1982_mcelhaney_the Use of Differential Scanning Calorimetry and Differential Thermal Analysis in...

8/13/2019 1982_mcelhaney_the Use of Differential Scanning Calorimetry and Differential Thermal Analysis in Studies of Mod

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Chemistry an d Physics of Lipids, 30 (1982) 229-259 229Elsevier/North-Holland Scientific Publishers Ltd.

T H E U S E O F D I F F E R E N T I L S C N N I N G C L O R I M E T R Y N DD I F F E R E N T I L T H E R M L N L Y S I S I N S T U D IE S O F M O D E L N DBIOLOGIC L ME MB R NES

RONALD N. McELHANEYDepartment o f Biochemistry, University o f Alberta, Edm onto n, Alberta T6G 2H7 Canada)

Differential scanning calorimetry (DSC), and to a lesser extent differential thermal analysis(DTA), are powerful yet relatively rapid and inexpensive thermodynamic techniques for study-ing the thermotropic phase behavior of lipids in model and biological membranes, without theintroduction of exogenous probe molecules. In this review the principles as well as the scopeand limitations of DSC and DTA are discussed f'trst. The application of these techniques to thestudy of the thermotropic phase behavior of aqueous dispersions of various single syntheticphospholipids are then summarized, and the effects of cholesterol, free fatty acids, lysophos-pholipids, drugs, anesthetics and proteins on the gel to fiquid-crystalline phase transitionsexhibited by these model systems are discussed. The phase mixing properties of model mem-branes consisting of mixtures of two or more synthetic or natural phospholipids are considerednext. Final/y, the thermotropic phase behavior of prokaryotic plasma membranes and of theplasma, microsomal and mitochondrial membranes of eukaryotic cells are reviewed, and theapplications of DSC and DTA to study the thermal behavior of specific membrane proteins,as well as the physical properties of the membrane lipid phase, are summarized.Keywords: differential scanning calorimetry; differential thermal analysis; lipids; biomem-branes.Introduction

Differential scanning calorimetry (DSC), and to a lesser extent differentialthermal analysis (DTA), are ther mody nami c techniques which have proven of greatvalue in studies of the thermotropic behavior of lipids in model and biologicalmem bra nes (for reviews, see Refs. 1- 3) . Historically DSC and DTA have been usedprimarily to study the thermally-induced transition of lipid bilayers and biologicalmem bra nes from a relatively ordered, crystalline-like state existi ng at lower temper-atures to a relatively disordered, fluid-like state at higher temperatures [1 - 3] . Thisgel to liquid- crystallin e phase tra nsit ion, which arises from a cooperative meltin gof the hydrocarbon chains of the phospholipid molecules [ 4- 7] , does not normallyresult in a gross molecul ar rear range ment to a differ ent liquid-cry stalline meso-phasic state; instead, the lamellar bilayer conforma tion is conserved throu ghout[8-10]. The lipid hydrocarbon chains are converted from a relatively rigid, ex-tended, largely all-trans con form ati on in the gel state to a more ori entati onally0009 -3084/82/0000-0000/ 02.75 1982 Elsevier/North-Holland Scientific Publishers Ltd.


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230 R.N. McElhaney DSC and DTA studies of lipids and biomembranesd i s o r d e r e d s t a t e c h a r a c t e r i z e d b y t h e p r e s e n c e o f a n u m b e r o f gauche c o n f o r m a -t i o n s a n d g r e a t ly i n c r e a s e d r a t e s o f i n tr a - a n d i n te r m o l e c u l a r m o t i o n s [ 1 1 , 1 2 ] . T h eh y d r o c a r b o n c h a i n m e l t i n g i s a c c o m p a n i e d b y a p r o n o u n c e d l a t e r a l e x p a n s i o n a n da concomi t an t dec rease i n b i l aye r t h i ckness , a s we l l a s by a smal l i nc rease i n t het o t a l v o l u m e o c c u p i e d b y t h e p h o s p h o l ip i d m o le c u le s [ 8 - 1 4 ] . T h e p h as e t r an s it io nt e m p e r a t u r e i s o f c o u r s e m a r k e d l y d e p e n d e n t o n t h e c h a in l e n g t h a n d th e c h e m i c a ls t r u c t u r e o f t h e l i p i d h y d r o c a r b o n c h a i n s , a s w e l l a s o n t h e n a t u r e o f t h e p o l a rh e a d g r o u p , t h e i n t e r a c t i o n s o f w h i c h a r e i n f l u e n c e d i n t u r n b y t h e p H a n d i o n i cc o m p o s i t i o n o f t h e a q u e o u s p h a s e [ 1 - 3 ] . T h e g e l t o li q u id - c r y st a ll in e ph a se tr a n s-i t i ons o f some s ing l e , pure p 'hosphol ip ids appear t o be sha rp , symmet r i ca l , f i r s t -o rd e r p rocesses [ 15 ] . Ho we ver , s i nce t he l ip ids in b io log ica l m em bran es t yp i ca l l ypossess apprec i ab l e f a t t y ac id and p o l a r head grou p he t e rog ene i t y , t he ge l t o l iqu id-c r y s ta l li n e p h a s e t r a n s it io n s o f b i o m e m b r a n e s a r e n o r m a l l y r a t h e r b r o a d a n d o f t e na s y m m e t r i c [ 1 - 3 , 7 - 1 2 , 1 6 ] . A l s o , s in c e l ip i d m o l e c u l e s w i t h i n t h e b i l a y e r u n d e r -go r ap id la t e r a l d i f (us ion a t t em pera tu res wi th in and ab ove t he t rans i t i on t empera -tu re , f r ac t i ona l c rys t a l l i za t i on wi th in t he b i l aye r p l ane , w i th i t s a t t endan t l a t e r a lp h as e s ep a r a ti o n , c a n o c c u r [ 1 - 3 , 1 7 ] .

The ge l t o l i qu id-c rys t a l l i ne l i p id phase t r ans i t i on i s ce r t a in ly t he mos t p romin-e n t a n d b e s t - u n d e r s t o o d t h e r m o t r o p i c e v e n t w h i c h o c c u r s i n m o d e l a n d b i o l o g i c a lmembranes . M oreover , t he b io log i ca l e f f ec t s o f t h i s t r ans i t i on have been r e l a t i ve lywell s tud i ed , pa r t i cu l a r l y in m ic roorgan i sms [1 ,3 ,18 ,19 ] . H ow ever , t he re i s som eevidence t ha t o the r l i p id -media t ed t he rma l even t s no t d i r ec t l y r e l a t ed t o t he me l t -i n g o f t h e h y d r o c a r b o n c h a i n s c a n a l s o o c c u r . D S C a n d o t h e r p h y s i c a l t e c h n i q u e sh a v e s u g g es te d t h e p o s si bl e p r e s e n c e o f p r e m e l t i n g a n d p r e f r e e z i n g p h e n o m e n a ,l i qu id- l i qu id phase sepa ra t i ons , g r a in boundary de fec t s i n t he so l i d s t a t e , and t heg lassy s t a t e i n some m ode l and b io log i ca l me mb ranes . P re l iminary s t ud i es suggestt ha t a t leas t some o f these even t s ma y in f luence t he fun c t i on as we l l a s t he s t ruc tu reof b io log i ca l me mb ranes ( fo r r ev iews , see Ref s . 1 and 3) . I n th i s b r i e f r ev i ew, how -ever , t he ge l t o l i qu id-c rys t a l l i ne phase t r ans i t i on wi l l be emphas i zed . M oreover ,s ince seve ra l exce l l en t r ev i ews on t he rm ot ro p i c even t s i n m ode l m em branes haveapp eared i n t he m id- to - la t e seven t ie s [1 -3 ] , t h i s a rt ic l e s wi ll f ocus p r imar i l y ont h e m o r e r e c e n t l i t e r a tu r e .

Princ ip les o f D SC and DTThe pr inc ip l e o f DSC is com para t i ve ly s imple . The sample and an ' i ne r t ' r e f e r -

e n c e ( a m a t e r i a l t h a t d o e s n o t u n d e r g o a p h a s e t r a n s i t i o n w i t h i n t h e t e m p e r a t u r erange o f i n t e r es t ) a r e s imul t aneous ly hea t ed a t i den t i ca l p r ede t e rmined r a t e s . Foro u r p u r p o s e s t h e s a m p l e w o u l d n o r m a l l y b e a s u s p e n si o n o f li p id o r m e m b r a n ei n w a t e r o r a n a q u e o u s b u f f e r , a n d th e r e f e re n c e c e ll w o u l d c o n t a i n t h e c o r r e s p o n d -ing so lven t a lone . Thus t he t em pera tu res o f t he samples and r e f e renc e cel ls in i t i a ll yi n cr e as e l i n e a r ly w i t h t i m e a n d t h e t e m p e r a t u r e d i f f e re n c e b e t w e e n t h e m is m a in -


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R.N. McElhaney DSC and DTA studies o f lipids and biomembranes 231t a i n e d a t z e r o . I f th e s a m p l e u n d e r g o e s a th e r m a l l y - i n d u c e d e v e n t , t h e c o n t r o ls y s t e m s e n s e s t h e r e s u l t i n g t e m p e r a t u r e d i f f e r e n t i a l b e t w e e n t h e s a m p l e a n d r e f e r -en ce ce ll an d su p p l i e s m o r e o r l e s s h ea t (p o w er ) t o th e sam p le cel l t o h o ld i t st e m p e r a t u r e e q u a l t o t h a t o f t h e r e f e r e n c e . T h e r e c o r d e d p a r a m e t e r i n D S C m e a s u r e -m e n t s i s t h u s t h e e x c e s s s p e c i f i c o r d i f f e r e n t i a l h e a t a s a f u n c t i o n o f t e m p e r a t u r e .I f t h e s a m p l e d o e s n o t u n d e r g o a t h e r m a l e v e n t , t h e n t h e r e c o r d e r s h o u l d t r a c e as t r ai g h t, h o r i z o n t a l b a s e li n e d e n o t i n g a z e r o d i f f e r e n ti a l p o w e r o u t p u t . I f a t h e r m a le v e n t o c c u r s , h o w e v e r , t h e r e c o r d e r p e n is d e f l e c t e d f r o m t h e b a s e l i n e , t h e d i r e c t io no f t h e d e f l e c t i o n d e p e n d i n g o n w h e t h e r t h e e v e n t i s e n d o t h e r m i c ( u p w a r d s ) o re x o t h e r m i c ( d o w n w a r d s ) , a n d t h e m a g n i t u d e o f t h e d e f l e c t i o n d e p e n d i n g o n t h em a g n i t u d e o f th e d i f f e r e n t i a l h e a t i n g r a t e . U p o n c o m p l e t i o n o f th e t h e r m a l e v e n t ,t h e r eco rd e r p en r e tu rn s to th e b ase l in e o r t o a n ew .b asel ine i f a ch an g e in th es p e c if i c h e a t o f t h e s a m p l e h a s o c c u r r e d .

T h e v a r i a ti o n o f e x c e s s s pe c i fi c h e a t w i t h t e m p e r a t u r e f o r a s i m p l e t w o - s t a t e ,f i r s t -o rd e r en d o th e rmic p ro cess , su ch as th e g e l t o l i q u id -c ry s t a l l i n e p h ase t r an s i t i o no f a s in g le, h ig h ly p u re p h o sp h a t id y lc h o l in e , i s i l l u s t r a t ed sch em at i ca l ly in F ig. 1 .F r o m s u c h a D S C t r a c e a n u m b e r o f i m p o r t a n t p a r a m e t e r s c a n b e d i r e c t l y d e t e r -m i n e d . T h e p h a s e t r a n s i t i o n te m p e r a t u r e , u s u a l ly d e n o t e d Tm o r Tc i s t h a t t e m p e r a -t u r e a t w h i c h t h e e x c e s s s p e c if i c h e a t r e a c h e s a m a x i m u m . F o r a s y m m e t r i c a l c ur v e ,Tm r e p r e s e n t s t h e t e m p e r a t u r e a t w h i c h t h e t r a n s it i o n f r o m t h e g el t o l iq u i d-c r y s ta l li n e s t a t e is o n e - h a l f c o m p l e t e . H o w e v e r , f o r a s y m m e t r i c t r a c e s w h i c h a r ec h a r a c t e r is t i c o f c e r t a i n p u r e p h o s p h o l i p i d s a n d m a n y b i o lo g i c al m e m b r a n e s , th eTm d o e s n o t r e p r e s e n t t h e m i d p o i n t o f t h e p h a s e t r a n s it i o n , a n d a T 1 /2 v a l ue m a yb e r e p o r t e d i n s te a d . T h e p e a k a r e a u n d e r t h e D S C t r a c e w h e n c a l i b r a t e d w i t h ak n o w n s t a n d a r d i s a d i re c t m e a s u r e m e n t o f t h e c a lo r i m e t r i c a l ly d e t e r m i n e de n t h a l p y o f t h e t r a n s i t io n , ~r/ca l , u s u a l l y e x p r e s s e d i n k c a l / m o l . T h e a r e a o f t h e

m

~HCAL

TIEMI~RATURE ~CFig. 1. The variation of excess specific heat with temperature during a two-state, e n d o t h e r m i cprocess. The s y m b o l s r e explained in the text.


