Some aspects of the structure change of lipid bilayers associated with the thermotropic phase transition

Physica 120B (1983)440-443 North-Holland Publishing Company

S O M E A S P E C T S O F T H E S T R U C T U R E C H A N G E O F L I P I D B I L A Y E R S ASSOCIATED

WITH THE T H E R M O T R O P I C PHASE TRANSITION Miscibility of different lipids in iiposome bUayers Yoshinori TOYOSHIMA, Takayoshi TAKEDA, Kozo AKABORI and Shigehiro KOMURA Faculty of Integrated Arts and Sciences, Hiroshima University, Higashisendamachi, Naka-ku, Hiroshima 730, Japan Dipalmitoylphosphatidylcholine with completely deuterized acyl chains (DDPPC) was synthesized to examine the temperature dependence of miscibility of two lipids in liposomes, which differ only by two methylene units in the lengths of their acyl chains, by means of neutron small angle scattering. The initiation and completion temperatures, Th and Tj (Th > Ti), of the phase change with regard to the chain ordering of the lipids were determined by the fluorescence method for the equimolar mixture of DDPPC and non-deuterized dimyristoylphosphatidylcholine(DMPC) and that of DDPPC and non-deuterized distearoylphosphatidylcholine(DSPC). Complete mixing of two lipids was observed by neutron small angle scattering only at a temperature above Th for DDPPC/DMPC, but was not observed for DDPPC/DSPC over the temperature range examined.

1. Introduction B i o l o g i c a l m e m b r a n e s c o n t a i n m i x t u r e s of different t y p e s of lipid a n d it is likely that t h e i r s p a t i a l d i s t r i b u t i o n is not h o m o g e n e o u s , p a r t i c u l a r l y in t h e r e g i o n s a r o u n d m e m b r a n e - b o u n d p r o t e i n s . E v i d e n c e for suggesting the e x i s t e n c e of a d o m a i n of c e r t a i n t y p e s of lipids in b i o l o g i cal m e m b r a n e s as well as in artificial lipid b i l a y e r m e m b r a n e s has a c c u m u l a t e d f r o m n u m e r o u s p h y s i c a l t e c h n i q u e s , including spin l a b e l i n g [1, 2], f l u o r e s c e n c e [3], c a l o r i m e t r y [4, 5], etc. F u r t h e r m o r e , t h e h e t e r o g e n e o u s d i s t r i b u t i o n of the m e m b r a n e lipids has b e e n c o n s i d e r e d to p l a y an i m p o r t a n t role in t h e f u n c t i o n s of biological m e m b r a n e s . H o w e v e r , d i r e c t o b s e r v a t i o n of the h e t e r o g e n e i t y in t h e l a t e r a l d i s t r i b u t i o n of the lipids b o t h in b i o l o g i c a l m e m b r a n e s a n d in artificial b i l a y e r s which consist of the m i x t u r e of different t y p e s of lipid has b e e n l i m i t e d to f r e e z e f r a c t u r e e l e c t r o n m i c r o s c o p i c s t u d i e s in which t h e s y s t e m suffers a d r a s t i c t e m p e r a t u r e c h a n g e in the s a m p l e p r e p a r a t i o n [6]. N e u t r o n small angle s c a t t e r i n g has its own p a r t i c u l a r a d v a n t a g e s for the i n v e s t i g a t i o n of lipid d i s t r i b u t i o n in b i o l o g i c a l a n d m o d e l m e m b r a n e s . T h e first a d v a n t a g e is that t h e m e a s u r e ments are carried out under the conditions s i m i l a r to t h o s e in vivo. T h e s e c o n d is b a s e d on

