1987_boggs_lipid intermolecular hydrogen bonding influence on structural organization and membrane...

8/13/2019 1987_Boggs_Lipid Intermolecular Hydrogen Bonding Influence on Structural Organization and Membrane Function

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Biochimica et Biophysica Acta 906 (1987) 353 -40 4 353Elsev ie r

BBA 85315

Lipid in termolecu lar hyd rogen bondinginf luence on s truc tura l organiza t ion and membrane func t ion

Joan M BoggsDepartment o f Biochemistry, Hospital for S ick Children, and Department of C linical Biochemistry, University of Toronto,Toronto Canada)

( R e c e i v e d 9 D e c e m b e r 1 9 86 )( R e v i s e d m a n u s c r i p t r e c e i v e d 1 5 M a y 1 9 87 )

on ten tsI . I n tr o du c ti o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 54I I. E v id e nc e f o r l ip i d i n te r m o le c u la r h y d ro g e n b o n d i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 55

A . G e n er a l c o ns id e ra ti on s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 5B . F a t ty a c id - a n io n c o m p le x es . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 57C . P r op e rt ie s o f g ly c er o l- b as e d l ip i ds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 58

1. P h a s e- t ra n s it io n t e m p e ra t u re . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 582. O t h er p r op e rt ie s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 03. H e x ag o na l- ph a se f o rm a t io n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 74

D . S p hi ng o li pi ds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 8E . I n te r m o le c u la r h y d ro g e n b o n d i n g b e tw e e n t w o d i ff e re n t l ip id s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 81F . H y d r og e n b o n d i n g o f c h o le s te r ol w i t h o t h e r l ip i ds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 82

I I I. I n f lu e n c e o f l i pi d i n t e rm 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 m e m b r a n e s t r u c tu r e a n d f u n c t i o n . . . . . . . . . . . . . . . . . . . . . . . 3 83A , L a m e ll a r t o n o n - la m e l la r p h a se t ra n s it io n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 83B . P h a se s e p a ra t io n o r d o m a i n f o r m a ti o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 83

1. L ip id m i sc ib il it y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 32 . A s y m m e t r ic d i s t ri b u t i o n i n s m a l l u n i l a m e l la r v e si cl es . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 853 . P r e fe r e n ti a l a s s o c ia t i on o f c h o le s t er o l w i t h d i f fe r e n t l i p id s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 86C . I n te r a ct io n s w i th p r o te i n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 88

D , O t h e r r ol es o f l ip id h y d ro g e n b o n d i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 89

A b b r e v i a t i o n s : L , l a u r o y l ; M , m y r i s t o y l ; P , p a l m i t o y l ; S ,s tearoyl ; E , e la idoyl ; O, o leoyl ; T , t e t radecyl ; H, hexadecyl ;P C , p h o s p h a t i d y l c h o l i n e ; P E , p h o s p h a t i d y l e t h a n o l a m i n e ;M e P E , m o n o m e t h y l p h o s p h a t i d y l e t h a n o l a m i n e ; M e 2 P E , d i -m e t h y l p h o s p h a t i d y l e t h a n o l a m i n e ; P S , p h o s p h a t i d y l s e r i n e ; P A ,p h o s p h a t i d i c a c i d ; P G , p h o s p h a t i d y l g l y c e r o l ; P M , p h o s p h a t i -d y l m e t h a n o l ; P I , p h o s p h a t i d y l i n o s i t o l ; l y s y l P G , l y s y l p h o s p h a -t i d y l g l y c e r o l ; M G D G , m o n o g l u c o s y l - o r m o n o g a l a c t o s y l d i -acy lg lycero l ; DGDG, d ig lucosyl - o r d iga lac tosy ld iacy lg lycero l ;D X P R , D X M G D G , D X D G D G , M e D X P E , M e 2 D X P E , a n dX Y P R , d i h y d r o c a r b o n c h a i n f o r m a n d m i x e d c h a i n f o r m o f

d i a c y lg l y c e ro l o r p h o s p h o l i p i d w h e r e P R = P C , P E , P S , P A ,P M , P G , a n d X , Y = L , M , P , S, E , O , T , a n d H a s d e f i n e da b o v e ; T o r T o , g e l t o l iq u i d c r y s ta l l i n e p h a s e - t r a n s i t i o ntemp era ture ; TCR crys ta l l ine to l iqu id-crys ta l l ine pha se- t ra ns i -t i o n t e m p e r a t u r e ; T H , l a m e l l a r t o h e x a g o n a l p h a s e - t r a n s i t i o nt e m p e r a t u r e ; D S C , d i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y ; S U V ,smal l un i lam el la r ves ic le.C o r r e s p o n d e n c e : J . M . B o g g s , D e p a r t m e n t o f B i o c h e m i s t r y ,H o s p i t a l f o r S i c k C h i l d r e n , 5 55 U n i v e r s i t y A v e n u e , T o r o n t o ,M 5 G 1 X 8 , C a n a d a .

0 3 0 4 - 4 1 5 7 / 8 7 / 0 3 . 5 0 1 98 7 E l s e v i e r S c i e n c e P u b l i s h e r s B .V . ( B i o m e d i c a l D i v i s i o n )


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354

IV Control mechani sms for memb rane function 389A Regulation of hydrogen bond ing by change in enviro nment 390B Regulation of hydrogen bond ing by enzymatic alteration of lipid composi tion 392

1 Respons e to changes in growth conditio ns 3922 Resp ons e to signals 395

V Summ ary 396Acknowledgements 397References 398

. In t r o d u c t i o n

The great variety of lipids present in mem-branes and the specificity of the lipid compositionof different types of membrane suggest that manyof these lipids have specific roles to play [1]. Inmammalian membranes this variety is achievedthrough a number of structural modifications inthe hydrocarbon chains and the polar head-groupjoined to the glycerol or sphingosine base of thecommon phospho- and sphingo-lipids. Furtherstructural modifications occur in the type of lin-kage ether or ester) of the hydrocarbon chains toglycerol and the presence of free hydroxyl groupson the sphingosine base and fatty acid chain ofsphingolipids. Rapid in situ modifications to lipidhead groups and hydrocarbon chains can alsooccur in response to various stimuli. These modifi-cations may affect the structural organization andfluidity of the membrane and the interactions oflipids with proteins, altering their behavior. Theymay also allow the lipid bilayer to respond dy-namically to changes in its environment and tocarry out certain dynamic functions of the mem-brane.

Examination of the physical properties andphase behavior of different lipids has helped inthe understanding of the molecular forces whichcontrol lipid behavior and the contribution ofdifferent structural modifications to these forces.Various forces which have been considered tocontrol lipid behavior include repulsive or attrac-tive forces between lipid head groups [1-8], themolecular shape and ratio between the volumes ofthe head group and hydrocarbon region [9-11]and hydration forces [12,13,113]. While theseforces are all important for the behavior and prop-erties of lipids and are interrelated, they are notidentical. This author considers that the repulsive

or attractive forces between lipid head groups arethe most important property of lipids, other thantheir amphipathic character. These forces arepartly responsible for the molecular shapes of thelipids and hydration forces, but go further instabilizing the specific molecular organizationtaken up by a particular lipid under differentconditions. Thus, consideration of these forces hasgreater predictive value for the effect of a particu-lar structural modification of a lipid molecule onits properties. The repulsive forces are primarilythe electrostatic repulsive forces between similarlycharged lipids while the attractive forces are elec-trostatic interactions between oppositely chargedgroups and intermolecular hydrogen bonding in-teractions between charged or neutral lipids whichhave hydrogen-donating and -accepting groups.

