Langmuir 1989, 5, 30-34
30
Monomolecular Film Behavior of a Homologous Series of 1,2-Bis(o-cyclohexylacyl)phosphatidylcholines at the Air/Water Interface B. Asgharian, D. K. Rice, and D. A. Cadenhead" Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 14214
R. N. A. H. Lewis and R. N. McElhaney Department of Biochemistry, T h e University of Alberta, Edmonton, Alberta, Canada T6G 2H7 Received April 26, 1988 Monomolecular film studies of a homologous series of 1,2-bis(w-cyclohexylacyl)phosphatidylcholines (w-PCs)have been carried out at the air/water interface over a temperature range of 3-40 "C. The bulky cyclohexyl rings produce a greater disturbance in the packing of the acyl chains than do either methyl is0 or anteiso groups. From the surface pressure/area per molecule isotherms we see that there is a very small transition region between the condensed and expanded states, even smaller than those found for iso- or anteiso-branched PCs, which are in turn smaller than those found for straight-chain PCs. The small transition region can be explained by a substantially expanded condensed phase and a similarly condensed expanded phase. As the chain length of the w-PC increases, so does the magnitude of the transition region, a finding consistent with a reduced effect of the cyclohexyl group on longer chain PCs. 1,2-Di-w-cyclohexyl PCs exhibit an odd-even chain effect in their condensed-state packing, depending on whether the straight-chain segment has an odd or even number of carbon atoms. In addition, results from stepwisedetermined isotherms suggest that the odd-numbered chains pack in a more stable arrangement than do their even-numbered counterparts. It is interesting to note that in reference to close-packed areas the PCs increased in the order straight chain < methyl is0 < methyl anteiso < w-cyclohexyl, while in reference to the lowest temperature at which an expanded state can form (To) PCs increase in the order straight chain < w-cyclohexyl < methyl is0 < methyl anteiso. Thus, while the condensed areas/molecule are larger, the chain-chain interactions are stronger for w-PCs than they are for is0 PCs or anteiso PCs. This means that while w-PCs occupy a greater area/molecule in the condensed state they have greater molecular interactions than in films of is0 or anteiso PCs with smaller areas/molecule. Even more important, these interactions explain why the w-PC expanded states form and are stable at higher temperatures. Such characteristics should help explain the high w-cyclohexyl Occurrence in the thermophilic bacterium Bacillus acidocaldarius where w-cyclohexyl chains are found in abundance.
Introduction w-Cyclohexyl fatty acids are found in abundance in membrane lipids of some bacterial microorganisms. They constitute at least 70 mol % of the membrane lipids of several strains of Bacillus a c i d o ~ a l d a r i u sand ~ - ~ the bacteriophage 4 N S l 1 which infects it.5 B. acidocaldarius is an acido thermophilic bacterium characterized by its tolerance to high temperature (65 "C) and acidity (pH 3). These fatty acids have also been found as significant components of the membrane lipids of the mesophilic bacterium Curtobacterium pusillum.6 Only odd-numbered fatty acyl chains (w-cyclohexylundecanoic and tridecanoic acids) are found to occur naturally. However, some of these fatty acids can also support normal growth and function of the microorganism Acholeplasma laidlawii B under conditions in which the cell membranes are made homogeneous with respect to such fatty acids.7 In this paper, a monomolecular film study is presented of a homologous series of w-cyclohexylphosphatidylcholines (w-PCs). In order to examine the effect of cyclohexyl rings on chain packing, the results have been compared with the monomolecular film studies carried out earlier in this laboratory for homologous series of straight-chain: methyl iso-branched? and methyl anteiso-branched'O PCs. We found that while w-cyclohexyl substitution produces a greater increase in the close-packed areas/molecule than does either is0 or anteiso methyl branching, it also achieves a greater chain-chain interaction, in chains of equivalent length, than either of the latter two perturbations. *Author to whom all correspondence should be addressed. 