t h e large d i f f e r e n c e b e t w e e n the H a n d D scatt e r i n g l e n g t h s for n e u t r o n s which d e v e l o p s the c o n t r a s t v a r i a t i o n a n d selective d e u t e r a t i o n m e t h o d s . H e r e , we p r e s e n t s o m e results o b t a i n e d f r o m the p r e l i m i n a r y w o r k s c o n c e r n i n g t h e miscibility of two kinds of lipid c o n t a i n i n g different hydrocarbon chains, d i p a l m i t o y l p h o s p h a t i d y l choline/dimyristoylphosphatidylcholine, and dipalmitoylphosphatidylcholine / distearoylphosp h a t i d y l c h o l i n e , in t h e b i l a y e r s t r u c t u r e . In o r d e r to distinguish d i p a l m i t o y i p h o s p h a t i d y l c h o l i n e from the others, dipalmitoylphosphatidylcholine with fully d e u t e r i z e d acyl chains w e r e s y n t h e s i z e d . A l t h o u g h , in g e n e r a l , two lipids which differ only by two m e t h y l e n e units in t h e lengths of their acyl chains h a v e b e e n b e l i e v e d to f o r m a n e a r l y ideal m i x t u r e , t h e p r e s e n t results s e e m to suggest that t h e h e t e r o g e n e i t y in t h e lipid d i s t r i b u t i o n d o e s exist even in such systems.

2. Experimental 2.1. Materials a n d preparation dipalrnitoylphosphatidylcholine

of

deuterized

L - ( a ) - g l y c e r o p h o s p h o r y l c h o l i n e ( G P C ) /3,ydimyristoyl-L-(a)-phosphatidylcholine (DMPC) and /3, y - d i s t e a r o y l - L - ( a ) - p h o s p h a t i d y l c h o l i n c

0378-4363/83/0000-0000/$03.00 © 1983 N o r t h - H o l l a n d a n d Y a m a d a Science F o u n d a t i o n

Y. Toyoshima et al. / Miscibility of lipids in liposome bilayers

(DSPC), and dicyclohexylcarbodiimide were obtained from Sigma Chem. Co. Deuterized palmitic acid, C15D3xCOOH (DPA), and D 2 0 were obtained from Merck Sharp & D o h m e C a n a d a Ltd. Deuterized /3, y-dipalmitoyl-L- (a)-phosphatidylcholine (DDPPC), fl, y-(C15D3xCO)2-L-(a )phosphatidylcholine, was synthesized by modifying the m e t h o d reported by D o r d o n and Jensen [7]. Briefly, to 1.2mmole G P C were added 2 . 1 m m o l e deuterized potassium palmitate and 2 . 5 m m o l e deuterized palmitic anhydride synthesized from D P A and dicyclohexylcarbodiimide. The reaction proceeded at 80°C under l0 2 m m H g for 72h. After this time the solid mixture was ground and washed with 25 ml portions of ethyl ether three times and then dissolved in 50 ml chloroform. With slight cooling at 10°C, the majority of the unwanted reactants crystallized and were then filtered out from the solution. W a t e r and methanol were added to the chloroform solution to a final ratio of 1:2:4, respectively. The b o t t o m phase was separated, dried through anhydrous sodium sulfate and e v a p o r a t e d to dryness. T h e residue, ca. 0.7 g, was dissolved in benzene solution containing 5% ethanol in volume and applied to thin layer c h r o m a t o g r a p h y on silica gel (Merck 60 F254). D D P P C , 0.45 g, was obtained as a white amorphous solid.

2.2. Fluorescence measurements T h e thermotropic phase transition behavior of the multi-lamellar liposome suspensions of the equimolar mixtures of D D P P C / D M P C and D D P P C / D S P C was examined by measuring the fluorescence intensity of distearyloxacarbocyanine (DSOCC) incorporated in the liposomes, according to the m e t h o d reported by the present authors [8]. Fluorescence m e a s u r e m e n t s were p e r f o r m e d on a Hitachi Model MPF-4 spectrofluorometer with a temperature controlled cell holder. T h e t e m p e r a t u r e profile of the fluorescence intensity at 505 nm was obtained for cooling and heating scans between 15 and 65°C at the rate of 0.5°C/min.

441

2.3. Neutron small angle scattering A simple neutron small angle scattering spect r o m e t e r has been assembled at Kyoto University R e a c t o r (KUR). Details of the instrument are described elsewhere in this proceedings ( K o m u r a et al. [15, 16])oThe wavelength of the neutrons, A, was 6.81 A with a resolution of AA/A = 23%. T h e samples were placed in a silica glass cell of 0 . 2 c m in thickness with a slit of 0.5 cm x 2.0 cm. T h e t e m p e r a t u r e of the samples was controlled by circulating thermostated water (-+0.1°C) and monitored by a thermocouple dipped in the cell.