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232 R.N. McElhaney, DS C and DTA studies of l ipids and biomernbranesp e a k c a n b e d e t e r m i n e d b y p l a n i m e t r y o r b y t h e c u t t i n g a n d w e i g h i n g t e c h n i q u e ;a l t e r n a t i v e l y , t h e c a l o r i m e t e r o u t p u t c a n b e d i g i t i z e d a n d t h e Trn a n d A H c a1 calcu-l a t e d b y a c o m p u t e r . S i n c e a t t h e p h a s e t r a n s i t i o n m i d p o i n t - t e m p e r a t u r e t h ec h a n g e i n f r e e e n e r g y ( A G ) o f t h e s y s t e m is z e r o , t h e e n t r o p y c h a n g e a ss o c i a te dw i t h t h e t r a n s i t i o n c a n b e c a l c u l a t e d d i r e c t l y f r o m t h e e q u a t i o n

a s = A ~ T m

w h e r e A S i s n o r m a l l y e x p r e s s e d in c a l / K m o l .T h e sh a rp n ess o r c o o p era t iv i ty o f t h e g e l t o li q u id -c ry s ta l l i n e p h ase t r an s i t i o n

c a n a l s o b e e v a l u a t e d f r o m t h e D S C t r a c e . T h e s h a r pn e s s o f t h e p h a s e tr a n s i ti o ni s o f t en ex p resse d as th e t em p er a tu re w id th a t h a l f -h e ig h t , A T 1 /2 , o r a s t h e t em p er -a t u r e d i f f e r e n c e b e t w e e n t h e o n s e t o r l o w e r b o u n d a r y o f t h e p h a se t r a n s i ti o n ,Ts, a n d t h e c o m p l e t i o n o r u p p e r b o u n d a r y , TI, o r A T = T - T s. T h e A T 1 /2 v a lu esm a y r a n g e f r o m < 0 . 1 C f o r v e r y p u r e s y n t h e t i c p h o s p h o l i p i d s t o a s m u c h a s 1 0 -1 5 C f o r b i o l o g i c a l m e m b r a n e s . F r o m t h e T m a n d A T 1 / 2 v a lu e s d e t e r m i n e d f o r ap a r t i c u l a r p h a s e t r a n s i t i o n , th e v a n ' t H o f f e n t h a l p y , A H v H , c a n be a p p r o x i m a t e l yd e t e r m i n e d f r o m t h e r e l a t i o n s h i p

A H v H ~-- 4 R T ~ / A T 1 / 2F r o m t h e r a t i o A H v H / A H c a l , t h e c o o p e r a t i v e u n i t ( C U ) ( i n m o l e c u l e s ) c a n b ed e t e r m i n e d [ 2 ] . T h e C U i s a m e a s u r e o f t h e d eg r e e o f i n t e r m o l e c u l a r c o o p e r a t i o nb e t w e e n p h o s p h o l i p i d m o l e c u l e s i n a b i l a y e r ; f o r a c o m p l e t e l y c o o p e r a t i v e , f ir s t-o r d e r p h a s e t r a n s i t i o n o f a n a b s o l u t e l y p u r e s u b s t a n c e , t h i s r a t i o s h o u l d a p p r o a c hi n f i n i ty , w h i le f o r a c o m p l e t e l y n o n - c o o p e r a t i v e p r o c e s s t h is r a t io s h o u l d a p p r o a c hu n i t y . A l t h o u g h t h e a b s o l u t e C U v al ue s d e t e r m i n e d s h o u l d b e r e g a r d e d as te n t a t i v e ,s i nc e t h i s p a r a m e t e r i s m a r k e d l y s e n si ti v e t o t h e p r e s e n c e o f i m p u r i t i e s a n d m a yb e l i m i t e d b y i n s t r u m e n t a l p a r a m e t e r s , c a r e f u l ly d e t e r m i n e d C U v a l ue s c a n b e u s e -fu l i n a ssessing th e p u r i ty o f sy n th e t i c p h o s p h o l ip id s an d in q u an t i t a t in g th e d eg reeo f c o o p e r a t i v i t y o f l i p id p h a s e t r a n s it i o n s.

D i f f e ren t i a l t h e rmal an a ly s i s i s su p er f i c i a l ly r e l a t ed to D S C a l th o u g h th em e a s u r e d p a r a m e t e r is d i f fe r e n t . I n D T A b o t h t h e s a m p l e a n d in e r t r e f e r e n c e a reh e a t e d t o g e t h e r i n a s i ng le b l o c k a t s o m e c o n s t a n t , p r e d e t e r m i n e d r a t e . I n th i sc a s e , h o w e v e r , t h e t e m p e r a t u r e d i f f e re n t i a l, a s o p p o s e d t o t h e d i f fe r e n t i a l h e a tf l o w , is r e c o r d e d a s a f u n c t i o n o f t h e t e m p e r a t u r e o f t h e r e f e r e n c e m a t e r i a l . A s i nD S C , t h e D T A t h e r m o g r a m p r o v i de s ac c u r a t e , c o m p a r a b l e i n f o r m a t i o n o n t h et r a n s i ti o n t e m p e r a t u r e , t h e t e m p e r a t u r e r a n ge o v e r w h i c h t h e tr a n s i ti o n o c c u r s ,a n d o n w h e t h e r t h e t r a n s i t io n i s e n d o t h e r m i c o r e x o t h e r m i c . A l s o, i n p r i n c ip l et h e p e a k a r e a s h o u l d b e r e l a t e d t o t h e e n t h a l p y o f t h e t r a n s it i o n . H o w e v e r , b e c a u s et h e D T A t h e r m o g r a m p e a k a r e a i s a ls o se n s it iv e t o t h e h e a t c a p a c i t y , t h e r m a ld i f fu s iv i ty an d g eo met ry o f t h e samp le , i t i s i n p rac t i ce d i f f i cu l t t o ca l ib ra t e th es y s t e m a n d t h u s t o d e t e r m i n e a c c u r a t e e n t h a l p y v a l ue s . DS C m i n i m i z e s th e d e p e n -d e n c e o f p e a k a r e a u p o n e x t r a n e o u s f a c t o r s s i n c e t h e h e a t d e l i v e r e d t o t h e e n t i r e


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R.N. McElhaney DSC and DTA studies o f lipids and biomembranes 233s a m p l e i s m e a s u r e d d i r e c t l y r a t h e r t h a n a s t h e t e m p e r a t u r e d i f f e r e n t i a l a t s o m ep o i n t w i t h in t h e s a m p l e . A l t h o u g h D T A w a s o f te n e m p l o y e d i n e a r l y s t u d ie sb e c a u s e c o m m e r c i a l D T A i n s t r u m e n t s w e r e m o r e s e n si ti v e a n d s ta b l e t h a n t h e lo w -sen s i t i v i ty D S C u n i t s t h en av a i l ab le , t h e p resen t av a i l ab i l it y o f h ig h - sen s i t i v i ty D SCi n s t r u m e n t s m a k e s D S C th e t e c h n i q u e o f c h oi c e .

I t m u s t b e s t r e ss e d t h a t f o r v a li d t h e r m o d y n a m i c p a r a m e t e r s t o b e o b t a i n e d b yD S C , t h e s y s t e m b e i n g s t u d i e d m u s t b e a t e q u i l i b r i u m t h r o u g h o u t t h e c a l o r i m e t r i cru n . A l th o u g h i t ap p e a r s th a t t h e g e l t o li q u id -c ry s taU in e p h ase t r an s i t i o n o f ce r t a ins in g le , p u r e s y n t h e t i c p h o s p h a t i d y l c h o l i n e s is q u it e r a p i d a n d n o t t h e r e f o r e k i n e t i-c a l ly l im i t e d e v e n a t m o d e r a t e s c a n r a t e s [ 1 5 ] , t h i s m a y w e l l n o t a l w a y s b e t h ec a s e. I t h a s b e e n d e m o n s t r a t e d , f o r e x a m p l e , t h a t t h e ' p r e t r a n s i t i o n ' o f s y n t h e t i cd i sa tu ra t ed p h o s p h a t id y lch o l in e s i s k in e t i ca l ly l im i t ed ev en a t t h e r e l a t iv e ly s lo wscan r a t e s o b ta in ab le w i th h ig h - sen s i t i v i ty D SC in s t ru m en t s [2 0 , 2 1 , L ew is e t a l. ,u n p u b l i s h e d d a t a ] . I t i s t h u s i m p o r t a n t t o e s t a b l is h t h a t t h e t h e r m o d y n a m i c p a r a -m e t e r s b e i n g m e a s u r e d a r e n o t s i g n i f i c a n t l y d e p e n d e n t o n s c a n r a t e , e i t h e r d u e t oa r a t e l im i t a t io n o f t h e p ro cess b e in g s tu d ied , o r t o th e rm al l ag s o r i n s t ru m en ta lr e s p o n s e p r o b l e m s i n t h e c a l o r i m e t e r i ts e l f. I t s h o u l d b e r e m e m b e r e d t h a t a n ys y s t e m a t e q u i l i b r i u m m u s t p r o c e e d i n a n e n d o t h e r m i c d i r e c t i o n w h e n t h e t e m p e r a -t u r e is i n c r ea s e d . H e a t c a n o n l y b e e v o l v ed w i t h i n c r e a s in g t e m p e r a t u r e i f th ep r o c e s s u n d e r s t u d y i s k i n e t i c a ll y r a t h e r t h a n t h e r m o d y n a m i c a l l y l im i t e d .

D S C is p r o b a b l y t h e s in gle m o s t p o w e r f u l t e c h n i q u e f o r t h e r o u ti n e m e a s u r e -men t o f g e l t o l i q u id -c ry s t a l l i n e p h ase t r an s i t i o n s in l i p id b i l ay e r s an d b io lo g ica lm e m b r a n e s . T h e i n s t r u m e n t a t i o n r e q u i r e d is c o m p a r a t i v e l y i n e x p e n s iv e , t h em e a s u r e m e n t s c a n n o r m a l l y b e m a d e r e l a t iv e l y q u i c k l y , a n d t h e in t e r p r e t a t i o n o fth e d a ta o b ta in ed i s f a i r ly s t r a ig h t fo r w a rd . A l so th e cu r ren t h ig h - sen s i t i v i ty D S Ci n s t r u m e n t s r e q u i re o n l y r e l a ti v e ly d i l u t e s u s p e n si o n s o f m a t e r i a l p e r m i t t i n g a c c u r a t ec o n t r o l o f t h e p H a n d i o n i c c o m p o s i t i o n o f th e a q u e o u s p h a s e . U n l i k e s o m e o t h e rt e c h n i q u e s w h i c h h a v e b e e n u t i l iz e d t o s t u d y l i pi d th e r m o t r o p i c b e h a v i o r , D S Ca c c u r a t e l y r e p o r t s t h e e n t i r e c o u r s e o f b r o a d p h a s e t r a n s it i on s i n c l u d in g t h e o n s e tan d co m p le t io n t em p era tu res a s w e l l a s t h e g en era l sh ap e o f t h e p h ase t r an s i t i o n .M o r e o v e r , u n l ik e a n u m b e r o f s p e c t r o s c o p i c t e c h n i q u e s , D S C d o e s n o t r e q u ir et h e i n t r o d u c t i o n o f f o r e ig n p r o b e m o l e c u l e s i n to t h e s y s t e m u n d e r s t u d y ; th i si s a d i s t in c t a d v an tag e s in ce ev id en ce is accu m u la t in g th a t t h ese p ro b es ma y lo ca li zea t p h a se b o u n d a r i e s a n d c a n c a u se m i c r o e n v i r o n m e n t a l p e r t u r b a ti o n s [ 3 , 2 2 - 2 7 ] .F i n a l l y , t h i s t e c h n i q u e i s u n i q u e i n p r o v i d i n g a d i r e c t a n d a c c u r a t e m e a s u r e m e n to f t h e t h e r m o d y n a m i c p a r a m e t e r s o f t h e l i p i d p h a s e t r a n s i t i o n u n d e r i n v e s t i g a t i o n .I t s h o u ld b e r e m e m b e r e d , h o w e v e r , t h a t a t h e r m o d y n a m i c t e c h n i q u e s u c h a s D S Cd o e s n o t p r o v i d e d i r e c t i n f o r m a t i o n a b o u t m o l e c u l a r s t r u c t u r e o r d y n a m i c s . H o w -e v e r , t h e c o m b i n a t i o n o f D S C w i t h a d i r e c t s t r u c t u r a l t e c h n i q u e s u c h a s X - r a yo r n e u t r o n d i f f r a c t i o n , o r w i t h a n a p p r o p r i a t e n o n - p e r t u r b i n g s p e c t r o s c o p i c te c h -n i q u e s u c h a s n u c le a r m a g e n t i c r e s o n a n c e ( N M R ) s p e c t r o s c o p y , p r o v i d e s a v e r yp o w e r f u l a p p r o a c h t o u n d e r st a n d in g t h e r m a l e v e n ts i n m e m b r a n e s y s te m s a t t h emo lecu la r l ev e l .