Some lipids are less hydrated than others andmany authors att ribute the physical properties andphase behavior of these lipids to their reducedhydration. However, those lipids which are lesshydrated are generally those with hydrogen bond-donating and -accepting groups and it seems rea-sonable to conclude that the reduced hydration isusually caused by the participation of the lipidhead groups in intermolecular hydrogen bondingwith each other rather than with water. Therefore,these interactions must ultimately be responsiblefor the phase behavior and other properties ofthese lipids which accompany the reduced hydra-tion. However, some structural modificat ions tolipids which make them less polar or more hydro-phobic may also cause increased dehydration,which can in turn, contribute to an increase in thestrength or probability of hydrogen bonding inter-actions.There is, of course, no absolute proof thatintermolecular hydrogen bonding occurs betweenthe head groups of lipids when in the presence of


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w a t e r. N M R , i n f ra r e d , o r R a m a n s p e c t r o s c o p ym i g h t b e a b l e , e v e n t u a l l y , t o p r o v i d e s u c h p r o o fb u t h a v e n o t y e t d o n e s o . H o w e v e r , t h e r e i s ac o r r e l a t i o n b e t w e e n t h e p h y s i c a l p r o p e r t i e s o f d i f -f e r e n t l ip i d s a n d t h e p r e s e n c e o r a b s e n c e o f p o t e n -t i a l h y d r o g e n - d o n a t i n g a n d - a c c e p t i n g g r o u p s .H i s t o r ic a l l y , i t w a s t h e u n u s u a l p h y s i c a l p r o p e r t i e so f w a t e r a n d a l c o h o l s w h i c h s u g g e s t e d t h a t t h e ya r e a s s o c ia t e d , le a d i n g t o t h e c o n c e p t o f h y d r o g e nb o n d i n g [ 1 5 ] . T h e p u r p o s e o f t h i s r e v i e w i s t od e m o n s t r a t e t h is c o r r e l a t i o n b e t w e e n t h e p h y s i c a lp r o p e r t i e s o f l i p id s a n d t h e i r m o l e c u l a r s t r u c t u r e ,u p d a t i n g t w o e a r l i e r r e v i e w s o n t h i s s u b j e c t [ 1 , 6 ] ,a n d t o d i s c u s s t h e s i g n i f ic a n c e o f i n t e r m o l e c u l a rh y d r o g e n b o n d i n g i n t e r a c t i o n s f o r l i p i d o r g a n i z a -t i o n a n d d y n a m i c f u n c t io n . A s p e c ia l m e n t i o ns h o u l d b e m a d e o f t h e w o r k o f T r ~ iu b le , E ib l ,P a s c h e r a n d t h e i r c o l l a b o r a t o r s , o n w h i c h t h i sr e v i e w h e a v i l y d e p e n d s .1 I E v i d e n c e f o r l i p i d i n t e r m o l e c u l a r h y d r o g e nb o n d i n gH A. General considerations

H y d r o g e n - d o n a t i n g g r o u p s o n l ip i d m o l e c u le si n cl u de N H ~ - , N H 2 , P O H , C O H , C O O H a n dH N C - - O , w h i le h y d r o g e n a c c e p t i n g g r o u p s i n c l u d es o m e o f t h es e a s w e ll a s P O - , C O O - , O C = O a n dC O C . T h e s t r e n g t h o f h y d r o g e n b o n d s g e n e r a ll yi n c r e as e s i n t h e o r d e r o f e l e c t r o n e g a t i v i ty o f t h ed o n o r , S < N < O , a n d , f o r t h e a c c e p t o r , i n t h eo r d e r e t h e r s < c a r b o n y l s < a m i n e s [ 15 ]. T w o c o m -m o n g r o u p s i n p h o s p h o l i p i d s , t h e P O 4 a n d N H ~ - ,m i g h t i n t e r a c t a s e i t h e r i o n p a i r s o r p r o t o n t r a n s -f e r c o m p l e x e s. H o w e v e r , t h e l a t te r a r e a p p r e c i a b l ym o r e s t a b l e t h a n t r u e i o n p a i r s a n d h a v e s m a l l e rd i s s o c i a t i o n c o n s t a n t s d u e t o h y d r o g e n b o n d i n gb e t w e e n t h e c a t i o n i c a n d a n i o n i c m o i e t i e s [ 1 5 ] .

X - r a y d i f f r a c t i o n s t u d i e s o f s i n g l e c r y s t a l s o ft h e p h o s p h o l i p i d , d i l a u r o y l - o L - p h o s p h a t i d y l e t h -a n o l a m i n e : a c e t i c a c i d [ 1 6 - 1 9 ] , a n d t h e s p h i n g o -l ip i d , c e r e b r o s i d e f l - D - g a l a c t o s y l - N - 2 - o - h y d r o x y -o c t a d e c a n o y l ) - o - d i h y d r o s p h i n g o s i n e ) [2 0], h a v es h o w n t h a t t h e m o l e c u l e s a r e p a c k e d i n s u c h aw a y t h a t 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 c a no c c u r . I n P E , e a c h a m i n e a n d p h o s p h a t e c a n p a r -t i c ip a t e in t w o h y d r o g e n b o n d s w i t h i n t e r m o l e c u -l a r N - O d i s t a n c e s o f 2 . 7 4 a n d 2 . 86 .A , a s s h o w n

355f o r a h e a d - o n v i e w i n F i g . 1 . I n c e r e b r o s i d e , t h es u g a r p o i n t s s i d e w a y s a w a y f r o m t h e c e r a m i d ep a r t b e c a u s e o f i n t ra m o l e c u l a r h y d r o g e n b o n d i n gb e t w e e n th e a m i d e N - H g r o u p a n d t h e o x y g e n s o ft h e g l y c o si d ic li n k a g e a n d t h e f a t t y a c i d h y d r o x y lg r o u p F i g . 2 , b o t t o m ) . T h i s a ll o w s it to in t e r a c tw i t h n e i g h b o r i n g m o l e c u l e s , f o r m i n g a n 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 n e t w o r k v i a th ea m i d e g r o u p a n d h y d r o x y l s o n t h e s u g a r, f a t t ya c i d c h a i n a n d s p h i n g o s i n e c h a i n F i g . 2 , t o p ) .

H o w e v e r , i t i s o f t e n q u e s t i o n e d w h e t h e r i n t e r -m o l e c u la r h y d r o g e n b o n d i n g b e t w e e n l i p id h e a dg r o u p s c o u l d t a k e p l a c e i n t h e p r e s e n c e o f w a t e r .J e n c k s [ 2 1 ] p o i n t s o u t t h a t i t w o u l d r e q u i r e t h a tt h e h y d r o g e n b o n d s o f e a c h o f t h e p a r t i c i p a t i n gg r o u p s w i t h w a t e r b e b r o k e n , a s i n d i c a t e d i n E q n .1.A - H . - O H 2 + B . . H O H --, A - H . . B + H O H - . O H 2 1 )w h e r e A - H is t h e h y d r o g e n - d o n a t i n g g r o u p a n d Bi s t h e h y d r o g e n - a c c e p t i n g g r o u p o n t w o l i p idm o l e c u l e s . T h e s t a b i l i t y o f s u c h h y d r o g e n b o n d sd e p e n d s o n t h e d i f f e r e n c e s i n t h e s t a b i li t ie s o f t h eb o n d s o n t h e t w o s i de s o f E q n . 1 , n o t o n t h e

Fig. 1. H ea d group pac king of 1,2-dilauroyl-DL-phosphatidyl-ethanolamine : acetic acid determined from single crystal X-raydiffraction analysis by Elder, Hitchcock, Ma son and Shipley[17], viewed perpendicular to the bilay er interface. Dashedlines show the intermolecular hydrogen bo nd s between thenitrogen of one moleculeand O-13 and O-14 of two neighbor-ing m olecules. The acetic acid m oleculeshave been omitted forclarity. Reproduced from R ef. 17 with permission of the authorand The Royal Society.