3. Results and discussion

In earlier works [9, 10], we demonstrated that Arrhenius type plots of the fluorescence intensity (If) of D S O C C incorporated in D P P C single lamellar liposomes, In If vs. T -~, exhibited an anomalous behavior reflecting the phase transition with regard to the chain ordering of D D P C molecules. The plots were clearly divided into three parts. It was confirmed that the low and high t e m p e r a t u r e parts in the plots corresponded to the m o n o p h a s e regions, i.e., solid and fluid phases, respectively, and the intermediate part corresponded to the state of coexistence of the lipids in solid and fluid states. Fig. 1 shows the plots of In If vs. T 1 with the data obtained in methanol and in D M P C , D D P P C / D M P C , and D D P P C / D S P C multilamellar liposomes. In the methanol solution, the plots follow a straight line, while in D M P C liposomes a very abrupt change appears in the plots around the calorimetrically observed main transition temperature. In the multilamellar liposomes composed of the equimolar mixtures of D D P P C / D M P C and those of D D P P C / D S P C , the plots exhibited two characteristic inflection points. Judging from our previous results, it is reasonably supposed that the temperatures corresponding to these two points, Th and T~ (Th > T~), are the initiation and completion temperatures of the phase change between the solid and fluid states. Consequently, above Th both lipids are in the fluid state and

442

Y. Toyoshima et al. / Miscibility of lipids in liposome bilayers °C 60

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3.2

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Fig. 1. Temperature dependence of fluorescence intensity in the multilamellar liposomes of DMPC and of the equimolar mixtures of D D P P C / D M P C and D D P P C / D S P C and in methanol. A: DMPC, B: D D P P C / D M P C , C: DDPPC/DSPC, D: methanol.

below Tj in the solid state. Between Th and T~, the lipids in the solid and fluid states coexist in the liposome bilayers. The values of Th and T~ determined from fig. 1 are 33.5 and 27.0°C for DDPPC/DMPC and 48.0 and 43.0°C for D D P P C / D S P C , respectively. The phase diagram for D P P C / D M P C has been determined, using DSC [11[, spin labeling [12], and fluorescence [13]. It is a little surprising that the values of Th and T~ obtained in the present works for the D D P P C / D M P C mixture are in good agreement with those obtained for the equimolar mixture of non-deuterized D P P C and D M P C by the fluorescence method, irrespective of the full deuteration of the acyl chains of DPPC. It may be mentioned here that the values of Th and T~ determined by p r o b e techniques, i.e., spin labeling and fluorescence, are consistently somewhat lower than those measured calorimetrically, but the shapes of the phase diagrams are closely similar. Neutron small angle scattering experiments were carried out on the equimolar mixtures of

D D P P C / D M P C and D D P P C / D S P C at three temperatures, higher than Th, between Th and Ti and below T~ for each mixture. The equimolar mixture of D D P P C / D M P C and that of D D P P C / D S P C lyophilized from benzene solution under vacuum were suspended in the mixture of D20 and H 2 0 containing 1 m M tris (hydroxymethyl)aminomethane-HC1 at p H 7.5. The molar ratio of D 2 0 / H 2 0 was adjusted such that the scattering amplitude, /7, of the solvent was equivalent to the mean value of /7 of each lipid mixture, as follows. The values of /7 of D D P P C , D M P C , and D S P C are 5.52× 10-14, o 3 0.275 × 10-14, and 0.86× 10- ~ 4 c m A - , respectively, therefore the mean values of /7 of the D D P P C / D M P C and of D D P P C / D S P C are 2.90 × 10-14 and 2.85 x 10 -14 cm ,~-3, respectively, while /7 of the mixture of D20 and H 2 0 satisfies the relation / 7 = ( - 0 . 5 6 2 + 6 . 9 7 x ) x 1 0 - 1 4 c m A , where x is the mole fraction of D 2 0 in the mixture. Using these values and the above relation, the mole fraction of D20 in the solvent was determined as 0.496 and 0.489 for the mixtures of D D P P C / D M P C and D D P P C / D S P C , respectively. U n d e r these conditions, it is expected that the excess scattering from each lipid mixture vanishes over the whole range of scattering vector, Q, if the spatial distribution of the lipids in the liposome bilayers is fully homogeneous. Fig. 2 illustrates the plots of the excess scattering intensity against Q, d2~/dO vs. Q, with the data obtained from the D D P P C / D M P C suspension at three temperatures. Fig. 3 shows the corresponding plots for the D D P P C / D S P C suspension. In these plots, the scattering due to the solvent was subtracted from the total scattering. As shown in fig. 2, the excess scattering from the D D P P C / D M P C suspension was not observed at temperatures above Th, 45°C, over the whole range of Q examined, suggesting that the two lipids are completely miscible at this temperature. When the sample was held at 32°C which corresponds to the t e m p e r a t u r e between Th and T~, the excess scattering due to spatial fluctuation of the scattering density appeared at the lower part of the Q-range. This fact strongly suggests that the two phases with different D D P P C / D M P C ratio appear in the liposome