0743-7463/89/2405-0030$01.50/0
Materials and Methods The synthesis and purification of the 0-PCs have been described el~ewhere.~ Each w-PC showed a single spot when examined by thin-layer chromatography. The solvent used for spreading the films was a 9:l volume ratio mixture of n-hexanelethanol due to the somewhat polar nature of PCs; 99 mol 7% pure n-hexane (Fisher Scientific, Fairlawn, NJ) was further purified by passing it through a column of alumina and then subjecting it to a final distillation. However, the absolute ethanol (Pharmco,Publicker Ind. Inc., Dayton, NJ) was used as supplied. The substrate water used was deionized and particulate free (Nano Pure System Sybron/Barnstead, Boston, MA) and subsequently quadruply distilled: once from alkaline permanganate, once from dilute sulfuric acid, and twice from itself (pH -5.6). The film balance system for measuring surface pressure (T) as a function of molecular area (A)has been previously described." For direct data collection and evaluation, the film balance was interfaced to a Data General Micronova computer. For continuous compression isotherms, the compression rate was 2-2.5 A'/ (1)DeRosa, M.; Gambacorta, A,; Minale, L.; Bu'lock, J. D. J. Chem. SOC.1971, 1334. (2) DeRosa, M.; Gambacorta, A.; Minale, L.; Bu'lock, J. D. Biochem. J. 1972, 128, 751. (3) Blume, A.; Dreher, R.; Poralla, K. Biochim. Biophys. Acta 1978, 512, 489. (4) Oshima, M.; Ariga, T. J. Biol. Chem. 1975, 250, 6963. (5) Sakaki, Y.; Oshima, M.; Yamada, K.; Oshima, T. J.Biochem. 1977, 82, 1457. (6) Suzuki, K.; Saito, K; Kawaguchi, A.; Okuda, S.; Komagata, K. J. Gen. Appl. Microbiol. 1981, 27, 261. (7) Lewis, R. N. A. H.; McElhaney, R. N. Biochemistry 1985,24,4903. (8) Suzuki, A.; Cadenhead, D. A. Chem. Phys. Lipids 1985, 37, 69. (9) Rice, D. K.; Cadenhead, D. A.; Lewis, R. N. A. H.; McElhaney, R. N . Biochemistry 1987,26, 3205. (10)Balthasar, D. M.; Cadenhead, D. A.; Lewis, R. N. A. H.; McEIhaney, R. N. Langmuir 1988, 4 , 180. (11) Cadenhead, D. A. Ind. Eng. Chem. 1969,61, 22.
0 1989 American Chemical Society
Monomolecular Films of 1,2-Bis(w-cyclohexylacyl)PCs
56 48
Langmuir, Vol. 5, No. 1, 1989 31
56
A 3.0
~
n
4.2
A 0 2 10 9. 3 1
v 4 21 2.1 0
v 5.7
-
0 6. 3
n
r\
*
48
E
\o
22.8
+ 23. 6
40
m
PI
t 32
x
-0
"
E
24 16 8
40
A R E A < A z / m o 1e c u 1e) Figure 1. Surface pressure (*)/area per molecule isotherms at stated temperatures ("C) for l,2-bis(o-cyclohexyldodecanoyl)phosphatidylcholine (w-12-PC) on a water substrate (pH 5.6).
r------
AREA
55
70
85
100
tA2/rno 1e c u 1e)
Figure 3. Surface pressure (n)/area per molecule isotherms at stated temperatures ("C) for 1,2-bis(~-cyclohexyltetradecanoy1)phosphatidylcholine (w-14-PC)on a water substrate (pH 5.6).
A 27. 6
n
0 28.4 V 29.2 4 30.3 31.3
48
*
E
\o
40-
(0
PI
C 32-
x
2
E
24
-
16
-
80-
AREA
I
1 e>
Figure 2. Surface pressure (*)/area per molecule isotherms at stated temperatures ("C) for 1,2-bis(w-cyclohexyltridecanoyl)phosphatidylcholine (r-13-PC) on a water substrate (pH 5.6). (molecule.min). In stepwise compression isotherms, the pressure was alllowed to relax to a pressure plateau for at least 10 min before the next compressional step was initiated.
