Molecular packing in steroid-lecithin monolayers, part II: Mixed films of cholesterol with dipalmitoylphosphatidylcholine and tetradecanoic acid

Chemistry and Physics of Ltpids 25 (1979) 315-328 © Elsevier/North-Holland Scientific Publishers Ltd.

MOLECULAR PACKING IN STEROID-LECITHIN MONOLAYERS, PART II: MIXED FILMS OF CHOLESTEROL WITH DIPALMITOYLPHOSPHATIDYLCHOIJNE AND TETRADECANOIC ACID

F. Mi~ILLER-LANDAUand D.A. CADENHEAD

Department of Chemistry, State University of New York at Buffalo, Buffalo, HY 14214 (U.S.A.)

Received June 6th, 1979

accepted July 26th, 1979

Mixed film studies of the systems cholesterol/tetradecanoic acid and cholesterol/dipalmitoylphosphatidylcholine have been carried out over the entire compositional range at 21°C. When compared on an acyl chain basis the condensing effects were found to be essentially independent of which host-lipid was u ~ e d . The phase change of the host lipid was shifted to higher pressures, then broadened and eliminated. Maximal condensation occurred at just above 42 tool%for the cholesterol/DPPC system. In both systems the two components were initially found to be miscfole at all proportions. The results are interpreted in terms of the molecular packing of cholesterol with acyl boundary layers, one significantly, one weakly affected. Maximum condensation is a result of packing that provides maximum cholesterol/acyl chain contact. Consideration is given to both long term stability of such mixed monolayers and the behaviour of the corresponding bilayer states.

Introduction In part I o f this series [I] the isotherms of pure films o f cholesterol, 3-doxyl cholestane (3-DC), 3-doxyl-17-hydroxyl-androstane (3-DA), tetradecanoic acid and dipalmitoylphosphatidylcholine (DPPC) were reviewed. In this article we will concem ourselves with our main objective: a better understanding o f the behavior o f cholesterol in admixture with potentially condensible host lipid molecules. We achieve this improved understanding thxough mixed f'dm studies of cholesterol with both tetradecanoic acid and DPPC. At f'trst it might seem unnecessary to produce fresh data o f this type in view o f the considerable amount already in existence (for reviews see ref. 2 - 4 ) . Closer inspection however, reveals that most of these studies were carried out within limited concentration ranges and in some cases with poor precision. The studies reported here were carried out with the maximum possible precision for the system and over the complete concentration range with about twenty intermediate concentrations. Our starting point in this endeavor was to attempt to understand the condensing behavior o f cholesterol on the physical state o f the host lipid, selecting two quite 315

316 F. Mfiller.Landau,DM. Cadenhead,Molecularpacking in steroid4ecithin monolayers11 different hosts, both of which exhibited an expanded and a condensed state in their compressional isotherm. As before we found the physical state of the host lipid was important [5], the condensing effect being maximized when the host lipid showed a partially expanded state. We also found that the fatty acid and DPPC host molecules behaved in a very similar way when the condensation results were compared on a 'per chain' basis, an indication that the polar groups play little or no role in such condensations. Finally, observation of the host lipid-liquid expanded/liquid condensed (LE/LC) phase change, and the way it shifts and is eliminated with cholesterol, and of the concentration at which maximal condensation was obtained, have led us to postulate that such condensations are best explained on a molecular packing basis.

Materials and methods

The automated f'tim balance which records simultaneously and continuously surface pressure (n) and surface potential (AIr) as a function of area/molecule (A) has already been described [6,7] as have general experimental procedures for establishing film component, spreading solvent and substrate purity and for obtaining the n/A, AV/A isotherms.

Preparation of multicomponent films The mixed fdms were obtained by applying pre-mixed solutions. Since the amounts of some materials available were limited, a method was developed in which the single component solutions were mixed in small quantities within an Agla syringe, immediately before spreading. This method was subsequently found to be superior in reproducibility and accuracy to the mixing of bulk solutions in larger quantities. The procedure required the determination of the dead volume of the syringe used: V0 at the micrometer reading R 0. The syringe was then filled with the first component and exhausted to a certain desired micrometer reading R~, wiping the attached teflon tip of the syringe. The syringe was then dipped into a solution of the second component and filled to a second point. Enough material within the teflon tube was then exhausted to just exclude any air bubbles and to accurately flU the translucent tube. This provided the micrometer reading R2. Subsequently, the plunger was again withdrawn to provide room for light mechanical mixing. The volumetric ratio of the solutions was then given by:

Vl

v~ - [ ( R I - R 0 ) + V0]/(R2--R1).

