed Monomolecular Film Behavior of Methyl Iso- and Anteiso- Branched-Chain and 0-Cyclohexyl Phosphatidylcholines with Cholesterol B. Asgharian, D. M. Balthasar,? D. A. Cadenhead,* and D. K. Rice* Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 14214 Received May 7, 1990. I n Final Form: July 13, 1990 This study presents the results obtained for several series of mixed monomolecular films of cholesterol with methyl iso-, methyl anteiso-, and w-cyclohexyl phosphatidylcholines, and these are compared with previously obtained cholesterol/straight-chain phosphatidylcholine data. The amounts of cholesterol required to eliminate the phosphatidylcholine liquid expandedlliquid condensed phase transition decrease in the order straight-chain > methyl iso-branched > methyl anteiso-branched > w-cyclohexyl alicyclic but increase with increasing chain length for both the methyl iso-branched and the methyl anteiso-branched series. In contrast there is little evidence of a chain length effect with the alicyclic w-cyclohexyl phosphatidylcholines. Since branching and w-cyclohexyl substitution expand the condensed state and condense the expanded state, thus reducing the extent of the liquid expanded/liquid condensed phase transition in the order straight-chain > methyl iso-branched > methyl anteiso-branched > w-cyclohexyl alicyclic, it is clear that such substitution reduces the need for cholestrol to eliminate the transition. The increased need for cholestrol with increasing chain length for branched phosphatidylcholine phase transition elimination reflects the reduced effect of such branching with increasing chain length. It is clear that the presence of chain-branching and alicyclic chains in bacterial membranes can at least partially offset the lack of cholesterol in producing a close-packed but liquid crystalline state.
Introduction Cholesterol is the major sterol present in mammalian cell membranes and is a major lipid in erythrocyte and myelin membranes. T o understand the function of cholesterol in membranes, many studies have focused on lipid/cholesterol model membrane systems and a substantial number have utilized monomolecular film balance On the basis of these studies cholesterol appears to promote a state of intermediate fluidity by producing a more condensed, but not rigid, expanded state and a gellike, but fluid, condensed state. Corresponding effects of cholesterol on phospholipids have been observed by using other model membrane system~.~,~J’-’~ For example, NMR studies have shown increased orientational
* To whom all correspondence should be addressed. t Present address: Nabisco Brands Technology Center, East Hanover, NJ 07936. Present address: Moore Research Center, 300 Lany Blvd., Grand Island, NY 14072. (1)Leathes, J. B. Lancet 1925,208,853. (2) DeBernard, L. Bull. SOC.Chim. Bid. 1958,40, 161. (3)Demel, R.A.; van Deenen, L. L. M.; Pethica, B. A. Biochim. Biophys. Acta 1967,135,11. (4)Chapman, D.; Owens, N. F.; Phillips, M. C.; Walker, D. A.Biochim. Biophys. Acta 1969,183,458. (5),Demel, R. A.;Guerts van Kessel, W. S. M.; van Deenen, L. L. M. Biochrm. Biophys. Acta 1972,266, 26. (6)Demel, R. A.; Bruckdorfer, K. R.; van Deenen, L. L. M. Biochim. Biophys. Acta 1972,255,311. (7)Muller-Landau, F.;Cadenhead, D. A. Chem. Phys. Lipids 1979,25, 315. (8)Albrecht,O.; Gruler,H.;Sackman, E. J.ColloidInterfaceSci. 1981, 79,319. (9)Smith, I. C. P. In NMR: Principles and Applications toBiomedical Research; Pettegrew, J. W., Ed.; Springer-Verlag: New York, Berlin, 1989;pp 124-156. (10)Smith, I. C.P.; Dufourc, E. J. In Analysis of Sterols and Other Significant Steroids; Academic Press: New York, 1989;Chapter 15,pp 301-318. (11)Mabrey, S.; Mateo, P.; Sturtevant, J. M. Biochemistry 1978,17, 2464. (12)Estep, T. N.; Mountcastle, D. B.; Biltonen, R. L.; Thompson, T. E. Biochemistry 1978,17,1984. Schmidt, G.; Ibel, K.; Sackmann, E. Biochemistry 1985, (13)Knoll, W.; 24, 5240.
