260
Bioct~imica et Biophysica Acta, 464 (1977) 260 275 © Elsevier/North-Holland Biomedical Press
BBA 77577 LIPID P H A S E T R A N S I T I O N S IN M O D E L B I O M E M B R A N E S T t t E E F F E C T O F IONS ON P I t O S P H A T I D Y L C H O L I N E B I L A Y E R S
D. CHAPMAN a, W.E. PEEL a, B. KINGSTON b and T.H. LILLEY b a Department o f Chemistry, Chelsea College, University o f London, London, SW3 6LX and b Department o f Chemistry, Universily o f Sheffield, Sheffield $3 7HF (U.K.)
(Received March 13th, 1976) (Revised manuscript received September 17th, 1976)
Summary Differential scanning c a l o r i m e t r y has been used t o s t u d y the e n d o t h e n n i c phase b e h a v i o u r o f some m o d e l b i o m e m b r a n e s (i.e. p h o s p h a t i d y l c h o l i n e - w a t e r systems) in the presence o f a wide range o f alkaline, alkaline earth and heavy metal salts. Studies and c o m p a r i s o n s were m a d e o f b o t h cation and anion effects. Shifts o c c u r in the t e m p e r a t u r e s o f b o t h the pre-transition and main transition e n d o t h e r m s . T h e observed shifts are smaller t h a n t h o s e which have been r e p o r t e d for charged lipids, and no evidence has been f o u n d for the f o r m a t i o n o f specific c o m p l e x e s . E l e c t r o n microscopic studies on freezef r a c t u r e d dispersions o f p h o s p h a t i d y l c h o l i n e - w a t e r - s a l t systems show t h a t with some salts the typical rippled surface observed with L-a-dimyristoyl phosphat i d y l c h o l i n e , w h e n in the gel state, is replaced by a s m o o t h surface.
Introduction
Previous studies o f pure lipids and lipid-water systems using infrared spect r o s c o p y [1], differential thermal analysis, differential scanning c a l o r i m e t r y [2--5] and NMR s p e c t r o s c o p y [6] s h o w e d the existence o f a t h e r m o t r o p i c phase transition associated with h y d r o c a r b o n chain melting involving increased r o t a t i o n a l and translational m o t i o n s o f these chains. The t e m p e r a t u r e s at which these e n d o t h e r m i c phase transitions o c c u r have been s h o w n t o be d e p e n d e n t u p o n the head group, the alkyl chain length and the degree and t y p e o f uns a t u r a t i o n present [7]. T h e presence o f a third c o m p o n e n t such as cholesterol [ 8 , 9 ] , proteins [ 1 0 - - 1 2 ] , drugs [ 1 3 - - 1 5 ] and salts [ 1 1 , 1 6 ] can cause significant shifts in the phase transition t e m p e r a t u r e or eliminate t h e phase transition. Interest in these systems is great because of their relevance to b i o m e m b r a n e systems.
261 In this paper we examine the effects of various cations and anions on the e n d o th er mie phase transitions o f two phosphatidylcholine-water systems. Previous studies of ion effects have been reported [11,16] but are of a less extensive nature than those r e por t ed here. Materials and Methods The lipids, L-a-dimyristoyl phosphatidylcholine and L-a-dipalmitoyl phosphatidylcholine used in the calorimetric experiments were obtained from Koch-Light Laboratories Ltd. The dimyristoyl phosphatidylcholine used in the electron microscopic studies was obtained from Fluka. Purity was checked by thin-layer chromatography. The salts used were usually of Analar quality but in some cases reagent grade was used. The solutions were prepared volumetrically using deionised distilled water. Samples were prepared by homogenising weighed amounts of the appropriate lipid and solution above the gel-liquid crystalline transition temperature (approx. 50°C) using a vortex mixer. Some samples were homogenised by centrifuging them backwards and forwards through a constriction in the sample tube. The different m et hods of sample preparation did not significantly alter the results obtained. Calorimetric measurements were p e r f o r m e d using a Perkin-Elmer DSC-1B on samples weighing approx. 8 mg contained in hermetically sealed aluminium pans with a heating rate of 8K/min. Some of the results were checked using a Perkin-Elmer DSC-2 with a heating rate of 5 K/min, no significant difference being obtained in the results from the two instruments. The temperature scale w a s calibrated using the transition temperatures of cyclohexane (279.8 K), sodium sulphate decahydrate (305.6 K) and indium (429.8 K), and was accurate to -+0.2 K. The indium transition was also used to calibrate the enthalpy per unit area. A minimum o f three different samples were prepared for each system studied and each sample was investigated at least twice. The extrapolated onset te m pe r at ur e was taken as the transition temperature. Enthalpy and e n t r o p y data for the observed transitions were calculated from the area below the en d o t he r m s by standard methods, the curve areas being measured with a fixed arm planimeter. Dispersions of dimyristoyl phosphatidyleholine in excess solution (usually approx. 1 : 2 or 1 : 3 by weight), homogenised as described above, were quenched from 15°C into liquid Freon at - 8 0 ° C and then liquid nitrogen. Fracturing and etching, using a Nanotech freeze-fracture apparatus, were carried out at --99°C following by Pt/C shadowing and carbon replication at --110° C. Results Fig. 1 shows the calorimetric curves for the dimyristoyl phosphatidylcholine-water system as a function of water content. The e n d o t h e r m associated with the ice.melting transition at approx. O°C has been o m i t t e d from the diagrams. At first only one transition occurs but with increasing water c o n t e n t this transition narrows and a second transition appears slightly lower in temperature. This has been called the pre-transition e n d o t h e r m , the ot her being the
262
E
[iJ
2;o
'
38o
3~o
Temper@ture (K) F i g . 1. C a l o r i m e t r i c c u r v e s f o r t h e d i m y r i s t o y l p h o s p h a t i d y l c h o l i n e - w a t e r concentration, c = wt. lipid/wt, lipid + wt. water.
system
as a f u n c t i o n
of lipid
main transition endotherm. The characteristic behaviour of these features with changing water c o n t e n t has been reported elsewhere [5]. There is an abrupt change in the temperatures at which the two transitions occur at weight fractions of lipid of approx. 0.8. This also coincides with the appearance of the ice-melting e n d o t h e r m in the calorimetric curves. For the dipalmitoyl phosphatidylcholine-water system this change is approximately paralleled by changes in the enthalpy associated with the main transition (Table I). In view of this marked dependence of transition temperature on the water content, the studies involving ionic solutions were perform ed at concentrations well removed from this critical region, a 1 : 1 by weight lipid/solution mixture being used. Table II shows the dependence of the pre and main transition temperatures (T~ and To) o f the dipalmitoyl phosphatidylcholine-water system on the presence o f some heavy metal salts at low concentrations. It is apparent that salt concentrations as low as 10-3M are inducing perturbations in the phase transitions, with a given salt affecting the two lipid transitions in an approximately similar fashion. Table III illustrates the effect of some heavy metal salts at concentrations up to 1 M on the transition temperatures of dimyristoyl phosphatidylcholinewater dispersions. All of the salts investigated increased the transition t e m p e r a t u r e o f both endotherms but it is apparent that the efficacy of the
263
various salts differs. The very large effects of ZnCI~ and SAC12 are particularly notable and it is also significant that in the systems containing these salts the pre-transition appeared as a shoulder on the main transition endotherm. TABLE I T H E E N T H A L P Y ( A / l ) A N D E N T R O P Y (AS) C H A N G E S A S S O C I A T E D W I T H T H E P R E - (p) A N D M A I N (c) P H A S E T R A N S I T I O N S FOR THE DIPALMITOYL PHOSPHATIDYLCHOLINE-WATER S Y S T E M AS A F U N C T I O N O F T H E W E I G H T F R A C T I O N O F T H E L I P I D Weight fraction of dipalmitoyl phosphat i d y l c h o l i n e (C) 0.99 0.833 0.768 0.714 0.666 0.625 0.5 Excess w a t e r Excess water
Attp
ASp ( e a l - K -1 • t o o l -1)