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234 R.N. McE lhaney, DSC and D TA studies o f lipids and biomembranesD S C a n d D T s t u d ie s o f m o d e l m e m b r a n e sStud ies o f s ing le phosp hol ip ids

W h e n a p u r e p h o s p h o l i p i d i s d is p e r s e d i n w a t e r b y m e c h a n i c a l a g i t a ti o n , m o s tf o r m c l o s e d s p h e r ic a l s t r u c t u r e s c o m p o s e d o f a n u m b e r o f c o n c e n t r i c b i l a y e r s h e e t ss e p a r a t e d b y a q u e o u s s p a c e s . T h e s e m u l t i l a m e l l a r s u s p e n s i o n s o f p h o s p h o l i p i d s( l i p o s o m e s ) u s u a l l y e x h i b i t o n e o r m o r e h i g h l y c o o p e r a t i v e , r e v e r si b le t h e r m a lp h ase t r an s i t i o n s . T h e p h o s p h a t id y l ch o l in es (P C s) co n ta in in g tw o id en t i ca l , ev en -c h a i n s a t u r a t e d f a t t y a c y l g r o u p s h a v e b e e n t h e m o s t i n te n s i v e ly st u d i e d b y D S Can d o th e r t ech n iq u es . A h ig h - sen s i t i v i ty D S C t r ace o f a mu l t i l amel l a r su sp en s io no f 1 ,2 -d ip a lmi to y l p h o sp h a t id y lch o l in e (D P P C ) is p rese n ted in F ig. 2 . T h e ma jo rc h a i n - m e l t in g t r a n s i ti o n i s c h a r a c t e r i z e d b y a s h a r p , s y m m e t r i c , r e la t iv e l y e n e rg e t i c ,f i r s t- o r d e r e n d o t h e r m i c t r a n s i t i o n . A s e c o n d , b r o a d e r e n d o t h e r m i c t r a n s i ti o n o flo w er en th a lp y o ccu r s ab o u t 7 C b e lo w th e g e l t o l i q u id -c ry s t a l l i n e t r an s i t i o n , t h ed i s ta n c e b e t w e e n t h is p r e t r a n s i t i o n t e m p e r a t u r e ( T p ) a n d th e m a i n t r a n s i t io n T i n )ap p ear in g to d ec rease w i th in c reas in g ch a in l en g th , w i th th e Tp ~ Tm fo r a ch a inl e n g t h o f 2 2 c a r b o n s . W e h a v e r e c e n t ly e s t a b l is h e d , h o w e v e r , t h a t t h e o b s e r v e dTp fo r a se r i es o f s imp le d i sa tu ra t ed P C s i s m ark ed ly scan r a t e - d ep en d en t , p ro g res -s iv e ly m o re so fo r PC s w i th ch a in l en g th s ab o v e 1 6 ca rb o n s ; t h u s th e Tp valuesc u r r e n t l y r e p o r t e d f o r t h e s e c o m p o u n d s a r e c o n s i d e r a b l y h i g h e r t h a n t h e t r u eeq u i l ib r iu m v a lu es , p a r t i cu la r ly fo r t h e lo n g er ch a in l en g th P C s . A p lo t o f Tp valuesex t ra p o la t ed to ze ro scan r a t e v e r su s th e ch a in l en g th o f t h e P C s r ev ea l s t h a t t h eA T m - - T p r each es a lo ca l m in im u m a t 1 7 ca rb o n s a n d in c reases s l ig h t ly a t l o n g er

i i | i iT pT22

l i i | i3 2 3 4 3 6 3 8

T p

~ --Tc

T E M P E R A T U R E , ~Fig. 2. A typical high-sensitivity DSC trace of a multilamellar, aqueous suspension of dipalmi-toyl phosphatidylcholine. The pretransition and rdaln transition temperatures are denoted byTp and Tin, respectively. The inset shows the pretransition on expanded excess specific heat andtemperature scales.


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R.N. M cElhaney DS C and DTA studies o f l ipids and biomembranes 235cha in l eng ths (Lewi s e t a l . , unpubl i shed da t a ) . PCs con t a in ing sa tu ra t ed f a t t y acy lcha ins o f le ss t ha n 14 ca rbons do n o t exh ib i t t he p re t r ans i t i on . A l so , t he p re -t r ans i t ion is abo l i shed b y small am oun t s o f cho l es t e ro l , f a t t y acids o r o th e r com -p o u n d s [ 2 ] . In t e r e s ti n g l y t h e T p va lues a t l east o f the m edium -cha in PCs exh ib i t anodd-even a l t e rna t i on whi l e t he T m values fa l l on a s ingle smooth curve [28, Lewise t a l., unpubl i shed da t a ] .

T h e t h e r m o d y n a m i c p a r a m e t e r s a s s o c i a t e d w i t h t h e p r e t r a n s i t i o n a n d m a i nt r a n s it io n o f a s erie s o f s im p l e d i s a t u r a t e d P C s h a v e b e e n d e t e r m i n e d b y a n u m b e rof wo rker s ( see Ref . 2 , Lewi s e t a l . , unp ubl i shed da t a ) . The p re t r ans i t i on is cha rac t e r -i z ed b y A H c a l v a lu e s o f a b o u t 1 . 0 - 1 . 8 k c a l / m o l a n d A H c al d o e s n o t v a r y m a r k e d l ywi th cha in l eng th . Th e CU o f th i s t rans i t i on i s r e l a t ive ly l a rge , t yp i ca l l y o f t he o rde r o fsevera l hu nd red mo lecu l es , com parab l e t o va lues fo r t he m a in t r ans i t i on ; t he magni-t u d e o f t h e C U d o e s n o t a p p e a r t o b e m a r k e d l y c h a in l e n g t h - d e p e n d e n t . T h e m a i nt r ans i t i on i s cha rac t e r i zed by AHca1 va lues w hich i nc rease p rogress ive ly wi th cha inl e n g t h f r o m a b o u t 1 .7 k c a l / m o l f o r d i la u r o y l P C ( D L P C ) t o a b o u t 1 0 .6 k c a l / m o lfor d i s t ea roy l PC (DSPC) ; A S va lues a lso inc rease p rogress ive ly wi th i nc reas ingc h a i n l e n g t h [ 4 ] . A l i n e a r r e l a ti o n s h i p b e t w e e n A H c a1 a n d T m h a s b e e n r e p o r t e dfor t he ser ie s o f even-cha in PCs f rom 12 t o 22 ca rbons [2 ] . How ever , i n ou r hand ssuch a p lo t is no t l i nea r and AHca va lues t en d t o p l a t eau a t t he l onger cha in l eng ths(Lewis e t a l. , unp ubl i she d da t a ) . T he C U va lues r epor t e d fo r t he ma in t r ans i t ionr a n ge f r o m s ev er al h u n d r e d t o o v e r a t h o u s a n d m o l e c u l e s [ 2 , 1 5 ] , t h e se d i f f e r e n c e spro bab ly be ing due l a rge ly t o d i f f e r ence s i n t he r e l a t i ve pur i t i e s o f t he sampless t u d i e d . T h e C U v a l u e o f t h e m a i n t r a n s i ti o n a ls o d o e s n o t a p p e a r t o b e m a r k e d l yc h a i n le n g t h - d e p e n d e n t .

T h e t h e r m o t r o p i c p h a s e b e h a v i o r o f s im p l e d i a c y l PC s c o n t a in i n g t w o i d e n t ic a ln - s a t u r a t e d , m e t h y l i so- o r a n t e i s o - b r a n c h e d sa tu ra t ed , o r c i s - and t rans -m ono-u n s a t u r a t e d a n d cis- a n d t r a n s - c y c l o p r o p y l f a t t y acy l g roups o f seve ra l d i f f e r en tc h a in le n g th s ha s re c e n t l y b e e n s t u d ie d b y D T A [ 2 9 - 3 1 ] . T h e Trn va lues o f t he 18-ca rbo n m em ber s o f t h i s s e rie s o f PCs dec reased i n t he fo l l owing orde r : n - sa tu ra t ed( 5 4 . 8 C ) > m e t h y l i s o - b r a n c h e d ( 3 6 . 5 C ) > m e t h y l a n t e i s o - b r a n c h e d (18.7C) -t r a n s - c y c l o p r o p a n e (16 .3C) > t r a n s - m o n o - u n s a t u r a t e d (12 .9C ) ~>c i s - c y c l o p r o p a n e(--0.5 C ) 2> c i s - m o n o - u n s a t u r a t e d ( - -15 .8C) . The e f f e c t o f t h e i n t r o d u c t i o n o f ac i s - d o u b l e b o n d o r c i s - c y c l o p r o p y l g r o u p o n t h e T m of s imple d i acy l PCs hasr e c e n t ly b e e n c o n f i r m e d b y D S C [ 3 2 ] . T h e c o rr e s p o n d e n c e b e t w e e n t h e m e lt in gp o i n t s o f t h e a n h y d r o u s f r e e f a t t y a c i d s a n d t h e Trn va lues o f t he co r r espon dingfu l l y hydra t ed PC brayer d i spe r s ion i s on ly f a i r . I n con t r as t t o t he d i -n - sa tu ra t edP C s, n o p r e t r a n s it i o n s w e r e o b s e r v e d f o r t h e b r a n c h e d c h a in , u n s a t u r a t e d o r c y c l o -p r o p a n e f a t t y a c i d -c o n t a in i n g P C s; m o r e o v e r , t h e m e t h y l i s o - a n d a n t e is o - b r a n c h e dPCs exh ib i t ed an odd-even a l t e rna t i on i n t he i r T m va lues i nd i ca t i ng t ha t t he i r f a t t yacy l ch a ins a r e t i l t ed i n t he ge l s ta t e . A n inc rease i n cha in l eng th f ro m 16 t o 18ca rbons i nc reases T m by 13 .3C a nd 14 .5C for the n - sa tu ra t ed and me thy l i so-bran che d , b y 16 .6C and 16 .9C for the t r a n s - m o n o - u n s a t u r a t e d a n d c y c l o p r o p y l ,by 19.4C and 19.7C for the c i s - m o n o - u n s a t u r a t e d a n d c y c l o p r o p y l , a n d b y


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236 R. N . M c Elhane y DS C and DT A s t ud i e s o f l ip i ds and b i ome mbrane s2 1 . 7 C f o r t h e m e t h y l a n t e i s o - b r a n c h e d PCs . A l though accura t e AHca1 valuesc o u l d n o t b e o b t a i n e d in t h es e D T A s t u d ie s , i t a p p e a r e d th a t t h e A H c a1 va lues werer e d u c e d i n t h e m e t h y l i s o - b r a n c h e d and pa r t i cu l a r l y i n t he a n t e i s o - b r a n c h e d PCsas com pare d t o t he i r n - sa tu ra t ed ana logs , suggesti ng t he ex i s t ence o f a le ss o rde redge l phase . T h i s conc lu s ion has r ece n t ly been p a r t i a ll y sup po r t ed by t he r e su lt s o fan X- ray d i f f r ac t i on s tudy [33] . Accura t e 2XHca and CU va lues fo r t hese se r i e s o fPCs a re cur r en t l y be ing ob t a ined by h igh- sens it i v it y DSC.

