calorimetry of archaeal tetraether lipid—indication of a novel metastable thermotropic phase in...

Chemistry and Physics of Lipids

94 (1998) 1–12

Calorimetry of archaeal tetraether lipid—indication of a novelmetastable thermotropic phase in the main phospholipid from

Thermoplasma acidophilum cultured at 59°C

Marc Ernst a, Hans-Joachim Freisleben a,b,*, Emmanouil Antonopoulos a,Lutz Henkel a, Walter Mlekusch a,c, Gilbert Reibnegger a,c

a Gusta6-Embden-Zentrum der Biologischen Chemie, Laboratorium fur Mikrobiologische Chemie,Johann Wolfgang Goethe-Uni6ersitat Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt/Main, Germany

b Graduate Program Biomedicine, Faculty of Medicine, Uni6ersity of Indonesia, Salemba Raya No. 4, Jakarta 10430, Indonesiac Medizinisch-Chemisches Institut und Pregl-Laboratorium, Karl-Franzens-Uni6ersitat, Harrachgasse 21, A-8010 Graz, Austria

Received 17 March 1997; received in revised form 2 January 1998; accepted 5 January 1998

Abstract

The main glycophospholipid (MPL) from the archaeon Thermoplasma acidophilum is composed of a di-isopranol-2,3-glycerotetraether. The fraction of pentacyclizations of its hydrocarbon chains increases with the growth tempera-ture of the source organism (39 and 59°C), the respective lipids being named MPL39 and MPL59. MPL has a mainphase transition between −15 and −30°C. Non-hydrated and hydrated samples of MPL59 have been studied bydifferential thermal analysis (DTA). Non-hydrated MPL59 does not exert any phase transition. Computer simulationof an unhydrated MPL molecule with four pentacycles and another without pentacyclations demonstrates similarbehavior, i.e. the MPL molecules form coils with both polar ends getting closely together. The molecule withoutpentacyclation coils faster than that with pentacycles. With hydrated samples, DTA scanning conditions were varied.Under certain conditions, the shape of the calorimetric scans, i.e. occurrence of an additional (endotherm) phasetransition peak at +17°C and enthalpy changes of the phase transitions indicate a (metastable) solid-analogue phasein MPL59 in addition to the well-known liquid–crystalline phase. Only lipid samples from T. acidophilum with a highdegree of acyclic hydrocarbon chains (MPL39) had thus far been reported to form a metastable solid-analogue phase(Blocher, D., Gutermann, R., Henkel, B., Ring, K., 1990. Biochim. Biophys. Acta 1024, 54–60). A phase transitionmodel is presented for MPL59 which includes the existence of a metastable solid-analogue phase. © 1998 Publishedby Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Thermoplama acidophilum ; Tetraether lipid; Calorimetry; Differential thermoanalysis

Abbre6iations: DSC, differential scanning calorimetry; DTA, differential thermal analysis; MPL, main phospholipid fromThermoplasma acidophilum.

* Corresponding author.

0009-3084/98/$19.00 © 1998 Published by Elsevier Science Ireland Ltd. All rights reserved.