(kcal • mo1-1)
1.3 1.6 1.7
AH c (kcal - tool -1)
AS c ( c a l " t o o l 1 • K I)
--
5.9
17.7
---
6.5 8.6
20.0 28.0
-
9.5
31.0
4.3 5.3 5.5
9.4 9.1 9.5 8.7 9.7
30.4 29.4 32.8 27.6 * 3 0 . 7 **
* Ref. 32. ** R e f . 3 3 . T A B L E II THE PRE-TRANSITION ENDOTHERM AND MAIN ENDOTIiERM TRANSITION TEMPERATURE. ¢ OF DIPALMITOYL PHOSPHATIDYLCHOLINE-HEAVY M E T A L S A L T S O L U T I O N S Y S T E M S (C =
0.5) Solution
Tp (K)
Water
10 -3 1 0 -3 1 0 -3 10-3 10 3 10 -3 1 0 -3
M M M M M M M
HgC12 ZnCI 2 SnC12 CuCI 2 CdC12 lead acetate CuS04
308.5 309.9 309.7 307.9 308.5 308.2 309.4 307.8
Tc (K)
314.5 316.7 315.6 ' 314.5 314.0 314.0 316.5 314.0
T A B L E III THE PRE-TRANSITION ENDOTHERM AND MAIN ENDOTHERM TRANSITION TEMPERATURE~ OF DIMYRISTOYL PHOSPHATIDYLCHOLINE--HEAVY M E T A L S A L T S O L U T I O N S Y S T E M S (C = O.5) Solution
Water 0 . I M ZnCI 2 0.1 M SnCI 2 I M CdCl 2 1 M AgNO 3 1 M lead acetate 1 M CuSO 4 1 M thallium acetate
Tp
Tc
(K)
(K)
287.2 --290.0 290.4 287.7 291.4 287.3
296.7 302.4 301.2 297.7 298.7 297.7 297.9 296.9
264 TABLE
IV
THE PRE-- TRANSITION ENDOTttERM AND MAIN ENI)OTHERM TRANSITION TEMPFRATURES OF DIMYRISTOYL PHOSPHATIDYLCHOLINE-ALKALINE, ALKAI,INE EARTH AN[) GUANAI ) I N I U M ( G u ) S A L T S O L U T I O N S Y S T E M S (C = 0 . 5 ) Sohttion
"I'1) (K)
Tc (K)
[ M LiCI
287.2
295.9
1 M NaCI 1 M KC1 1 M RbCI
289.2 288.6 288.2
297.8 297.4 297.6
1 M CsCl 1 M NaF
286.7 289.9
297.0 298.2
1 M NaBr
284.1
296.7
I M Nal
-
294.3
l M NaCNO 1 hi s o d i u m a c e t a t e
287.6 290.6
297.2 297.6
1 M KCN
287.0
297.2
1 M NII4CI 1 M tetramethylammonium
290.2 289.2
299.3 297.4
chloride
1 M CaC12 1 M MgC12 1 M GuSCN 0.1 M GuC1
287.1
301.7 298.8 292.9 296.7
286.1 281.3
296.6 296.3
292.7
0.2 M GuCI I. M GuC1
Table IV illustrates t h e effects o f a c o m p r e h e n s i v e range o f salts o n t h e transition t e m p e r a t u r e s o f t h e d i m y r i s t o y l p h o s p h a t i d y l c h o l i n e - w a t e r s y s t e m . F o r 1 M CaC12, N a S C N and G u S C N no pre-transition e n d o t h e r m s were observable, and for 1 M NaI this e n d o t h e r m was so broad that it was n o t possible to obtain a reliable e s t i m a t e o f t h e transition temperature. Table V gives the e n t r o p y and e n t h a l p y values associated with the phase transitions for s o m e o f t h e salts studied. TABI, E V Atf
AND AS DATA
Estimated
errors:
FOR
DIMYRISTOYL
PHOSPHATIDYLCHOLINE/AQUEOUS
SALT SOLUTIONS
A t t e ~ 0 . 9 k c a l - r e e l -1 ; A S e -+- 2 e a l . r e e l -1 - K - 1 .
AHp
ASp
AH c
( k e a l • t o o l -1 )
( c a l - t o o l -1 - K -1 )
(kcal . tool -I )
AS c (eal - reel
6.7
22.4 *
l M N[t4Cl
1.2
4.1
7.2
23.7
1 M tetramethylammonium chloride 1 M sodium acetate 1 M NaBr 1 M NaCNO i M NaF 1 M NaI 1 M KCN 0 . [ M Z nC 12 I M MgC12
0.8 1.0 0.8 0.7 0.9 -0.7 0.7 1.2
2.1 3.6 2.7 2.5 3.2 -2.6 2.4 4.0
6.6 6.5 7.4 6.8 6.8 6.9 7.1 7.2 7.6
22.4 22.1 25.0 22.9 23.0 23.8 24.1 24.5
1 M CaCI 2
--
--
8.6
Solution
Water
I . K-1 )
25.5 2 8 . 8 **
• Ref. 32. ** T h e l a r g e v a l u e s o f from a pre-transition
AH e and AS e produced by this salt are probably coincident with the main endothenn.
due to some contribution
265 Figs. 2a and 2b show the marked effect of various NaSCN concentrations on the dimyristoyl phosphatidylcholine-water system at 50 weight percent lipid. At SCN- concentrations > 0 . 2 M no pre-transition endotherm is observable and indeed as the concentration of salt approaches this value the endotherm is broadening. The main transition temperature increases rapidly at low concentrations and thereafter increases more slowly. The most significant effect however, is the way in which the endotherms diverge as the salt concentration increases. In view of the importance of possible hydration effects the dipalmitoyl phosphatidylcholine-water-ZnC12 system was studied in more detail. The mol ratio of dipalmitoyl phosphatidylcholine/ZnC12 was kept constant at 3 : 1 and the water content of the system was varied. The results for the main transition endotherm are shown in Fig. 3. It can be seen that ZnC12 modifies the effect
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transition
tern
266
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Fig. 3. T h e d e p e n d e n c e o f t h e m a i n e n d o t h e r m t r a n s i t i o n t e m p e r a t u r e on w a t e r c o n c e n t r a t i o n f o r th~ d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e - w a t e r (full line) a n d d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e - w a t e r - Z n C l 2 ( d a s h e d line) s y s t e m s . R a t i o d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e / Z n 2+ w a s 3 : 1 t h r o u g h o u t .
Fig. 4. F o r l e g e n d see o p p o s i t e page.
267
b'ig. 4. F r e e z e - f r a c t u r e s u r f a c e s : a, d i m y r i s t o y l p h o s p h a t i d y l e h o l i n e - w a t e r : b, d i m y r i s t o y l p h o s p h a t i d y l choline-1 M GuCl;c, dbnyristoyl phosphatidyleholine-1 M NaSCN. C = 0.25. Magnification X 50000. ( A t t h e s e c o n c e n t r a t i o n s GuC1 d o e s n o t r e m o v e t h e p r e - t r a n s i t i o n w h i l s t N a S C N d o e s r e m o v e t h i s t r a n s i tion).
268 of water on dipalmitoyl phosphatidylcholine since the abrupt change in transition temp er atu r e has shifted from 30% water to 55% water by weight. However, there was no significant alteration in the heat absorbed during the transition. Electron microscopic studies on the freeze-fractured surfaces of dispersions o f dimyristoyl phosphatidylcholine with various salt solutions have shown that in cases where the pre-transition e n d o t h e r m has been removed, the ripples usually observed on the surfaces of the liposomes have also been removed. Electron micrographs of the different surfaces observed are shown in Fig. 4. Discussion Previous results on the interaction of metal ions with charged and uncharged lipids using a variety of physical techniques have been interpreted using double layer t h e o r y [17,18], dehydr a t i on effects [19], complex formation [11,16, 20--24] and structure making and breaking effects [21,25]. It is to be expect ed that the interaction of metal ions with uncharged phosphatidylcholine should produce only small effects and the work of Simon et al. [16] and the results reported here show this is so. The transition temperatures are shifted by 6°C at the most and the enthalpy change associated with the transition is hardly affected by the presence of salt (Table V). The behaviour observed experimentally of the effects of salts on the transition temperatures in phosphatidylcholine-water systems is apparently rather complex. T h e r m o d y n a m i c arguments developed in the appendix give an insight into the sort of behaviour we would expect for the effect of solutes on a phase transition. It is shown t ha t the t em pe r at ure change in a phase transition depends on four u n k n o w n parameters for a given salt. In view of this any precise and unambigous interpretation of the effect of salts on phase transitions is no t possible at present and all one can hope for is some correlation with the experimentally observed effects and observations on some simpler systems. Eqns. 17 and 18 (Appendix) indicate that both anions and cations may affect phase transitions and it is convenient to consider first the order of cation effects by investigating salts with a c o m m o n anion. The experimental results on systems of metal chlorides show t hat the t e m p e r a t u r e of the main transition in the dimyristoyl phosphatidylcholine-water system is in the order (NMe~, t e t r a m e t h y l a m m o n i u m ion) NH~ > Na+~ K t ~ Rb+~ NMe~ > Cs+> G u t > Li t for mo n o v alen t cations and Zn 2+ ~> Sn 2+ > Ca 2. > Mg 2+ > Cd 2+ for divalent cations. The order observed for the pre-transition e n d o t h e r m is NH; > Na t ~ NMe; > K*> Rb+> L i t > Cs*> Gu* for the monovalent ions. No pre-transition endotherms were observed for ZnCI> SnC12 and CaC12 whilst the order for MgC12 and CdC12 was Mg 2. >Cd 2.. It seems likely that for the first three salts the pre-transition e n d o t h e r m has been shifted so high in temperature that it is overlapping the main transition.