M ul t i lamel l a r suspens ions o f d iph y t a no yl PC a re unusua l i n exh ib i t i ng no ca lo r i -m e t r i ca l l y de t ec t ab l e ge l t o l i qu id-c rys t a l li ne phase t r ans i t i on ove r t he t em pera tu rerange - -120C to + 120C . NM R s tud i es r evea l ed t ha t t h i s unusua l PC undergoes agradua l dec rease i n o r i en t a t i on a l o rd e r and a g radua l i nc rease in t he r a t e s o f mo le -c u l a r m o t i o n o v e r a w i d e t e m p e r a t u r e r an g e . It a p p e ar s th a t t h e p r e s e n c e o fm u l t i p l e m e t h y l b r a n c h e s a l o n g t h e h y d r o c a r b o n c h a i n s m a i n t a i n s t h i s P C i n a ne x p a n d e d a n d o n l y p a r t ia l ly o r d e r e d s ta t e e v en a t v e r y l o w t e m p e r a t u r e s [ 3 4 ] .T h e t h e r m o t r o p i c b e h a v i o r o f m i x e d - a c i d s a t u r a t e d P C s c o n t a i n i n g m y r i s t a t e ,pa lm i t a t e o r s t ea ra t e cha ins has r ecen t ly been i nves t i ga ted by conven t iona l [35]and h igh- sens i t i v i t y DSC [36 ,37] . I n each s tudy t he i n t e r es t i ng obse rva t i on wasm a d e t h a t t h e T p T m a n d A H c a1va lues o f e ach i somer i c pa i r o f PCs were d i f f e r en t ,w i th t he i somer hav ing t he l onger ch a in a t pos i t i on 1 o f sn -g lyce ro l exh ib i t i ngs ign i f i can t l y l ower T p T m a n d A H c a1 va lues . When the f a t t y ac id cha in l eng th i npos i t i on 2 i s con s t an t , i nc reas ing t he cha in l eng th a t pos i t i on 1 wi ll i nc rease T p a n dT i n but wi l l no t a l t e r t he AHca 1 o f t h e m a i n t r a n s it i o n . O n t h e o t h e r h a n d i n c r e a s-ing t he cha in l eng th a t p os i t i on 2 wi th a con s t an t cha in l eng th a t pos i t i on 1 a lsoincreases T p a n d T i n but i n add i t i on , a l so i nc reases AHca1 b y a b o u t 0 . 5 k c a l / m o lfor each CH segme nt adde d , j us t a s p rev ious ly obse rved fo r sa tu ra t ed PCs wi thiden t i ca l acy l cha ins . These r e su l t s can be r a t i ona l i zed by examin ing t he s t ruc tu reof PCs in b i l aye r s in b o t h t he ge l and l iqu id-c rys t a ll i ne s t a t e s . Both NM R [3 8 -4 0 ]a n d n e u t r o n [ 4 1 ] a n d X - r ay [ 4 2 , 4 3 ] d i f f r a c ti o n st u d ie s h a v e s h o w n t h a t i d e n t ic a lacy l cha ins a t t he 1 - an d 2-pos i t ions o f t he g lyce ro l bac kbo ne never the l es s haved i f f e r e n t c o n f o r m a t i o n s . T h e a c y l c h a i n a t p o s i t i o n 2 b e g i n s r o u g h l y p a r a l l e l t ot h e b i l a y e r s u rf a c e b e f o r e b e n d i n g a t C 2 t o o r i e n t t h e h y d r o c a r b o n c h a i n p e r p e n d i-cu l a r t o t he b i l aye r p l ane , whereas t he f a t t y acy l cha in a t po s i t i on 1 i s pe rpe ndicu l a rt o t h e b i l a y e r th r o u g h o u t i ts l e n g t h ; th u s , t h e e f f e c ti v e l e n g t h o f t h e f a t t y a c y lc h a i n a t t h e 1 - p o si ti o n is a b o u t 1 . 8 - 1 . 9 A o r a b o u t 1 .5 C - C b o n d l e n g th s lo n g e rthan t he same f a t t y ac id e s te r i f ied t o pos i t i on 2 . Th ere fo re , s a tu ra t ed m ixed-ac idPCs wi th t he shor t e r cha in ac id a t pos i t i on 1 t end t o min imize t he i n t r i ns i c cha inl e n g th m i s m a t c h d u e t o t h e d i f f e ri n g c o n f o r m a t i o n s o f th e t w o f a t t y a c y l c h a in s ,r e su l t i ng i n a more s t ab l e b i l aye r wi th a h igher T m a n d A H c a1 tha n i s the case forthe cor r esponding pos i t i ona l i somer i n which t he i n t r i ns i c cha in l eng th mi smatchi s i n f ac t accen tua t ed . In t e r es t i ng ly , a l l t he mixed-ac id sa tu ra t ed PCs i somersexa m ined exh ib i t a p re t r ans i t i on exc ep t 1 -my r i s toy l -2 - s t ea roy l PC . Al so , t he p re -t r ans i t i on AHca1 va lues fo r a ll pos i t i ona l i somers exa m ined a re l ower t han t hoser e p o r t e d f o r s a t u r a t e d P C s c o n t a i n i n g i d e n t i c a l f a t t y a c i d s , e x c e p t f o r t h e i s o m e r1- s t ea roy l -2 -myr i s toy l PC which has a cons ide rab ly h igher AHca1.


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R.N. McElhaney, DSC and DTA studies of lipids and biomembranes 237T h e t h e r m o t r o p i c b e h a v i o r o f s e v e r a l m i x e d - a c i d s a t u r a t e d - u n s a t u r a t e d P C s

c o n t a i n i n g o le ic a n d e la id i c a c id s h a v e b e e n e x a m i n e d b y D S C a n d D T A [ 6 , 4 4 - 4 8 ] .For 1 -pa lmi toy l -2 -o l eoy l PC , r ep or t ed T m v a l u e s r a n g e f r o m - - 5 C t o + 3 C a n dAH ca va lues f rom 8 .0 t o 8 .1 k ca l /m ol ; com parab l e va lues fo r t he r eve r sed i some rl - o l e o y l - 2 - p a lm i t o y l PC a r e - - l l C a n d 6 . 7 k c a l / m o l , q u a li t at i v e ly t h e r e s u lt se x p e c t e d f r o m a c o n s i d e r a t i o n o f t h e d i f f e re n t i a l e f f e c ti v e c h a i n l e n g t h e f f e c t j u s td i scussed . I t i s no t ewor thy t ha t t he T m va lues fo r t hese mixed-ac id , s a tu ra t ed-uns a tu ra t ed PCs f a ll we ll be low the average fo r t he cor r e spon ding d i sa tu ra t ed an dd i - u n s a t u r a t e d c o m p o u n d s Tm ~- 4 1 C a n d - - 1 4 t o - - 2 2 C f o r D P PC a n d d i o l e o y lPC (DOPC) , r e spec t i ve ly [49] ) . On the o the r hand , t he ZXHca fo r 1 -pa lmi toy l -2 -o l eo y l PC (8 .0 kca l /mo l ) fa ll s nea r t he ave rage fo r DPPC and DOPC (a bo ut 8 .5 kca l /t oo l and 7 .6 kca l /m ol , r e spec t i ve ly [4 9] ) , whi le t he cor r espon ding va lue fo r 1 -o l eoy l -2 -pa lm i toy l PC (6 .7 kca l /mo l ) i s be low tha t o f DOPC. In con t r a s t , t he T mof 1 -pa lmi toy l -2 -e la idoy l PC (35C) li e s c lose r t o t he T m o f D P P C t h a n t o t h a t o fd i e l a id o y l PC ( D E P C ) ( 9 . 5 - 1 3 C [ 4 9 ] ) ; u n f o r t u n a t e l y , a A H ca1 value for thi s l ip idi s no t ava i l ab l e , nor has t he r eve r se i somer been s tud i ed . For 1 - s t ea roy l -2 -o l eoy lP C , r e p o r t e d T m va lues r ange f rom +3C to 13C and AHca1 va lues f rom 4 .3 t o5 .3 kca i /mol ; a T m value i s n ot available fo r 1-o leoyl -2 -s tearoy l PC, bu t a AHca1o f 6 . 7 k c a l / m o l h a s b e e n r e p o r t e d . A ga in , t h e T m values fa l l wel l belo w the averagefor t he cor r esponding d i sa tu ra t ed and d i -unsa tu ra t ed ana logs Tm -~ 54C forDSPC) and t he AHca1 va lues f al l we l l be low those o f DOPC. In t e r es t ing ly , t he T mfo r 1 - s t ea roy l-2 -e l a idoy l PC (26C) f a lls c lose r t o t ha t o f DEPC than t o DSPC andwel l be low the T m of 1 -pa lmi toy l -2 -e la idoy l PC , desp i t e i ts g r ea t e r ave rage cha inl e n g t h , a ga in i ll u s tr a ti n g t h e e f f e c t o f t h e d i f f e r e n t c h a in c o n f o r m a t i o n s a t p o s i t i o n s1 and 2 ; t he AHca1 fo r 1 - s t ea roy l-2 -e l a idoy l PC (8 .4 k ca l /m ol ) aga in f a ll s be low theaverage for DSPC and DEPC (AHca1 f o r D E P C = 7 . 3 - 1 0 . 0 k c a l /m o l , [ 4 9 ] ) . I tt h u s a p p e a r s t h a t i n t h e a b s e n c e o f i n tr in s ic c h a i n l e n g t h m i s m a t c h c o m p e n s a t i o n ,t he ge l s t a t e s t ab i l i t y o f a t l eas t t he mixed -ac id sa tu ra t ed-c / s -unsa tu ra t ed PCs m orec lose ly r e sembles t ha t o f the co r r espon ding d i -unsa tu ra t ed t ha n t ha t o f t he d isa tu -r a t ed ana logs . A lso t he mixed -ac id sa tu ra t ed-un sa tu ra t ed PCs , l i ke t he d i -unsa tu ra t edspec i es , do no t ex h ib i t p r e t r ans i t i ons .

T h e t h e r m o t r o p i c b e h a v i o r a n d o t h e r p h y s ic a l p r o p e r t ie s o f 1 ,3 -D P PC h a v er e c e n t l y b e e n s t u d ie d b y D S C, N M R a n d m o n o l a y e r t e c h n i q u e s a n d c o m p a r e d t othose o f the na tu ra l l y occur r i ng i somer 1 ,2 -DPPC [50 ] . Th e Tp a n d T m o f t h e1 , 3 -i so m e r w e r e re d u c e d b y a b o u t 1 5 C a n d 3 - 4 C , r e s p e c ti v e l y , c o m p a r e d t o t h e1 2 - i som er whereas t he AHca1 of t he ma in t r ans i t i on was i nc reased by abo ut1 k c a l /m o l . T h e 1 ,3 -D P PC f o r m e d m o r e e x p a n d e d m o n o l a y e r f i lm s a t a ll p r e ss u re sa n d t h e h y d r o c a r b o n i n t e ri o r s o f th e b i l a y e rs w e r e m o r e d i s o r d e r e d th a n f o r 1 ,2 -DPPC. In 1 ,3 -DPPC b i l aye r s bo th f a t t y acy l cha ins were found to beg in pa ra l l e l t othe b i l aye r su r f ace , and a r e b en t pe rpe ndicu l a r t o i t a f t e r t he C-2 ' s egm ent . Thee f f e c ti v e le n g t h s o f b o t h p a l m i t o y l c h a in s a r e th u s s h o r t e n e d , p r e s u m a b l y r e su l ti n gi n r e d u c e d i n t e r m o l e c u l a r i n t e r a c t i o n s w h i c h w o u l d a c c o u n t f o r t h e l o w e r Tp andT m va lues o f t he 1 ,3 - isomer , fo r i ts ex pan de d c ross - sec ti ona i a rea and fo r i t s m ored i s o r d e r e d h y d r o c a r b o n c h a in s . T h e b a sis f o r t h e i n c re a s e d h e a t c o n t e n t o f th e


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238 R.N. McElhaney, DSC and DTA studies of lipids and biomembranesmain t r ans i t ion o f t he 1 ,3 - isomer i s , how ever , unc l ea r .

