High sensitivity differential scanning calorimetry of the bilayer to hexagonal phase transitions of diacylphosphatidylethanolamines

Chemistry and Physics o f Lipids, 36 (1985) 387-393 Elsevier Scientific Publishers Ireland Ltd.

387

HIGH SENSITIVITY D I F F E R E N T I A l . SCANNING CALORIMETRY OF THE BILAYER TO HEXAGONAL PHASE TRANSITIONS OF DIACYI.PHOSPHATIDYLETHANOLAMINES

RICHARD M. EPAND

Department o f Biochemistry, McMaster University, Health Sciences Centre, Hamilton, Ontario, LSN 3Z5 (Canada) Received September 27th, 1984 accepted January 9th, 1985

revision received December 17th, 1984

The bilayer to hexagonal phase transition of dioleoylphosphatidylethanolamine has been detected for the f'trst time by differential scanning calorimetry. The observed transition is dfpendem on scan rate. This dependence can be explained by assuming that at rapid scan rates, the rate of conversion of bilayer to hexagonal phase is too slow at low temperatures for equilibration to take place. At higher temperatures the rate of interconversion becomes more rapid. The transition is observed to occur at 14°C using a scan rate of 0.74 K/min while it is centered at 8°C using a scan rate of 0.19 K/min. The enthalpy of the transition is 290 ± 40 cal/mol lipid and the transition is characterized by a ACp of --9 ± 1 mcal K-t (g lipid)-~. The bilayer to hexagonal phase transition of dielaidoylphosphatidylethanolamine and of l-palmitoyl-2oleoylphosphatidylethanolamine occurs at 65.6°C and 71.4°C, respectively, with a corresponding transition enthalpy of 450 ± 20 and 400 ± 30 cal/mol lipid. The transitions of?these phosphatidylethanolamines, occuring at higher temperatures, are independent of scan rate and show a higher degree of cooperativity than that of dioleoylpho~hatidylethanolamine.Compared with the gel to liquid-crystalline transition of bilayer phospholipids the transition to hexagonal phase has a much lower entha~py.

Keywords. hexagonal phase; phase transition; differential scanning calorimetry; phosphatidylet hanolam inc. Introduction The conversion of phospholipid bilayers into a hexagonal phase has attracted considerable interest because of the marked alteration of membrane properties which it promotes [1,2]. Many of the previous studies of the bilayer to hexagonal phase transition have utilized naturally-occurring phospholipids including phosphatidylethanolamines [2,3 and references therein]. In order to accurately characterize phase transitions it is preferable to utilize synthetic phospholipids containing a single molecular species. Recent studies using synthetic phosphatidylethanolamines have evaluated their polymorphic behaviour in mixtures with phosphatidylcholines [4,5]. However, only a few studies have been done on the bilayer to hexagonal phase transition of single synthetic phospholipid preparations [ 6 - 9 ] and there has 0009-3084/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

388 been only one report assigning an enthalpy to this transition [6]. Differential scanning calorimetry has proven to be a very useful method for studying the thermotropic phase behaviour of phospholipids [10]. Unfortunately, one of the more readily available synthetic phospholipids known to undergo a bilayer to hexagonal phase transition, dioleoylphosphatidylethanolamine, did not exhibit a transition enthalpy which is sufficiently large to be detected by scanning calorimetry [11]. With recent improvements in sensitivity, stability and baseline reproducibility of differential scanning calorimeters, we can now demonstrate the bilayer to hexagonal phase transition of dioleoylphosphatidylethanolamine and compare it to the bilayer to hexagonal phase transition of other synthetic phosphatidylethanolamines. Materials and Methods Dioleoylphosphatidylethanolamine, dielaidoylphosphatidylethanolamine and l-palmitoyl-2-oleoylphosphatidylethanolamine were obtained from Avanti Polar Lipids. The dry lipid was suspended in 20 mM PIPES, I mM EDTA, 150 mM NaCI and 0.02 mg/ml NaNa (pH 7.40) by vortexing at a temperature above the gel to liquid-crystalline phase transition temperature of the lipid. In the case of dioleoylphosphatidylethanolamine, the vortexing was done at room temperature, above the bilayer to hexagonal phase transition temperature of this lipid. The other lipids were vortexed while still in the bilayer phase. The resulting suspension was degassed under vacuum and loaded into an MC-2 scanning calorimeter (Microcal Co.. Amherst, MA), totally filling a 1.3-ml fixed volume cell. Results and Discussion The observed calorimetric scans of dioleoylphosphatidylethanolamine were dependent on the length of time the samples were incubated at 0°C. Suspensions of dioleoylphosphatidylethanolamine in buffer at room temperature were cooled in the calorimeter to 0°C and left for various periods of time before scanning. The observed transition enthalpy was greater after longer times of incubation at 0°C. Markedly lower transition enthalpy values were observed after equilibration of the sample at 0°C for less than two hours. Since thermal equilibration of the calorimeter is achieved in shorter times, this result suggested that the conversion of the hexagonal phase of dioleoylphosphatidylethanolamine to the bilayer phase, known to occur in the region of 10-15°C for this phospholipid [I 2]. is a relatively slow process, as has previously been observed [5,12]. The kinetics of this conversion. i.e., the dependence of the transition enthalpy on the length of time of equilibration at O°C, was investigated. It was found that the rate of conversion varied erratically, presumably depending on the exact thermal history of the sample or the presence of minor impurities to act as nucleation sites. The scans obtained after complete equilibration at 0°C were reproducible. Scans reported in this paper