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356B~ A Ba A

, ~ ) O -

Fig. 2. Conformation and bilayer packing of the head group ofcerebroside ( fl-D-galactosyl-N-(2-D-hydroxyoctadecanoyl)-D-dihydrosphingosine) as determined b y P ascher and Sundell [20]using single crystal X-ray diffraction analysis. Upper p art, thehead groups o f molecules in one half of the bilayer are viewedparallel to the bilayer. There are two independent molecules Aand B in the unit. T he A molecules are half a unit edge abovethe B molecules. The dotted l in e indicates the intra- andintermolecular hydrogen bond systems between molecules B>A B 3 and A 2. One system originates at the amide N -H groupof molecule A (A -N ) and proceeds via the hydroxyl groupsA - O 2 ' to B 1 - O 4 t o A - O 2 t o B 3 - O 2 t o A 2 - O l ' . T h eother hydrogen bon d system starts at B3 -O 6 and continuesvia A -O 3 to A 2-O 1' . The lengths of the hydrogen bonds are2.63-2.77 A except for that from B1-O 4 ' ' to A -O 2 which i s3.02 ~,. Each cerebroside molecule participates in eight hydro-gen bonds, form ing a t ight two-dimensional network o f hydro-gen bonds throughout the polar boundaries of each layer. Anintramolecular hydrogen bond between A -N and A -O1 is alsoshown. The intramolecular hydrogen bonds between the amideN -H and the oxygens of the glycosidic l inkage (O 1) and thefatty acid hydroxyl group ( 02 ') are shown more clearly in thelower part . R eproduced from Ref. 20 with permission from theauthors an d Elsevier Scientific Publishers Ireland, L td.

a b s o l u t e s t r e n g t h o f th e b o n d i n A - H - B [ 21 ]. Ad e t a i l e d a n a l y s is o f a ll e n t h a l p y a n d e n t r o p y c o n -t r i b u ti o n s f r o m b o n d b r e a k i n g a n d b o n d f o r m in g ,t o t h e f r ee e n e r g y o f f o r m a t i o n o f a l i p id b i l a y e r

w o u l d b e n e c e s s a r y i n o r d e r t o e v a l u a t e t h e s t a b i l -i t y o f A - H B . T h i s h a s n o t b e e n d o n e a n d w i lln o t b e a t t e m p t e d h e r e .

H o w e v e r , s o m e p r o p e r t i e s o f a l ip i d b i l a y e rw h i c h w o u l d f a v o r i n te r m o l e cu 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 p p r o p r i a t e l ip i d s c a n b e p o i n t e d o u t .T h e l i p i d m o l e c u l e s a r e a l r e a d y i n t e r m o l e c u l a r l ya s s o c i a t e d a s a r e s u l t o f h y d r o p h o b i c e f f e c ts a n dt h e V a n d e r W a a l ' s i n t e r a c t i o n s b e t w e e n t h e a c y lc h a i n s . T h e l i p i d h e a d g r o u p s m a y a l r e a d y b ec o r r e c tl y p o s i t io n e d f o r in t e r m o l e c u l a r h y d r o g e nb o n d i n g t o t ak e p la c e. C o r r e c t o r i e n t a t i o n m a y b ef a c i li t a te d b y e l e c t r o s t a t ic i n t e r a c t i o n s b e t w e e n t h eh e a d g r o u p s T h u s , t h e d e c r e a s e i n e n t r o p y w h i c hd i s f a v o r s i n t e r m o l e c u l a r a s s o c i a t i o n o f t w o m o l e -c u l e s i n s o l u t i o n w o u l d n o t b e a f a c t o r . S m i t h a n dT a n f o r d [ 22 ] h a v e s u g g e s t e d t h a t i n t e r m o l e c u l a rh y d r o g e n b o n d i n g c a n b e e x t r a o r d in a r i l y s t a b lew h e n t h e p a r ti c i p a t in g g r o u p s h a v e l o n g a l k y lc h a in s T h u s , t h e li p id b i l a y e r c a n b e t h o u g h t o f a sa la r g e m o l e c u le o r p o l y m e r i n w h i c h ' i n t r a 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 r in t e r s u b u n i t h y d r o g e nb o n d i n g t a k e s p l a c e r a t h e r t h a n c o n v e n t i o n a l 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 . T h e r e i s m u c hm o r e e v i d e n c e t h a t t h e f o r m e r c a n o c c u r i n w a t e r ,a s i n p r o t e i n s , o l i g o n u c l e o t i d e s a n d c o m p l e xc a r b o h y d r a t e s , t h a n t h e l a tt e r. H o w e v e r , it s h o u l db e m e n t i o n e d t h a t 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 a p p e a r s t o o c c u r b e tw e e n i o n i z ed a n d u n -i o n i z ed s p e c ie s o f o r t h o p h o s p h o r i c a c i d in w a t e r[ 23 ]. A n o t h e r f a c t o r w h i c h m a y h e l p s t a b il iz e A - H - B b e t w e e n b i l a y e r l i p i d s i s th e r e l e a s e o f h y d r o -g e n - b o n d e d w a t e r f r o m t h e s u rf a c e o f t h e b il a y e rw h i c h w o u l d c o n t r i b u t e t o a n i n c r e a s e i n t h e e n t -r o p y a n d h e l p c o m p e n s a t e f o r th e e n e r g y n e ce s -s a r y to b r e a k t h e h y d r o g e n b o n d s b e t w e e n t h el i p i d a n d w a t e r .

T h e r e la t i v e ly r a p i d a x i a l r o t a t i o n o f P E a n do t h e r l ip i ds f o r w h i c h i n t e r m o l e c u l a r h y d r o g e nb o n d i n g h a s b e e n p o s tu l a t ed , a n d t h e m o t i o n o ft h e h e a d g r o u p s , e v e n i n t h e g e l p h a s e [ 2 4 , 2 5] ,i n d i c a te t h a t i n t e rm o l e c u l a r h y d r o g e n b o n d s b e -t w e e n t h e l i pi d h e a d g r o u p s c a n n o t b e l o n g li v ed .D e u t e r i u m a n d 1 3 C - N M R s t u di e s s h o w t h a t i n t h eg e l p h a s e o f P E a x i a l d i f f u s i o n o c c u r s a t a r a t e o f1 0 5 - 1 0 6 s - 1 [ 2 6, 27 ]. I n t h e g e l p h a s e o f c e r e b r o -s i d e , h o w e v e r , a x i a l d i f f u s i o n i s s l o w e r , 1 0 2 s - 1[281.V i n o g r a d o v a n d L i n n e l l [1 5 ] p o i n t o u t t h a t i n


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order to be considered structurally significant theaverage lifetime of a given arrangement of mole-cules must exceed, by a factor of about 10, thevibrational period of the bonds which form thestructure, The period of an O-H stretching vibra-tion is about 1 0 - 1 4 S while that of an intermolecu-lar hydrogen bond which absorbs at 200 cm-1 isabout 2.10-13 s. The lifetime of hydrogen bondsin liquid water is about 10 -11 s. It seems likelythat the lifetime of intermolecular hydrogen bondsbetween lipids in a bilayer could be of these ordersof magnitude. Thus, individual hydrogen bondsbetween two lipid molecules need not be 'strong'and fixed for long periods of time; they are prob-ably easily broken but then reform with anothermolecule. They are probably not of a fixedstoichiometry. Each proton and acceptor may beinvolved in several hydrogen bonds simulta-neously. Thus, bilayers may be stabilized over afairly wide range of ratios of donor to acceptor.Protons may be exchanged from one molecule toanother and be transported laterally along thebilayer [29]. Protons can diffuse along the surfaceof a lipid monolayer of PE 20-times faster than inthe bulk phase [30]. This ready availability in thelipid bilayer of other molecules for interchange ofprotons and rapid reformation of hydrogen bondsmay also contribute to the stability of intermolec-ular hydrogen bonding in the lipid bilayer. Thus,although these bonds may not be long lasting,they stabilize a particular arrangement of lipidmolecules in which the bonds can readily reform.

Theoretical studies show that it is necessary toinclude intermolecular hydrogen bonding forcesbetween lipid head groups in order to account forthe high transition temperature of PE and PA[4,31-33]. They show further that these forcesincrease the enthalpy by only a small percentage.The enthalpy of the transition is determined prim-arily by changes in the Van der Waals' attractiveforces and the increase in rotational isomeric en-ergy during the phase transition. Other theoreticalstudies do not take the intermolecular forces intoaccount specifically, but do make use of experi-mentally determined parameters, such as thesmaller head group volume or the closer packingof PE, which are probably a result of the inter-molecular interactions [9,14]. Nagle [32] has esti-mated that only one hydrogen bond of length 2.8

357

for every 40 molecules is necessary to increasethe transition temperature by 10-12 Cdeg (relativeto the neutral state). This is consistent with tran-sient and /o r weak hydrogen bonds and the knownrotational and translational mobility of PE, but isenough to inhibit lateral expansion of the lipidand stabilize the gel phase.H B. Fatty acid anion complexes