Results .rr vs A isotherms for the five w-PCs containing 0-cyclohexylacyl chains of 12-16 linear carbon segments, 1,2bis(o-cyclohexyldodecanoyl) PC (0-12-PC), 1,2-bis(wcyclohexyltridecanoyl) PC (w-13-PC), 1,2-bis(w-cyclohexyltetradecanoyl) PC (w-14-PC), 1,2-bis(w-cyclohexylpentadecanoyl) PC (w-15-PC), and 1,2-bis(w-cyclohexylhexadecanoyl) PC (w-16-PC),are shown in Figures 1-5 over the temperature range in which they show an expanded/condensed phase transition. An w-11-PC was also examined, but the isotherms showed only expanded f i i s at all temperatures examined down to 3 OC. Generally the isotherms are more expanded in the condensed states and more condensed in the expanded states, with smaller transition intermediate regions than are found for the straight-chain PCs, the is0 PCs, or the anteiso PCs. The condensed-state molecular area, just before collapse, is between 53 and 54 A2.
AREA (A2/mo 1 1e > Figure 4. Surface pressure (r)/area per molecule isotherms at stated temperatures ("C) for 1,2-bis(w-cyclohexylpentadecanoy1)phosphatidylcholine (w-15-PC)on a water substrate (pH 5.6).
The expanded/condensed phase transition was observed over a very small temperature range of 5-6 "C in all five 0-PCs. This should be compared with 8-10 "C for is0 and anteiso PCs and with 25 OC for straight-chain PCs. For the w-PC homologous series, the expanded states and the intermediate regions of the members possessing longer chains exhibit larger molecular areas when they are similarly compared a t reduced temperatures ([ T - To]/ T o ) , where the w-PC is in a physical state equivalent to that of the is0 or anteiso PC. The temperature dependencies of the compressional onset of the expandedjcondensed transition (7rJ for the five 0-PCs are shown in Figure 6. By use of the least-squares method, 7rt was found to be better expressed by a higher order of curvature (2nd and 3rd) rather than a linear fit. Figure 7 shows the chain length dependency of To, the lowest temperature a t which an expanded state can exist.I2 For the homologous series (12) Kellner, B. M. J.; MUer-Landau, F.; Cadenhead, D. A. J.Colloid Interface Sci. 1978, 66, 597.
32 Langmuir, Vol. 5, No. 1, 1989
56 -
Figure 5. Surface pressure (,)/area per molecule isotherms at stated temperatures ("C) for 1,2-bis(w-cyclohexylhexadecanoy1)phosphatidylcholine (w-16-PC)on a water substrate
(pH 5.6).
E
e
A R E A t A * / m o 1e c u 1e > Figure 8. Stepwise (equilibrium)compressional isotherm for the
odd straight-chain (o-13-PC) lecithin (broken line) versus the continuous compressional data (solid line).
2ot 15
0,
\ 0
10
E5
10
30
20
40
T /=c Figure 6. Temperature dependency of the compressional onset of the expanded/condensed transition ( ~for 3 the five w-cyclohexyl PCS: 0 ,w-12-PC;A w13-PC; 0,w-14-PC; 0, W-ISPC; V,W-16-PC. AREA (Az/mo 1 = c u 1 e > Figure 9. Stepwise (equilibrium) compressional isotherm for the
401
/
30
0 I
I
I
18
20 Cn
22
Figure 7. Chain-length dependency of To (the lowest temperature
at which an expanded state can exist'*) for the homologous series of 0-PCs w-12-PC through w-16-PC. The abscissa is labeled in terms of C,, the total number of carbon atoms per chain including the cyclohexyl group. of w-PCs, no odd-even chain-length effect on the transition temperature was observed such as was reported by Lewis and McEIhaney' for the corresponding T , of aqueous bilayer dispersions of these compounds.
even straight-chain (w-14-PC)lecithin (broken line) versus the continuous compressional data (solid line).