PII S0009-3084(98)00004-8

M. Ernst et al. / Chemistry and Physics of Lipids 94 (1998) 1–122

1. Introduction

Thermoplasma acidophilum is a thermo-acidophilic archaeon growing between 39 and59°C at pH 1–2 (Darland et al., 1970;Freisleben et al., 1994). It lacks a cell wall sothat its only permeability barrier is the cyto-plasma membrane composed of membrane-span-ning tetraetherlipids, which were previouslycharacterized by Langworthy (1977) as diiso-pranyl-2,3-glycero-tetraethers, modulated by dif-ferent headgroups attached to the 1-positions ofthe two glycerols. The main phospholipid (MPL)was identified as containing a hexose on one endand a phosphoric acid esterified to another glyc-erol on the other (Langworthy et al., 1982). Itschemical formula was given by Strobl et al.(1985) as 2,3,2%,3%-tetra-O-dibiphytanyl-di-sn-glycerol-1-glycosyl-1-phosphoryl-3% -sn-glyceroland recently, the sugar component was identifiedindependently by two groups as being b-L-gu-lose (Freisleben et al., 1996; Swain et al., 1997).Fermentor cultivation in order to obtain highyields of MPL was optimized at 59°C and pH 2(Freisleben et al., 1994). Its molecular mass wasgiven as 1638 g/mol for the sodium salt, whichcorresponds well with the recently calculated andexperimentally determined formula mass for theundissociated molecule of 1617.4 g/mol(Freisleben et al., 1996).

Suspended in water, MPL forms monolayersof low viscosity within the hydrophobic core,which is certainly due to its repetitively methyl-branched hydrocarbon chains (Langworthy etal., 1982). The rotational and space-filling be-havior of these methyl side groups is comparableto that of unsaturated fatty acid chains in com-mon phosphoester lipids.

By adaptation to raised growth temperature,the C40-biphytanyl chains show an increasingnumber of pentacyclizations, which reduce therotational freedom of the hydrocarbon chains.At the minimal growth temperature of 39°C(MPL39) acyclic biphythanyl chains prevail,whereas at the optimal growth temperature of59°C (MPL59) the degree of pentacylation ishigher (Langworthy, 1977). The exact pattern ofpentacyclations was recently determined

(Freisleben et al., 1996), differing from that pre-sented by Langworthy (1977) (Table 1).

Formerly, MPL59 had not shown ther-motropic phase transition between 0 and 70°C;only a small and broad endothermal transition(DH=14 kJ/mol) was found at sub-zero temper-atures between −30°C and −5°C (Blocher etal., 1984). MPL39 however, exhibits complexthermotropic behavior, which includes a sub-zero endotherm, followed by an exotherm be-tween −10 and 0°C and another endothermbetween 10 and 20°C. The transitions of MPL39have been rationalized by the postulation oftransitions between three different phases: ametastable solid-analogue phase, a stable solid-analogue phase and a liquid–crystalline phase(Blocher et al., 1990). The time constant for theformation of the stable solid-analogue phase wasfound to be in the order of minutes. Increasingcholesterol concentration was shown to have astabilizing effect on the metastable solid-ana-logue phase and the time constant for the for-mation of the stable solid-analogue phase wasprolonged (Blocher et al., 1991a). Mixtures ofMPL with varying degrees of pentacyclation, i.e.mixtures of MPL39 and MPL59, also exhibitedkinetic effects for a metastable phase (Blocherand Ring, 1991).

All former calorimetric measurements were ac-complished under comparable lyotropic condi-tions with a fixed buffer volume of 20 m l addedto each sample and the thermotropic behaviorwas determined (Blocher et al., 1984, 1985a,b,1990, 1991a,b; Freisleben et al., 1992). Non-hy-drated and hydrated samples were then scannedand the conditions were varied differently fromformer measurements in order to detect kineticeffects for a metastable phase possibly also exist-ing in MPL59.

2. Materials and methods

All chemicals were of analytical grade, pur-chased from Merck (Darmstadt, Germany), orSigma (Deisenhofen, Germany). The organic sol-vents were from Baker (Groß-Gerau, Germany;Resi quality).

M. Ernst et al. / Chemistry and Physics of Lipids 94 (1998) 1–12 3

2.1. Growth of T. acidophilum and isolation ofthe main phospholipid (MPL)

T. acidophilum, stem 1728, was obtained fromthe Deutsche Sammlung von Mikroorganismenund Zellkulturen (DSM), Gottingen, Germanyand cultured at 59°C and pH 2, according toFreisleben et al. (1994) using a Biostat 50D fer-mentor, Braun, Melsungen, Germany. Cells wereharvested in the late exponential growth phase,washed, concentrated, centrifuged and lyop-hilized (Freisleben et al., 1994).