(1)

The translucent flexible teflon needle used with the syringe was of the thinnest inner diameter available (0.03 cm). Its olephobic nature, together with a short airspacing between solutions in the tube, prevented gross contamination of the

F. Mailer-Landau, D.4. Cadenhead, Molecular packing in steroid-lecithin monolayers I1 317

stock solutions during f'tlling. The maximum loss of solution through the plunger during the time needed for the complete handling was found to be less than 10-2 ml (out of 0.5 ml), but the compositional error deriving from this is of much smaller order, since both components were affected in a similar way. Evaporation loss with this method was negligible because there was little air/solution interface, much less

DPPC

WITH

CHOLESTEROL AT 21°C

45

0.7

40

0.6

35

0.5...

30

0.4 -J

E

I.-IJJ

laJ

o.3

25 u.J

Cholesterol 0.0 MOLE- % t2.0 MOLE- % 21.8 MOLE-% '~ 32.4 MOLE- % X 40.2 MOLE-% ~> 52.8 MOLE-% ~( 60.4 MOLE- % Y 69.6 MOLE- % 0 79.2 MOLE-% Z 90.4 MOLE-% • loo, o MOLE-%

o_ 2(3

1C

B

-0.1

0.0

\ O0

I

20

• \1 6O 80 100 AREA MOLECULE (A°z)

4O

I

120

Fig. I. Pressuze/area (left hand ordinate) and potential/area (right hand ordinate) for indicated mixed f'rims of cholesterol/DPPC. The symbols are for identification purposes only, all data were continuously recorded.

318 F. MiiUer.Landau, D.A. Cadenhead, Molecular packing in steroid4ecithin monolayers I I

than with any other method of mixing. Comparison measurements carried out by mixing different weights of the two components in one solvent gave identical results. Within a given set of experiments reproducibility was within -+0.5 )[2/molecule for a given surface pressure, and within -+5 mV for a given area/molecule. In general, for differing sets of determinations, these values were -+1.0 ~2/molecule and -+10 mV.

Results The results shown here are for the DPPC/cholesterol system. Similar results (not shown) were obtained for the tetradecanoic acid/cholesterol system. Figure 1 shows the force/area and potential/area plots for selected concentrations of the system at 21°C, all of which were determined as a single set. Additional concentrations were determined, but are not shown for the sake of clarity in the figure. Two main observations can be made from the force/area data: (1) that the LE/LC phase change of DPPC is initially shifted to higher pressures with the addition of cholesterol ( 0 - 9 mol% cholesterol) and then gradually eliminated ( 9 - 2 2 mol% cholesterol); (2) the collapse pressures of films having 4 0 - 1 0 0 mol% cholesterol vary in a near linear fashion. (The true collapse pressures of DPPC rich films were not determined. With the experimental set-up used here the aqueous substrate level was set just above the edge of the trough to avoid leakage under the barrier. At high pressures (approx. 50 dynes/cm) this procedure resulted in •m loss over the edge). While condensation effects are readily observed in Fig. 1 they are best quantified in the corresponding mean molecular area plots shown in Fig. 2. At all compositions over the whole compression range there is a marked reduction of the molecular areas over those of the corresponding pure component ftlms. Film condensation is greatest at pressures in which DPPC is liquid expanded, i.e., below 5 dyne/cm (Fig. 2). Above 44 mol% cholesterol the mean molecular area relationship is close to linear. In Table I (right hand side) a summary is made of the salient observations from the mixed trim data for both host lipids. From the surface potential values in Fig. 1, apparent surface dipole moments (or the vertical components of such moments) ta± (mDebye) were calculated from the formula: lax = A V / 2 r t r • n = 2.653 • 10-Is • AV. A

(2)

where AV = surface potential (mV), ~rx = 3.1415, circular constant, n = number of film molecules per square centimeter, and A = area per molecule (~2/molecule). The resultant mean dipole moment versus area/molecule plots are shown in Fig. 3. In contrast to the mean molecular area plots of Fig. 2, the mean dipole plots show relatively little deviation from lineatity except at low pressures.