However, an odd-even effect with the condensed-state areas was observed. For all five w-PCs, the even-numbered chains occupy areas about 1 A2 larger than their oddnumbered counterparts. This was further examined with isotherms obtained by using multiple samples. A t least two samples were used for each w-PC data determination, and the results were always consistent. It was also studied by measuring equilibrium isotherms. This was done by analog recording of the * / A plots and stepwise compression. After each interval, the compression was stopped and the film pressure left to relax until no further relaxation was observed. Figures 8 and 9 show the typical odd-even equilibrium isotherms (dotted line) of an odd (w-13-PC) and an even (w-14-PC) lecithin a t about 3 OC above their respective Tovalues in contrast with the continuous compressional isotherms (solid lines). As can be seen, no relaxation is observed for the expanded state of either oddor even-numbered PCs, and relaxations are first observed above the transition, with the larger relaxation being observed for odd-numbered-chain w-PCs. Independent of chain length, a t 3 OC above their respective Tovalues the highest pressure obtained was about
Monomolecular Film of 1,2-Bis(o-cyclohexytacyl)PCs
Langmuir, Vol. 5. No.1. 1989 33
35 dyn/cm. With continuing compreasion no further pressure inmaw waa obtained, and after each compreasion the pressure relaxed back to the original value, regardless of aren, indicating the formation of a new (collap& phase. The molecular areas at this pressure were consistent with the close-packed arena obtained by continuous compreasion just before collapse and showed an odd-even effect. The equilibrium spreading pressure (q.J of C,, o-PC was found to be 4.5 dyn/cm at 25 OC (T,value of 34.6 OC).' This is substantially higher than that of DPPC, which has a urn 0 below its T, of 41.5 0C.L3 Thus the typical w-P8 has a much greater tendency to spread at the air/ water interface, but in spite of stepwise compreasions and delaw to allow for all obvious relaxation. the Dhvsid states above 4.5 dyn/cm must be regarded t& me&table at or below 25 OC.
-
Discussion
Transition M o n s and ToValues. The p m n c e of a cyclohexyl ring at the hydrocarbon chain terminal of the PCs obviously produces a large disturbance in chain packing. The closest packed condensed-state areas of the
Figure 10. Cory-Pauling-Koltun model of o-ryclohexylhendecanoic acid in the closest-packed arrangement. T h e two views depicted are taken at right angles to one another.
0-PCs studied here are all 10-12 A*/molecule larger than those o k ~ e for d straightchain PCs. The lwsely packed condensed states of these o-PCs clearly indicate weaker van der Waals forces. This permits the formation of an expanded phase with the expenditure of legs energy, and thus such states appear at lower temperatures than they do for the corresponding straightchain PCs; indeed, lower To values are found for the o-PCs. The isotherms (Figures 1-5) clearly show how introduction of a o-cyclohexyl moiety into a P C affecta the expanded states. At moderate surface pressures (15 dyn/cm and higher), the transition is essentially wiped out. This is particularly obvious in isotherms of shorter chain w-PCs. Furthermore, the expanded states observed here have lower areas/molecule than do straight-chain, methyl h b r a n c h e d or methyl anteisc-branched PCs. Studies of o-PC liposome^^'^'^ confirm that these lipids are more resistant to permeation by small molecules and have higher microviscosity than straight-chain PCs in the liquidcrystalline state. The bulky cyclohexyl ring reduces the conformational freedom in an expanded or liquid crystalline state and results in an ordering effect that permits the hydrocarbon chains to pack closer. This also causes the observed lower molecular areas for the liquid expanded state in the monolayer. This, coupled with the more expanded condensed state, not only reduces the intermediate transition region but also explains why the transition is observed over a very short temperature range of 5-6 OC (less thermal energy required for the transition). This is an even smaller transition region than those observed for methyl i m and methyl anteisc-branched PCs.BJo The very small phase transition region characteristic of w-PCs probably explains why Kannenberg et al.'6 did not observe a phase transition in their monolayer study of o-12-PC. The isotherms presented by these workers are either fully expanded (15, 19, or 22 "C) or fully condensed (1 "C), presumably because they did not record isotherms in the critical temperature range of 2-7 OC. The study of Kannenberg et al.l6 is the only other monolayer study of this class of phospholipids.