Lipids were extracted from freeze-dried cellsas described (Freisleben et al., 1994). MPL wasisolated and purified chromatographically overDEAE-cellulose and silica columns using chloro-form–methanol gradients (Freisleben et al.,1994). Analysis was carried out by high perfor-mance thin layer chromatography (HPTLC) andhigh performance liquid chromatography(HPLC) using various solvent systems. MPL wasobtained at 99% purity.

For sample preparation, MPL was dissolvedin chloroform/methanol, pipetted into differen-tial thermal analysis (DTA)-pans in 5 m l frac-tions and dried under a stream of nitrogen gas.After application of the lipid, the samples werekept in a vacuum for 24 h in order to removeresidual organic solvent. 1.78–9.59 mg MPLwere applied per pan, the lipid mass was deter-mined gravimetrically (DmB0.01 mg). Finally,between 0 and 20 m l buffer (380 mM/1 Na ca-codylate/HCl, pH 7.0) was added and the panssealed. Moreover, the mixtures contained 50% ofthe cryoprotectant ethyleneglycol (12.5 M/l).This amount of cryoprotectant is necessary be-cause of the low temperatures of the sub-zerophase transition of MPL. The influence of cry-oprotectants was investigated previously (Cura-tolo, 1985; Miller and Bach, 1986; Blocher,1990). The samples were exposed to repeatedfreeze–thaw cycles in order to facilitate completehydration of the sample and DTA was per-formed with a Mettler TA3000/DSC30 instru-ment equipped with a liquid nitrogen coolingdevice. Heating and cooling scans were runrepetitively at scan rates between dT/dt=0.02and 0.25 K/s in order to confirm the equilibrium

of the sample. Details of the sample preparationfor calorimetry were given elsewhere (Blocher etal., 1984, 1985a,b, 1990).

Mass percent (%) of MPL was calculated ac-cording to Eq. (1):

PMPL=100MMPL

MMPL+Mbuffer

(%) (1)

where P is the percentage of MPL and M is themass as determined by means of a Mettler ana-lytical scale.

2.2. Molecular mechanics and dynamics

In order to explore the behavior of the lipidmolecules with and without pentacyclations,molecular models were constructed by the pro-gram Hyperchem Release 4 (Hypercube, Water-loo, Canada). Since these calculations wereperformed only to give a qualitative picture oflipid behavior, simplified model lipids were usedlacking both the hexose moiety and the phos-phate structure. By molecular mechanics usingthe MM+ force field, geometries of stretchedstarting conformations were optimized with re-spect to energy. The conformations thus ob-tained were then subjected to a 100 ps moleculardynamical simulation in vacuo at 310 K. Thetime step used was 0.001 ps. For the sake ofvisualization, two characteristic intramolecularatom–atom distances were recorded throughoutthe simulations.

3. Results

Differences in the degrees of pentacyclationbetween MPL39 and MPL59 are shown in Table1. It can be seen that the degree of pentacycla-tion is gradually higher in MPL59 but not basi-cally different from that in MPL39. Table 1shows the percentage of lipid molecules with adefined number of pentacycles as determined byliquid chromatography–mass spectroscopy (LC–MS) correlated to the total amount of lipidmolecules (Freisleben et al., 1996).


Table 1Degree of pentacyclation in MPL39 and MPL59 (%)

MPL39 (%)Pentacycles MPL59 (%)

0 6.690.55.590.51 17.390.4 9.291.22 23.094.0 9.490.5

17.191.423.490.5314.891.34 19.091.3

21.690.35 10.891.011.790.23.690.96

1.290.57 4.790.38 0.890.30.590.4

Fig. 1. Transition temperature (Tm) versus mass percentages ofMPL between 7.7 and 60.5%. , exotherm occurring incooling scans at −27°C; �, endotherm occurring in heatingscans at −15°C; �, second endotherm in heating scans at+17°C. Details are given in the text.