F. Miiller-Landau, D.A. Cadenhead, Molecular packing in steroiddeeithin monolayers 11319 DPPC WITH CHOLESTEROL AT 21°C SURFACE PRESSURE

11C

© /~ -~×

'lOG

• [] Z Y }{

9C

2.0 4.0 7.0 10.0 12.0 15.0 22.0 27.0 35.0 44.0

DYNE/CM DYNE/ CM DYNE/CM DYNE/CM DYNE/CM DYNE/ CM DYNE / CM DYNE/ CM DYNE/ CM DYNE / CM

55

50

E 45

0

C O

C: El

~r

4O ~

e- 8(;

(/)

-J

LI,.I .J

c.)

70

~~5 " _ j' 0

,~ 60

30,,,

I,LI --.I 0

IJJ rr"

z

;

z

,~ 5C

25 ,,,

t.tJ

4C

20

I

1 8.7%

22%

15

50%

41.4~

2O

I

I

0.2 0.4 0.6 0~.8 MOLE FRACTION CHOLESTEROL

1.6 l°

Fig. 2. Mean molecular area plots (DPPC, left hand ordinate; cholestezol, right hand ordinate) as a function of the cholesterol/DPPC film composition at indicated pressures. Data were calculated from the pressure/area data of Fig. 1.

Discussion An acyl chain/cholesterol model In a recent review of lecithin/cholesterol condensations [4] a strong emphasis was placed on the role of hydrophobic interactions with a minor, and somewhat ill-defined role, ascribed to possible polar group interactions. For the data des-

4.5

33.3

100

Increamn8 direct contact between cholesterol molecules

Pure cholesterol film

2:1

26. 1

12.5

0

Minimum for each cholesterol to be mrrounded by 7 hydrocarbon chains

All hydrocarbon chains can be accommodated in direct proximity to cholesterol

All hydrocarbon chains can be accommodated within two layers from cholesterol

no cholesterol

Mol% cholesterol in reference to hydrocarbon chain concentration

3:1

7:1

21 : I

No. of hydrocarbon chains per cholesterol molecule 0

22.2

Little or no additional condensation for higher cholesterol concentrations

100.0

50.0

System maximally condensed 4 1 . 4

The transition disappears

Very little additional 8.7 inca'ease of transition premure and inflection getring less pronounced as film becomes more condensed with increasing cholesterol concentration

Initially linear increase of the transition pressure with increasing cholesterol concentration

Mol% cholesterol in reference to phospholipid systems (2 hydrocarbon chains)

CHOLESTEROL IN LIPID SYSTEMS WHERE THE PURE LIPID COMPONENT EXHIBITS A LIQUID EXPANDED/LIQUID CONDENSED PHASE TRANSITION

TABLE I

St

O

F. MFdler.Landau, D.A. Cadenhead, Molecular packing in steroid4ecithin monolayers II 321

DPPC WITH CHOLESTEROL AT 210C 0.90

0.50

0.85

0.45 hi )--

hi

0.40

0.80

I-I,-Z I.kl

UJ

~E 0.75 0

, ,~''~

~,s j

3.35 o

=E

UJ ..J

W ._1

s s

o 0.7O

-

t o•

0.30 LIJ C.)

1.1.1

0.25 ~-

0.6~ O0

0 ~ + X

.../~

0.6(:

Q [] Z Y

Q55

0.500

2.0 4.0 7.0 10.0 12.0 15.0 22.0 27.0 35.0 44.0

DYNE/CM DYNE/CM DYNE/ CM DYNE/CM DYNE / CM DYNE/CM DYNE / CM DYNE/CM DYNE/ CM DYNE/ CM

(/)

~0.20

0.15

, 0.2

, t t 1,( 0.10 0.4 0.6 0.8 MOLE FRACTION CHOLESTEROL

Fig. 3. Mean dipole moment plots (DPPC, left hand ordinate; cholesterol, right hand ordinate) as a function of the cholesterol/DPPC film composition at indicated pzessure~ Data were calculated from the potential/and data of Fig. 1 according to equation 2.