T h e t h e r m o t r o p i c b e h a v i o r o f t h e c o m p l e t e s e r i e s o f p o s i t i o n a l i s o m e r s o f d i -cis-octadecenoyl P C s h a s b e e n s t u d i e d b y D S C a n d D T A [ 5 1 ] . A p l o t o f T m versusd o u b l e b o n d p o s i t io n f e ll o n a s m o o t h U - s h ap e d c u r v e w i t h a m i n i m u m a t t h ec e n t e r o f t h e h y d r o c a r b o n c h a i n. T m v a lu e s r a n ge d f r o m + 4 5 C a n d + 4 1 C f o r th eA 1 7 a n d A 2 i so m e r s , r e s p e c ti v e l y , t o - -2 1 C f o r t h e A 9 a n d A 1 0 i s o m e r s . T h eAHca1 v a lu e s w e r e a ls o h i g h e r w h e n t h e d o u b l e b o n d w a s lo c a t e d n e a r e i t h e r e n d o ft h e c h a i n ( 9 . 6 k c a l / m o l ) t h a n n e a r t h e c h a i n c e n t e r ( 7 . 5 - 7 . 7 k c a l / m o l ) ; A S v a lu e sd id no t va ry s i gn i f i can t l y wi th double bond pos i t i on . A s imi l a r va r i a t i on i n T mw i t h d o u b l e b o n d l o c a t i o n w a s o b s e r v e d f o r th e m o r e li m i t e d n u m b e r o f 1 - s t ea r o y l-2 , c i s -oc t adecan oyl PC i som ers exam ined . In t e r es t i ng ly , o f t he s ix t een pos i t i ona li s o m e r s o f d i , c i s - o c t a d e c e n o y l P C s t u d i e d , o n l y t h e A 1 5 a n d A 1 6 i s o m e r s ( a n dposs ib ly t he A17 i some r ) exh ib i t ed a p re t r ans i t i on .

T h e e f f e c t o f t h e p r e s e n c e o f a n u m b e r o f a l k a l i n e , a l k a l i n e e a r t h a n d h e a v ymeta l s a l t s on t he phase behav ior o f PCs have been i nves t i ga t ed by DSC and DTA[ 5 2 - 5 5 ] . T h e Tp a n d Tm values , and thei r cor r esp on din g z~t- /ca values , we re

l i tt l e a f f e c t e d b y m o n o v a l e n t c a t i o n s i n c lu d i n g N a a n d K, even a t concen t r a t i onso f 1 M . T h e p r e s e n c e o f 1 M M g2+ inc rea sed Tp, T m and AHcaI fo r t he m a in t r ans i -t i on s l igh t l y , whi l e Ca ~ con cen t r a t i ons o f >1 0 mM resu l t ed i n a subs t an t ia l i n -crease in Tp and a less marked increase in Tm, unt i l t he two t r ans i t i ons merged a tC a ~ c o n c e n t r a t i o n s > 2 5 0 m M ; i n c r ea s in g C a 2+ c o n c e n t r a t i o n a b o v e 1 m M a ls or e d u c e d t h e A H c a1 o f t h e p r e t r a n s i ti o n , b u t i n c r e a s e d t h is v a l u e a t c o n c e n t r a t io n sa b o v e 1 0 r a M . T h e t h e r m o t r o p i c b e h a v io r o f t h e z w i t te r i o n i c P C s a r e g e n e ra l lym uch l es s sens i ti ve t o t he p resence o f ca t ions t han a r e t he nega t i ve ly cha rged phos -phol ip ids .

In mul t i l amel l a r d i spe r s ions o f phosphol ip ids hav ing i den t i ca l f a t t y acy l cha ins ,t h e n a t u r e o f t h e p o l a r h e a d g r o u p is a ls o i m p o r t a n t i n d e te r m i n i n g t h e r m o t r o p i cb e h a v i o r . C a l o r im e t r i c s tu d i e s o f a n u m b e r o f p h o s p h a t i d y l e t h a n o l a m i n e s ( P E s )a t p H v a lu e s n e a r n e u t r a l i ty h a v e b e e n r e p o r t e d [ 5 3 , 5 6 - 6 1 ] . S i m p l e d i s a t u ra t e dP E s d o n o t e x h i b i t p r e t r a n s it io n s a n d t h e m a i n t r a n s i ti o n is m a r k e d l y a s y m m e t r ic ,i n con t r as t t o t he s imple d i sa tu ra t ed PCs . The T m va lues o f t he d i sa tu ra t ed PEsare a lways cons ide rab ly g rea t e r t han fo r t he cor r esponding PCs , t he AT m b e i n ggrea t es t fo r t he d f l auroy l spec ies (~ 30 C ) and progress ive ly dec reas ing as t he cha inl eng th i nc reases ATm fo r DSPE and DSPC ~ 17C) . A l tho ugh the T m f o r D E P E( 3 5 - 3 8 . 5 C ) i s a l s o c o n s i d e r a b l y h i g h e r t h a n f o r D E P C ( 9 . 5 - 1 3 C ) , t h e T m valuesfor d ipa lmi to l eoy l PE ( - -33 .5C) and DOPE ( - -16C) a r e on ly s l i gh t l y h igher t hanf o r d i p a l m i t o l e o y l P C ( -- 3 5 .5 C t o - - 3 6 C ) a n d D O P C ( - -1 4 C t o - - 2 2 C ) . T h eA H c a v a lu e s f o r t h e d i s a tu r a t e d P E s , h o w e v e r , a r e c o m p a r a b l e t o t h o s e o f t h ecor r esponding d i sa tu ra t ed PCs , whi l e t he AHca1 va lues fo r t he d i -unsa tu ra t ed PEsare on ly about ha l f a s l a rge as fo r t he cor r esponding PCs . The CUs fo r t he d i sa tu -r a t ed PEs a r e a l so on ly ab ou t ha l f a s l a rge as t hose o f t he cor r espo nding d i sa tu ra t edPCs [62 ] . The i nc rease d s t ab i l i t y o f d i sa tu ra t ed PE in t he gel s t a t e appear s to bed u e t o b o t h t h e s m a ll er siz e o f t h e P E a s o p p o s e d t o t h e P C h e a d g r o u p [ 6 2 ] , a n dt o t h e a b il it y o f t h e p r o t o n a t e d a m i n o g r o u p o f P E t o h y d r o g e n b o n d t o t h e p ro -


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R.N. McElhaney, DSC and DTA studies o[ lipids and biomembranes 239t o n a t e d a m i n o a n d n e g a t i v e l y c h a r g e d p h o s p h a t e g r o u p s o f a d j a c e n t P E m o l e c u l e s[ 6 1 , 6 3 ] . T h e o b s e rv a t io n t h a t t h e d e p r o t o n a t i o n o f t h e D P PE a m i n o g ro u p s , b yinc reasing t he pH to 12 , dec reases Trn f r o m 6 3 C t o 4 1 C ( t h e T m f o r D P P C )s u p p o r t s t h e l a t te r v i e w [ 6 4 ] . A t p H < 8 s y n t h e t i c a n d n a t u r a l l y o c c u r r i n g u n s a t u -r a t ed PEs undergo a sha rp , b i laye r - t o -hexag ona l (H l l ) phase t r ans i t i on a t a t em pera -t u r e a b o v e [ 5 8 ] , o r s o m e t i m e s w i t h in [ 6 1 ] t h e g e l t o l iq u i d -c r y s ta l li n e t e m p e r a t u r er a ng e o f th e s e P E s . T h e e n t h a l p y o f t h i s t r an s i ti o n is l o w a n d i t c a n n o t a l w a y s b ed e t e c t e d b y c o n v e n t i o n a l D S C.

C a l o r im e t r ic s t u d i es o f t h e n e g a t iv e l y ch a r g e d p h o s p h o l i p id p h o s p h a t i d y l g l y c e r o l( P G ) h a v e b e e n c a r ri e d o u t a n d t h e e f f e c t o f v a r ia t io n s in f a t t y a c y l g r o u p c o m p o s i -t i o n , p H a n d i o n ic e n v i ro n m e n t o n t h e t h e r m o t r o p i c b eh a v i or o f t h is c o m p o u n dh a v e b e e n i n v e s ti g a te d [ 6 6 - 7 0 ] . W h e n d i sp e r se d in w a t e r a t n e u t r a l p H , d i s a tu r a t e dPGs have Tin, AHca1 a n d A S v a l u e s a l m o s t i d e n t i c a l t o t h e c o r r e s p o n d i n g P C s ;m oreo ver , t hese d i sa tu ra t ed PGs a l so ex h ib i t a p re t r ans i t i on w hich i s l ack ing i n t hed i -unsa tu ra t ed ana log DOPG. Al so l i ke t he cor r esponding PCs , PG the rmot rop i cb e h a v i o r is o n l y s l ig h t ly a f f e c t e d b y t h e n a t u r e a n d c o n c e n t r a t i o n o f m o n o v a l e n tca t i ons p resen t in t he aque ous phase . H ow ever , i n t he p resen ce o f Ca ~ o r M gz~ PGf o r m s a h i g h - m e l t in g , m e t a s t a b l e c o m p l e x i n c o n t r a s t t o t h e P C s. T h e p h a se b e h a v -i o r o f t h e P G s is a ls o m a r k e d l y d e p e n d e n t o n t h e p H a n d t h u s t h e c h a r g e o f t h ep o l a r h e a d g r o u p . A t p H 2 , t h e T m of DPPG inc reases f rom 41C (pH 7) t o 61C ,which has been asc r ibed t o a r ed uc t i o n o f t he r epu ls ive fo rces be tw een ad j acen tn e g a t i v e ly c h a r g e d p h o s p h a t e g r o u p s . I n t e r e s ti n g l y , p a rt ia l p r o t o n a t i o n o f t h ep h o s p h a t e o f P C h a s a s im i la r e f f e c t o n i ts t h e r m o t r o p i c b e h a v i o r d e s p it e t h e p r e -s e n c e o f a p o s i ti v e l y c h a r g e d q u a t e r n a r y a m i n e g r o u p . T h e s i m i l a ri ty in t h e t h e r m o -t rop i c beha v ior o f PG and PC has l ed t o t he sugges t i on t ha t PC behaves i n e f f e c t a sa nega t i ve ly cha rged r a the r t han a zwi t t e r i on i c l i p id a t neu t r a l pH, i t s behav iorbe ing l i t tl e a f f e c t ed by t he p resence o f i ts pos i t i ve ly cha rged bu t sh i e lded aminem o i e t y [ 6 4 ] .

The phase t r ans i t ion cha rac t e r i s ti c s o f d iph osph a t idy lg lyce ro l s ( ca rd io li p ins ,C L ) a n d t h e i r d e p e n d e n c e o n i o n ic e n v i r o n m e n t h a v e r e c e n t l y b e e n s t u d ie d b y D S C[ 7 1 ] . I n g e n e r al , t h e i r t h e r m o t r o p i c b e h a v io r Tm and AHca va lues pe r ch a in) isve ry s imi la r t o t ha t o f t he cor r espo ndin g PGs and PCs, and a p re t r ans i t i on oc cur r i ngs o m e 5 - 1 5 C b e l o w th e m a i n t r a n s i ti o n a n d h a v in g a n e n t h a l p y a b o u t 1 0 o f t h a to f t he ma in t r ans i t i on i s a l so obse rved . The Tm o f m e t a l s a lts o f D P C L w as f o u n dt o e x h i b i t a b i p h a si c d e p e n d e n c e o n t h e u n h y d r a t e d i o n i c r a di u s, w i t h t h e T m be inghighe s t for DPCL-Ca 2+ and decre as ing for ions o f e i ther sm al ler or larger radi i .