Table I. T,Valnea for Stnight- and BmncbedGh.in PCs
To.OC
mhm
pel
chain
straight
b
chain8
brancheds
15.5
9.1 17.2
antaim
branched" UCyclohexyP
IS -"
16 17 18 19 20 21
'Taken
32.1
5.0 15.0
322
ns 11.3 19.5 27.3 35.0
30.0 35.0 M the
straightchain -ent
plus three.
With CoreyPauling-Koltun (CPK) molecular models, it is easy to demonstrate that the most stable close packing of the w-cyclohexyl chain will occur when the all-trans hydrocarbon chain is in the equatorial position of the cyclohexyl ringwith three of the ringcarbons contiguous with the main hydrocarbon chain. The other three remaining ring carbons are offset on one side of the hydrocarbon chain (Figure 10). In this arrangement the o-PCs have an effective chain length equal to the straight-chain segment plus three carbon atoms. A greater insight into the magnitude and nature of the effectof bulky substitution on hydrocarbon chain packing can be achieved if we compare the Tovalues of this homologous o-PC series with those of some straight,8methyl iso? and methyl antehbranched"' PCs. Table I compares the To values on the basis of the effective chain length. When comparing the Tovalues we observe that the o-cyclohexyl substitution has caused the transition to shift to lower temperatures when compared to straight-chain PCs but that the effect is less than that observed for methyl iso- and anteiso-branched PCs. The relative decrease of Toin this series is in the order straight > o-cyclohexyl > methyl is0 > methyl anteiso. At the same time, the condensed-state areas of these hydrocarbon chains in PCs increase in the order straight chain < methyl is0 < methyl anteiso < w-cyclohexyl. The relative increase in transition energy (AH) is in reverse order from that of the condensed areas. accord in^ to the two-dimensional a d o g u e of the Chpeyron equatioi
Asgharian et al.
34 Langmuir, Vol. 5, No. I , 1989
at comparable reduced temperatures, decreases much more rapidly and results in the observed transition energy order. This shows that, even though bulky w-PCs occupy larger areas in the condensed states than the corresponding methyl-branched PCs, a higher absolute temperature is required for an w-PC to transform from a liquid condensed state to a liquid expanded state. This means that w-cyclohexyl hydrocarbon chains pack in such a way as to increase the chain-chain interactions, even though the molecules occupy larger areas/molecule. X-ray studies on the homologous series of w-cyclohexyl fatty acids and alcohols in the solid state suggest that the hydrocarbon chains have a 60' tilt to the normal to the end-group planes.17J8 This is in contrast with the 30° tilt observed for saturated straight-chain hydrocarbon compounds in the solid state. A higher degree 6f chain tilt in a monolayer may well facilitate an improved chain-chain interaction of these bulky w-cyclohexyl chains and hence increase the trasition temperature from that which is expected based on its condensed-state molecular area. Diffraction studies on Langmuir-Blodgett multilayers of these compounds should provide considerable insight into the chain packing and tilt angles. Odd-Even Packing Effects. Within the homologous series of w-PCs, an odd-even alternation of the condensed-state areas was observed. This clearly shows a somewhat different packing for these components in the condensed monolayer, depending on whether the hydrocabon chains have an odd or even effective number of carbon atoms. This phenomenon is fairly common for long-chain paraffinic compounds in the solid state and results in an odd-even discontinuity in the melting points.lg Such effects have been found for liposomes of branched-chain PCs based on differential scanning calorimetry re~ulta.~ In monolayers, however, this has not been observed before, though it has been looked for.1° It could be that the effect has not been previously found since a sufficiently large homologous series has not been studied. In addition, the effect would probably be exaggerated in the case of compounds having a large bulky substituent such as an w-cyclohexyl group. In the solid state this effect arises naturally from the difference in the possible endgroup interactions of compounds which have odd- and even-numbered long-chain paraffinic chains that are in a crystalline or quasi-crystalline lattice with the long chains tilted to the end-group planes.*O In monolayers, however, not enough data are available to explain the nature of the difference in condensed-state packing modes for odd and even chains. For odd-numbered chains, due to greater relaxation of the surface pressure in intermediate region of isotherms recorded via stepwise compression, one could predict that even-numbered-chain w-PCs have a more stable condensed state. The fact that no odd-even effect was observed with To values suggests similar packing of the molecules in the expanded state regardless of odd or even chain length. Comparison with Lipid Bilayers. Aqueous multilamellar dispersions of an extensive series of o-PCs were studied by Lewis and McElhaney7 using differential scanning calorimetry and 31Pnuclear magnetic resonance spectroscopy. These workers report that the T, values (17)Ishizawa, A. Bull. Chem. SOC.Jpn. 1971, 44, 845. (18) Ishizawa, A. Bull. Chem. SOC.Jpn. 1971, 44, 846. (19) Broadhurst, M. G.J. Res. Natl. Bur. Stand. ( U S )1962, A66,241. (20) Lewis, R. N. A. H.; McElhaney, R. N. Biochemistry 1985, 24, 2431.