Complex formation between fatty acids andtheir soaps in water [34-36] may be stabilized byintermolecular hydrogen bonding, and representthe simplest example of the effects of such hydro-gen bonding [29]. Fat ty acids are in a state ofpartial dissociation over a wide pH range, bothbelow and above their transition temperatures[29,36,37], as shown by Haines and Heller in Fig.3 for oleic acid. The pH titration curve of oleicacid has two plateaus [29,36]. At the first one, atpH 9.5, where the fat ty acid becomes partiallyprotonated, a phase transition from micelles offatty acid to acid-anion liposomes occurs. At thesecond plateau, at pH 7.5, where the fatty acid isprotonated by more than 80 , a phase transitionto oil droplets occurs. Oligolamellar liquid-crystal-line phase liposomes, which are relatively imper-

100908070

~- 60--q 50

30

o ~ o

~ _ ~ He l l ~ ~ p h a s ehange iio ~ |6 7 8 9p H

01020

~ 30 i

Liposomes ~.~5 0o g

70 :~8090

~0 ;1 i'~ 100F i g . 3 . T i t r a t i o n c u r v e e ) o f a q u e o u s d i s p e r s i o n o f o l e i c a c i d50 mM), l abe led wi th [1 -13C] laur ic ac id , f rom pH 12 to 6 and

p e r c e n t d i s s o c i a t i o n A ) a s d e t e r m i n e d f r o m t h e c h e m i c a l s h i f tu s i n g 1 3 C - N M R b y T . H a i n e s a n d M . H e l l e r [ 2 9 ] . T h e h i g h e ri n f l e c t i o n p o i n t i n t h e t i t r a t i o n c u r v e i s a s s o c i a t e d w i t h at r a n s i t i o n f r o m a n i o n m i c e l l e s t o a c i d - a n i o n l i p o s o m e s . T h el o w e r i n f l e c t i o n p o i n t r e f l e c t s a p h a s e c h a n g e f r o m l i p o s o m e st o o i l d r o p l e t s o f t h e a c i d f o r m . R e p r i n t e d f r o m R e f . 2 9 w i t h

p e r m i s s i o n f r o m t h e a u t h o r .


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58

meable to small molecules, are formed only overthe pH range where significant percentages ofboth the anion and acid are present. Below thetransition temperature, the acid-anion complexforms crystals over this pH range [37]. Intermolec-ular hydrogen bonding between the acid and an-ion is undoubtedly involved in crystal formationbelow the transition temperature. The formationof liposomes and stabilization of the acid andanion over a wide pH range in the liquid-crystal-line phase suggests that hydrogen bonding occursin this phase also, as concluded by Haines [29].However, the formation of liquid-crystalline phasebilayers over this pH range has also been attri-buted to reduction of the surface charge densityand repulsive forces when a certain percentage ofthe fatty acid is in the protonated state [38].

Evidence that formation of bilayers stabilizesboth the anion and acid, i.e., that it both lowersthe intrinsic pK first plateau) and raises it sec-ond plateau), would help to support the conclu-sion that intermolecular hydrogen bonding occurs.The occurrence of intramolecular hydrogen bond-ing between the acid and anion of the cis form ofa dicarboxylic acid, maleic acid, was inferred fromthe lower p K 1 for dissociation of the free acid toform the monoanion and the higher pK 2 for dis-sociation of the monoanion to the dianion, com-pared to the trans form, fumaric acid, in whichintramolecular hydrogen bonding is not possible[21]. It is difficult to distinguish the effects ofelectrostatic repulsion from intramolecular hydro-gen bonding on the pK values of simple dicarbo-xylic acids, but studies of more complex alkylatedsuccinic acids support the involvement of in-tramolecular hydrogen bonding [45].

Although the apparent pK1 of a long chainfatty acid in a bilayer is considerably higher thanfor a carboxylic acid monomer in solution pK 4),this is due partly to the high negative surfacecharge density of the bilayer resulting in a surfacepH which is lower than the bulk pH. The intrinsicpK values are not known. However, the pH de-pendence of dissociation of the fatty acid-anionbilayer can be compared to that of the fatty acidin a bilayer of similar surface charge density wherehydrogen bonding cannot occur, or where it oc-curs exclusively between another compound andeither the anion form or the acid form of the fatty

acid. Some data are available for the latter situa-tion on a fatty acid/ alcoh ol mixture dodecanoicacid and dodecanol) and a fatty acid/phospho lipidmixture palmitic acid and DPPC).

The dodecanoate/dodecanol mixture alsoforms bilayers at pH values where the fatty acid isionized and could hydrogen bond with the alcohol[36]. The pK of the fatty acid in the acid/alcoholmixture is 7 and only one plateau occurs. Thus,the fatty acid becomes completely ionized at alower pH than in the acid/anion mixture suggest-ing that the anion form is stabilized by hydrogenbonding with the alcohol. In DPPC on the otherhand, where hydrogen bonding could only takeplace between the protonated form of the fattyacid and the phospholipid, the pH at which thepalmitic acid begins to dissociate is higher, aboutpH 8 [39] compared to about 7 for the acid anionmixture [29]. The pK of the fatty acid in DPPCwas reported as 10.2, but insufficient data weregiven to determine whether the titration curveexhibits two or only one plateau. However, thehigher pH at which dissociation begins suggeststhat the acid form is stabilized by intermolecularhydrogen bonding with the anionic phosphate ofDPPC. Comparison with the fatty acid/anionmixture then suggests that hydrogen bonding be-tween the acid and the anion stabilizes both formsallowing them to persist over a wider pH rangethan in the absence of hydrogen bonding.H C . Properties o f glycerol based lipidsII C1. Phase transition temperaturesOne of the most important properties of lipidswhich is suggestive of intermolecular hydrogenbonding in the gel phase is the high gel to liquid-crystalline phase-transition temperature of lipidswith compatible hydrogen bond-donating and-accepting groups. An increase in the phase-transi-tion temperature indicates stabilization of the gelphase. Intermolecular hydrogen bonding interac-tions which are stronger in the gel phase wherecloser lipid packing occurs, would stabilize thisphase. If the intermolecular hydrogen bondinginteractions can occur equally well in either phase,as may occur for some lipids with large headgroups, it would have no effect on the transitiontemperature, although it might have effects on


7/52

other lipid properties. A charged head group onthe other hand would cause repulsion of the lipidmolecules and would stabilize the liquid-crystal-line phase where the lipids are less closely packed,resulting in a decrease in the transition temper-ature.If the phase-transition temperatures of only afew lipids with different polar head groups arecompared it may be difficult to distinguish thepossible effects of head group size or other factorsfrom that of their intermolecular hydrogen-bond-ing potential. However, if a large variety of lipids,including a number of synthetic lipid analogues,with different head groups in different ionizationstates are considered, the conclusion that inter-molecular hydrogen bonding is involved seemsinescapable.

The phase-transition temperatures of a numberof synthetic glycerol-based lipids containingpalmitic acid at neutral pH are shown in Table I.The ionization state of each lipid at this pH isindicated by the charges present on the head

T A B L E IE F F E C T O F T H E P O L A R H E A D G R O U P O N T H E T E M -P E R A T U R E O F T H E G E L T O L I Q U I D - C R Y S T A L L IN EP H A S E T R A N S I T I O N , To, A T N E U T R A L p HL i p i d C h a r g e a T c o C ) R e f .D P P A - ) 6 5 4 0D H M G D G b 0 ) 6 3.6 4 1D P P E - + ) 6 3 4 0D P P S - - + ) 5 4 4 2 , 4 3D P P G - ) 4 1 4 4D P P M - ) 4 4 8D P P C - , + ) 4 1 7 3 , 5 5C a r d i o l i p i n _ ) c 4 0 1 7 9D i p a l m i t i n 0 ) 5 0 , 6 3 d 4 7a N u m b e r a n d t y p e o f c h a r g e d g r o u p s , n o t n e t c h a r g e .b T h i s f i p id h a s n o t b e e n p r e p a r e d w i t h p a l m i t o y l c h a i n s .H o w e v e r , c o m p a r i s o n o f t h e d i a c y l a n d d i a l k y l f o r m s c o n -

t a i n i n g C l S c h a i n s s u g g e s t s t h a t t h e e t h e r l i n k a g e m a y i n -c r e a s e t h e Tc b y o n l y 3 . 5 C d e g [ 4 1 , 4 8 ], s i m i l a r t o i t s e f f e c t o nP E .

c C h a r g e p e r t w o c h a i n s . C o n t a i n s f o u r p a l m i t o y l c h a i n s .a T h e f i r st t ra n s i t i o n i s d u e t o a m e t a s t a b l e p h a s e . T h e s e c o n d

i s t h a t o f a s t a b l e c r y s t a l l i n e t o i s o t r o p i c l i q u i d t r a n s i t i o n[ 4 7 ] . T h u s , i t i s n o t s t r i c t l y c o m p a r a b l e t o t h e g e l t o l i q u i d -c r y s t a l l i n e p h a s e t r a n s i t i o n s o f t h e o t h e r l i p i d s l i s t e d . T h ee n t h a l p y o f t h e s e c o n d t r a n s i t i o n i s t w i c e t h a t o f t h e f i r s tB o g g s , u n p u b l i s h e d d a t a ) .