3.1. Hydration of MPL

Up to about 10 mg of MPL were either nothydrated or hydrated with various amounts of 5,10 or 20 m l of buffer (Table 2).

Independently of the mass percentage of MPLbetween 60.5% and 7.7% (Table 2), hydratedMPL exhibits the well-known phase transition atTm= −15°C in the heating curves. In the coolingscans, Tm is between −27 and −30°C, while inheating curves, a second transition is detected atTm= +17°C (Fig. 1). The exotherm in the cool-ing curves and the endotherm at −15°C in theheating scans exert heat flows with DH between10 and 20 J/g, independently of the mass percent-age of MPL below 60.5% (Figs. 1 and 2). In thesample with only 7.7% MPL, DH increases toover 30 J/g, which value may be due to insuffi-

ciently precise registration of our DTA measuringsystem. The newly detected endotherm at +17°Ctends to a DH of almost zero in the sample with60.5% of MPL (Fig. 2).

With non-hydrated MPL samples, no phasetransitions could be detected due to the lack ofassembly and non-cooperativity. This observationis in good agreement with the predictions of themolecular simulations. Fig. 3a shows the starting

Fig. 2. Heat flow (DH, J/g) versus mass percent of MPL. �,DH of exotherm occurring in cooling scans at −27°C; �, DHof endotherm occurring in heating scans at −15°C; �, DH ofsecond endotherm in heating scans at +17°C. Details aregiven in the text.

Table 2Hydration of MPL

Buffer (m l)a MPL (mass%)MPL (mg)

0 6.39 1009.59 60.557.035 53.13.32 35.456.9210 37.8

10 5.88 33.610 24.43.78

23.520 7.3120 3.71 14.1

1.7820 7.7

a 380 mM/l Na cacodylate/HCl, 12.5 M/l ethyleneglycol, pH7.0.


Fig. 3. Computer simulations showing the conformation of MPL in vacuo at 310 K in a time range of 100 ps. (a) depicts thestretched starting conformations used, as well as distances of two carbon atoms in the middle of the hydrocarbon chains and thedistances of two oxygen atoms in the glycerol–ether bonds throughout the simulation (these atoms are separately marked). Lipid1 refers to a model compound without pentacyclations and lipid 2 denotes a model compound with maximum number ofpentacyclations; (b) shows the conformations of lipid 1 (without pentacyclations) and those of lipid 2 containing four pentacyclesper biphytanyl chain at 310 K in the time range of 100 ps.

conformations of the model lipid without (‘lipid 1’)and with pentacyclations (‘lipid 2’) together withthe 100 ps trajectories of two intramolecular dis-tances. These distances change dramaticallythroughout the simulation, indicating the thermalinstability of the stretched conformations in vacuo.Fig. 3b emphasises this statement by demonstratingselected conformations of the molecules after spe-cified times of molecular dynamical simulation. Theextended initial conformations quickly start to formcoils; according to the atom–atom trajectories andaccording to the conformations, the lipid withoutpentacyclations seems to rearrange its conforma-tion more rapidly and more vividly than the lipidwith pentacyclations. From this data, assembly ofunhydrated MPL to membraneous aggregations

and cooperativity in phase transitions cannot beexpected (Boggs, 1987). All transitions in the heat-ing and cooling curves, if they occurred, were atconstant temperatures (Tm; Fig. 1). All scan ratesin Figs. 1 and 2 were dT/dt=2 K/min (0.033 K/s).

Occurrence of additional enthalpy changes (en-dotherms) in the heating curves appears to distin-guish between the values of the exotherms in thecooling curves. The latter only correlate well withthe main transition endotherms. In the furthercourse of our experiments, scan rates were variedin order to reveal the conditions under which theendotherm at 17°C occurs and endotherms andexotherms follow the rules of thermodynamics. Allexperiments were accomplished with several sam-ples and representative scans are depicted.