T h e p h a se b e h a v i o r o f n a t u r a l a n d s y n t h e t i c d i s a t u r a t e d p h o s p h a t i d y l s e ri n e s( P S s ) , a n d h o w t h i s b e h a v i o r i s i n f l u e n c e d b y p H a n d c a t i o n c o n c e n t r a t i o n , h a sbeen i nves t i ga t ed by severa l g roups u t i l i z i ng DSC and o the r phys i ca l me thods[ 6 6 , 6 8 , 7 2 ] . A t n e u t r a l p H , w h e r e t h e P S m o l e c u l e b e a rs tw o n e g a t iv e a n d o n epos i t ive cha rge , t he d i sa tu ra t ed PSs exh ib i t T rn va lues cons id e rab ly h igher t hano b s e r v e d f o r t h e c o r r e s p o n d i n g P C s , P G s o r C L s , b u t l o w e r t h a n f o r t h e c o r r e s p o n d -ing PEs. Th e /XHca values are s imi lar to those o f the ot he r ph osp hol ip ids discusseda n d a p r e t r a n s i t io n d o e s n o t o c c u r . A t l o w p H w h e r e th e f r e e c a r b o x y l g r o u p o f


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240 R . N . M c E l h a n e y D S C a n d D T A s t u d ie s o f l ip i ds a n d b i o r n e m b r a n e sD P P S i s p r o t o n a t e d a n d t h e m o l e c u l e i s t h u s z w i t t e r i o n i c , t h e T m i s e l eva t ed t o7 2 C a n d t h e A H c a t o a b o u t 9 k c a l / m o l ; a t h ig h e r p H w h e r e t h e c a r b o x y l g r o u pb e c o m e s f u l l y i o n i z e d , T m f all s t o 55C an d A Hca to ab ou t 3 kca l /m ol . Whi le t heincrease in T m a n d A H c a1 u p o n p r o t o n a t i o n o f t h e c a r b o x y l g r o u p o f P S c a n b eq u a l i ta t iv e l y e x p l a i n e d b y a d i m i n u t i o n o f t h e r e pu l si v e fo r c e s a c ti n g b e t w e e n t h enega t i ve ly cha rged head group s a t neu t r a l pH, t he r e l a t ive ly h igh T r n of t h i s phos -phol ip id suggests t ha t , a s fo r PEs , i n t e rmolecu l a r hydro gen bond ing mak es a ma jorco n t r i b u t i o n t o b i l aye r s t ab i l i t y . A l thou gh the phase behav ior o f PS is r a the r i nsen-s it iv e t o t h e n a t u r e a n d c o n c e n t r a t i o n o f t h e m o n o v a l e n t c a ti o n s p r e s e n t , Ca2~ an dto a lesser extent Mg2. fo rm t i gh t l y packed , wa t e r - exc lud ing , quas i - c rys t a l l i nec o m p l e x e s w i t h P S w h i c h d o n o t m e l t b e l o w 1 00 C .

A l t h o u g h p r e s e n t i n b i o l o g i c a l m e m b r a n e s i n o n l y t r a c e a m o u n t s , t h e t h e r m o -t r o p i c p h a s e b e h a v i o r o f p h o s p h a t i d i c a c id ( P A ) a s a f u n c t i o n o f p H h a s b e e ns t u d ie d e x t e n s i v e l y b y D S C a n d o t h e r te c h n i q u e s [ 6 6 , 6 8 , 7 3 - 7 5 ] , s in c e t h is p h o s-p h o l i p i d i s u n i q u e i n h a v i n g t w o d i s s o c i a b l e p r o t o n s o n i t s p h o s p h a t e g r o u p . A tneu t r a l pH wh ere t he d i sa tu ra t ed PAs have a ne t nega t i ve cha rge o f one , t hesec o m p o u n d s e x h i b i t T m v a lu e s w h i c h a r e e v e n h i g h e r t h a n t h o s e o f t h e c o r r e s p o n d -i n g P E s a l t h o u g h t h e A H c a1 va lues ar e simil ar t o t hose o f PE an d o f t he o the rp h o s p h o l i p i d s . T h e h i g h T m of PA, desp i t e i t s nega t i ve cha rge a t pH 7 , can a lso bee x p l a i n e d b y i n t e r m o l e c u l a r h y d r o g e n b o n d i n g b e t w e e n a d j a c e n t m o l e c u l e s s i n c ea t t h i s pH one d i s soci ab le p r o to n i s s t il l p r esen t . As t he pH i s low ered , t he T mac tua l l y i nc reases s l i gh t l y , r each ing a l oca l maximum near pH 4 ; a t t h i s pH, t hef'trs t p r o t o n i s o n l y 5 0 d i s so c i a te d a n d h y d r o g e n - b o n d i n g o p p o r t u n i t i e s a r em a x i m i z e d . A f u r t h e r d e c r ea s e i n p H r e s u l ti n g i n c o m p l e t e p r o t o n a t i o n o f P A ,resul t s in a decrease in T m o f a b o u t 9 C a n d a l so a m a r k e d r e d u c t i o n in ~ H c a 1v a lu e s . S i m i la r ly , w h e n t h e s e c o n d p h o s p h a t e p r o t o n is c o m p l e t e l y d is s o c ia t ed a tpH 11 , t he T m also dec reases dras t ica l ly , by ab ou t 25C, as doe s the ZkHcal,p r e s u m -a b l y d u e t o t h e r e p u l s i o n o f t h e d o u b l y n e g a t i v e l y c h a r g e d p h o s p h a t e h e a d g r o u p sa n d t h e i r i n a b i l i t y t o f o r m h y d r o g e n b o n d s w i t h a d j a c e n t P A m o l e c u l e s . I n t e r -es t i ng ly , a t h igh pHs d i sa tu ra t ed PAs exh ib i t a p re t r ans i t i on as do t he d i sa tu ra t edPCs and PGs a t neu t r a l pHs . Recen t ev idence i nd i ca t es t ha t t he p re t r ans i t i one x h i b i t e d b y t h e se p h o s p h o l i p i d s r e su l ts f r o m a r e a r r a n g e m e n t o f t h e l a t ti c e p ac k -i n g o f t h e h y d r o c a r b o n c h a i n s f r o m a p l a n a r b i la y e r , i n w h i c h th e c h a i n s a re t il te dwi th r e spec t t o t he b i l aye r p l ane , t o a r i pp l ed b i l aye r in w hich t he b i l aye r p l ane isl o c a l l y e v e r y w h e r e c u r v e d a n d t h e h y d r o c a r b o n c h a i n t i l t i s a l t e r e d o r a b o l i s h e d[ 2 8 , L e w is e t a l ., u n p u b l i s h e d d a t a ] . I n o r d e r t o s t u d y t h e t h e r m o t r o p i c b e h a v io ro f t h e se p h o s p h o l i p i d s a t e x t r e m e s o f p H t h e d i e t h e r r a t h e r t h a n t h e d i e s te r f o r m swere u t i l i zed , s i nce t he fo rmer a r e r e s i s t an t t o hydro lys i s o f t he f a t t y acy l cha insa t ac id i c and basi c pHs . The d i e the r pho sphol ip ids genera l l y have s imi la r bu tno t i den t i ca l T m a n d A H c a v a lu e s t o t h e c o r r e s p o n d i n g d i e s te r c o m p o u n d s[56 , 7 3 -7 5 ] . The r eader is r e f e r r ed t o an exce l l en t r ecen t rev i ew for a fu l le r d iscus -s io n o f t h e e f f e c t o f in t e r m o l e c u l a r h y d r o g e n b o n d i n g o n t h e p r o p e r t i e s o f l ip i d si n m o d e l a n d b i o l o g ic a l m e m b r a n e s [ 7 6 ] .The t he r m ot ro p i c beha v ior o f small s ing le b i l aye r vesic le s o f a pa r t i cu l a r phos -


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R... M cElhaney DSC and DT A s tudies o f l ipids and biomembranes 241phol ip id d i f f e r s s i gn i f i can t l y f rom tha t o f t he same l i p id i n a mul t i l amel l a r d i spe r -s ion . The phase behav ior o f small p hosp hol ip id ves ic le s p repa red by u l tr a son i ci r r ad i a t i on has b een s tud i ed b y DSC in con junc t i on wi tl 't o th e r phys i ca l t echn iques[7 7 -8 1 ] . I t was in i t i a l ly r epo r t ed t ha t t he ca lo r im e t r i c cha rac t e r i s ti c s o f son i ca t edand unso n i ca t ed l ip id d i spe r s ions were i nd i s ti ngu i shab l e by con ven t iona l DSC, andl a t e r t ha t son i ca t ed d i sa tu ra t ed PC ves i c l e s exh ib i t ed a l e s s en tha lp i c p re t r ans i t i ona n d a d o w n w a r d - s h i f t e d , s o m e w h a t b r o a d e n e d m a i n p h a s e tr a n s i ti o n w i th o n l y as l igh t l y r educed AHca [80 ] . Ho we ver , s t ud i es em ploy ing h igh- sens i ti v i ty DSCdem ons t r a t ed t ha t in f r e sh ly son i ca t ed vesi cl es o f d i sa tu ra t ed PCs t he p re t r ans i t i oni s absen t and t ha t t he AHca1 of t he ma in t r an s i t i on is d r as t ica l l y r educed [78 ,79 ,81 ] . The b road er t h e rm ot ro p i c phase t r ans i t ion exh ib i t ed by t he son i ca t ed ves ic le sw a s s h o w n t o r e s u l t m o s t l y f r o m t h e r e d u c e d A H c a 1 r a t h e r t h a n f r o m a m a r k e dd e c r e a s e i n t h e C U [ 8 1 ] . T h e r e s u l t s o b t a i n e d w i t h c o n v e n t i o n a l D S C w e r e p r o b -ab ly due t o a t ime-dependen t aggrega t i on o f un i l amel l a r ves i c l e s i n to l a rge r mul t i -l amel l ar vesi cle s p ro m ote d by t he h igh li p id con cen t r a t i ons r equ i r ed . A l so ves ic l efus ion was shown to be enhan ced b y cyc l i ng t h rou gh the l i p id phase tr ans i t i on o rby t he f r eez ing o f t he aque ous ph ase , a s we ll a s by an i nc reased med ium osm ola r i t y ,t he add i t i on o f f a t t y ac ids , o r t he p resence o f d iva l en t ca t i ons such as ca lc ium[ 7 9 , 8 0 ] . A n u m b e r o f o t h e r p h y s i c a l t e c h n i q u e s h a v e i n d i c a t e d t h a t t h e s m a llr ad ius o f curv a ture o f so n i ca t ed pho spho l ip id vesic le s l eads t o a l e ss o rd e red o r i en-t a ti o n a n d t o a g r e a te r f r e e d o m o f m o t i o n o f th e p h o s p h o l i p id h y d r o c a r b o n c h ai nst h a n a re f o u n d i n l a rg e r v e si cl es [ I - 3 ] .S t u d i e s o f p h o s p h o l i p i d m i x t u r e s

A l t h o u g h s tu d i e s o f t h e t h e r m o t r o p i c p h a se b e h a v i o r o f s in g l e - c o m p o n e n t m u l ti -l amel l a r p hosp hol ip id vesic le s a r e necessa ry and va luab l e , t hese sys t ems a re no tr ea l i s t i c mode l s fo r b io log i ca l membranes which normal ly con t a in a t l eas t s eve ra ld i f f e r en t t y pes o f phospho l ip ids and a va r i e ty o f f a t t y acy l cha ins. As a f ir s t s t ept o w a r d u n d e r s t a n d i n g t h e i n t e r a c ti o n s o f b o t h t h e p o l a r a n d ap o l a r p o r t i o n s o fd i f f e r en t l ip ids p resen t in , m ix tures , D SC s tud ies o f va rious b ina ry ph osph ol ip idsys t ems have been ca r r i ed ou t . Phase d i agrams can be con s t ruc t ed by spec i fy ingthe onse t and c om ple t i o n t em pera tu res fo r t he phase t r ans i t ion o f a ser ie s o f mix-t u r e s a n d b y a n i n s p e c t io n o f t h e s h a p es o f t h e c a l o r im e t r i c tr a c e s. A c o m p a r i s o nof t he obse rved t r ans i t i on curves wi th t he t heo re t i ca l curves supp or t s a l it e r a l i n t e r -p r e t a t i o n o f th e p h a s e d ia g r am s o b t a i n e d b y D S C [ 2 ] .