*
order in phospholipid/cholesterol bilayers above the main gel/liquid crystalline transition ( Tm),9while below the T , value cholesterol prevents the ordering and crystallization of the acyl c h a i n ~ . ~ J ~ Cholesterol/phospholipid interactions in model membranes were found to be primarily of a nonspecific dispersive n a t ~ r e Mixed . ~ ~ ~ monolayer studies of cholesterol/ tetradecanoic acid and cholesterol/dipalmitoylphosphatidylcholine (DPPC),7 carried out over the entire compositional range a t 21 “C, found that the condensation results were very similar when compared on a “per chain” basis. These results suggested that maximum condensation could be associated with maximum cholesterol/ acyl chain contact. Albrecht et a1.8observed similar results for the DPPC/cholesterol system a t 25 OC. Although eubacterial membranes, which commonly contain branched-chain or alicyclic fatty acids, do not contain chole~terol,’~ considerable insight into the action of cholesterol can be gained by studying branched-chain or alicyclic PC/cholesterol systems. To date only very limited information has been reported on such interactions. One such study has examined diisoheptadecanoyl PC (DIHPC) and dianteisoheptadecanoyl PC (DAHPC) in admixture with cholesterol over a limited concentration range.I5 It was found that cholesterol could condense these branched-chain PCs, but the magnitude of the effect was smaller than that observed for the DPPC/cholesterol ~ y s t e m .To ~ further investigate the effect of branching and the presence of a ring in the hydrocarbon chain on cholesterol/lipid interactions, a wide range of methyl iso-, methyl anteiso-branched,and w-cyclohexyl PCs have been examined in admixture with cholesterol in the present study.
Materials and Methods The procedures for the synthesis and purification of the branched-chain PCs have been described elsewhere (14)Rohmer, M.; Bouvier-Nave, P.; Ourisson, G. J. Gen. Microbiol. 1984,130, 1137. (15) Kannenberg, E.; Blume, A.; McElhaney, R. N.; Poralla, K. Biochim. Biophys. Acta 1983, 733,111.
0 1991 American Chemical Society
Mixed Monomolecular Cholesterol Films [methyl iso-branched PCs (iPCs) (16), methyl anteisobranched PCs (aiPCs) (17), w-cyclohexyl PCs (wPCs) (IS)]. Cholesterol (Sigma grade 99+ % ) was obtained from Sigma Chemical Corp. (St. Louis, MO). The cholesterol used was always recrystallized from ethanol shortly before use since it has been found to oxidize over extended periods of time (19). The spreading solvent for all experiments was a 9:l volume ratio mixture of n-hexanelethanol. Certified ACS n-hexane (99 mol 96 pure; Fisher Scientific, Fair Lawn, NJ) was further purified immediately before use, first by passage through a freshly packed alumina column and then by distillation. Absolute ethanol was used as supplied. The substrate water used (pH 5.6) was deionized, particulate free (Nanopure system, SYBRON/ Barnstead, Boston, MA), and subsequently quadruply distilled, once from alkaline permanganate, once from very dilute sulfuric acid, and, finally, twice within a two-stage quartz distillation apparatus (Model 3140, Heraeus-Schott, Hanau, West Germany). Mixing of the two components was carried out in the Agla micrometer syringe used to deposit the film, according to the procedure described by Muller-Landau and Cadenhead,7 since this was found to give significantly better reproducibility. The film balance used for measuring surface pressure (*) as a function of molecular area ( A ) has been previously described in detail.20 For direct data collection and storage, the film balance was interfaced to a Data General MicroNova computer21 (Data General Corp., Westboro, MA). For each homologous series, all mixed film s / A isotherms were obtained 3-4 "C above the respective lowest temperature a t which an expanded state can be observed for that particular lipid (TO), ensuring that each branched-chain and w-cyclohexyl PC was in a similar physical state regardless of acyl chain length. This also ensured that both expanded and condensed states would be observed in each lipid isotherm.
Results iPC/Cholesterol Mixed Films. Three systems were examined which consisted of cholesterol in admixture with an iPC of effective acyl chain length 16 (DIHPC), 17 (diisooctadecanoylphosphatidylcholine,DIOPC) and 19 (diisoeicosanoylphosphatidylcholine,DIEPC) carbon atoms. The * / A isotherms for the DIHPC/cholesterol system over the entire compositional range are shown in Figure 1. The transition of DIHPC is first shifted to slightly higher pressures and then broadened and was eliminated as the proportion of cholesterol in the film is increased. The transition pressure increased about 1 dyn/cm with increasing cholesterol concentration and was no longer detectable a t 16 mol 94 cholesterol. A plot of the transition onset (at) versus mole fraction of cholesterol for each iPC/ cholesterol mixed system is shown in Figure 2A. It is clearly seen for this homologous series that as the acyl chain length is increased, both the cholesterol concentration necessary to eliminate the transition and the at shift increase. The mean molecular area plots ( A vs the mole fraction of cholesterol (x&I)at constant T ) for the DIHPC/ (16) Lewis, R. N. A. H.; McElhaney, R. N. Biochemistry 1985, 24, 2431. (17) Lewis, R. N. A. H.; Sykes, B. D.; McElhaney, R. N. Biochemistry 1987,26,4036. (18) Lewis, R. N. A. H.; McElhaney, R. N. Biochemistry 1985, 24, 4903. (19) Cadenhead, D. A.; Kellner, B. M. J.; Balthasar,D. M. Chem. Phys. Lipids 1982,31, 87. (20) Cadenhead, D. A. Ind. Eng. Chem. 1969,61, 22. (21) Asgharian, B.; Cadenhead, D. A. J. Colloid Interface Sci. 1990, 134, 522.