269
The d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e - w a t e r s y s t e m shows t h a t in the presence o f divalent metal chlorides the main transition e n d o t h e r m decreases in the order Hg2*> Zn2+> Sn2+> Cu2+> Cd > and the pre-transition t e m p e r a t u r e s f o l l o w the o r d e r Hg 2+> Zn 2 . > Cu 2+> Cd 2 . > Sn 2+ T h e r e f o r e f o r a given p h o s p h a t i d y l c h o l i n e - w a t e r - m e t a l chloride system the o r d e r for b o t h the main and pre-transition e n d o t h e r m s is the same. T h e r e is a rough c o r r e l a t i o n b e t w e e n the orders observed and the m a g n i t u d e s o f the association c o n s t a n t s o f metal ions with simple phosphates. The association c o n s t a n t s e x p e r i m e n t a l l y observed [26,27] vary with the p h o s p h a t e used but f o r m o n o v a l e n t cations the o r d e r is L i * > N a * > K +> Cs* and for divalent cations Hg 2+ > pb 2÷ > Cd 2* > Zn :+ > Cu 2+ >
Ca 2 + >
Mg 2+
The a g r e e m e n t is only a p p r o x i m a t e and we t h e r e f o r e consider o t h e r possible i n t e r a c t i o n mechanisms. S o m e aspects o f previously r e p o r t e d lipid-metal ion interactions have been i n t e r p r e t e d o n the basis o f the k n o w n s t r u c t u r e making and breaking effects o f the ions on w a t e r [ 2 1 , 2 5 ] . A t t e m p t s to correlate o u r wider range o f cations with p h o s p h a t i d y l c h o l i n e in a similar m a n n e r were unsuccessful. I n d e e d t h e e f f e c t o f urea, at a c o n c e n t r a t i o n o f 1 M, on the transition t e m p e r a t u r e s was virtually negligible although it is a well-known s t r u c t u r e breaker. in view o f t h e fact t h a t all ions in solution are solvated it is necessary to take a c c o u n t o f possible h y d r a t i o n effects in i n t e r p r e t i n g the results r e p o r t e d here, and the results d e p i c t e d in Fig. 3 c o n f i r m this. It is n o t e w o r t h y t h a t for salts with a c o m m o n anion the ratio (charge)2/radius, (an a p p r o x i m a t e measure o f the solvation interaction), predicts fairly accurately the relative orders o f m a g n i t u d e o f the effects p r o d u c e d (Table VI) with the above ratio increasing with the ability o f the metal ion to raise t h e transition t e m p e r a t u r e s . However, t h e positions o f Li* and Hg 2+ are anomalous. It s h o u l d be n o t e d also t h a t the anionic e f f e c t is by no means small and considering the main transition e n d o t h e r m with s o d i u m salts the t e m p e r a t u r e o f the transition decreases as follows SCN- > F - > C1- ~ a c e t a t e - > C N O
> Br- > I-
whereas the o r d e r observed for the pre-transition is acetate
> F- > C1- > C N O - > Br- > SCN-
T h e r e is a reasonable c o r r e l a t i o n in these t w o series e x c e p t for the e x t r e m e change o f position o f SCN-. The effectiveness o f anions in d e s t r o y i n g the s t r u c t u r e o f w a t e r and lowering the h y d r o g e n b o n d strength is given by ref. 28 I- > SCN- > Br
> C1- ~ CNO- > F -
270 TABLE
VI
RATIO
OF (CttARGE)2/IONIC
Data from 53rd edition Itandbook
RADIUS
FOR METAL
of Chemistry
Ion
Radius (A)
C u 2+
0.96
4.17
Ag + Z n 2+ Cd 2+
1.26 0.74 0.97
0.794 5.42 4.13
n g 2+
1.1
3.64
TI + S n 2+
1.49 0.84
0.67 4.76
P b 2+
1.21
3.31
Li + Na + K+
0.68 0.97 1.33
1.47 1.03 0.753
Rb + Cs + Mg 2+ Ca 2+
1.47 1.67 0.66 0.99
0.68 0.6 6.06 4.04
IONS STUDIED
and I'hysics.
( C h a r g e ) 2 / R a d i us (,\ J)
There is a correlation between the structure-breaking efficiency of the anions and their ability to lower the transition temperatures. This may be explained by the anions interfering with the hydr ogen-bonded structure around the polar head group and hence altering the packing of the alkyl chains. The very large lowering of the pre-transition t em perat ure in the case of SCN may be due to the fact that this ion is known to be adsorbed preferentially at interfaces [29] and therefore may be producing a negatively charged lipid-water interface, This could lead to both longitudinal and lateral repulsion in the bilayer structure, the latter tending to alter the packing of the alkyl chains so leading to a lowering of the pre-transition temperature. Preliminary X-ray measurements have indicated that the long spacings are increasing with increasing SCN- concentration. It is difficult to explain the observed increase of the main e n d o t h e r m transition with increasing SCN- concentration. It may be that the reorientation of the head group at the pre-transition temperature allows the formation of a new more stable hydrogen bond network, involving SCN-. If the " m e l t i n g " and lateral separation of the alkyl chains at the main transition leads to a disruption o f such a hydrogen bond network additional thermal energy will be required and so T c will be raised provided there is no significant change in ASc. This idea of additional hydrogen bonds has been used by Simon et al. [16] to e x p l a i n the increase in AH,~ observed for dipalmitoyl phosphatidylcholine + 1 M KSCN. Simon et al. [16] also found that the main transition t em perat ure decreased for dipalmitoyl phosphatidylcholine + 1 M KSCN. In view of the fact that the t e m p er atu r e changes are small and different chain length lipids and cations were used we consider that the discrepancy is not significant. The pre-transition phase change is associated with an increase in the mobility
271 o f t h e polar head group and r e c e n t w o r k has s h o w n that the transition is also associated with a change in the angle o f tilt o f the alkyl chains [30]. A simult a n e o u s r e d u c t i o n in the area per polar h e a d g r o u p at the lipid-water i n t e r f a c e occurs. Replicas o f f r e e z e - f r a c t u r e d surfaces o f d i m y r i s t o y l p h o s p h a t i d y l c h o l i n e w a t e r dispersions in the gel phase, which Luzzati terms PB' [ 3 1 ] , have s h o w n rippled surfaces u n d e r the e l e c t r o n m i c r o s c o p e (Fig. 4). T h e ripples are t h o u g h t t o be due to u n d u l a t i o n s in the surface o f the liposomes caused by the longitudinal d i s p l a c e m e n t required in packing the tilted chains. Our calorimetric and e l e c t r o n m i c r o s c o p i c results s u p p o r t the idea t h a t the pre-transition is associated with the presence o f tilted chains, since dispersions o f d i m y r i s t o y l p h o s p h a t i d y l c h o l i n e - w a t e r c o n t a i n i n g salts which r e m o v e the pre-transition e n d o t h e r m s h o w no ripples in their f r e e z e - f r a c t u r e d surfaces. This indicates t h a t the chain tilt, which is a r e q u i r e m e n t for the presence o f ripples, has been altered by the presence o f t h o s e salts which r e m o v e t h e pre-transition endot h e r m . This also occurs u p o n a d d i t i o n o f 7% decane which gives a vertical-chain phase. Conclusions T h e effects generally observed are smaller t h a n those which have been observed for charged lipids. T h e effects o f cations appears to correlate a p p r o x i m a t e l y with a ratio, (charge)2/radius, which measures their solvation interaction. N o t w i t h s t a n d i n g the d i f f i c u l t y o f q u a n t i t a t i v e i n t e r p r e t a t i o n s some qualitative conclusions can be drawn a b o u t the effects o f cations on p h o s p h a t i d y l c h o l i n e - w a t e r systems. It w o u l d seem t h a t Hg 2÷, Pb 2+ and Zn 2. c o n f e r increased stability t o t h e r m a l change on gel phase dispersions o f d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e whereas Cd > and Cu 2+ have a small destabilising effect. With the d i m y r i s t o y l p h o s p h a t o c y l e h o l i n e - w a t e r system it is f o u n d t h a t Sn 2÷ and Zn 2+ have a very m a r k e d e f f e c t in increasing gel phase stability whereas o t h e r heavy metals have r a t h e r less effect. The heavy metal salts increase gel phase stability. O f the c o m m o n alkaline and alkaline earth salts it m a y be c o n c l u d e d t h a t Ca 2. and Mg 2÷ give rise to increased stability as do Na + and K ÷ b u t t o a lesser extent. Anionic effects can be e x p l a i n e d qualitatively in terms o f s t r u c t u r e - b r e a k i n g effects on the organisation o f the polar head group region, leading t o an alteration in the packing o f the alkyl chains. The a n o m a l o u s effects o f the SCN- are t h o u g h t to be due to its great t e n d e n c y to a c c u m u l a t e at the polar lipid-water interface. Appendix We shall consider t h e b e h a v i o u r we c o u l d e x p e c t for the effects o f solutes on a phase transition. S u p p o s e t h a t we consider a phase transition for a m o n o disperse system a -~ /3