T h e t h e r m o t r o p i c b e h a v i o r o f m i x t u r e s o f t w o P C s d i f f e r i n g i n t h e n a t u r e o fthe i r f a t t y acy l con s t i t uen t s has been i nves t iga t ed . D i sa tu ra t ed PCs d i f f e r i ng byo n l y t w o c a r b o n s i n t h e l e n g t h o f t h e i r h y d r o c a r b o n c h a i n s e x h i b i t a l m o s t i d e a lbehav ior i n b ina ry phosphol ip id -wa te r d i spe r s ions ( see F ig . 3 ) . A l though a d i f f e r -e n c e i n c h a i n l e n g t h o f f o u r c a r b o n s p r e s e n ts a s y s t e m c o n s i d e r a b l y r e m o v e d f r o midea l i t y , i so the rmal me l t i ng o f t he sh or t e r - cha in PC is no t obse rved an d a s i gn i fi can tdegree o f l a t e r a l phase sepa ra t i on d oes n o t occ ur (bu t s ee Ref . 83) . A d i f f e r ence i nh y d r o c a r b o n c h a i n l e n g t h o f s i x c a r b o n s r e s u l t s i n m o n o t e c t i c b e h a v i o r , w i t h t h ec h a i n -m e l t in g o n s e t t e m p e r a t u r e r e m a i n i n g c o n s t a n t o v e r m o s t o f t h e c o n c e n t r a t i o n


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242 R.N. McElhaney DSC and DTA studies o f lipids and biomernbranes

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X D P PFig. 3. (A) - - , observed calorimetric transition curves for two mixtures of DMPC andDPPC. - - - - , transition curves calculated on the basis of the ideal phase diagram in B. (B)- - , phase diagram constructed from initiation and completion temperatures derived fromthe observed transition curves. - - - - , the ideal phase diagram. (Reproduced from Mabreyand Stur tevant (1978) [2] with permission from Plenum Press).


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R .N . M cE l h an ey D SC and D TA st u d i e s o f i p i d s a n d b i omemb r a n e s 243r a n ge ; a r e g io n o f p r o n o u n c e d la te r al p h a se s e p a ra t io n i s ev id e n t f r o m t h e DS Ct ra c es s e e F ig . 4 ) [ 4 6 ,5 2 ,5 7 ,8 2 ,8 3 ] . M o n o t e c t i c b eh a v io r i s a l s o o b s e r v e d f o r t h esystems DOPC plus DMPC, DPPC or DSPC, ind icat ing that d i -c i s -mono-unsaturatedPCs and d isa turated PC o f wha tever chain length are large ly imm isc ib le in the ge ls ta te [ 8 2 ] . I n c o n t r a s t , DE P C a n d DM P C a r e n e a r ly p e r f e c t ly m i s c ib l e in a l l p r o p o r -

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Fig. 4. (A) - , d as h e d b as e l in e s : ob s e r ve d c a l or i m e t r i c t ran s i t ion curves for D L P C - D S P Cm i x t ~ e s of mole f ract i on DSPC (X DSPC) = 0 .191 and 0 .819. - - - - , sol id base l ine: observedcal orim etri c cu rve for X DSPC - '- 0.4 98. (B) P h as e d i agr am cons t ruc ted f rom the ca lo r im et r i ct r an s i t i on curves . (Repr oduce d f r om Mabrey and Sturtevant (1978) [2 ] w i t h p e r m i s s i on f r o mPlen um Press .


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244 R . N . M c E l h a n e y D S C a n d D T A s t u d i e s o f li p id s a n d b i o m e r n b r an e st i o n s , w h i l e D E P C an d D P P C m ix l e s s i d ea l ly an d ex h ib i t a so l id - so l id imm isc ib i l i t yg a p a t m o l e f r a c t i o n s o f D P PC f r o m a b o u t 0 . 3 0 t o 0. 5 5 [ 8 3 ] . I n ge n e ra l , t h esmal l e r t h e d i f f e ren ce in th e T m v a lu es o f t h ese s imp le d i acy l P C s , t h e m o re n ea r lyi d ea l i s t h e i r m i x i n g b e h a v i o r . P ha s e d i a g r a m s f o r t h e b i n a r y s y s t e m s 1 - p a l m i t o y l-2 -o leo y l P C : D P P C an d l - s t ea ro y l -2 -o leo y l P C : D P P C h av e a l so b een co n s t ru c te du s in g D SC [ 4 7 ] . T h e P O P C - D P P C s y s t e m e x h ib i te d b e h a v i o r th a t w a s f a r f r o mi d e a l b u t t h e r e w a s l i t t l e g e l - s t a t e i m m i s c i b i l i t y a t a n y c o m p o s i t i o n , w h e r e a s t h eS O P C - D P P C s y s t e m e x h i b i t e d a p p r e c i a b l e ge l s t a t e im m i s c i b i fi t y p a r t i c u l a r ly a tD P PC c o n c e n t r a t i o n s o f le ss t h a n 5 0 m o I . O f t h e s e l a t t e r t w o b i n a r y s y s te m s , t h em o r e n e a r l y i d e a l b e h a v i o r w a s e x h i b i t e d b y t h e P C p a i r w i t h t h e l a r g e st d i ff e r e n c ein T m v a lu es , i n d ica t in g th a t t h e m isc ib i l it y o f PC s in b f l ay e r s m ay b e in f lu en ced b ym o r e s u b t l e s t r u c t u ra l v a r i a t io n s a s w e l l a s b y d i f f e re n c e s in c h a i n - m e l t in g t e m p e r a -tu res .

A n u m b e r o f c a l o ri m e t r i c s tu d i e s o f b i n a r y m i x t u r e s o f t w o d i f f e r e n t p h o s p h o -l ip id s co n ta in in g e i th e r s im i l a r o r d i s s im i l a r f a t ty ac id s h av e b ee n d o n e . F o r b in a rym i x t u r e s o f d i s a t u r a t e d P G - N a a n d P C s w i t h i d e n t i c a l a c y l c ha i ns n e a r l y i d e a l m i x -in g w as o b se rv ed . M ix in g s im p le P G s an d P C s w i th in c reas in g ly la rg e d i f f e ren ces in th ec h a in l e n g th s o f t h e i r s a t u r a t e d f a t t y a c y l g r o u p s , o r m i x i n g d i s a t u r a t e d w i t h d i-u n s a t u r a t e d l ip i d s , p r o d u c e d i n c re a s in g l y n o n -i d e a l b e h a v i o r b u t n o m o r e s o t h a nf o r a b i n a r y m i x t u r e o f P C s c o n t a i ni n ~ t h e s a m e f a t t y a c id s . T h e s e re s u lt s i n d i c a tea h i g h d e g r e e o f m i s c i b i li t y o f t h e P G a n d P C h e a d g r o u p s e i t h e r in t h e p r e s e n c e orab sen c e o f C a 2+. In t e res t in g ly , sma l l am o u n t s o f P C w ere fo u n d to ab o l i sh th ef o r m a t i o n o f h i g h - m e l t in g m e t a s t a b l e P G - C a 2+ o r P G - M g 2+ c o m p l e x e s o b s e r v e dw i t h t h e p u r e P G s a l o n e [ 6 9 ] .

S e v er a l g r o u ps h a v e s t u d i e d b i n a r y m i x t u r e s o f P C a n d P E [ 5 2 , 8 4 , 8 5 ] . M i x t u r e so f t h e s e t w o p h o s p h o l i p i d s h a v i n g i d e n t ic a l f a t t y a c y l g r o u p s , s u c h a s D M P C -D M P E , ex h ib i t ed q u i t e n o n - id ea l b eh av io r . A l th o u g h th ese l i p id s seem ed misc ib l ein th e l i q u id -c ry s ta l l i n e p h ase , t h e so l id u s cu rv es o f t h ese p h ase d i ag rams sh o w eda m i n i m u m a t a b o u t 2 0 m o l , i n d i c at i n g ge l s t a t e i m m i s c i b i li t y i n t hi s c o m p o s i t i o nr a n g e. S o m e g e l - st a te i m m i s c i b il i ty w a s a l so o b s e r v e d i n D M P E - D P P C a n d D M P E -D S P C m i x t u r e s ; h o w e v e r , t h o s e m i x t u r e s , p a r t i c u l a r l y t h e l a t t e r o n e , e x h i b i t e dm o r e n e a r l y i d e a l b e h a v i o r t h a n d i d t h e D M P E - D M P C s y s t e m . S i n c e t h e A T mv a lu e s a ls o d e c r e a s e d a s t h e c h a i n le n g t h o f t h e P C c o m p o n e n t o f t h e D M P E - P Cm i x t u r e s i n c r e as e d f r o m 1 4 - 1 8 c a r b o n a t o m s , t h e s e re s u lt s s u g ge s t t h a t t h e r e l a ti v ec h a i n -m e l t i n g t e m p e r a t u r e s r a t h e r t h a n t h e a b s o l u t e r e l a ti v e ch a i n l e n g th s a r e o fp r i m a r y i m p o r t a n c e i n d e t e r m i n i n g P E - P C m i s c i b i li t y . N e v e r t h e le s s , e v e n w h e nt h e T m v a lu e s o f b o t h c o m p o n e n t s a r e c lo s e ly m a t c h e d , P E - P C m i x t u r e s d i sp la yc o n s id e r a bl y m o r e n o n - id e a l it y t h a n c o m p a r a b l e P C - P C o r P C - P G m i x t u r e s ,i n d ic a t i ve o f s o m e p o l a r h e a d g r o u p i m m i s c i b il i ty .

T h e p h a s e b e h a v i o r o f m i x t u r e s o f D P PC w i t h b o v i n e b r a i n P S [ 6 6 , 8 6 ] h a sb e e n s t u d i e d b y D S C in c o n j u n c t i o n w i t h o t h e r t e c h n i q u e s . C o m p l e x a n d s o m e -w h a t n o n - id e a l p h a s e m i x i n g o c c u r s , b u t m o r e q u a n t i t a t i v e c o n c l u s i on s a b o u t t h emisc ib i l i t y o f p o la r h ead g ro u p s a re d i f f i cu l t s ince th e n a tu ra l P S i s a l r ead y a


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R.N. McElhaney DSC and DTA s tudies o f l ipids and biomembranes 245m i x t u r e o f m o l e c u l e s d i ff e r in ~ i n t h e l e n z t h a n d d e g r ee o f u n s a t u r a t io n o f th e i rf a t t y acy l cha ins . Ca lor ime t r ic st ud i es o f egg PC-ce rebros ide [87] and sy n the t i cPC-ganglios ide [48 ,88] mix tures have a lso been pub l i shed . Fo r a sum ma ry ofc a l o r im e t r i c a n d o t h e r s tu d i e s o f t h e t h e r m o t r o p i c p h a s e b e h a v i o r o f n a t u r a l a n ds y n t h e t i c s p h i n g o m y e l in s (S M s) a n d o f t h e i r m i x i n g p r o p e r t i e s w i th c h o l e s te r o land pho sphol ip ids , t he r eader i s r e f e r r ed t o a r ecen t com prehens ive rev i ew [891 .

R e c e n t t h e o r e t ic a l a n a l y s e s o f t h e p h a se d i ag r am s o f b i n a r y m i x t u r e s o f d is a tu -r a t e d P C s o f d i f f e r e n t c h a in l e n g th s a n d o f P C - P E m i x t u r e s , i n d i c at e s t h a t m i c ro -scopic mix ing m ay be s igni f icant ly non - ideal in the l iquid crys ta l l ine as wel l ast h e g e l p h a s e s, e v e n th o u g h n o m a c r o s c o p i c la t e ra l p h a s e s e p a r a ti o n s o c c u r [ 9 0 , 9 1 ] .Thus , mic roscop i c c lus t e r s o f l i ke l i p id molecu l es may ex i s t i n t he ge l and l i qu id-c rys t a l l i ne phases a l t hough genera l l y more r andom mix ing wi l l occur i n t he l i qu id-c rys t a ll i ne phase . In t e r es t i ng ly , the p rop ens i t y fo r t he se l f- a s soc ia t ion o f twodi f f e r en t l i qu ids i n t he ge l and l i qu id-c rys t a ll i ne s t a te s is no t a lways r e l a t ed , ne i t he ris th i s p r o p e n s i t y a l w a ys c o r r e l a t e d w i t h t h e m a c r o s c o p i c p h a se b e h a v i o r o f t h a tp a r t i c u l a r s y s t e m . S o m e i n d i r e c t n o n - c a l o r i m e t r i c e v i d e n c e f o r p h o s p h o l i p i dclus ter ing in the l iquid-crys ta l l ine s ta te i s available ( see Ref . 7 6 fo r a review) .