Langmuir, Vol. 7,No. 2, 1991 273
AREA methyl-branched > w-cyclohexyl. These observations can be interpreted in terms of an increasingly more condensed expanded state and an increasingly more expanded condensed state.24 For methyl iso- and anteiso-PCs, the cholesterol concentration necessary for maximum condensation and the magnitude of the effect are similar. In all systems, the magnitude of the condensation is smaller a t higher pressures because, in the expanded state, the hydrocarbon chains themselves occupy smaller areas. Figure 2 shows another distinct difference between w-cyclohexyl and methyl branching on mixed film behavior. Within the series of w-cyclohexyl PC/cholesterol systems ((313 to C I ~ )at , shifts in a similar manner irrespective of chain length and in all cases the transition is eliminated at 11-12 mol 5% cholesterol. This is a significantly different picture from that observed for the methyl iso- and anteisoPCs. The two latter mixed film systems show both an increase in slope for plots of at versus cholesterol concentration and that higher cholesterol concentrations are required to eliminate the transition as the chain length increases. These effects are believed to be the result of both the branched-chain behavior and the length of the
Discussion Evidence for a t least partial miscibility in all branchedchain and alicyclic PC/cholesterol systems examined here is given, firstly, by the observed increase in at (Art) as
(22) Heckl, W.; Cadenhead, D. A.; MBhwald, H. Langmuir 1988, 4, 1352. (23) Cadenhead, D. A.; Phillips, M. C. Adu. Chem. Ser. 1968, No. 84, 131. (24) Asgharian, B.; Rice, D. K.; Cadenhead, D. A.; Lewis, R. N. A. H.; McElhaney, R. N. Langmuir 1989,5, 30.
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Figure 6. Surface pressure (*)/area per molecule isothermsfor w-14-PC/cholesterolat 22 O C at the concentrations indicated.
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276 Langmuir, Vol. 7, No. 2, 1991
Asgharian et al.
PC molecule, or methyl branch position, relative to cholesterol. With CPK models, it can be shown that cholesterol penetrates up to about the 14 carbon atom of a phospholipid hydrocarbon chain. For longer chain phospholipids (>14 carbon atoms in the main chain), cholesterol sees essentially a straight chain. Therefore, the amount of cholesterol required to eliminate the transition should be directly related to the fluidity and structure of the chains themselves. For methyl iso- and anteiso-PCs, as the PC chain length increases, the expanded state areas increase significantly (4-5 A2/molecule per CH2 g r o ~ p l ~ ~The t~~). expanded phase behavior of longer chain methyl iso- and anteiso-PCs is expected to approach, but not reach, the behavior of straight-chain PCs, as the chain length increases. It has been shown for w-PCs that the effect of increasing chain length on increasing expanded state areas is greatly reduced, with each CH2 increment leading only to a 2-2.5 A2/molecule increase.24 It should also be noted that, based on the number of carbon atoms, the w-cyclohexyl group represents a much larger portion of the total hydrophobic chain (27-32 90) than either the isopropyl (iPC, 15-18'~~)or sec-butyl (aiPC, 18-22%) groups. Thus, while w-cyclohexyl groups do not directly interact with cholesterol, they clearly contribute in a condensation process by themselves reducing the mobility of the phospholipid chains. While similar qualitative statements may be made for methyl iso- and anteiso-PC/cholesterol systems, the magnitude of the reduction of the extent of the LE/LC transition due to introduction of w-cyclohexyl groups is much greater and produces a greater uniformity of effect which is almost independent of chain length. It would seem that, while both methyl-branching and w-cyclohexyl substitution will increase the spacing of the lower portions of the acyl chains, the cyclohexyl group appears to enhance, rather than diminish, upper-chain interactions so that a condensed region is produced at the ends of the chains. Particularly interesting is the fact that when the aiPC and iPC are branched a t equivalent carbons of the mainchain, the cholesterol concentration necessary to eliminate the transition is nearly identical (see Table I). This also supports the postulate that the action of cholesterol on these methyl-branched PCs is highly dependent on the branch position relative to cholesterol. For the shortest member of the two series, DIHPC and DAOPC, the branch point is the fifteenth carbon atom. Thus, the branched region, or region of largest increase in dis0rder,~7lies just above the cholesterol molecule, and cholesterol should be able to exert a fairly large ordering effect on a major portion of the acyl chains. As the chain length increases, the ordering effect of cholesterol on the total PC chain length decreases and there is a corresponding decrease in the condensing ability of cholesterol. The limited data reported on various mixed films of chain-substituted PCs and cholesterol make it clear that cholesterol will condense branched-chain PCs, although the effect is reduced relative to straight-chain PCs. Their reduced condensability can be understood in terms of the ~
~~
~~~
~
(25) Rice, D. K.; Cadenhead, D. A.; Lewis, R. N. A. H.; McElhaney, R. N . Biochemistry 1987, 26, 3205. (26) Balthasar, D. M.; Cadenhead, D. A.; Lewis, R. N. A. H.; McE1haney, R. N. Langmuir 1988, 4, 180. (27) Macdonald, P. M.; McDonough, B.; Sykes, B. D.; McElhaney, R. N. Biochemistry 1983, 22, 5103.