Fig. 3. (Continued)

3.2. Variation of heating scan rates

The samples were cooled at the instrumentalmaximum cooling rate (i.e. at least dT/dt=30K/min) to −45°C and then heated to +35°C atvarying scan rates dT/dt=1, 2, 3, 4, 6 and 8K/min (0.017, 0.033, 0.05, 0.067, 0.1 and 0.133K/s). In Fig. 4, the heating scans are shown for asample containing 3.78 mg MPL and 10 m l ofbuffer corresponding to 24.4% MPL.

The endotherm at −15°C increased with in-creasing scan rates and concomitantly, the peak at+17°C disappears (Fig. 4a–f). Whereas in scan(a), roughly half the lipid undergoes phase transi-tion at −15°C, the other half at +17°C (the twopeaks cover similar areas), in scans (e) and (f)

MPL exhibits only the main endotherm phasetransition at −15°C.

3.3. Variation of cooling scan rates

The samples were cooled at varying scan ratesdT/dt=2, 6, 10, 15 K/min (0.033, 0.1, 0.167, 0.25K/s) and at the instrumental maximum rate, i.e. atleast 30 K/min (0.5 K/s). The heating scans wereaccomplished at dT/dt=2 K/min (0.033 K/s). InFig. 5, the heating scans are shown for a samplecontaining 5.88 mg MPL and 10 m l of buffercorresponding to 33.6% MPL.

The endotherm at +17°C only occurs withincreasing cooling rates (Fig. 5c–e). These resultswere not altered by lowering the end point of the


cooling rates from −45 to −160°C (not shownfor this sample, but can be seen for another onefrom Fig. 6e).

3.4. Variation of the starting temperature

The samples were cooled from +35 to −25,−30, −40, −50 and −150°C at a scan rate ofdT/dt=90 K/min (0.3, K/s) and from these tem-peratures the samples were heated at a scan rateof dT/dt=2 K/min (0.033 K/s). In Fig. 6, theresults are demonstrated with a sample containing3.71 mg MPL and 20 m l of buffer correspondingto 14.1% MPL.

Fig. 5. Heating scans of a sample containing 5.88 mg MPLand 10 m l buffer corresponding to 33.6% MPL (scan ratedT/dt=2 K/min (0.033 K/s). The samples were cooled to−60, −90, and −160°C (the scans shown were cooled to−60°C prior to heating) at varying scan rates dT/dt=2 (scan(a)), 6 (b), 10 (c), and 15 (d) K/min, corresponding to 0.033(a), 0.1 (b), 0.167 (c), and 0.25 (d) K/s) and at the maximuminstrumental scan rate (scan (e)).

Fig. 4. Heating scans of a sample containing 3.78 mg MPLand 10 m l buffer corresponding to 24.4% MPL. The sampleswere cooled to −45°C at instrumental maximum cooling rateand then heated to +35°C at various scan rates dT/dt=1(scan (a)), 2 (b), 3 (c), 4 (d), 6 (e), and 8 (f) K/min, correspond-ing to 0.017 (a), 0.033 (b), 0.05 (c), 0.067 (d), 0.1 (e), and 0.133(f) K/s.

Starting from −25°C (heating scan (a)), Tm ofthe cooling scan at −27°C (see Fig. 1) was notyet reached. Hence, no transition peak occurs inthe heating scan. Heating scan (b) starts at −30°C, i.e. in the range of the exotherm of thecooling scan (Tm at −27°C). The endotherm ofthe main phase transition (Tm, at −15°C) ispresent in the heating scan, demonstrating hys-teretic behavior of MPL with DT=12°C betweencooling and heating scans. Starting at −40°C(heating scan (c)) the exotherm of the coolingscan is almost accomplished. In this scan, a slightsecond endotherm occurs at +17°C in the heat-ing scan, in addition to the main phase transitionpeak at −15°C. The starting temperatures ofheating scans (d) and (e) (−50 and −150°C,respectively) are clearly below the exotherm of thecooling scan. The endotherm at +17°C is fullyexpressed in both heating scans. Heating scan (e)differs from all other scans (a–d) by an inverse(upward) direction of the curve from the startingtemperature at −150 to −30°C.