389 were taken after overnight equilibration at --0.5°C. The conversion of the hexagonal to bilayer phase of dielaidoylphosphatidylethanolamine and of l-palmitoyl2-oleoyiphosphatidylethanolamine occured more rapidly, presumably because of the higher temperatures used. Only in the case of dielaidoylphosphatidylethanolamine could a reduction in the enthalpy of the bilayer to hexagonal phase transition be observed in repeated scans. If after the first scan samples of this lipid were recooled to 50°C and a second scan made after 30 rain of thermal equilibration, the resulting enthalpy was decreased by about 20%. While some reduction in enthalpy could be detected with dielaidoylphosphatidylethanolamine, due to incomplete conversion of hexagonal to bilayer phase, the effects were much smaller than those observed with dioleoylphosphatidylethanolamine. The bilayer to hexagonal phase transition of dioleoylphosphatidylethanolamine is dependent on scan rate (Fig. !). The observed higher transition temperature at faster scan rates can be explained if the rate of conversion of bilayer to hexagonal phase has a half-time much longer than l0 min at temperatures below about 8°C but the rate becomes more rapid above 10°C. At a scan rate of 0.74 K/rain the sample does not have sufficient time to equilibrate until temperatures above 10°C are reached, while at a scan rate of 0.19 K/min the sample is probably close to equilibrium throughout the scan. The transition enthalpy was independent of scan rate and equal to 290 -+ 40 cal/mol. The precision of this measurenlent is poorer than we have found with other phospholipids possibly because of some of

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Fig. I. Differential scanning calorimetry tracings of a suspension of 7 mg/ml of dioleoylphosphatidylethanolamine in 20 mM PIPES, 1 mM EDTA, 150 mM NaC! and 0.02 mg/ml NaN 3 (pH 7.40). Upper curve, scan rate 0 . 7 4 K/rain, lower curve scan rate 0.19 K/rain.

390 the following factors: the low value of the enthalpy, the observed sensitivity of the transition to scan rate. the slow conversion to bilayer phase, the difficulty of extrapolating a baseline from the low temperature side of the transition so close to the freezing point of water, the inaccuracy in drawing a baseline for a transition with a ACp. The baseline was constructed by extrapolating the linear region of the scan before or after the transition to the temperature corresponding to the midpoint of the transition. The two extrapolated baselines were joined by a vertical line. From the scans at 0.74 K/rain the ZlCp of the transition was calculated to be ---9 +- 1 mcal K -~ (g lipid) -~. The heat capacity of gel state lipid bilayers increases rapidly as the temperature is raised to approach the phase transition temperature [13,14]. An analogous effect probably occurs with dioleoylphosphatidylethanolamine as the temperature is raised to a region where the bilayer becomes marginally stable allowing the lipid molecules to take on increased motions, possibly with accompanying changes in hydrophobic hydration [14]. In contrast to dioleoylphosphatidylethanolamine, dielaidoylphosphatidylethanolamine and I-palmitoyl-2-oleoyiphosphatidylethanolamine have gel to hexagonal phase transitions which are independent of scan rate. Examples of these transitions measured at a scan rate of 0.19 K/rain are shown in Fig. 2. Because these transitions (Fig. 2) are much sharper than those of dioleoylphosphatidylethanolamine (Fig. 1), the ACp of the transition could not be readily determined with the low sensitivity required to keep the transition on scale. These phospholipids also exhibit a gel to liquid-crystalline phase transition at lower temperatures. For dielaidoylphosphatidylethanolamine it occurs at 37.2°C with a transition enthalpy of 8.8 Kcal/mol. The gel-to-liquid-crystalline phase transition of l-palmitoyl-2-oleoylphosphatidylethanolamine is at 24.8°C with an enthalpy of 5.2 Kcal/mol. The phase transitions of these phospholipids show a high degree of cooperativity with a van't Hoff enthalpy of 1800 and 950 Kcal/mol for the dielaidoylphosphatidylethanolamine and the I-palmitoyl-2-oleoylphosphatidylethanolamine, respectively. This is evidence of the high degree of purity of these lipid samples. The phase transition temperature of the dielaidoytphosphatidylethanolamine agrees reasonably well with the transition temperature of 38°C previously reported [12]. Our value for the transition temperature of I-palmitoyl2-oleoylphosphatidylethanolamine, however, is in discrepancy with the value of 36°C reported by Dekker, et al. [8]. Both gel to liquid-crystalline phase transitions show a small degree of asymetry, with a shoulder on the low temperature side of the transition peak, a characteristic of gel to liquid-crystalline phase transitions of phosphatidylethanolamines [ 15]. The shape of the transition curve obtained for dioleoylphosphatidylethanolamine at a scan rate of 0.19 K/min deviates somewhat from that for a single van't Hoff transition (Fig. 1). Nevertheless, the breadth of the transition must be close to that for an infinitely slow scan rate. Since incubation of the lipid at 2°C or below completely converts the sample to the bilayer phase, the bilayer to hexagonal transition must begin above 2°C. The transition is completed by about 15°C at the