3 5 9

group, e.g. PE - + ). There does not seem to beany relationship between the size or net charge onthe head group and the phase-transition tempera-ture since, of the three lipids with the smallestnegatively charged head groups, one, DPPA, hasthe highest transition temperature, while the othertwo, DPPM and cardiolipin, are among thosehaving the lowest. The transition temperature ofDPPA with its negative charge is similar to thoseof three lipids with varying head group size andcharge, DPPE with a net neutral charge but twoionized groups, and dipalmitin and DHMGDG,with neutral and unionized head groups. The tran-sition temperature of DPPC with a large headgroup and net neutral charge is similar to those ofthe negatively charged DPPG, cardiolipin andDPPM.

Examination of the transition temperatures of anumber of synthetic analogue lipids with differenthead groups, shown in Table II, indicates that thesize of the head group has little effect on thetransition temperature as pointed out by Eibl [7,8].Although there is a decrease in the transitiontemperature of lipids with long alkyl chains in thehead group, this is probably caused by penetrationof the chain into the bilayer causing a fluidizingeffect. It is more pronounced at low pH values.This fluidizing effect does not occur if there is acharged group at the end of the chain, sinceinsertion of up to ten methylenes between thephosphate and the quaternary amine of PC de-creases the transition temperature by only a fewdegrees. 14N- and 31p-NMR spectra of such PCanalogues indicate that an increasing number ofmethylene groups changes the average orientationof the C-N bond and its dynamics [52]. Mono-layer studies indicate that lipids with large headgroups have a larger molecular area at low surfacepressures than lipids with small head groups, butall can be compressed to a similar molecular areaat high surface pressures. The large alkyl headgroups can change conformation and extend per-pendicularly away from the bilayer into the aque-ous phase, if necessary, to fit into the area availa-ble to them in a closely packed monolayer orbilayer, as indicated by a 2H-NMR study [49].

The transition temperatures of most of the lipidsindicated in Table II, are similar to those of thelowest melting lipids given in Table I. Their simi-


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3 6 0

T A B L E I IE F F E C T O F M O D I F I C A T IO N S T O P O L A R H E A D G R O U P O F S Y N T H E T I C L I P ID A N A L O G U E S O N T H E T R A N S I T I O NT E M P E R A T U R E O F T H E G E L T O L I Q U I D C R Y S T A L L I N E P H A S E T R A N S I T I O N , Tc a

C H 2 - O RR O - C H

C H 2 - XH e a d g r o u p X p H 7 p H 1 R e f .

ch arg e T~ o C ) T~ o C )P O 4 - - C H 3 D P P M ) - ) 4 4 5 3 8 ,4 9P O 4 - C H 2 - C H 3 - ) 4 1 5 1 7 , 49P O 4 - - C H 2 ) 2 - C H 3 - ) 4 0 - 4 2 3 8 7 , 49P O 4- - C H 2 ) 3 - C H 3 - ) 3 9 - 4 2 3 4 7 , 49P O 4 - - C H 2 ) 5 - C H 3 - ) 3 3 2 8 7P O 4 - C H 2 ) 7 - C H 3 - ) 2 2 2 8 7P O 4- - C H 2 ) 2 - N C H 3 ) ~ - D P P C ) - + ) 4 2 4 9 7 ,5 0 bP O 4 - C H 2 ) 5 - N C H 3 ) f - + ) 4 0 5 9 7 , 50P O 4 - C H 2 ) 8 - N C H 3 ) ~ - + ) 4 0 7 ,5 0P O 4 - C H 2 ) l o - N C H 3 ) ~- - + ) 3 8 7 , 50P O 4 - - C H 2 ) 2 - N C H 3 ) ~ - C H 2 ) 2 - N C H 3 ) ~ - + + ) 4 0 8P O 4 - C H 2 ) 2 C C H 3 ) 3 - ) 4 2 7N C H 3 ) ~ + ) 4 2 8P O 4 - C H 2 - C H O H - C H 2 O H D P P G ) - ) 4 1 6 1 4 6 ,4 4P O ~ - - C H 2 - C H 2 O H - ) 4 1 4 6P O 4 - C H 2 - C H 2 - C H 2 O H - ) 4 1 ~ 4 6

I n t h e p r e s e n c e o f N a C 1 . R , p a l m i t i c a c i d. C o m m o n n a m e i s i n d i c a t e d i n p a r e n t h e s e s , i f t h e r e i s o n e .b T r a n s i t i o n t e m p e r a t u r e s f o r o t h e r o d d a n d e v e n v a l u e s o f n a l s o r e p o r t e d i n t h e s e R e f s .

B y a n a l o g y w i t h t h e s i m i l a r i t y o f Tc f o r a l ip i d c o n t a i n i n g t hi s h e a d g r o u p a n d t w o m y r i s t o y l c h a i n s t o D M P G .

larity regardless of head group size and net charge,which varies from - 1 to + 1, indicates that thelow transition temperature of these lipids is due tocharge repulsion and not head group size. The factthat DPPC and its longer chain analogues with anet neutral charge also have this low transitiontemperature indicates that they behave as lipidswith a net repulsive charge [7] . Thus, thequaternary ammonium group does not help toreduce the lateral repulsive effect of the negativelycharged phosphate, although it does reduce thesurface charge of PC bilayers as seen from theaqueous phase. This is also supported by thesimilarity in t ransition temperature of the PC ana-logue with a C CH3) 3 group and net charge of- 1 to that of PC with the N CH3) ~- group andnet charge of 0.Protonation of the negatively charged phos-phate of these lipids raises the transition temper-ature to 53-61C as a result of loss of chargerepulsion. The PC analogue with five methylene

groups has a similar transition temperature at lowpH indicating that the large head group, now witha net positive charge, can be accommodated in thegel phase without destabilizing it. The transitiontemperature of DPPC at low pH is not as high.This may be because the positive charge on thehead group of DPPC causes more repulsion thanfor the analogue with five methylene groups, orbecause of incomplete protonation of DPPC. Thenearness of the positively charged quaternary am-monium group to the phosphate of PC may lowerthe intrinsic pK of the phosphate, or the increasein positive charge of the bilayer surface as thephosphate becomes protonated may repel protonsresulting in a higher surface pH and a decrease inthe apparent pK. Addition of palmitic acid toDPPC at pH 1 causes phase separation of twopopulations of DPPC, i) a complex of fatty acidand DPPC - + ) with a transition temperature of65 C see subsection II-E), and ii) another lipiddomain which melted at 61.4C [53]. This latter


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p o p u l a t i o n m a y b e a p o p u l a t i o n o f D P P C + )w h i c h i s c o m p l e t e l y p r o t o n a t e d , e i t h e r p u r e o rm i x e d w i t h p a l m i t i c a c id . T h e o c c u r r e n c e o f tw op o p u l a t i o n s o f D P P C s u p p o r t s t h e c o n c l u s i o n t h a tD P P C i s n o t c o m p l e t e l y p r o t o n a t e d a t l o w p H .T h i s r e s u l t a ls o su g g e s t s t h a t t h e t r a n s i t i o n t e m -p e r a t u r e o f D P P C + ) m a y b e c l os e t o 6 1 C ,s i m i l a r t o t h a t o f t h e P C a n a l o g u e w i t h f i v e m e t h -y l e n e s a t l o w p H .