observed upon heating these compounds are lower than those of linear saturated PCs of equivalent effective chain length2' but higher than those of the methyl is0 PCs previously studied by these workers.20 However, the enthalpy of the chain-melting transition is lower than for either the linear or methyl iso-branched PCs. Similar calorimetric results on a more restricted number of w-PCs were previously presented by Kannenberg et al.15 and by Endo et al.14 Lewis and McElhaney7 also reported the existence of very loosely packed gel states for these phospholipids, with the even-numbered w-PCs exhibiting even more disorder in the gel state than their odd-numbered counterparts. All of these calorimetric and spectroscopic data are consistent with the monolayer results presented here. Blume et alS3studied the influence of the synthesis and incorporation of branched-chain and w-cyclohexyl fatty acids on the phase transition temperature of B. subtilis membrane lipids. These workers reported that membrane lipids enriched with w-cyclohexyl fatty acids exhibited a slightly lower T, than the normal methyl iso- and anteiso-branched-enriched lipids. From this result Blume et aL3 concluded that w-cyclohexyl fatty acids confer no special advantage to growth a t higher temperatures. Rather, these workers postulated that w-cyclohexyl fatty acids might enhance growth a t high temperature by providing a good interaction with the hopanoids present in the B. acidocaldarius membrane, and some evidence for this view was subsequently provided by Benz et a1.I6 However, the results of the present study demonstrate that, in addition to having high Tovalues and hence expanded-state stability at higher temperatures, the areas per molecule of the o-PCs above their phase transition temperatures are smaller than those of the corresponding linear or branched-chain PCs in their expanded states. This indicates that the strengths of the chain-chain interactions are greater for the w-PCs. This finding is supported by the results of Endo et al.14and Kannenberg et al.,15 who reported that the permeability of liquid crystalline w-PC bilayers to water and nonelectrolytes is much lower than that of bilayers composed of linear saturated PCs. It is also supported by the fluorescence polarization studies of Sunamoto et al.122 who demonstrated that w-PC bilayers are more ordered above (but less ordered below) their T, values than are linear saturated PCs. Thus, despite their relatively disordered gel states and their reduced T, values, it is clear that w-PCs form more closely packed, highly ordered, less permeable bilayers in the liquid crystalline state than do linear saturated or branched-chain PCs. Therefore, w-cyclohexyl fatty acids should directly contribute to the thermal stability of biological membranes regardless of their ability to interact with any hopanoids which may also be present.
Acknowledgment. R.N.A.H.L. and R.N.M. acknowledge the financial support of the Medical Research Council of Canada and the Alberta Heritage Foundation for Medical Research. D.A.C. would like to thank Barbara Marsh for her assistance in typing this manuscript. Registry No. w-12-PC, 96332-11-5; w-13-PC, 81326-18-3; w14-PC, 97352-46-0; w-15-PC, 97352-47-1; w-16-PC, 97352-48-2. (21) Lewis, R. N. A. H.: Mak, N.; McElhaney, R. N. Biochemistry
1987,26, 6118.
(22) Sunamoto, J.; Iwamoto, K.; Inove, K.; Endo, T.; Nojima, S. Biochim. Biophys. Acta 1982, 685, 283.