Because a l t e r a t i ons i n pH and i on i c concen t r a t i on can cause s i gn i f i can t changesin t he T m par t i cu l a r l y o f ac id i c phosphol ip ids , i so the rmal phase t r ans i t i ons can bei n d u c e d i n p u r e p h o s p h o l i p i d s b y v a r i a ti o n i n p H o r d iv a le n t c a t i o n c o n c e n t r a t io n .S imi l a rl y an i so the rmal l a t e ra l phase sepa ra t i on i n l iqu id-c rys t a l li ne mix tures o f azwi t ter ionic l ip id such as PC and an acidic l ip id such as PG, PS or PA, can a l so beindu ced by changes i n pH or by t he add i t i on o f Ca2+; i n t h is case domains o f ge l o rquas i - c rys ta l l ine ac id i c phospho l ip ids can p resu m ably be fo rm ed , l eav ing t he neu t r a lphosph01ip id i n sepa ra t e f l u id do mains . Ca lor ime t r ic an d o th e r ev idence fo r Ca 2*-i n du c e d p ha se se p ar at io n s in P S - P C [ 6 6 , 6 8 , 9 2 - 9 4 ] , P A - P C [ 6 6 , 9 5 - 9 7 ] a n dP G - P C [ 6 7 - 6 9 ] m i x t u r e s h a s b e e n p u b li sh e d . A p p a r e n t l y M g~* is no t as e ffec t ivea t i nduc ing l a t e r a l phase sepa ra t ions i n such b ina ry m ix tures [66 ,93 ,97 ,981 .E f f e c t o f c h o l e s te r o l o n t h e t h e r m o t r o p i e b e h a v io r o f p h o s p h o l ip i d s

T h e o c c u r r e n c e o f c h o l e s t e ro l a n d r e l a t e d s t er o ls i n th e m e m b r a n e s o f e u k a r y o t i cc ells h a s p r o m p t e d m a n y i n v e st ig a t io n s o f t h e e f f e c t o f c h o l e s te r o l o n t h e t h e r m o -t r o p i c p h a s e b e h a v i o r o f p h o s p h o l i p id s [ 1 - 3 ] . S t u d ie s u t il iz i ng c a l o r i m e t r ic a n do t h e r p h y s ic a l t e c h n i q u e s h a v e e s ta b l is h e d t h a t c h o l e s te r o l c a n h a v e p r o f o u n de f f e c t s o n t h e p h y s i ca l p r o p e r t i e s o f p h o s p h o l i p i d b i la y e r s a n d p r o b a b l y p l a y s a nim po r t an t ro l e in con t ro l l ing t he f l u id i t y o f b io log ica l me mb ranes ( fo r a r ev i ew,see Ref . 9 9) . Ch oles t e ro l i nduces an ' i n t e rme dia t e s t a t e ' i n pho spho l ip id mo lecu l esw i t h w h i c h i t i n t e r a c t s , i n cr e as in g t h e f l u i d it y o f t h e h y d r o c a r b o n c h a i n s b e l o w , a n ddecreas ing t he f l u id i t y above t he ge l t o l i qu id-c rys t a l l i ne phase t r ans i t i on t empera -t u r e .

A n u m b e r o f g r o u p s h a v e s t u d i e d P C - - c h o le s t e ro l m i x t u r e s u s in g c o n v e n t i o n a lDSC [ 1 3 ] . A l tho ugh the re was genera l agreem ent t ha t t he p resence o f i nc reas ingconcen t r a t i ons o f cho l es t e ro l b roadened t he ge l t o l i qu id-c rys t a l l i ne phase t r ans i -


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246 R.N. McElhaney DSC and DTA studies o f lipids and biomembranest i o n o f P C a n d r e d u c e d i t s A H c a1 , t he re was d i sagreement on t he d i r ec t i on o f t hesh i f t i n t he T m a n d o n t h e a m o u n t o f c h o l e s te r o l n e c e s s a r y t o c o m p l e t e l y a b o li shthe t r ans i t i on . Severa l r ecen t s t ud i es o f DPPC - -cho l es t e ro l sys t em s emp loy ing h igh-sens i t iv i t y DSC appe ar t o have r eso lved t h i s con t rover s y and ha ve revea l ed add i -t io n a l i n f o r m a t i o n a b o u t t h e n a t u r e a n d s t o i c h i o m e t r y o f c h o l e s t e r o l - p h o s p h o l i p i di n te r a c ti o n s [ 1 0 0 - 1 0 2 ] . A t c h o le s te r o l c o n c e n t r a ti o n s b e t w e e n 0 - 2 0 m o l , t h eD S C t r a c e o f t h e m a i n t r a n s it i o n c a n b e r es o l v ed i n t o t w o c o m p o n e n t s ( s e e F ig . 5 ).T h e s h a r p c o m p o n e n t o r i g i n a l l y c e n t e r e d a t t h e Trn o f pure DPPC sh i ft s t o s l i gh t lyl o w e r t e m p e r a t u r e s a s c h o l e s t e r o l i s a d d e d a n d i t s A H c a 1 dec reases l i nea r ly wi thi n cr e as in g c h o l e s t e ro l c o n t e n t , f a ll in g f r o m a n i n it ia l v a lu e o f 8 - 8 . 5 k c a l / m o l t oz e r o a t a b o u t 2 0 m o l . T h e b r o a d c o m p o n e n t e x h i b i ts a s li g h tl y h i g h e r T m w h i c hs h if ts t o h i g h e r t e m p e r a t u r e s a n d is b r o a d e n e d a s c h o l e s t e r o l c o n t e n t i n c re a s esa b o v e 2 0 m o l ; t h e A H c a1 o f t h i s b r o a d c o m p o n e n t i n it ia l ly i n c re a se s a n d t h e nd e c r e a s e s w i t h i n c r e a s i n g c h o l e s t e r o l c o n t e n t , r e a c h i n g z e r o a t a b o u t 5 0 m o lcho l es t e ro l . A r ecen t d i l a tomet r i c s t udy has p rov ided suppor t fo r t h i s ana lys i s[103] . Essen t i a l l y s imi l a r f i nd ings have been r epor t ed fo r PE-cho les t e ro l [1031a n d S M - - c h o l e s te r o l I 1 0 5 ] s y s te m s , e x c e p t t h a t i n t h e s e l a tt e r m i x t u r e s th e T mo f t h e b r o a d c o m p o n e n t s h if ts t o l o w e r r a t h e r t h a n t o h ig h e r t e m p e r a t u r e s . T h ee x i s t e n c e ' o f b o t h a s h a r p a n d a b r o a d t r a n s it i o n at c h o l e s te r o l c o n c e n t r a t io n s o f< 20 too l appear s t o be due t o a l a t e ra l phase sepa ra t i on i n to cho l es t e ro l - f r eea n d c h o l e s t e ro l - e n r ic h e d d o m a i n s , r es p e c t iv e l y [ 1 0 0 - 1 0 6 ] . T h e r e a s o n t h a tcho l es t e ro l dec reases t he T m of PE and SM whi l e i nc reas ing t he T m o f P C m a y b ed u e t o t h e i n t e r m o l e c u l a r h y d r o g e n b o n d i n g p r e s e n t i n P E a n d S M b i la y e r s , w h i c hm a y b e p a r t i a ll y d i s r u p t e d b y t h e i n s e r t io n o f c h o l e s t e r o l m o l e c u l e s . In c i d e n t a l l y ,

) -I - -

Q . =( D

I II

X H L ~ M O L E D M L io 1 7 2 4 . 2 4

0 2 0 4 20 2 5 8 3 0 3

0 3 2 9 7 4 6. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 . 5 4 6 6 . 0 9

I I 1 I 1 L II 0 2 0 3 0 4 0 5 0 6 0 tO

CF i g . 5 . DS C t r aces o f f i ve m i x t u res o f cho l es t e ro l w i t h m u l t t l am e l l a r d i s pe r s i ons o f DMP C( R e p r o d u c e d f r o m abrey and S t u r t e v a n t ( 1 9 7 8 ) [ 2 ] w i t h p e r m i s s i o n f r o m P l e n u m P r e ss ).


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R.N. McElhaney DSC and DTA studies of lipids and biomembranes 247t h e i n c o r p o r a t i o n o f s m a ll a m o u n t s o f c h o l e s t e r o l ( a b o u t 3 - 7 t o o l ) i n t o P C o rSM d i spe r s ions co m ple t e ly abo l ishes t he i r p r e t r ans i t i ons .

S e v e ra l c o n v e n t i o n a l D S C s t u d ie s o f b i n a r y m i x t u r e s o f tw o p h o s p h o l i p i d sexh ib i t i ng ge l phase imm isc ib i li t y have i nd i ca t ed t h a t cho l es t e ro l m ay p re fe ren t i a l l yassoc i a te wi th d i f f e r e n t l ip id c la sses in such mix tures [58 ,10 1 ,1 02 ] . When cho l -e s te r ol w as a d d e d t o m o n o t e c t i c m i x t u r e s o f P C - P C , P C - P G , P C - P S o r P S - P E ,cho l es t e ro l p r e fe ren t i a l l y i n t e r ac t ed wi th t he l ower -mel t i ng l i p id a s i nd i ca t ed by ad e c r ea s e in A H c a a n d a b r o a d e n i n g o f t h e p h a s e t r a n s i ti o n . I n o t h e r b i n a r y s y s t e m s ,h o w e v e r , c h o l e s t e r o l e x h i b i t e d a p r e f e r e n c e f o r a p a r ti c u l a r p h o s p h o l i p i d w h e t h e ro r n o t t h a t l i p i d w a s t h e l o w e r o r h i g h e r -m e l t in g c o m p o n e n t . T h e o r d e r o f p r e f e r -ence o f cho les tero l th us es tabl i she d was SM >> PS ---- PG > PC >> PE. C holes terold o e s n o t a p p e a r , h o w e v e r , t o s h o w a n y c a l o r i m e t d c a l l y d e t e c t a b l e p r e f e r e n t i a la f f in i ty in se ve ra l P C - P E - c h o l e s t e r o l [ 1 0 4 ] a n d P C - S M - c h o l e s t e r o l [ 1 0 9 ]s y s te m s w h i c h , w h i le n o n - i d e a l, w e r e n o t m o n o t e c t i c . S in c e t h e e x t r a c t e d p h o s p h o -l ip i d s f r o m e u k a r y o t i c m e m b r a n e s w i t h o u t c h o l e s t e ro l s h o w s in gle r e l a ti v e ly b r o a dt r a n s it io n s a n d n o m o n o t e c t i c b e h a v i o r , i t s ee m s u n l ik e l y t h a t i n b i o lo g i ca l m e m -b r a n e s p r e f e r e n ti a l c h o l e s t e r o l - p h o s p h o l i p i d i n t e r a c t i o n s o f s u f f ic i e n t s t r e n g t h a n dspec i f i c i t y occu r , i nduc ing t he fo rm at ion o f l a rge cho l es t e ro l - f ree and cho l es t e ro l -r i ch dom ains as apparen t l y occur s in ce r t a in non-co crys t a l l iz i ng b ina ry m ix tureso f s y n t h e t i c p h o s p h o l i p i d s . C h o l e s t e r o l - p o o r o r c h o l e s t e r o l - r i c h m i c r o d o m a i n s o rc lus te r s wh ich a r e a l so enr i ch ed o r de p l e t ed o f ce r t a in phosph ol ip id cl as ses m ay ,h o w e v e r , o c c u r .Effect o f small molecules on the thermot ropic behavior of phospholipids

A number o f l i p id - so lub l e smal l molecu l es i nc lud ing drugs l i ke t r anqui l i ze r s ,an t i depressan t s , n a rco t i c s and anes the t i c s , p ro duc e b io log i ca l e f f ec t s i n l iv ingc ells . A l t h o u g h s o m e o f t h e s e c o m p o u n d s a r e k n o w n t o p r o d u c e t h e i r c h a ra c t e ri s ti ce f f e c t s b y i n t e r a c t i n g w i t h s p e c i f i c m e m b r a n e p r o t e i n s , o t h e r s s e e m t o i n t e r a c tr a t h e r n o n - s p e c i fi c a ll y w i th t h e l ip i d b i l a y e r o f m a n y b i o lo g i ca l m e m b r a n e s . T h eef f ec t o n t he ge l t o l i qu id-c rys t a l li ne phase t r ans i t i on p rof i l e o f syn the t i c PCs ofo v e r o n e h u n d r e d h y d r o p h o b i c s m all m o l e c u le s p r o d u c i n g b i o lo g i c al e f f e c t s h a v enow been s tud i ed by DSC ( fo r a r ev i ew see Ref . 110) . A t l east fou r d i f f e r e n t t ypesof modi f i ed t r ans i t i on p rof i l e s can be d i s t i ngu i shed . In so-ca l l

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