(1)
where a and fi are the forms which arc stable at low and high t e m p e r a t u r e s ,
272
respectively. P,(T)
At any temperature
we have
=&3(T)
(2)
where pi(T) is the chemical potential of i at temperature T. If we consider the situation when water is the only other species present then expanding chemical potentials and rearranging gives K”(T)
=
fpW(T) = exp f,"
_
1
g”(T) -- PTiY (T) RT
CT)
(3)
where f;(T) and f,“(T) are the fractions of fl and CYpresent at temperature T. respectively, K”(T) is the equilibrium constant for the equilibrium and /LT.” denotes the standard chemical potential of i in water. Application of the GibbsHelmholtz equation gives a___ In _~ K”(T) i a (I/T)
= __AH*‘V(T) R I’
(4)
where Ai?%” is the standard enthalpy change associated with the transition 0: --f fl at temperature T. If some solute is present as well as the water we may write by analogy wit,h Eqns. 3 and 4 ;Ks( ‘j”)
=
@!?=exp f&(T)
_
[
-1
@“(T) -- &T(T) _ ~_~ RT
(5)
(6) The terminology in Eqns. 5 and 6 is such that the superscript “s” denotes that solution is present. When the equilibrium constants K”(T) and K”(T) are unity then the corresponding transition temperatures are T\,> and T, and we have from Eqns. 3 and 5
and neglecting the temperature change phase transitions using Eqn. 6 gives
/_I?( T,)
of the heat capacity
associated
with the
- /A?“‘(T,) = A&+‘( T,)
for an i’th species with of species i in its standard
AFT ,tr( Ts) being the standard states from water to solution,
A/_@( T,) - A/.@‘( T,) = -T, It is found
experimentally
(9)
[$
-- $1
for small
free energy of transfer then Eqn. 8 becomes
AIPw(T,) molecules
(10) at not
too
high
concentrations
273 o f solute and can be s h o w n to be generally true for any disperse system t h a t
RT
(ki .~e~)
(11)
where k~ ~ is a c o n s t a n t which d e p e n d s u p o n the n a t u r e o f i and s and e~ is the m o l a r c o n c e n t r a t i o n o f solute s. The c o n s t a n t k~_~ is strictly speaking t e m p e r a t u r e d e p e n d e n t although investigations on simple systems indicate t h a t t h e t e m p e r a t u r e d e p e n d e n c e is small and as an a p p r o x i m a t i o n we shall assume t h a t it is i n d e e d constant. C o n s e q u e n t l y f r o m Eqns. 10 and 11 writing A T = T w - - T ~ and T~Tw~- ~,2_wweget
AT
=
[
LAH
- -. .R. . ~(T,,,)_I , _] (Its s--k~, s)c~
(12)
Thus at low c o n c e n t r a t i o n s o f solute we should e x p e c t a linear variation o f the transition t e m p e r a t u r e associated with the a -~ ~ phase change with c o n c e n t r a tion o f a d d e d solute. The p r o p o r t i o n a l i t y c o n s t a n t depends u p o n t w o terms. T h e first o f these is the usual sort o f term o b t a i n e d for colligative p r o p e r t i e s and is calculable. The s e c o n d is a c o m p o s i t e term and d e p e n d s u p o n the d i f f e r e n c e b e t w e e n the e x t e n t o f the i n t e r a c t i o n o f the solute with the a and forms participating in the general equilibrium. It should be n o t e d that it is n o t necessary t o assume t h a t the solute S actually binds in an associative way t o a n d / o r ft. T h e ] t s terms allow for the fact that r a t h e r longer range perturbations o f the solute on c~ and ~ can exist than would be c o n s i d e r e d in an association t r e a t m e n t . However, if one wishes to use a chemical m o d e l invoking association b e t w e e n the solute S and a and ~ t h e n it is easy t o show t h a t Ap~tr(Ts) . . . .
RT~ In (1 + K~,~a~)
(13)
where Ko.~ is the equilibrium c o n s t a n t for the process + S ~ ~S
(14)
and a~ is the activity o f species S. If we assume ideality and low c o n c e n t r a t i o n s o f S t h e n Eqn. 13 b e c o m e s
Ap~o , t r (T~)=--RT~K~c~
(15)
with an analogous expression for ~P~ A ~ , t ~t~.~J. , T h e r e f o r e from Eqns. 10 and 15 A T = LAH~,W(Tw) ] (K~,
--
K~s)e~
(16)
T h e first t e r m in parentheses on the right-hand side o f Eqns. 12 and 16 is positive and c o n s e q u e n t l y w h e t h e r o n e observes an increase or decrease in the t e m p e r a t u r e at which the phase transition occurs depends u p o n the d i f f e r e n c e b e t w e e n the i n t e r a c t i o n o f the solute S with the ~ and a forms. A stronger i n t e r a c t i o n (i.e. m o r e association) o f the solute with the ~ f o r m t h a n with the a form will decrease the transition t e m p e r a t u r e and vice versa. Obviously if the e x t e n t o f i n t e r a c t i o n o f the solute S with b o t h species participating in the phase change is the same t h e n no p e r t u r b a t i o n in the phase transition
274
temperature will occur. Eqn. 12 and Eqn. 16 to a greater extent have considerable limitations particularly with regard to their range of applicability. This is at present indeterminate, but one can confidently predict non-linearity in AT vs. c~ plots at unspecifiable but higher concentrations arising from higher order interactions of solutes with the species participating in the equilibrium and from neglect of the variation of the heat capacity with temperature associated with the transitions. Furthermore it has been assumed that the h~.s terms are temperature independent and this will not be quite correct. Eqns. 12 and 16 are, however, useful as limiting cases. It should be noted that if the solute S is a strong electrolyte such that 1 mol of salt gives rise to n M mol of cation M and nx tool of anion X Eqns. 12 and 16 become -2 R Tw AT . . . . . . .
AH~'~(T~)
[(nMh~_M+nxh~_x)--(nMk~
M+n×k~_x)]G
(17)
nxK~x)]Cs
(18)
--Rv AT
= -
[(/2M]~3 M + r/xK#x)
-- (nMK~M
+
where Cs is the stoichiometric salt concentration. Consequently the temperature change of a phase transition depends upon four unknown parameters for any given salt. Acknowledgements We wish to acknowledge financial support for this research from the Ministry of Defence, the Multiple Sclerosis Society and the Wellcome Trust. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
B y r n e , P. and C h a p m a n , D. ( 1 9 6 4 ) N a t u r e 202, 987- 9 8 8 C h a p m a n , D. and Collin, D.T. ( 1 9 6 5 ) N a t u r e 20G, 189 C h a p m a n , D. ( 1 9 6 5 ) The S t r u c t u r e of Lipids, M e t h u e n , L o n d o n C h a p m a n , D., B y r n e , P. and Shipley, G.G. ( 1 9 6 6 ) Proc. R. Soc. L o n d . Set. A, 290, 115 142 C h a p m a n , D., Williams, R.M. a n d L a d b r o o k e , B.D. ( 1 9 6 7 ) C h e m . Phys. Lipids 1, 4 4 5 4 7 5 C h a p m a n , D. and S a l s h u r y , N.J. ( 1 9 6 6 ) Trans. F a r a d a y Soc. 62, 2 6 0 7 - - 2 6 2 1 C h a p m a n , D. ( 1 9 6 8 ) Biological M e m b r a n e s ( C h a p m a n , D., ed.), Vol. 1, A c a d e m i c P r e s s , N e w Y o r k L a d b r o o k e , B.D., Williams, R.M. and C h a p m a n , D. ( 1 9 6 8 ) Biochim. Biophys. A e t a 1 50, 3 3 3 - - 3 4 0 Hinz, H.J. a n d S t u r t e v a n t , J . M . ( 1 9 7 2 ) J. Biol. C h e m . 247, 3 6 9 7 3 7 0 0 Hsu, M. and C h e n , S.I. ( 1 9 7 3 ) B i o c h e m i s t r y 12, 3 8 7 2 - - 3 8 7 6 C h a p m a n , I)., U r b i n a , J. a n d K e o u g h , K.M. ( 1 9 7 4 ) J. Biol. C h e m . 249, 2 5 1 2 - - 2 5 2 1 P a p a h a d j o p o u l o s , D., Mosearello, M., Eylar, F,H. and Isae, T. ( 1 9 7 5 ) Biochim. Biophys. Aeta 401. 317 335 Cater, B.A., C h a p m a n , D., H a w e s , S. and S a v i l l e , J. ( 1 9 7 4 ) Biochim. Biophys. A e t a 363, 54---69 Hill, M,W. ( 1 9 7 4 ) Biochim. Biophys. A e t a 3 5 6 , 117 124 P a p a h a d j o p o u l o s , D., J a e o b s o n , K., Poste, G. and S h e p h e r d , G. ( 1 9 7 5 ) Biochim. Biophys. A c t a 39,t, 504--519 S i m o n , S.A., Lis, L.J., K a u f f m a n , J.W. and M c D o n a l d , R.C. ( 1 9 7 5 ) Bioehim. Biophys. A c t a 3 7 5 , 317--326 Verkleij, A.J., De K r u y f f , B., V e r v e r g a e r t , P . t t . J . T h . , T o c a n n e , J.F. a n d Van D e e n e n , L.L.M. ( 1 9 7 4 ) B i o c h i m . Biophys. A c t a 339, 4 3 2 - - 4 3 7 T r a u h l e , H. and Eibl, H. ( 1 9 7 4 ) P r o e . Natl. Aead. Sei. U.S. 71, 2 1 4 - 219 G o d i n , N.G. a n d Ng, T.W. ( 1 9 7 4 ) Moi. P h a r m a c n l . 9, 8 0 2 819 G o t t l i e b , M.H. and Eanes, E.D. ( 1 9 7 2 ) Bioohys. J. 12, 1533 1 5 4 8 E h r s t r o m , M., E r i k s o n n , L . E . G . , Israelaehvili, ,I. a n d E h r e n b e r g , A. ( 1 9 7 3 ) B i o c h e m . Biophys. Res. C o m m u n . 55, 3 9 6 - 4 0 2