3.5. Isotherms

The temperature was held constant for differenttimes (5, 10 and 30 min) once at 0°C in theheating period and once between cooling andheating at −45°C. These isotherms had no effecton the phase transitions (not shown). However, aspecial temperature program is demonstrated withthe sample containing 3.78 mg MPL and 10 m l ofbuffer corresponding to 24.4% MPL (Fig. 7).Cooling at a scan rate of 2 K/min (0.033 K/s)from +30 to −45°C, followed immediatelyby heating at 2 K/min to 0°C (Fig. 7a andb) exhibits the behavior described above. After 10min of isotherm measurement at 0°C (Fig. 7c)and cooling again to −45°C at the same scanrate as before, the heating curve (Fig. 7d),scanned immediately, showed both transitions (at−15 and +17°C). In comparison to Fig. 5a (alsocooled and heated at a rate of dT/dt=2 K/min),the phase transition at +17°C can be seen in Fig.7d.

Fig. 7. Temperature program: The special instrumental tem-perature program is demonstrated with a sample containing3.78 mg MPL and 10 m l buffer corresponding to 24.4% MPL.The sample was cooled from +35 to −45°C (scan (a)) andheated again to 0°C (b). At 0°C the temperature was heldconstant for 10 min (c). Following this, the sample was cooledagain to −45°C and then heated to +35°C (d). All scans ofthe temperature program were recorded at a scan rate ofdT/dt=2 K/min (0.033 K/s). Details are given in the text.

4. Discussion

No phase transitions could be detected withnonhydrated MPL samples, possibly due to thelack of both assembly and cooperativity. Com-puter calculations clearly demonstrate that unhy-drated MPL does not tend to remain in thestretched conformation, but immediately starts toform ‘coils’, regardless of whether or not the MPLcontains pentacycles. Coiling occurs only slightlyfaster in MPL without pentacycles. However, af-ter 40–60 ps MPL with a high degree of pentacy-clation also exerts a globular shape. Thesecalculations simulate a vacuum, i.e. an unhy-drated, gas-analogue phase, in which the lipid hasno tendency for assembly. This situation resem-bles the unhydrated MPL in the DTA experi-ments in which no aggregation, no cooperativityand hence, no phase transition can be observed.

Formerly, the general interpretation wouldhave expected clear differences between the be-

Fig. 6. Heating scans of a sample containing 3.71 mg MPLand 20 m l buffer corresponding to 14.1% MPL (scan ratedT/dt=2 K/min (0.033 K/s). The samples were cooled from+35 to −25°C (scan (a)), −30°C (b), −40°C (c) −50°C(d), and −150°C (e) at a scan rate of dT/dt=2 K/min (0.033K/s) prior to heating. Details are given in the text.


havior of non-pentacycled and pentacycled MPL.Pentacyclations were thought to inhibit the abovecoiling and keep MPL in an upright (more or lessstretched, although possibly twisted) conforma-tion, even at the water–air interface (Strobl et al.,1985). We now must consider that coiling mightnot only occur in the case simulated in our com-puter model, but also (and possibly in particular)at the water–air interface, with both polar head-groups in the aqueous phase (‘horseshoe’conformation).