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 h a s l o n g b e e nh e l d r e s p o n s i b l e f o r t h e h i g h t r a n s i t i o n t e m p e r -a t u r e o f P E a t n e u t r a l p H . T h e p H d e p e n d e n c e o ft h e p h a s e - t r a n s i t i o n t e m p e r a t u r e s o f P E a n d P Sa r e s h o w n i n F i g s . 4 a n d 5 . T h e s e w e r e d e t e r m i n e db y S e d d o n e t al . [ 8 5] a n d C e v c e t al . [ 42 ], r e s p e c -

160 rPEt~14 3 ] 2 tM NoCI

a120T

A 8 01 L~ 60[ * C ) 0 t ' 0 ~ ~ ~ ~ ~ ~

l b 2p H

Fig . 4 . pH d ependen ce o f the ge l to l iqu id-c rys ta l l ine ) an dlamel la r to hexagona l [2 ) phase - t rans i t ion tem pera tu res o fd idodecy lphosph a t idy le thano la rn ine d ispe rs ions in 2 .4 M N aCI,de te rm ined by Seddon , Cev c and M arsh [85] . Al l tempera tu resw e re d e te rmin e d b y D S C e x c e p t th e d a ta p o in t i n p a re n th e s esa t p H 9 . 2 8 w h ic h w a s d e te rmin e d f ro m a n X - ra y c o n t in u o u stempera tu re scan . The dashed l ine ind ica tes the appearance o fadd i t iona l d i f f rac t ion l ines in the reg ion o f the lamel la r tohexagona l t rans i t ion . The TH va lues ly ing above the so l id l inea t p H v a lu es f ro m 4 to 7 w e re o b ta in e d w i th p h o s p h a te b u f fe r.Some buffe rs y ie lded a cons tan t va lue o f Tr~ over th is rangewhile ace t ic and fo rmic ac id buffe rs abo l ished the lameUar tohexagona l ca lo r imetr ic t rans i t ion . Reproduced from Ref . 85w i th p e rmis sio n f ro m th e a u th o r s a n d th e A me r ic a n C h e mic a lSociety.

361

tively. Transition temperatures at selected pH val-ues for these lipids in different ionization statesand containing palmitoyl chains are given in Ta-ble III. At pH values up to 9 for PE and 4 for PS,under the conditions used, these two lipids eachhave a hydrogen bond donating NH~ group anda hydrogen bond accepting PO4- group. In thesepH ranges, their phase-transition temperatures areat a maximum (Figs 4 and 5A, respectively), and

T A B L E I I IP H A S E - T R A N S I T I O N T E M P E R A T U R E S , T o, O F R E P U L -S IV E L Y C H A R G E D A N D I N T E R A C T I V E S TA T E S O FG L Y C E R O L - B A S E D P H O S P H O L I P I D S W I T H C 16C H A I N S

L ip id C h a rg e T C ) p H R e fs .Repuls ive ly charged s ta tes

D P P C - + ) 4 2 > 3 7 3 , 5 5D P P E - ) 41 12 7DP PG - ) 41 > 4 44D P P A - - ) 4 5 1 1 5 4D P P M - ) 4 4 > 6 8D P P S - - ) 3 2 1 3 4 2card io l ip in _ ) a 40 7 179

In te rac t ive s ta tesD P P E - + ) 64 7 40DP PE - , + 1 ) b 66 1 4 ,7 ,56-

or 0) 58,85D P P S _ _ + ) c 55 7 42, 43D P P S - + ) 69 ~ 65 1 42~ 6 2 dDP PA - ) 71 7 54DP PA - ) 73 4 54D P P G - ) 61 2 44,60or 0) eD P P M - )D H M G D G 0) fD ip a lmi t in 0 )

61 4 864 4150 ,63 g 47

a See Tab le I , foo tno te c .b B y e x t ra p o la t io n f ro m me a s u re d v a lu e s fo r P O P E , D H P Ea n d D D P E . C o mp le te p ro to n a t io n ma y n o t b e a c h ie v e d .c Par t ia l ly repu ls ive and pa r t ia l ly in te rac t ive .d Tem pera tu re decreases with inc reas ing hydra t ion . A T~ of

72 o C was repor ted f o r th is charge s ta te by M acDo na ld e t a l .[43].e I t has been assumed tha t DPPG was in the neu tra l s ta te a t

low pH. How ever , a s d iscussed in the tex t , i t may be in the- ) s tate . E ven i f in the neu tra l s ta te , i t shou ld be ab le toin te rac t in te rmolecu la r ly by hydrogen bonding .r See Tab le I , foo tn o te b .g See Tab le I , foo tno te d .


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5 0 t D M P S

q r r : ~ - 4 ~ - ~ , . . . . . ;_G / I ' ] " F T I ' " ~ - ' J ~ ' T ~ , ~ T 4 - T~ J ,_ I 1" F " T r ' . 1 t q

3O0 1 2 3 z . 5 6

p H

o ~ 7 o r

: i2O

' , , . 1DMPS

pHF i g . 5. p H d e p e n d e n c e o f t h e g e l t o l i q u id - c r y s t a ll i n e p h a s e - t r a n s i t i o n t e m p e r a t u r e o f D M P S A ) a t a c i d i c p H a n d B ) a t a l k a l in e p H ,d e t e r m i n e d b y C e v c , W a t t s , a n d M a r s h [ 42 ]. T h e i o n i c s t r e n g t h w a s c o n s t a n t t h r o u g h o u t a t J = 0 .1 . T r a n s i t i o n t e m p e r a t u r e s w e r ed e t e r m i n e d b y m o n i t o r i n g t h e h e i g h t o f t h e E S R s p e c t r u m o f a p a r t i t i o n i n g s p i n l a b e l. I n A ) t r a n s i t i o n te m p e r a t u r e s O ) w e r e s e e no n l y o n t h e f i r s t h e a t i n g s c a n a n d p r o b a b l y r e f l e c t a l e s s h y d r a t e d o r c r y s t a l l i n e s t a t e . T h e i n t e r m e d i a t e s e t o f t e m p e r a t u r e s w a so b s e r v e d o n s u b s e q u e n t s c a n s o f d i s p e r s io n s w h i c h w e r e n o t i n c u b a t e d a t h i g h t e m p e r a t u r e . T h e l o w e s t s e t o f t r a n s i t io n t e m p e r a t u r e si n d i c a t e d a t lo w p H t ) w e r e o b t a i n e d f o r d i s p e r s i o n s i n c u b a t e d a t 9 0 o C b e f o r e s c a n n i n g a n d p r o b a b l y r e f l e c t g r e a t e r h y d r a t i o n o ft h e s a m p l e . I n B ) a t p H 1 1 . 5 - 1 4 , o ) c o r r e s p o n d s to t h e m a i n t r a n s i t i o n t e m p e r a t u r e a n d O ) t o t h e p r e t r a n s i t i o n t e m p e r a t u r e , o n l yo b s e r v e d f o r P S i n t h e - - ) s t a te . R e p r o d u c e d f r o m R e f . 4 2 w i t h p e r m i s s i o n f r o m t h e a u t h o r s a n d t h e A m e r i c a n C h e m i c a l S o c i e ty .

providing they have the same fatty acid chainlengths, are similar to each other Table III) andto the other lipids having high phase-transitiontemperatures in Table I. The transition tempera-tures of both are at a minimum at high pH wherethe amine is deprotonated and they have acquireda net negative charge - 1 for PE and - 2 for PS)Figs. 4 and 5B, respectively). DPPE in the - )

state has a similar transition temperature to theother negatively charged lipids in Table II whileDPPS with two negatively charged groups has alower transition temperature.

Although these two lipids at high pH still havea hydrogen bond donating NH 2 group, the netnegative charge on the lipid causes repulsion sothat the stability of the lipid in the gel phasedecreases. Intramolecular hydrogen bond ing be-tween NH 2 and PO4 or COO- for PS) probablyoccurs instead of intermolecular hydrogen bond-ing. Addition of hexadecyl amine to DPPE, in a2:1 molar ratio, at a high pH value where thehexadecyl amine is neutral and the PE has a netnegative charge, restores the high transition tem-perature, indicating that the N H 2 group of aneutral molecule can hydrogen bond intermolecu-larly with the PO4- of PE - ) , although the NH2

group of PE - ) in pure PE cannot [53]. Theseresults indicate that charge repulsion between hy-drogen-donating and accepting molecules may in-hibit intermolecular hydrogen bonding betweenthem, even if suitable hydrogen-donating and-accepting groups are available. Alternatively, theNH 2 of PE - ) may no longer be favorablyoriented to interact with the PO4- of a neighboringmolecule if there is no longer any electrostaticattraction between them.The transition temperature of PS decreases atpH 4-7 where the carboxyl becomes deprotonatedand the lipid is in its - - + ) state with a netcharge of - 1 [42,43]. The fact that the transitiontemperature of PS - - + ) is intermediate be-tween that of the - + ) states of PE and PS andthe - ) state of PE Table III) indicates that thenegatively charged carboxyl weakens the hydrogenbonding interactions rather than abolishing themcompletely. Shielding of the negative charge of PS- - + ) at high ionic strength increases its transi-tion temperature to a similar value as for the- + ) state [42] indicating that when the lateralrepulsion is decreased, the hydrogen bonding in-teractions are strengthened and the stability of thegel phase increases.