cribed here, a comparison o f the condensing effect of cholesterol on both host lipids (tetradecanoic acid and DPPC), is most revealing when carried out in relation to the aeyl chain concentration (Table I). The temperature of the respective host lipid films was chosen in such a way that DPPC and the fatty acid in their pure t'rims had equivalent physical states. The similarity of the condensing behavior on a per chain basis, places specific significance on the hydrophobic interaction between cholesterol and the acyl chains. The effect appears essentially independent

322 F. Ma//er-Landau,D.A. Cadenhead,Molecularpacking in steroidJecttMn monolayers H of whether the polar head group is a carboxyl group or is the more complex head group of lecithin. Significantly, the mean molecular dipole moment plots for the cholesterol/DPPC system (Fig. 3) show a near linear dependence over the whole concentration range. This is in agreement with the proposition that there is little interaction between the polar groups of the steroid and the lipid that would require a reorientation of the polar moiety. From these observaUons we have evolved a model for condensation effects, based on the molecular packing of cholesterol and acyl chains. Table I gives the characteristic concentrations for the proposed model and relates them to the observed phenomena of the mixed systems. The equivalent molar concentrations for the same hydrocarbon chain concentration for both the fatty acid system and the phospholipid system are shown. The cholesterol area/molecule is fixed and the area/close-packed alkane chain is approx. 20A 2. 0-8. 7 mol% (cholesterol/DPPCsystem). At a concentration of close to 21 hydrocarbon chains per cholesterol all chains can be grouped within two layers from the cholesterol molecules. In a well mixed system with positive interaction between the sterol and the neighboring hydrocarbon chains, this would be the concentration where no undisturbed hydrocarbon chain-chain interaction would be left. Therefore, it is not surprising that up to this concentration the condensing efficiency (AA/AobsJ per added cholesterol molecule increases lineady (Fig. 4). The motional freedom of the hydrocarbon chains would either be directly affected by immediate proximity to a cholesterol surface (nearest neighbor interaction) or indirectly by interaction with chains that are involved in such a direct interaction (next to nearest neighbor interaction). In other words, we are proposing that each cholesterol molecule has associated with it two layers o f boundary lipid, one significantly, one slightly affected. It is tempting to expand this model to other rigid hydrophobic imunity molecules. Certainly we would expect to see similar effects with steroid molecules that resemble cholesterol. Even more importantly, it would not seem unreasonable to consider an integral protein as such an impurity and anticipate that it too would have two similar boundary lipid layers due to non-specific lipid/protein interactions. t.0 AA

Aicleol

Q5

I

°

Mote % Cholesterol F~. 4. The efficiency of the cholestem! condensation( ~ l ~ l / A o ~

cholesteml/DPPC as a function of film oompodtton at 21"(2.

"

for DPPC) for the system

F. Miiller-Landau, D,4. Cadenhead, Molecular packing in steroid-lecithin monolayers II 323

The experimental observations are that, up to 4.5 mol% cholesterol (based on a single acyl chain reference), or up to 8.7 tool% for the cholesterol/DPPC system, the pressure of the observed LE/LC phase change of the hydrocarbon film increases in a near linear fashion. Little or no pressure change occurs at higher cholesterol concentrations. This behavior is especially pronounced in the fatty acid system (Fig. 5). The dependency below 4.5 mol% cholesterol clearly implies the existence of a homogenous two-component system [8]. The subsequent invariance of the LE/LC phase change to further addition of cholesterol has been taken as necessary evidence for the formation of a two-phase system for the higher cholesterol concentration range. Accordingly, the concept of a two-phase system has been presented [8]. This would explain the constancy of the transition pressure. It does n o t readily account for the fact that between a molar ratio of 4.5 mol% and 12.5 tool% (acyl chain reference) the transition broadens and disappears. Such an observation would suggest the presence of a single phase system. Moreover, from both our measurements and the isotherms shown by Motomura et al. [8], the typical condensation in the hydrocarbon chain/cholesterol system at the kink point is only 7% of the area of the corresponding pure Film molecular contribution (see Fig. 2). Subsequently, with increasing cholesterol concentration, the condensation reaches 15-30% depending on the compressional state of the film. This large additional condensation clearly implies strong molecular interactions only possible in a miscible system. Hitherto kink points, such as the one seen in Fig. 5, have generally been taken as evidence for phase boundaries and have been plotted as such into phase diagrams. It is certainly true that they reflect changes in the motional pattern of the hydrocarbon chain moiety but they do not have to define phase boundaries. In the model presented here and its application to the cholesterol systems studied, it is shown that abrupt changes in the motional parameters do not necessarily indicate true

6" c

n

hi

a. ,,,=.