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Fig. 2. Differential scanning calorimetry tracings of a suspension of 9 mg/ml of dielaidoylphosphatidylethanolamine (DEPE) or 10 mg/ml l-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) in 20 mM PIPES, l mM EDTA, 150 mM NaCI and 0.02 mg/ml NaN 3 (pH 7.40). Heating scan rate 0.19 K/min. slowest scan rate used. At this temperature the rate o f conversion o f bilayer to hexagonal phase is rapid enough for a major fraction o f the lipid to be converted in this temperature range at a scan rate o f 0.74 K/min. Assuming that the transition is first order, we can estimate the size o f the van't Hoff entha|py, AHvH. The values obtained for this and the other lipids (Table I) were calculated from:

AHvH = 4 RT~ Cex'max A HcaI where To is the temperature of maximum excess heat capacity; Cex,max is the maximal excess heat capacity and AHca ! is the calorimetric enthalpy. The cooperative unit equals AHvH/AHoI.

392

TABLE I PROPERTIES OF THE BILAYER TO HEXAGONAL PHASE TRANSITION OF PHOSPHATIDYLETHANOLAMINES Tc is the temperature of maximum excess heat capacity. AHcaI, the calorimetric enthalpy and &Hv/./, the van't Hoff ent halpy.

Pho sphat idylet hanolamine

Tc (°C)

AHcai (cal/mol)

AH VH (Kcal/mol)

Cooperat ire Unit (molecules)

DioleoylDielaidoyl1-Paimitoyl-2-oleoyl-

8 65.6 71.4

290 ± 40 450 ± 20 400 _*30

100 900 1000

350 2000 2500

The A H v H for the bilayer to hexagonal phase transition o f dielaidoyl- and Ipalmitoyl-2-oleoyl-phosphatidylethanolamines (Table I) are comparable in size to those of gel to liquid-crystalline transitions (this work and Ref. 15). The dioleoylphosphatidylethanolamine transition is somewhat less cooperative. The increased broadening of the transition to hexagonal phase for this lipid could result from kinetic effects if the system were not at equilibrium even at the slowest scan rates. This is not a problem for the other phosphatidylethanolamines studied because their transitions to the hexagonal phase occur at higher temperatures where the rates of reaction are more rapid. The presence of trace impurities can also lead to transition broadening, although this is unlikely to be a major factor for transitions with a A H v H o f only 100 Kcal/mol. An additional factor for transitions to the hexagonal phase appears to be the size of the macromolecular lipid aggregate. Scans of dielaidoyl- and l-palmitoyl-2-oleoyl-phosphatidylethanolamines at concentrations one tenth, those shown in Fig. 2, exhibited transitions to the hexagonal phase at the same temperature as with the more concentrated samples but were somewhat broader. These more dilute suspensions were only moderately turbid and settled slowly, while the more concentrated suspensions contained larger aggregates of lipid which settled rapidly. Since the liquid crystalline to gel transition for dioleoylphosphatidylethanolamine occurs below the freezing point o f water, these samples were never cooled to the gel state and therefore may not be as aggregated. Thus, several factors may contribute to the breadth of the bilayer to hexagonal phase transition of dioleoylphosphatidylethanolamine. The enthalpy of the bilayer to hexagonal phase transition is much smaller than that for the gel to liquid crystalline state transition. As with the gel to liquidcrystalline state transitions, the enth',dpy of the bilayer to hexagonal phase transition is larger for transitions having a higher transition temperature (Table I). Somewhat larger bilayer to hexagonal phase transition enthalpys were reported [6] for 1,2-dihexadecylglycerophosphoethanolamine ( A H = 1.22 Kcal/mol, Tc = 88°C) and l-hexadecyl-2-palmitoyl glycerophosphoethanolamine ( A H = 0.75 Kcal/mol, Tc = 102°C).

393 Our study shows that differential scanning calorimetry can be used to monitor the bilayer to hexagonal phase transition of phospholipids. It is more sensitive and rapid than 3tp-NMR or X-ray scattering. These other methods, along with freezefracture electron microscopy, are still necessary to define the nature of the transition. However, to accurately study the temperature dependence of the transition and the effect of added substances, differential scanning calorimetry may be the method of choice.

Acknowledgement This work was supported by a grant from the Medical Research Council of Canada.

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