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T h e l o w p h a s e - t ra n s i ti o n t e m p e r a t u r e o f D P P Ga t n e u t r a l p H , s i m i l a r t o D P P M a n d o t h e r r e p u l -s ively charg ed l ip ids in Table II , suggests that i tdoes no t i n t e rac t i n t e rmolecu la r ly even though i th a s h y d r o g e n b o n d - d o n a t i n g a n d - a c c e p t i n gg r o u p s . I n a n X - r a y c r y s t a l l o g r a p h y s t u d y o fs o d i u m 1 2-dimyris toyl-sn-glycero-phospho-rac-g lyce ro l , Pascher e t a l . [61 ] have rec en t ly dem on-s t r a t e d t h a t 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 s o fl eng th 2 .7 and 2 .9 ,~ occu r be tween the g lyce ro lO - 1 5 a n d O - 1 6 a n d t h e p h o s p h a t e O - 1 3 a s s h o w nin F ig . 6B. The phosphog lyce ro l head g roup ex -t ends pa ra l l e l to t he b i laye r su r face F ig . 6A) a ss h o w n b y N M R f o r t h e h y d r a t e d g e l p h a s e [ 6 2] .However , i n excess wa te r , t he repu l s ive fo rcesm u s t d o m i n a t e o v e r t h e h y d r o g e n b o n d i n g a t t r a c -t i ve fo rces a s fo r PE - ) so t ha t t he s t ab i l i ty o fthe ge l phase dec reases . In t ramolecu la r hydrogenb o n d i n g m a y t a k e p l a c e i n s t e a d w h e n P G i s h y -d r a t e d . S cr e e ni n g o f t h e c h a r ge o f P G - ) a t h i ghion ic s t reng th ra i ses t he t rans i t i on t empera tu re byon ly 6 Cdeg [69 ], mu ch l e ss t han the 18 -20 Cde g

inc rease found on low er ing the pH [44 ,63 ,64 ]. Th i ssugges ts t ha t PG - ) does no t i n t e rac t i n t e rmolec -u l a r l y b y h y d r o g e n b o n d i n g e v e n w h e n t h e r e p u l -s ive cha rge i s sc reened . I t m igh t be a rgued in s t eadtha t i n t e rmolecu la r hydrogen bond ing does occu r ,bu t t o an equa l ex t en t i n bo th t he ge l and l i qu id -c rys t a ll i ne phases o f PG - ) and , the re fo re , i tdoes no t s t ab i l i ze one phase re l a t i ve t o t he o the r .However , t he fac t t ha t l ower ing the pH ra i ses t het rans i t i on t empera tu re by 18 -20 Cdeg ind i ca t estha t t he ge l phase o f PG a t l ow pH i s s t ab i li zed byi 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 c o n s i d e r a b l ymo re t han the ge l phase o f PG - ) .

T h e m o n o s o d i u m f o r m o f P A a l s o h a s a n e g a -t i ve cha rge bu t a much h igher t rans i t i on t empera -tu re t han PG. X-ray d i f f rac t i on o f s ing l e c rys t a l so f th e m o n o s o d i u m f o r m o f D M P A s h o w s t h at t h ehead g roups a re i n t e rd ig i t a t ed i n to ad j acen t b i -l aye rs F ig . 7 ) w i th t he pho sph a te g roups l i nkeda l t e rna t e ly f rom one b i l aye r t o ano the r by sho r thydrog en bond s o f l eng th 2 .5 A in to l i nea r s t randsl a t e ra l l y sepa ra t ed by row s o f N a + F ig . 8 ) [65 ]. In

2 I S I

1 0 4 ~

A BF i g . 6. P a c k i n g a n d i n t e r a c t i o n s o f t h e p h o s p h o g l y c e r o l h e a d g r o u p s o f D M P G v i e w e d a ) p a r a l l e l a n d b ) p e r p e n d i c u l a r t o th e b i l a y e ri n t e r f a c e. D e t e r m i n e d b y s i n g le c r y s t a l X - r a y d i f f r a c t i o n a n a l y s i s o f s o d i u m 1 2-dimyr i s toy l - sn-glycero-phospho-rac-glycerol b yP a s c h e r , S u n d e ll , H a r l o s a n d E i b l [6 1 ] . T h e r e a r e t w o i n d e p e n d e n t m o l e c u l e s A a n d B ) i n t h e u n i t w h i c h a r e m i r r o r i m a g e s w i t hr e s p e c t t o t h e c o n f i g u r a t i o n a n d c o n f o r m a t i o n o f t h e i r g l y c e r o l h e a d g r o u p s . T h e p o l a r g r o u p s i n t e r a c t l a t e r a l l y b y a n e x t e n s i v en e t w o r k o f h y d r o g e n , i o n ic a n d c o o r d i n a t i o n b o n d s w i t h t h e s o d i u m i o n s . 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 s a r e i n d i c a t e d b y d o t t e dl i n e s a n d i o n i c a n d c o o r d i n a t i o n b o n d s a r e i n d i c a t e d b y b r o k e n l i n e s . T h e c o n t a c t d i s t a n c e s a r e g i v e n i n ,~. A s e q u e n c e o f t w oh y d r o g e n b o n d s s t a r t s a t t h e g l y c e r o l h y d r o x y l o x y g e n O 1 5 ) o f m o l e c u l e A . T h e b o n d s a r e d i r e c t e d v i a h y d r o x y l o x y g e n 0 1 6 ) o fm o l e c u le B a n d t e r m i n a t e d a t a p h o s p h a t e o x y g e n 0 1 3 ) o f a n o t h e r A m o l e cu l e. A c o r r e s p o n d i n g s e q u en c e o f h y d r o g e n b o n d s , b u tw i t h o p p o s i te o r i e n t a t i o n i n t h e b i la y e r pl a n e, r u n s b e t w e e n m o l e cu l e s B - A - B . R e p r o d u c e d f r o m R e f . 61 w i t h p e r m i ss i o n f r o m t h e

a u t h o r s a n d E l s e v i er S c i e n c e P u b l is h e r s .


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A

b aF i g . 7 . M o l e c u l a r p a c k i n g a n d c o n f o r m a t i o n o f m o n o s o d i u m D M P A s e e n a l o n g t w o d i f f e r e n t a x e s p a r a l l e l t o t h e b i l a y e r a sd e t e r m i n e d b y s i n g l e c r y s t a l X - r a y d i f f r a c t i o n a n a l y s i s b y H a r l o s , E i b l , P a s c h e r a n d S u n d e l l [ 6 5 ] . T h e p h o s p h a t e g r o u p s o f t w oa p p o s i n g b i l a y e r s p r o j e c t a l t e r n a t e l y i n t o t h e b i l a y e r i n t e r f a c e a n d f o r m a s i n g l e p h o s p h a t e g r o u p l a y e r c o m m o n t o b o t h b i l a y e r s .T h u s , t w o s o d i u m p h o s p h a t e g r o u p s o f o p p o s i t e ly o r i e n t e d m o l e c u l e s d e t e r m i n e t h e m o l e c u l a r c r o ss s e c t i o n 4 3 .3 ~ 2 ) o f o n e D M P Am o l e c u l e i n t h e p l a n e o f t h e b i l a y e r . R e p r o d u c e d f r o m R e f . 6 5 w i t h p e r m i s s i o n f r o m t h e a u t h o r s a n d E l s e v i e r S c i e n t i f i c P u b l i s h e r s ,

I re land , L td .