5

I0 Mole %

15

Chol.este~l

Fig. 5. The shift of the onset of the LE/LC phase change of tetradecanoic acid foz the cholesterol/tetradecanoic acid system as a function of film composition.

324 F. Miiller.Landau,D.A. Cadenhead, Molecularpacking in steroid4ecithin monolayers 11 phase boundaries, but instead can result from changes in the population of a homogeneously mixed f'tlm.

8. 7-22 mol~ (cholesterol/DPPC system). Below 8.7 mol% cholesterol, the motional character of the individual hydrocarbon chains is heterogeneous depending on their distance from the nearest cholesterol molecule. At the point where the hydrocarbon chains that are unassociated with cholesterol are eliminated, the LE/LC phase change assumes a constant value. It subsequently disappears with the disappearance of all chains not directly in contact with cholesterol (all but nearest neighbors). This way as the cholesterol concentration increases, there is a successive disappearance of hydrocarbon chains in the more expanded states. This explanation does not involve a phase boundary in the conventional sense, and yet still accounts for the quasi-phase behavior of the particular molecular arrangement. As already indicated, at an acyl chain/cholesterol ratio of 7 : 1, all hydrocarbon chains can be accommodated in direct contact with the cholesterol molecule, and it is at about this concentration that we observe that the phase change of the hydrocarbon chain host fdm is eliminated. Previously this group [9] had reported the elimination of the DPPC phase change with cholesterol at 23 reel% cholesterol. In this work for the cholesterol/DPPC system a value of 2 2 - 2 3 reel% cholesterol was obtained, confirming the previous value and establishing the elimination of the LE/LC phase change when the acyl chain/cholesterol ratio reaches 7 : 1. A similar model was recently proposed by Martin and Yeagle [10] for bilayers, based on numerous experimental observations [11-13] at about 20-25 mol% in bilayers. 41.4 mol% (cholesterol/DPPC system). At an acyl chain/cholesterol ratio of approx. 3 : 1 there are just enough hydrocarbon chains to surround each individual cholesterol molecule with 7 acyl chains, though now each acyl chain is not exclusively assigned to one cholesterol molecule (41.4 mol% cholesterol (cholesterol/ DPPC system)). It is approximately at this point that maximal condensation of the system is reached. Engelman and Rothman [14] proposed a model that would account for a mixed phase in which each cholesterol would be surrounded by just seven acyl chains (see Fig. 7a of Part III of this series). From the model it is clear that acyl chain]cholesterol contacts are maximized. They indicate in their article that the model has a ratio of lecithin to cholesterol of approximately 2 : 1. A similar conclusion was reached by Martin and Yeagle [10]. Re-examination of the model shows that the PC/cholesterol ratio is in fact 1.5 : 1 i.e., it has an acyl chain/cholesterol ratio of 3 : 1. This occurs because several of the acyl chains are shared by three, not two, cholesterol molecules. Engelman and Rothman's argument was based on the cross-sectional area of cholesterol and the acyl chains at the interface and on their wide angle X-ray observations of an apparent two-phase region in their bilayer system below about 33 mol% cholesterol. The two phases were postulated to be an ordered gel phase of pure lecithin and a liquid-like mixed lecithin/cholesterol phase.