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22 23 24 25 26 27 ' 28 29 30 31 32 33
Y a b u s a k i , K . K . a n d Wells, M.A, ( 1 9 7 5 ) B i o c h e m i s t r y 14, 1 6 2 - - 1 6 6 Lis, L.J., K a u f f m a n , J.W. and Shriver, D.F. (19"/5) Biochim. Biophys. A c t a 406, 453- 4 6 4 I n o k o , Y., Y a m a g u c h i , T., F u r u y a , K. a n d Mitsui, T. ( 1 9 7 5 ) Biochim. Biophys, A c t a 413, 2 4 - - 3 2 M c L a u g h l i n , S., Bruder, A., C h e m S. and Moser, C. ( 1 9 7 5 ) Biochim. Biophys. A c t a 394, 3 0 4 - - 3 1 3 Stability c o n s t a n t s of m e t a l - i o n c o m p l e x e s , C h e m i c a l S o c i e t y Special P u b l i c a t i o n s ( 1 9 6 4 ) (Sillen, L.G, and Martell, A.E., eds.), Vol. 17 Stability c o n s t a n t s , S u p p l e m e n t No. 1, C h e m i c a l S o c i e t y Special P u b l i c a t i o n s (tti}gfeldt, E. and Martell, A . F . , eds.), Vol. 25 Davies, J., O r m o n r o y d , S. and S y m o n s , M.C.R. ( 1 9 7 1 ) Trans. F a r a d a y Soc. 67, 3 4 6 5 3 4 7 3 ttatefi, Y. and H a n s t e i n , W.G. ( 1 9 6 9 ) Proc. Natl. A c a d . Sci. U.S. 62, 1 1 2 9 - - 1 1 3 6 R a n d , P., C h a p m a n , D. a n d Larsson, K. ( 1 9 7 5 ) Biophys. J. 15, 1 1 1 7 - - 1 1 2 4 T a r d i e u , A., L u z z a t i , V. and R e m a n , F.C. ( 1 9 7 3 ) J. Mol. Biol. 75, 7 1 1 - - 7 3 3 Phillips, M.C., Williams, R.J.P. and C h a p m a n , D. ( 1 9 6 9 ) C h e m . Phys. Lipids 3, 23.1--244 Hillz, H.J. and S t u r t e v a n t , J.M. ( 1 9 7 2 ) J. Biol. C h e m . 247, 6 0 7 1 - - 6 0 7 5
Bioct~imica et Biophysica Acta, 464 (1977) 260 275 © Elsevier/North-Holland Biomedical Press
BBA 77577 LIPID P H A S E T R A N S I T I O N S IN M O D E L B I O M E M B R A N E S T t t E E F F E C T O F IONS ON P I t O S P H A T I D Y L C H O L I N E B I L A Y E R S
D. CHAPMAN a, W.E. PEEL a, B. KINGSTON b and T.H. LILLEY b a Department o f Chemistry, Chelsea College, University o f London, London, SW3 6LX and b Department o f Chemistry, Universily o f Sheffield, Sheffield $3 7HF (U.K.)
(Received March 13th, 1976) (Revised manuscript received September 17th, 1976)
Summary Differential scanning c a l o r i m e t r y has been used t o s t u d y the e n d o t h e n n i c phase b e h a v i o u r o f some m o d e l b i o m e m b r a n e s (i.e. p h o s p h a t i d y l c h o l i n e - w a t e r systems) in the presence o f a wide range o f alkaline, alkaline earth and heavy metal salts. Studies and c o m p a r i s o n s were m a d e o f b o t h cation and anion effects. Shifts o c c u r in the t e m p e r a t u r e s o f b o t h the pre-transition and main transition e n d o t h e r m s . T h e observed shifts are smaller t h a n t h o s e which have been r e p o r t e d for charged lipids, and no evidence has been f o u n d for the f o r m a t i o n o f specific c o m p l e x e s . E l e c t r o n microscopic studies on freezef r a c t u r e d dispersions o f p h o s p h a t i d y l c h o l i n e - w a t e r - s a l t systems show t h a t with some salts the typical rippled surface observed with L-a-dimyristoyl phosphat i d y l c h o l i n e , w h e n in the gel state, is replaced by a s m o o t h surface.
Introduction
Previous studies o f pure lipids and lipid-water systems using infrared spect r o s c o p y [1], differential thermal analysis, differential scanning c a l o r i m e t r y [2--5] and NMR s p e c t r o s c o p y [6] s h o w e d the existence o f a t h e r m o t r o p i c phase transition associated with h y d r o c a r b o n chain melting involving increased r o t a t i o n a l and translational m o t i o n s o f these chains. The t e m p e r a t u r e s at which these e n d o t h e r m i c phase transitions o c c u r have been s h o w n t o be d e p e n d e n t u p o n the head group, the alkyl chain length and the degree and t y p e o f uns a t u r a t i o n present [7]. T h e presence o f a third c o m p o n e n t such as cholesterol [ 8 , 9 ] , proteins [ 1 0 - - 1 2 ] , drugs [ 1 3 - - 1 5 ] and salts [ 1 1 , 1 6 ] can cause significant shifts in the phase transition t e m p e r a t u r e or eliminate t h e phase transition. Interest in these systems is great because of their relevance to b i o m e m b r a n e systems.
261 In this paper we examine the effects of various cations and anions on the e n d o th er mie phase transitions o f two phosphatidylcholine-water systems. Previous studies of ion effects have been reported [11,16] but are of a less extensive nature than those r e por t ed here. Materials and Methods The lipids, L-a-dimyristoyl phosphatidylcholine and L-a-dipalmitoyl phosphatidylcholine used in the calorimetric experiments were obtained from Koch-Light Laboratories Ltd. The dimyristoyl phosphatidylcholine used in the electron microscopic studies was obtained from Fluka. Purity was checked by thin-layer chromatography. The salts used were usually of Analar quality but in some cases reagent grade was used. The solutions were prepared volumetrically using deionised distilled water. Samples were prepared by homogenising weighed amounts of the appropriate lipid and solution above the gel-liquid crystalline transition temperature (approx. 50°C) using a vortex mixer. Some samples were homogenised by centrifuging them backwards and forwards through a constriction in the sample tube. The different m et hods of sample preparation did not significantly alter the results obtained. Calorimetric measurements were p e r f o r m e d using a Perkin-Elmer DSC-1B on samples weighing approx. 8 mg contained in hermetically sealed aluminium pans with a heating rate of 8K/min. Some of the results were checked using a Perkin-Elmer DSC-2 with a heating rate of 5 K/min, no significant difference being obtained in the results from the two instruments. The temperature scale w a s calibrated using the transition temperatures of cyclohexane (279.8 K), sodium sulphate decahydrate (305.6 K) and indium (429.8 K), and was accurate to -+0.2 K. The indium transition was also used to calibrate the enthalpy per unit area. A minimum o f three different samples were prepared for each system studied and each sample was investigated at least twice. The extrapolated onset te m pe r at ur e was taken as the transition temperature. Enthalpy and e n t r o p y data for the observed transitions were calculated from the area below the en d o t he r m s by standard methods, the curve areas being measured with a fixed arm planimeter. Dispersions of dimyristoyl phosphatidyleholine in excess solution (usually approx. 1 : 2 or 1 : 3 by weight), homogenised as described above, were quenched from 15°C into liquid Freon at - 8 0 ° C and then liquid nitrogen. Fracturing and etching, using a Nanotech freeze-fracture apparatus, were carried out at --99°C following by Pt/C shadowing and carbon replication at --110° C. Results Fig. 1 shows the calorimetric curves for the dimyristoyl phosphatidylcholine-water system as a function of water content. The e n d o t h e r m associated with the ice.melting transition at approx. O°C has been o m i t t e d from the diagrams. At first only one transition occurs but with increasing water c o n t e n t this transition narrows and a second transition appears slightly lower in temperature. This has been called the pre-transition e n d o t h e r m , the ot her being the
262
E
[iJ
2;o
'
38o
3~o
Temper@ture (K) F i g . 1. C a l o r i m e t r i c c u r v e s f o r t h e d i m y r i s t o y l p h o s p h a t i d y l c h o l i n e - w a t e r concentration, c = wt. lipid/wt, lipid + wt. water.