In hydrated samples, varying mass percentagesof MPL, between 7.7 and 60.5%, exhibit the well-known phase transition at −15°C and differencesbetween heating and cooling curves (Fig. 1), al-though Tm is constant in both heating and coolingscans. First, occurrence of a second transitionpeak in the heating curves did not appear tofollow the law of thermodynamics, since theexotherms in the cooling curves only correlatedwell with the main transition endotherms in theheating curves. We knew from our experimentswith MPL39 (i.e. MPL with lower degree of pen-tacyclations) that the occurrence of metastablephases is mainly a kinetic phenomenon. Hence, inthe further course of our experiments scan ratesand other scan conditions were varied in order toreveal the interrelations between the endotherm at+17°C, the endotherm at −15°C and theexotherm at −27°C.

The two endotherms occurring in the heatingscans correlate well, as is clearly demonstrated inFig. 4. Under these instrumental (scanner andregistration) conditions the endothermal enthalpychanges are additive, i.e. the heat flow decreasesin the phase transition peak at −15°C, when DHin the peak at +17°C increases (Fig. 4f–a). Fromthis data it is obvious that the occurrence of thesecond transition peak follows the rules of ther-modynamics. To detect kinetic effects, it was nec-essary to apply complicated scanning conditionsand programs, which may result in less ‘accurate’registrations of the curves. Hence, it must beattributed to instrumental registration insufficien-cies, if in some cases the heat flows do not appearto correspond well enough. This may also be thecase in a phenomenon observed in Fig. 2, wherethe newly detected endotherm at +17°C tends to

a DH of almost zero in the sample with 60.5%MPL. For the above mentioned reasons, it cannotyet be decided whether or not this is a lyotropiceffect.

Apart from these considerations, the new re-sults provide the background for a phase transi-tion scheme of MPL59 (Fig. 8) similar to thatestablished for MPL39 (Blocher et al., 1990). Slowcooling from phase C to phase A results in aphase transition at −30°C and slow heating fromphase A to phase C exerts a phase transition at−15°C (case 1) This has been the accepted hys-teretic behavior for MPL59 up to the present.

Rapid cooling from phase C results in concomi-tant transition to phase A and phase B/B%. Whenslowly heated, the phase transition from phase Ato phase C occurs at −15°C as in case 1. How-

Fig. 8. Models for phase transitions of MPL: A, metastablesolid-analogue phase; B, stable solid-analogue phase with a B%phase (B/B%); C, liquid-analogue phase; GT, glass transition(nomenclature according to Blocher et al., 1985a). Slow scanrates are denoted by slim arrows, rapid scans by bold ones.Case 1: slow cooling from phase C to phase A results in aphase transition at −30°C; slow heating from phase A tophase C exerts a transition at −15°C. This is the well knownhysteresis of MPL59. Case 2: rapid cooling from phase Cresults in concomitant transition to phase A and phase B/B%.When slowly heated, the transition from phase A to phase Coccurs at −15°C, as in case 1. However, the transition fromB/B% to phase C is at +17°C. Case 3: rapid cooling fromphase C results in concomitant transition to phase A andphase B/B%, as in case 2. When quickly heated, the phasetransitions from phase A and phase B/B% to phase C occur at−15°C.


ever, the phase transition from B/B% to phase C isat +17°C (case 2).

Rapid cooling from phase C results in concomi-tant transitions to phase A and phase B/B% as incase 2. When quickly heated (case 3), the phasetransitions from phase A and phase B/B% to phaseC occur at −15°C.

The thermotropic properties of MPL39 haverecently been described in detail (Blocher et al.,1990). These properties comprise endothermalmelting of a metastable solid-analogue to theliquid–crystalline phase (A to C) between −30and −10°C, exothermal recrystallization ofacyclic lipid in a stable solid-analogue phase (B/B%) between −10 and 5°C and melting of phase(B/B%) between 5 and 20°C. The extent of theformation of (B/B%) is limited by the time span ofsweeping over the transition (A–C) and the pre-treatment of the sample. The heatflow of the threetransitions overlap and partially offset each other.Upon addition of cholesterol to MPL39, the onsetof the sub-zero transition (A–C) and the exother-mal transition (A or C–B/B%) were shifted tolower temperatures and their heatflows were re-duced (Blocher et al., 1991a).