F. Mi~ller.Landau, D,4. Cadenhead, Molecular packing in steroid4ecithin monolayers II 325

Examination of Fig. 2 shows that maximum condensation of a liquid expanded f'flm occurs at about 43 mol% cholesterol (chol./DPPC ratio) rather than the 41.4 reel% which the model predicts. We believe that constitutes a reasonable margin of error, in that packing is unlikely to be precisely as the model suggests. Such discrepancies may well increase with increasing temperature requiring additional cholesterol to achieve maximum condensation. From the minima in Fig. 2, it is also evident that the amount of cholesterol required for maximal condensation decreases slightly with increasing pressure. The important feature here is that maximum condensation can be associated with maximum cholesterol/acyl chain contact. Perhaps because of a failure to recognize that the Engelman/Rothman model applied to a 4 1 - 4 2 reel% cholesterol/PC ratio rather than the maximum of 35 mol% they suggest, Martin and Yeagle [10] have postulated cholesterol dimer complexes to account for experimental observations at higher ( 4 0 - 5 0 reel%) cholesterol ratios in bilayers. While such explanations are not inconsistent with some of the effects seen, such dimer formation would presumably result in a decrease in fiflm condensation rather than an increase. For this reason we do not feel that dimer formation can adequately explain our results. With the air/water interface, in this concentration range, at least on initial compression we are dealing with a well mixed system. This is a necessary conclusion reached from the mean molecular area plot and the phase behavior of the mixed system as evidenced in the pressure versus area isotherms. At the same time an intimate mixture of boundary layer surrounded cholesterol and free acyl chains would be consistent with the apparent two-phase X-ray observations. Thus it appears possible to use an expanded model, on the basis of the one proposed by Engeiman and Rothman, in which nearest and next nearest neighbor interactions are taken into account. Above 41.4 molto (cholesterol/DPPC system). At higher cholesterol concentrations greater than 41.4 mol% (DPPC dependence), the strictly linear dependence in the mean molecular area plot could be interpreted in terms of a demixed, twophase system. But here the steady increase of the collapse pressure of cholesterol with increasing hydrocarbon chain concentration is evidence that cholesterol is not separating into a pure cholesterol phase. Similar findings have recently been reported by Snik et al. [15]. It appears that at cholesterol concentrations above the maximally condensed system, the mixed monolayer behaves as a near ideally miscible system. Our evidence thus indicates that the cholesterol/DPPC system is miscible over the entire concentration range. Possible metastable mixed films We have checked the stability of various film mixtures of cholesterol/DPPC at various temperatures and none are unstable in the sense that they all can be left for several hours. Typically a 1 : 2 phospholipid/cholesterol mixture, predicted by Darke et al. [16] to form a 1 : 1 complex plus excess cholesterol, remained stable for a period of 12 h (in a N2 atmosphere). Previous work of one of us [9]

326 F. M~_!__ie~.Zmutau, D.A. Cadenhead,Molecularpacking in steroid4ecithtn monolayers II showed that a cholesterol/DPPC mixture gave signs of segregation after only 30 min at a very low (approx. 0.01 dyne/cm) surface pressure. It should be noted, however, that a reasonable membrane packing pressure (approx. 20 dynes/cm, [17]) we were unable to detect segregation for times up to 16 h. In a recent cholesterol/DPPC study [18] at the air/water interface using sorbed monolayers of cholesterol/DMPC (dimyristoylphosphatidylcholine). Tajima and Gershfeld show an abrupt spreading pressure break somewhere in the region of 3 0 - 5 0 tool% cholesterol. The data were interpreted in terms of a 2 : 1 PC/cholesterol complex. Excess cholesterol or excess PC was thought to form a separate phase. It was also postulated that the complex represents a stable form and that miscible cholesterol/PC solvent spread mixtures at other compositions may be metastable. Should this indeed be the case it would seem that many of the mixtures we have studied may be metastable. Several comments can be made in this regard: (1) the Tajima and Gershfeld data [18] are equally well interpretable in terms of a 1.5 : 1 PC/cholesterol 'complex'. In other words our maximally condensed mixture may be a preferred packing ratio;(2) the other mixed films we have studied may be metastable, but they are not unstable. If metastable, the concern is as to whether such model membrane systems stay mixed for periods of time comparable to the turn-over time of cholesterol in the membrane [19]. Such would indeed seem to be the case; (3) if such films are metastable and collapse kinetics can be studied, such studies could have considerable significance. Typically, the 1 : 2 PC/ cholesterol film which remained stable for a 12 h period may subsequently undergo segregation to a stable 'complex' plus excess cholesterol. Presumably this excess cholesterol would squeeze out from a multilayer on top of the original monolayer as indicated in Part I of this series. Our observations indicate that it would not dissolve in the substrate. In a bilayer this would mean that cholesterol could build up between the monolayers of the bilayer [20]. Such a phenomenon could relate to atherosclerotic build-up; (4) if some of these monolayers are indeed metastable, it would seem that similar metastable mixtures are possible in the bilayer in view of the monolayer-bilayer correspondence discussed below. Monolayer/bilayer equivalence There is considerable confusion in the bilayer fiterature concerning the various effects seen at differing concentrations with phase segregations and molecular complexes being liberally evoked. (For a recent discussion see ref. 21). It would appear, however, that in spite of possible energetic differences between monolayer and bilayer, there is a considerable correspondence. In particular for the DPPC/ cholesterol system the recent DSC work of Estep et al. [22] and Mabrey et al. [23] as well as the NMR work of Haberkorn et al. [21] and Oldfield et al. [24] would seem to confirm our 22 and 42 mol% values (DPPC reference, Table I) in that at approximately these concentrations the sharp and broad components of the main DPPC transition are eliminated. It may also be that the compositionally more precise monolayer studies can continue to predict further significant changes