system
as a f u n c t i o n
of lipid
main transition endotherm. The characteristic behaviour of these features with changing water c o n t e n t has been reported elsewhere [5]. There is an abrupt change in the temperatures at which the two transitions occur at weight fractions of lipid of approx. 0.8. This also coincides with the appearance of the ice-melting e n d o t h e r m in the calorimetric curves. For the dipalmitoyl phosphatidylcholine-water system this change is approximately paralleled by changes in the enthalpy associated with the main transition (Table I). In view of this marked dependence of transition temperature on the water content, the studies involving ionic solutions were perform ed at concentrations well removed from this critical region, a 1 : 1 by weight lipid/solution mixture being used. Table II shows the dependence of the pre and main transition temperatures (T~ and To) o f the dipalmitoyl phosphatidylcholine-water system on the presence o f some heavy metal salts at low concentrations. It is apparent that salt concentrations as low as 10-3M are inducing perturbations in the phase transitions, with a given salt affecting the two lipid transitions in an approximately similar fashion. Table III illustrates the effect of some heavy metal salts at concentrations up to 1 M on the transition temperatures of dimyristoyl phosphatidylcholinewater dispersions. All of the salts investigated increased the transition t e m p e r a t u r e o f both endotherms but it is apparent that the efficacy of the
263
various salts differs. The very large effects of ZnCI~ and SAC12 are particularly notable and it is also significant that in the systems containing these salts the pre-transition appeared as a shoulder on the main transition endotherm. TABLE I T H E E N T H A L P Y ( A / l ) A N D E N T R O P Y (AS) C H A N G E S A S S O C I A T E D W I T H T H E P R E - (p) A N D M A I N (c) P H A S E T R A N S I T I O N S FOR THE DIPALMITOYL PHOSPHATIDYLCHOLINE-WATER S Y S T E M AS A F U N C T I O N O F T H E W E I G H T F R A C T I O N O F T H E L I P I D Weight fraction of dipalmitoyl phosphat i d y l c h o l i n e (C) 0.99 0.833 0.768 0.714 0.666 0.625 0.5 Excess w a t e r Excess water
Attp
ASp ( e a l - K -1 • t o o l -1)
(kcal • mo1-1)
1.3 1.6 1.7
AH c (kcal - tool -1)
AS c ( c a l " t o o l 1 • K I)
--
5.9
17.7
---
6.5 8.6
20.0 28.0
-
9.5
31.0
4.3 5.3 5.5
9.4 9.1 9.5 8.7 9.7
30.4 29.4 32.8 27.6 * 3 0 . 7 **
* Ref. 32. ** R e f . 3 3 . T A B L E II THE PRE-TRANSITION ENDOTHERM AND MAIN ENDOTIiERM TRANSITION TEMPERATURE. ¢ OF DIPALMITOYL PHOSPHATIDYLCHOLINE-HEAVY M E T A L S A L T S O L U T I O N S Y S T E M S (C =
0.5) Solution
Tp (K)
Water
10 -3 1 0 -3 1 0 -3 10-3 10 3 10 -3 1 0 -3
M M M M M M M
HgC12 ZnCI 2 SnC12 CuCI 2 CdC12 lead acetate CuS04
308.5 309.9 309.7 307.9 308.5 308.2 309.4 307.8
Tc (K)
314.5 316.7 315.6 ' 314.5 314.0 314.0 316.5 314.0
T A B L E III THE PRE-TRANSITION ENDOTHERM AND MAIN ENDOTHERM TRANSITION TEMPERATURE~ OF DIMYRISTOYL PHOSPHATIDYLCHOLINE--HEAVY M E T A L S A L T S O L U T I O N S Y S T E M S (C = O.5) Solution
Water 0 . I M ZnCI 2 0.1 M SnCI 2 I M CdCl 2 1 M AgNO 3 1 M lead acetate 1 M CuSO 4 1 M thallium acetate
Tp
Tc
(K)
(K)
287.2 --290.0 290.4 287.7 291.4 287.3
296.7 302.4 301.2 297.7 298.7 297.7 297.9 296.9
264 TABLE
IV
THE PRE-- TRANSITION ENDOTttERM AND MAIN ENI)OTHERM TRANSITION TEMPFRATURES OF DIMYRISTOYL PHOSPHATIDYLCHOLINE-ALKALINE, ALKAI,INE EARTH AN[) GUANAI ) I N I U M ( G u ) S A L T S O L U T I O N S Y S T E M S (C = 0 . 5 ) Sohttion
"I'1) (K)
Tc (K)
[ M LiCI
287.2
295.9
1 M NaCI 1 M KC1 1 M RbCI
289.2 288.6 288.2
297.8 297.4 297.6
1 M CsCl 1 M NaF
286.7 289.9
297.0 298.2
1 M NaBr
284.1
296.7
I M Nal
-
294.3
l M NaCNO 1 hi s o d i u m a c e t a t e
287.6 290.6
297.2 297.6
1 M KCN
287.0
297.2
1 M NII4CI 1 M tetramethylammonium
290.2 289.2
299.3 297.4
chloride
1 M CaC12 1 M MgC12 1 M GuSCN 0.1 M GuC1
287.1
301.7 298.8 292.9 296.7
286.1 281.3
296.6 296.3
292.7
0.2 M GuCI I. M GuC1
Table IV illustrates t h e effects o f a c o m p r e h e n s i v e range o f salts o n t h e transition t e m p e r a t u r e s o f t h e d i m y r i s t o y l p h o s p h a t i d y l c h o l i n e - w a t e r s y s t e m . F o r 1 M CaC12, N a S C N and G u S C N no pre-transition e n d o t h e r m s were observable, and for 1 M NaI this e n d o t h e r m was so broad that it was n o t possible to obtain a reliable e s t i m a t e o f t h e transition temperature. Table V gives the e n t r o p y and e n t h a l p y values associated with the phase transitions for s o m e o f t h e salts studied. TABI, E V Atf
AND AS DATA
Estimated
errors:
FOR
DIMYRISTOYL
PHOSPHATIDYLCHOLINE/AQUEOUS
SALT SOLUTIONS
A t t e ~ 0 . 9 k c a l - r e e l -1 ; A S e -+- 2 e a l . r e e l -1 - K - 1 .
AHp
ASp
AH c
( k e a l • t o o l -1 )
( c a l - t o o l -1 - K -1 )
(kcal . tool -I )
AS c (eal - reel
6.7
22.4 *
l M N[t4Cl
1.2
4.1
7.2
23.7
1 M tetramethylammonium chloride 1 M sodium acetate 1 M NaBr 1 M NaCNO i M NaF 1 M NaI 1 M KCN 0 . [ M Z nC 12 I M MgC12
0.8 1.0 0.8 0.7 0.9 -0.7 0.7 1.2
2.1 3.6 2.7 2.5 3.2 -2.6 2.4 4.0
6.6 6.5 7.4 6.8 6.8 6.9 7.1 7.2 7.6
22.4 22.1 25.0 22.9 23.0 23.8 24.1 24.5
1 M CaCI 2
--
--
8.6
Solution
Water
I . K-1 )
25.5 2 8 . 8 **
• Ref. 32. ** T h e l a r g e v a l u e s o f from a pre-transition
AH e and AS e produced by this salt are probably coincident with the main endothenn.
due to some contribution
265 Figs. 2a and 2b show the marked effect of various NaSCN concentrations on the dimyristoyl phosphatidylcholine-water system at 50 weight percent lipid. At SCN- concentrations > 0 . 2 M no pre-transition endotherm is observable and indeed as the concentration of salt approaches this value the endotherm is broadening. The main transition temperature increases rapidly at low concentrations and thereafter increases more slowly. The most significant effect however, is the way in which the endotherms diverge as the salt concentration increases. In view of the importance of possible hydration effects the dipalmitoyl phosphatidylcholine-water-ZnC12 system was studied in more detail. The mol ratio of dipalmitoyl phosphatidylcholine/ZnC12 was kept constant at 3 : 1 and the water content of the system was varied. The results for the main transition endotherm are shown in Fig. 3. It can be seen that ZnC12 modifies the effect
(a) 288
z 2~7
R o
\
,~i 2R6
\
\
~\o
\
i2~ 5
\
R. o \
283[__
o'.1
o.$5
o.%
o:2
1.s
2:o
Thiocyanate concentrqtion (M) (b)
8 BOO E
299 298
J
jo-
o p ~ °/
297
£
o.s
1.o
Thiocyanate concentration ( b4)
2is
3.0
Fig. 2. T h e d e p e n d e n c e o f (a) t h e p r e - t r a n s i t i o n e n d o t h e r r a a n d ( b ) t h e m a i n e n d o t h e r m peratures of dimyristoyl phosphatidylcholine on NaSCN concentration. C = 0.5.
transition
tern
266
//t/l/l! /
c~
~ 317 E £ 8 3~6 315 o
I O__o_O
o
Z 314
i
Weight
_1
lrGction
c
Fig. 3. T h e d e p e n d e n c e o f t h e m a i n e n d o t h e r m t r a n s i t i o n t e m p e r a t u r e on w a t e r c o n c e n t r a t i o n f o r th~ d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e - w a t e r (full line) a n d d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e - w a t e r - Z n C l 2 ( d a s h e d line) s y s t e m s . R a t i o d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e / Z n 2+ w a s 3 : 1 t h r o u g h o u t .
Fig. 4. F o r l e g e n d see o p p o s i t e page.
267
b'ig. 4. F r e e z e - f r a c t u r e s u r f a c e s : a, d i m y r i s t o y l p h o s p h a t i d y l e h o l i n e - w a t e r : b, d i m y r i s t o y l p h o s p h a t i d y l choline-1 M GuCl;c, dbnyristoyl phosphatidyleholine-1 M NaSCN. C = 0.25. Magnification X 50000. ( A t t h e s e c o n c e n t r a t i o n s GuC1 d o e s n o t r e m o v e t h e p r e - t r a n s i t i o n w h i l s t N a S C N d o e s r e m o v e t h i s t r a n s i tion).