The calorimetric data were interpreted as indi-cating that already cyclic MPL (i.e. with pentacy-cles) quenches the sub-zero phase temperaturetransition (A–C) of MPL without pentacyclationand that cyclic MPL and cholesterol would havean additive effect on the temperature of maximalheatflow and DH. Varying flexibility of merelymethylbranched chains and those containing pen-tacycles were thought to contribute to different(metastable) phases in MPL39 and MPL59.

MPL59 showed in these investigations only abroad transition at sub-zero temperatures,MPL39 and MPL49 (i.e. MPL from cells grownat 49°C) exhibited complex thermotropic proper-ties which included competing transitions betweena metastable and a stable gel phase as well as aliquid–crystalline phase (Blocher and Ring, 1991).

It has further been postulated that this phase(B) is characterized by the formation of a networkof hydrogen bonds between the headgroups ofMPL (Blocher et al., 1990) so that certain effects,such as reduction of the melting temperature,enthalpy change and cooperativity, may resultfrom the limitation of the size of domains.

In the cited investigations, MPL39 was foundand interpreted to behave as other hydratedlipids, e.g. phosphatidylcholines, lecithins withshort isobranched fatty acids, phosphatidylglyc-erols, phosphatidylethanolamines, phos-phatidylserines, glycosyldiglycerides,sphingomyelins, cerebrosides and cerebroside sul-fates (Blocher et al., 1990). Furthermore, the verysimilar tetraether lipids from Sulfolobus solfatari-cus were found to form different phases andmetastable polymorphism depending on the po-larity of the headgroups and on the flexibility ofthe hydrocarbon chains (Gliozzi et al., 1986; Gu-lik et al., 1986; Luzatti and Gulik, 1986).

All these peculiarities were not found and inter-preted for MPL59. Blocher et al. (1990) still sup-posed that the thermotropic properties of MPL39differ significantly from those of the correspond-ing MPL59 and assumed that the latter only existsin the A and C phases, as well as the subphasebelow glass transition, because no indication ofmetastable polymorphism had been found underthe experimental conditions used. However, theauthors (Blocher et al., 1990) already raised thequestion (citation): ‘‘It is possible that MPLS9shows a similar phase transition at higher temper-atures (around 40°C), but that the right condi-tions for its transformation have not yet beenfound…’’ (end of citation).

The suitable conditions for this transition arereported here. The temperature is not around40°C but rather, is similar to that of MPL39(around 20°C), exactly at 17°C. In addition, onthe basis of our computer calculations in thevaccum, a gas-analogue phase can be postulated,which confirms similar behavior of (unhydrated)MPL39 and MPL59. In light of the new data,former interpretations concerning phase behaviorof MPL59 and formation of domains and mixedphases must be reconsidered, especially if differ-ences between MPL39 and MPL59 were workedout.

The difficulty in finding the right conditions todetect the metastable phase and its transition maybe because with rapid as well as with slow scanrates, phase transition always occurs at one tem-perature. Only the combination of rapid coolingand slow heating scan rates made the secondphase transition visible (Fig. 8, case 2).


It could not be shown for either MPL39 orMPL59 whether phase separation occurs, for ex-ample between highly pentacyclated and non-cy-clated lipids and whether or not occurrence of themetastable phase is connected with phase separa-tion. At present, compressed and expandedmonofilms of these lipids at the water (buffer)–airinterface are being re-investigated with modernLangmuir film balance equipment, including X-ray diffraction and atomic force monitoring.These investigations will provide further insightsand probably answer the question concerningphase separations.

Acknowledgements

This work was supported by the DeutscheForschungsgemeinschaft Fr 856/2-1 and the Jubi-laumsfond der O8 sterreichischen Nationalbanlk,project 5085.

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