F. Miiller.La~.d~, D.A. Cadenhead, Molecular packing in steroid4ecithin monolayers H 327

for the bilayer. Typically, Mabrey et al. [23] find a slight decrease in the T c with the first l0 tool% cholesterol added and comment that broadening initiates at about 5 mol% cholesterol. These findings effectively overlap our 8.7 tool% cholesterol (DPPC mixed system) composition where the LE/LC phase change ceases to shift and begins to broaden. Hydrophobic or polar interactions

The fact that we obtain near identical behavior on a per acyl chain basis, irrespective of whether the polar group is a carboxyl group of the more complex head group of lecithin, indicates that the condensation is essentially due to hydrophobic interactions. This is substantiated by the way in which the mean dipole moment versus composition plots show little deviation from ideality. This last piece of information could, of course, be explained by an aqueous substrate compensation effect, but this would seem unlikely over such a wide compositional range. In the next paper this question will again be taken up when both the polar group of the steroid and is hydrophobic portion will be modified. Thus, the systems 3-DC/DPPC and 3-DA/DPPC will be examined in detail and compared to the results presented here for the cholesterol/DPPC system. In addition the question of the effective failure of epicholesterol and other steroids to condense expanded phospholipids will be reviewed and a new explanation offered. Molecular interactions and fluidity

Recently, molecular packing models for cholesterol/lipid and intrinsic protein/ lipid systems have been proposed by Chapman and coworkers [25,26]. These models differ from that proposed here in that we observe effects interpretable in terms of boundary lipid, whereas the absence of attractive forces in the Chapman models exclude such a possibility. It should be noted that where 'impurity' molecules, such as cholesterol, are capable of spacefilling [27] enhanced dispersive interactions can result (to be published). Even at maximum condensation, cholesterol/DPPC films are still fluid. This may be demonstrated by the movement of purified talc particles deposited on the film when gently blown. This in turn suggests that exchange between boundary lipid and surrounding lipid is possible, and can explain discrepancies between ESR and NMR spectral interpretations in the observation of boundary lipid in bilayer lipid/protein systems [26].

Conclusion

(1) Mixed monomolecular films of cholesterol and DPPC appear initially miscible in all proportions. The hydrocarbon chains of the lecithin, or other host lipid molecule, are divisible into three groups based on their packing abilities: nearest neighbors (strong interactions), next-nearest neighbors (weak interactions) and those

328 F. M~/er-Landau, D.A. Cadenhead, Molecular packing in steroid-lecithin monolayers H which are essentially unaffected. The observations with cholesterol m a y have significance for other ' i m p u r i t y ' molecules such as integral proteins. (2) Maximal condensation occurs with cholesterol at approximately a molar ratio o f 3 : 1 acyl chains/cholesterol. The effect is explained in terms o f a molecular packing model where acyl chain/cholesterol contacts are maximized. (3) Cholesterol condensation effects on expanded host lipid tllms are thought to arise primarily through hydrophobic interactions in that when the two acyl chains o f DPPC are taken into account, the quantitative condensation effects are very similar to those found for tetradecanoic acid having only one chain.

Acknowledgement We wish to acknowledge the financial assistance o f the Heart Lung and Blood Institute, through HL-12760 and HL-24535 in the completion o f this work.

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