268 of water on dipalmitoyl phosphatidylcholine since the abrupt change in transition temp er atu r e has shifted from 30% water to 55% water by weight. However, there was no significant alteration in the heat absorbed during the transition. Electron microscopic studies on the freeze-fractured surfaces of dispersions o f dimyristoyl phosphatidylcholine with various salt solutions have shown that in cases where the pre-transition e n d o t h e r m has been removed, the ripples usually observed on the surfaces of the liposomes have also been removed. Electron micrographs of the different surfaces observed are shown in Fig. 4. Discussion Previous results on the interaction of metal ions with charged and uncharged lipids using a variety of physical techniques have been interpreted using double layer t h e o r y [17,18], dehydr a t i on effects [19], complex formation [11,16, 20--24] and structure making and breaking effects [21,25]. It is to be expect ed that the interaction of metal ions with uncharged phosphatidylcholine should produce only small effects and the work of Simon et al. [16] and the results reported here show this is so. The transition temperatures are shifted by 6°C at the most and the enthalpy change associated with the transition is hardly affected by the presence of salt (Table V). The behaviour observed experimentally of the effects of salts on the transition temperatures in phosphatidylcholine-water systems is apparently rather complex. T h e r m o d y n a m i c arguments developed in the appendix give an insight into the sort of behaviour we would expect for the effect of solutes on a phase transition. It is shown t ha t the t em pe r at ure change in a phase transition depends on four u n k n o w n parameters for a given salt. In view of this any precise and unambigous interpretation of the effect of salts on phase transitions is no t possible at present and all one can hope for is some correlation with the experimentally observed effects and observations on some simpler systems. Eqns. 17 and 18 (Appendix) indicate that both anions and cations may affect phase transitions and it is convenient to consider first the order of cation effects by investigating salts with a c o m m o n anion. The experimental results on systems of metal chlorides show t hat the t e m p e r a t u r e of the main transition in the dimyristoyl phosphatidylcholine-water system is in the order (NMe~, t e t r a m e t h y l a m m o n i u m ion) NH~ > Na+~ K t ~ Rb+~ NMe~ > Cs+> G u t > Li t for mo n o v alen t cations and Zn 2+ ~> Sn 2+ > Ca 2. > Mg 2+ > Cd 2+ for divalent cations. The order observed for the pre-transition e n d o t h e r m is NH; > Na t ~ NMe; > K*> Rb+> L i t > Cs*> Gu* for the monovalent ions. No pre-transition endotherms were observed for ZnCI> SnC12 and CaC12 whilst the order for MgC12 and CdC12 was Mg 2. >Cd 2.. It seems likely that for the first three salts the pre-transition e n d o t h e r m has been shifted so high in temperature that it is overlapping the main transition.
269
The d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e - w a t e r s y s t e m shows t h a t in the presence o f divalent metal chlorides the main transition e n d o t h e r m decreases in the order Hg2*> Zn2+> Sn2+> Cu2+> Cd > and the pre-transition t e m p e r a t u r e s f o l l o w the o r d e r Hg 2+> Zn 2 . > Cu 2+> Cd 2 . > Sn 2+ T h e r e f o r e f o r a given p h o s p h a t i d y l c h o l i n e - w a t e r - m e t a l chloride system the o r d e r for b o t h the main and pre-transition e n d o t h e r m s is the same. T h e r e is a rough c o r r e l a t i o n b e t w e e n the orders observed and the m a g n i t u d e s o f the association c o n s t a n t s o f metal ions with simple phosphates. The association c o n s t a n t s e x p e r i m e n t a l l y observed [26,27] vary with the p h o s p h a t e used but f o r m o n o v a l e n t cations the o r d e r is L i * > N a * > K +> Cs* and for divalent cations Hg 2+ > pb 2÷ > Cd 2* > Zn :+ > Cu 2+ >
Ca 2 + >
Mg 2+
The a g r e e m e n t is only a p p r o x i m a t e and we t h e r e f o r e consider o t h e r possible i n t e r a c t i o n mechanisms. S o m e aspects o f previously r e p o r t e d lipid-metal ion interactions have been i n t e r p r e t e d o n the basis o f the k n o w n s t r u c t u r e making and breaking effects o f the ions on w a t e r [ 2 1 , 2 5 ] . A t t e m p t s to correlate o u r wider range o f cations with p h o s p h a t i d y l c h o l i n e in a similar m a n n e r were unsuccessful. I n d e e d t h e e f f e c t o f urea, at a c o n c e n t r a t i o n o f 1 M, on the transition t e m p e r a t u r e s was virtually negligible although it is a well-known s t r u c t u r e breaker. in view o f t h e fact t h a t all ions in solution are solvated it is necessary to take a c c o u n t o f possible h y d r a t i o n effects in i n t e r p r e t i n g the results r e p o r t e d here, and the results d e p i c t e d in Fig. 3 c o n f i r m this. It is n o t e w o r t h y t h a t for salts with a c o m m o n anion the ratio (charge)2/radius, (an a p p r o x i m a t e measure o f the solvation interaction), predicts fairly accurately the relative orders o f m a g n i t u d e o f the effects p r o d u c e d (Table VI) with the above ratio increasing with the ability o f the metal ion to raise t h e transition t e m p e r a t u r e s . However, t h e positions o f Li* and Hg 2+ are anomalous. It s h o u l d be n o t e d also t h a t the anionic e f f e c t is by no means small and considering the main transition e n d o t h e r m with s o d i u m salts the t e m p e r a t u r e o f the transition decreases as follows SCN- > F - > C1- ~ a c e t a t e - > C N O
> Br- > I-
whereas the o r d e r observed for the pre-transition is acetate
> F- > C1- > C N O - > Br- > SCN-
T h e r e is a reasonable c o r r e l a t i o n in these t w o series e x c e p t for the e x t r e m e change o f position o f SCN-. The effectiveness o f anions in d e s t r o y i n g the s t r u c t u r e o f w a t e r and lowering the h y d r o g e n b o n d strength is given by ref. 28 I- > SCN- > Br
> C1- ~ CNO- > F -
270 TABLE
VI
RATIO
OF (CttARGE)2/IONIC
Data from 53rd edition Itandbook
RADIUS
FOR METAL
of Chemistry
Ion
Radius (A)
C u 2+
0.96
4.17
Ag + Z n 2+ Cd 2+
1.26 0.74 0.97
0.794 5.42 4.13
n g 2+
1.1
3.64
TI + S n 2+
1.49 0.84
0.67 4.76
P b 2+
1.21
3.31
Li + Na + K+
0.68 0.97 1.33
1.47 1.03 0.753
Rb + Cs + Mg 2+ Ca 2+
1.47 1.67 0.66 0.99
0.68 0.6 6.06 4.04
IONS STUDIED
and I'hysics.