reduced magnitude of the LE/LC transition of the chainsubstituted lipids themselves. The magnitude of the effect that cholesterol has on methyl-branched and w-cyclohexyl PCs appears to be less because these lipids have already accomplished by themselves much of what cholesterol can achieve. Why cholesterol is not found in bacterial membranes, where the occurrence of branching is common, poses an interesting question since, although the need for cholesterol is reduced, it is still capable of producing effects. Typically cholesterol is only found in eucaryotes which are usually aerobic and only evolved when an oxygen-containing atmosphere developed. However, some aerobic bacteria do synthesize unsaturated fatty acids by using an aerobic pathway and it is conceivable that they could have synthesized cholesterol had there been a need for it. What we have shown here is that, because of the nature of the fatty acid chains in bacteria, there is much less of a need for cholesterol. It is interesting that cholesterol films have been shown to undergo oxidation with time.19128,29Although the degree of oxidation in mixed films with PCs may be much less than in pure cholesterol films, without knowing the precise oxidized form that may arise, it is difficult to say to what degree oxidized forms of cholesterol will be capable of condensing admixed PCs. However, there would appear to be an agreement19s29,30 that, due to initial oxidation of the 5-6 double bond and enhanced tilting to satisfy polar group immersion requirements, all oxidized forms will be substantially less effective than cholesterol. The harsh bacterial environment of Bacillus acidocaldarius, for example, would not appear to be a suitable place for cholesterol. Instead, hopanoids, a fully saturated class of pentacyclic triterpenoids, are known to occur in about 50% of the bacteria examined.31 Thus, 15% of the total lipids of the thermoacidophilic B. acidocaldarius consists of tetrahydroxybacteriohopaneand a related g l ~ c o l i p i d . ~ ~ It has been suggested that these have an equivalent function as that of cholesterol in e u k a r y o t e ~ , ~although 3$~~ hopenoids appear less effective than cholesterol. This argument, however, is significantly weakened by the presence of A 5 and A5,7 mono- and diunsaturated sterols in the eucaryotic algae Cyanidium caldarium in that this grows at temperatures of 55-60 OC in moderately acidic hot ~prings.3~
Acknowledgment. We wish to acknowledge the financial assistance of the New York State Science and Technology Foundation under Grant CAT887 in the completion of this work. We also wish to thank Dr. R. N. A. H. Lewis and Dr. R. N. McElhaney for the synthesis of methyl-branched and alicyclic phosphatidylcholines and for their very helpful comments on this manuscript. (28) Kamel, A. M.; Weiner, N. D.; Felmeister, A. J. Colloid Interface Sci. 1971, 35, 163. (29) Balthasar, D. M. Ph.D. Thesis, State University of New York a t Buffalo, 1987. (30) Heckl, W. M. Ph.D. Thesis,TechnicalUniversitat Munchen, 1988. (31) Rohmer, M.; Bouvier,P.;Ourisson, G.Proc. N ~ t l . A c a d . S ~Ui .S A . 1979, 76, 847. (32) Langworthy, T. A.; Mayberry, W. R.; Smith, P. F. Biochim. Biophys. Acta 1976, 431, 550. (33) Kannenberg, E.; Poralla, K.; Blume, A. Z. Naturwissenschaften 1980, 67, 458. (34) Poralla, K.; Kannenberg, E.; Blume, A. FEBS Lett. 1980, 113, 107. (35) Ikan, R.; Seckbach, J. Phytochemistry 1972, 11, 1077.