Y. Toyoshima et al. / Miscibility of lipids in liposome bilayers

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DDPPC + DMPC • 45°C

o 32°C 32"C

~

" 20°C

0.2

-o 0.1

20"

I

I

I

[

0,1

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0.2 Q

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Fig. 2. Excess scattering intensity from the equimolar mixture of D D P P C / D M P C as a function of the scattering vector, Q, at three temperatures. T h e data were obtained for the suspension of 200rag lipids/ml. Q: 45.0°C, O: 32.0°C, A: 20.0°C.

bilayers at this temperature. When the temperature was lowered to 20°C which is below T], the excess scattering significantly decreased but did not disappear, contrary to our expectation. These results suggest that the two phases with 0.6

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443

different D D P P C / D M P C ratio still exist, when both D D P P C and D M P C are in the solid state with regard to their acyl chains. Furthermore, as shown in fig. 3, the excess scattering at low Q was observed in the D D P P C / D S P C suspension at all three temperatures, 58.2, 49.5, and 35.0°C, which correspond to above Th, between Th and Tl, and below Ti, respectively. These results may suggest that the equimolar suspension of D D P P C / D S P C in water is not fully miscible, irrespective of the physical states with regard to the ordering of their acyl chains. As mentioned in the introductory remarks, it has been generally believed that the two lipids which differ by only two methylene units in the lengths of their acyl chains are completely miscible, when both lipids are thoroughly in the solid state or in the fluid state, i.e., above Th or below T~ [14]. The discrepancy between the present results and the results previously obtained by the indirect method [12, 13] is still to be studied.

References [1] E.J. Shimshick and H.M. McConnell, Biochem. Biophys. Res. C o m m u n . 53 (1973) 446. [2] S.H. W u and H.M. McConnell, Biochemistry 14 (1975) 847. [3] H.J. Galla and E. Sackmann, Biochim. Biophys. Acta 339 (1974) 103. [4] K. Jacobson and D. Papahadjopoulos, Biochemistry 14 (1975) 152. [5] S. Mabrey and J.M. Sturtevant, Proc. Nat. Acad. Sci. U.S. 73 (1976) 3862. [6] W. K l e e m a n and H.M. McConnell, Biochim. Biophys. Acta 345 (1974) 220. [7] D.T. Gordon and R.G. Jensen, Lipids 7 (1972) 261. [8] K. Onugi, K. Kurihara, Y. T o y o s h i m a and M. Sukigara, Bull. C h e m . Soc. Jpn. 53 (1980) 1914. [9] H. "rakemoto, S. Inoue, T. Yasunaga, M. Sukigara and Y. Toyoshima, J. Phys. C h e m . 85 (1981) 1032. [10] S. Inoue, M. Nishimura, T. Yasunaga, H. T a k e m o t o and Y. Toyoshima, J. Phys. C h e m . 85 (1981) 1401. [11] D. C h a p m a n , J. Urbina and K.M. Keough, J. Biol. C h e m . 249 (1974) 2512. [12] E.J. Shimshick and H.M. McConnell, Biochemistry 12 (1973) 2351. [13] A.G. Lee, Biophim. Biophys. Acta 413 (1975) 413. [14] S. Mabrey and J.M. Sturtevant, M e t h o d s in M e m b r a n e Biology, Vol. 9 (Plenum Press, New York and London, 1978) p. 237. [15] S. K o m u r a et al., Physica 119B (1983) in press. [16] S. K o m u r a et al., Jpn. J. Appl. Phys. 22 (1983) 351.