( C h a r g e ) 2 / R a d i us (,\ J)
There is a correlation between the structure-breaking efficiency of the anions and their ability to lower the transition temperatures. This may be explained by the anions interfering with the hydr ogen-bonded structure around the polar head group and hence altering the packing of the alkyl chains. The very large lowering of the pre-transition t em perat ure in the case of SCN may be due to the fact that this ion is known to be adsorbed preferentially at interfaces [29] and therefore may be producing a negatively charged lipid-water interface, This could lead to both longitudinal and lateral repulsion in the bilayer structure, the latter tending to alter the packing of the alkyl chains so leading to a lowering of the pre-transition temperature. Preliminary X-ray measurements have indicated that the long spacings are increasing with increasing SCN- concentration. It is difficult to explain the observed increase of the main e n d o t h e r m transition with increasing SCN- concentration. It may be that the reorientation of the head group at the pre-transition temperature allows the formation of a new more stable hydrogen bond network, involving SCN-. If the " m e l t i n g " and lateral separation of the alkyl chains at the main transition leads to a disruption o f such a hydrogen bond network additional thermal energy will be required and so T c will be raised provided there is no significant change in ASc. This idea of additional hydrogen bonds has been used by Simon et al. [16] to e x p l a i n the increase in AH,~ observed for dipalmitoyl phosphatidylcholine + 1 M KSCN. Simon et al. [16] also found that the main transition t em perat ure decreased for dipalmitoyl phosphatidylcholine + 1 M KSCN. In view of the fact that the t e m p er atu r e changes are small and different chain length lipids and cations were used we consider that the discrepancy is not significant. The pre-transition phase change is associated with an increase in the mobility
271 o f t h e polar head group and r e c e n t w o r k has s h o w n that the transition is also associated with a change in the angle o f tilt o f the alkyl chains [30]. A simult a n e o u s r e d u c t i o n in the area per polar h e a d g r o u p at the lipid-water i n t e r f a c e occurs. Replicas o f f r e e z e - f r a c t u r e d surfaces o f d i m y r i s t o y l p h o s p h a t i d y l c h o l i n e w a t e r dispersions in the gel phase, which Luzzati terms PB' [ 3 1 ] , have s h o w n rippled surfaces u n d e r the e l e c t r o n m i c r o s c o p e (Fig. 4). T h e ripples are t h o u g h t t o be due to u n d u l a t i o n s in the surface o f the liposomes caused by the longitudinal d i s p l a c e m e n t required in packing the tilted chains. Our calorimetric and e l e c t r o n m i c r o s c o p i c results s u p p o r t the idea t h a t the pre-transition is associated with the presence o f tilted chains, since dispersions o f d i m y r i s t o y l p h o s p h a t i d y l c h o l i n e - w a t e r c o n t a i n i n g salts which r e m o v e the pre-transition e n d o t h e r m s h o w no ripples in their f r e e z e - f r a c t u r e d surfaces. This indicates t h a t the chain tilt, which is a r e q u i r e m e n t for the presence o f ripples, has been altered by the presence o f t h o s e salts which r e m o v e t h e pre-transition endot h e r m . This also occurs u p o n a d d i t i o n o f 7% decane which gives a vertical-chain phase. Conclusions T h e effects generally observed are smaller t h a n those which have been observed for charged lipids. T h e effects o f cations appears to correlate a p p r o x i m a t e l y with a ratio, (charge)2/radius, which measures their solvation interaction. N o t w i t h s t a n d i n g the d i f f i c u l t y o f q u a n t i t a t i v e i n t e r p r e t a t i o n s some qualitative conclusions can be drawn a b o u t the effects o f cations on p h o s p h a t i d y l c h o l i n e - w a t e r systems. It w o u l d seem t h a t Hg 2÷, Pb 2+ and Zn 2. c o n f e r increased stability t o t h e r m a l change on gel phase dispersions o f d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e whereas Cd > and Cu 2+ have a small destabilising effect. With the d i m y r i s t o y l p h o s p h a t o c y l e h o l i n e - w a t e r system it is f o u n d t h a t Sn 2÷ and Zn 2+ have a very m a r k e d e f f e c t in increasing gel phase stability whereas o t h e r heavy metals have r a t h e r less effect. The heavy metal salts increase gel phase stability. O f the c o m m o n alkaline and alkaline earth salts it m a y be c o n c l u d e d t h a t Ca 2. and Mg 2÷ give rise to increased stability as do Na + and K ÷ b u t t o a lesser extent. Anionic effects can be e x p l a i n e d qualitatively in terms o f s t r u c t u r e - b r e a k i n g effects on the organisation o f the polar head group region, leading t o an alteration in the packing o f the alkyl chains. The a n o m a l o u s effects o f the SCN- are t h o u g h t to be due to its great t e n d e n c y to a c c u m u l a t e at the polar lipid-water interface. Appendix We shall consider t h e b e h a v i o u r we c o u l d e x p e c t for the effects o f solutes on a phase transition. S u p p o s e t h a t we consider a phase transition for a m o n o disperse system a -~ /3
(1)
where a and fi are the forms which arc stable at low and high t e m p e r a t u r e s ,
272
respectively. P,(T)
At any temperature
we have
=&3(T)
(2)
where pi(T) is the chemical potential of i at temperature T. If we consider the situation when water is the only other species present then expanding chemical potentials and rearranging gives K”(T)
=
fpW(T) = exp f,"
_
1
g”(T) -- PTiY (T) RT
CT)
(3)
where f;(T) and f,“(T) are the fractions of fl and CYpresent at temperature T. respectively, K”(T) is the equilibrium constant for the equilibrium and /LT.” denotes the standard chemical potential of i in water. Application of the GibbsHelmholtz equation gives a___ In _~ K”(T) i a (I/T)
= __AH*‘V(T) R I’
(4)
where Ai?%” is the standard enthalpy change associated with the transition 0: --f fl at temperature T. If some solute is present as well as the water we may write by analogy wit,h Eqns. 3 and 4 ;Ks( ‘j”)
=
@!?=exp f&(T)
_
[
-1
@“(T) -- &T(T) _ ~_~ RT
(5)
(6) The terminology in Eqns. 5 and 6 is such that the superscript “s” denotes that solution is present. When the equilibrium constants K”(T) and K”(T) are unity then the corresponding transition temperatures are T\,> and T, and we have from Eqns. 3 and 5
and neglecting the temperature change phase transitions using Eqn. 6 gives
/_I?( T,)
of the heat capacity
associated
with the
- /A?“‘(T,) = A&+‘( T,)
for an i’th species with of species i in its standard
AFT ,tr( Ts) being the standard states from water to solution,
A/_@( T,) - A/.@‘( T,) = -T, It is found
experimentally
(9)
[$
-- $1
for small
free energy of transfer then Eqn. 8 becomes
AIPw(T,) molecules
(10) at not
too
high
concentrations
273 o f solute and can be s h o w n to be generally true for any disperse system t h a t
RT
(ki .~e~)
(11)
where k~ ~ is a c o n s t a n t which d e p e n d s u p o n the n a t u r e o f i and s and e~ is the m o l a r c o n c e n t r a t i o n o f solute s. The c o n s t a n t k~_~ is strictly speaking t e m p e r a t u r e d e p e n d e n t although investigations on simple systems indicate t h a t t h e t e m p e r a t u r e d e p e n d e n c e is small and as an a p p r o x i m a t i o n we shall assume t h a t it is i n d e e d constant. C o n s e q u e n t l y f r o m Eqns. 10 and 11 writing A T = T w - - T ~ and T~Tw~- ~,2_wweget
AT
=
[
LAH
- -. .R. . ~(T,,,)_I , _] (Its s--k~, s)c~
(12)
Thus at low c o n c e n t r a t i o n s o f solute we should e x p e c t a linear variation o f the transition t e m p e r a t u r e associated with the a -~ ~ phase change with c o n c e n t r a tion o f a d d e d solute. The p r o p o r t i o n a l i t y c o n s t a n t depends u p o n t w o terms. T h e first o f these is the usual sort o f term o b t a i n e d for colligative p r o p e r t i e s and is calculable. The s e c o n d is a c o m p o s i t e term and d e p e n d s u p o n the d i f f e r e n c e b e t w e e n the e x t e n t o f the i n t e r a c t i o n o f the solute with the a and forms participating in the general equilibrium. It should be n o t e d that it is n o t necessary t o assume t h a t the solute S actually binds in an associative way t o a n d / o r ft. T h e ] t s terms allow for the fact that r a t h e r longer range perturbations o f the solute on c~ and ~ can exist than would be c o n s i d e r e d in an association t r e a t m e n t . However, if one wishes to use a chemical m o d e l invoking association b e t w e e n the solute S and a and ~ t h e n it is easy t o show t h a t Ap~tr(Ts) . . . .
RT~ In (1 + K~,~a~)
(13)
where Ko.~ is the equilibrium c o n s t a n t for the process + S ~ ~S
(14)
and a~ is the activity o f species S. If we assume ideality and low c o n c e n t r a t i o n s o f S t h e n Eqn. 13 b e c o m e s
Ap~o , t r (T~)=--RT~K~c~
(15)
with an analogous expression for ~P~ A ~ , t ~t~.~J. , T h e r e f o r e from Eqns. 10 and 15 A T = LAH~,W(Tw) ] (K~,
--
K~s)e~
(16)
T h e first t e r m in parentheses on the right-hand side o f Eqns. 12 and 16 is positive and c o n s e q u e n t l y w h e t h e r o n e observes an increase or decrease in the t e m p e r a t u r e at which the phase transition occurs depends u p o n the d i f f e r e n c e b e t w e e n the i n t e r a c t i o n o f the solute S with the ~ and a forms. A stronger i n t e r a c t i o n (i.e. m o r e association) o f the solute with the ~ f o r m t h a n with the a form will decrease the transition t e m p e r a t u r e and vice versa. Obviously if the e x t e n t o f i n t e r a c t i o n o f the solute S with b o t h species participating in the phase change is the same t h e n no p e r t u r b a t i o n in the phase transition
274
temperature will occur. Eqn. 12 and Eqn. 16 to a greater extent have considerable limitations particularly with regard to their range of applicability. This is at present indeterminate, but one can confidently predict non-linearity in AT vs. c~ plots at unspecifiable but higher concentrations arising from higher order interactions of solutes with the species participating in the equilibrium and from neglect of the variation of the heat capacity with temperature associated with the transitions. Furthermore it has been assumed that the h~.s terms are temperature independent and this will not be quite correct. Eqns. 12 and 16 are, however, useful as limiting cases. It should be noted that if the solute S is a strong electrolyte such that 1 mol of salt gives rise to n M mol of cation M and nx tool of anion X Eqns. 12 and 16 become -2 R Tw AT . . . . . . .
AH~'~(T~)
[(nMh~_M+nxh~_x)--(nMk~
M+n×k~_x)]G
(17)
nxK~x)]Cs
(18)
--Rv AT
= -
[(/2M]~3 M + r/xK#x)
-- (nMK~M
+
where Cs is the stoichiometric salt concentration. Consequently the temperature change of a phase transition depends upon four unknown parameters for any given salt. Acknowledgements We wish to acknowledge financial support for this research from the Ministry of Defence, the Multiple Sclerosis Society and the Wellcome Trust. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
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