the monohydrate form of monosodium dilauroylphosphatidate, however, the phosphate headgroups are no longer interdigitated Pascher andSundell, personal communication). Hydrogenbonds of 2.5 A then link the phosphates of mole-cules in the same bilayer into linear rows sep-arated by rows of Na + and water molecules. Thehigh phase-transition temperature of PA in the- ) state suggests that intermolecular hydrogenbonding also occurs in excess water despite the

presence of the negative charge, as concluded byJacobson and Papahadjopoulos [66] and Eibl andBlume [54,67]. Interestingly, a thioanalog of DPPA1,2-dipalmitoyl-sn-glycero-3-thionphosphate) at

neutral pH had a transition temperature similar tothat of DPPG - ) and DPPC [348]. This wasattributed to weaker hydrogen bonding betweenmolecules of the thioanalog, since S-H hydrogen

bonds are weaker than O -H [15].The behavior of PA - ) is different from that

of PG - ) and PE - ) , since PA - ) apparentlycan participate in intermolecular hydrogen bond-ing in spite of its negative charge. However, in-tramolecular hydrogen bonding is impossible forPA, unlike the latter two lipids. Furthermore, thenumber of possible orientations of the head groupis more limited for PA so that it may not benecessary to have electrostatic attraction of twogroups as in PE - + ) in order to achieve theoptimal orientation for hydrogen bonding. How-ever, it seems unlikely that PA is organized into asregular an array in the hydrated gel phase as inthe crystal. Rotational motion of the lipid mayoccur in the gel phase. Transient hydrogen bond-ing may occur whenever two or more moleculesare correctly oriented. Such hydrogen bonding,


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B

F i g . 8 . P a c k i n g a n d i n t e r a c t i o n s o f t h e p o l a r p a r t o f D M P Av i e w e d A ) p a r a l l e l a n d B ) p e r p e n d i c u l a r t o t h e b i l a y e r i n t e r -f a c e, a s d e t e r m i n e d b y H a r l o s , E i b l , P a s c h e r a n d S u n d e U [ 65 ].T h e o r i e n t a t i o n i n A ) i s i d e n t i c a l to t h a t i n F i g . 7 A e x c e p t t h a tt h e p o l a r r e g i o n o f tw o b i l a y e r s i s sh o w n . I n B ) t h e p h o s -p h a t e s o f t w o a p p o s i n g b i l a y e r s a r e s h o w n , t h e p l a n e o f o n eb i l a y e r a b o ve t h a t o f t h e o t h e r . 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 sa r e i n d i c a t e d b y d o t t e d l i n e s a n d i o n i c b o n d s b y b r o k e n l i n e s .H y d r o g e n b o n d s f o r m b e t w e e n p h o s p h a t e o x y g e n s O 1 3 ) a n dO 1 4 ) o f m o l e c u l e s i n a p p o s i n g b i l a y e r s a n d l i n k t h e p h o s p h a t eg r o u p s o f a p p o s i n g l a y e r s a l t e r n a t e l y i n t o a z i g - z a g r i b b o ne x t e n d i n g a l o n g t h e b i l a y e r i n t e r f a c e i n t h e b d i r e c t i o n . T h e s er o w s o f p h o s p h a t e r i b b o n s a r e s e p a r a t e d b y r o w s o f s o d i u mi o n s i n t h e a d i r e c t i o n . R e p r o d u c e d f r o m R e f . 6 5 w i t h p e r m i s -s i o n f r o m t h e a u t h o r s a n d E l s e v i er S c i en t i fi c P u b l i s h e r s Ir e l a n d ,

L t d .

h o w e v e r , s h o u l d s t a b i l i z e t h e g e l p h a s e , w h e r e i th a s a g r e a t e r p r o b a b i l i t y o f o c c u r r i n g t h a n i n t h el i q u i d - c r y s t a l l i n e p h a s e .

T h e e f f e c t o f p H o n t h e t r a n s i ti o n t e m p e r a t u r eo f P A [ 8 , 5 4 , 6 7 ] , s h o w n i n F i g . 9 , s h o w s t h a t t h er e l a t i v e s t a b i l i z a t i o n o f t h e g e l p h a s e o v e r t h el iq u i d - cr y s ta U i n e p h a s e i s g r e a t e r f o r P A ( - ) t h a nf o r t h e c o m p l e t e l y i o n iz e d f o r m , P A ( - - ) . T h et r a n s i ti o n t e m p e r a t u r e i s h i g h o v e r t h e p H r a n g e4 - 1 0 w h e r e t h e s t a t e o f d i s s o c i a t i o n i s 0 . 5 t o 1 . 5

365

8 0 -U

?oo' / O - L ~ o/ / ~ o /

~ 60-,,,I

o_zn,-

1 2 H H G P

y - TT - GP

PROTON CONCENTRATION pH)Fig. 9. pH dependenc e of the gel to liquid-crystalline phase-transit ion temperature of DT PA (O) and DH PA (O ) deter-mined by Ei bl [8]. The lipid was in distilled w ate r at aconcentration of 1 m g/ml. The pH was adjusted by theaddition of dilute NaOH. The dotted lines indicate regionswhere two transitions were sometimes observed, a lower and anupper transition. Reproduced fro m Ref. 8 w ith permissionfrom the author and Academic Press, Inc.a n d h y d r o g e n b o n d - d o n a t i n g a n d - a c c e p t in gg r o u p s a r e p r e s e n t ( s e e F i g . 1 0 ) . W h e n t h e l i p i d i sc o m p l e t e l y i o n iz e d t h e t e m p e r a t u r e d r o p s b y a b o u t1 4 - 2 2 C d e g , a n d b e c o m e s s i m i la r t o t h a t o f t h er e p u l s i v e l y n e g a t i v e l y c h a r g e d l i p i d s i n T a b l e I I i ft h e c h a i n l e n g t h i s s i m i la r ( s ee T a b l e I I I f o r c o m -p a r i s o n o f li p id s w i t h p a l m i t o y l c h a i n s) . W h e n t h el ip i d is c o m p l e t e l y p r o t o n a t e d , t h e t e m p e r a t u r ed r o p s b y 6 - 1 4 C d e g ( d e p e n d i n g o n s p ec ie s o f P A ).H o w e v e r , t h e li p id i s t h e n i n a n a n h y d r o u s c r y s t a l -l i n e f o r m a n d t h e t r a n s i t i o n t e m p e r a t u r e m a y n ol o n g e r b e c o m p a r a b l e t o t h a t o f h y d r a t e d g e lp h a s e l i p i d s . T h e f a c t t h a t i t b e c o m e s d e h y d r a t e dw h e n c o m p l e t e ly p r o t o n a t e d m a y b e d u e t o g re a t e ri n t er m o l e c u l a r h y d r o g e n b o n d i n g i n t h is f o r m t h a nw h e n p a r t i a ll y i o n i z e d .

T h e m a x i m u m i n th e t r a ns i ti o n t e m p e r a t u r e o f


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3 6 6

CHARGE PHOSPH ATIDIC ACID

0 0

0 5

1 0

1

2 0

H H H H H0 O O O OP O -O R P O - O R P O - O R P O - O R P O - O R

o o oH H H H H

H H0'' H' 8 H' O O 0iPO OR PO OR PO OR PO OR PO OR

0 0 O . H OO ~ %H oOH N

. . H . e . H . e . . HO ' 0 0 ' O OPO OR PO OR PO OR PO OR PO OR

i i i i.o o H o o oH '

8 - ' ' - 8 8 8 -'t iPO OR PO OR PO OR PO OR PO ORi i. . .o o o . . o o

A 0 0 0 0 0O 0 0 O Oi iPO OR PO OR PO OR PO OR PO OR

Fig. 10. Depiction of the intermolecular hydrogen bondinginteractions in the pola r region of different ionization states o fPA by Eibl [8]. W hen com pletely deprotonated the hy drogenbonds are broken and stabilization is lost. Reproduced fromRef. 8 with permission fro m the a utho r and A cademic Press,Inc.

P A o c c u r s a t a s t a t e o f d i s s o c i a t i o n o f 0 . 5 . T h es m a l l d r o p i n t r a n s i t i o n t e m p e r a t u r e f r o m p H4 - 1 0 i s d u e t o t h e i n c r e a s i n g c o n c e n t r a t i o n o fn e g a t i v e l y c h a r g e d l i p i d a s t h e s t a t e o f d i s s o c ia -t i o n i n c r e a s e s t o 1 .5 . A t p H 4 t h e p K 1 ) t h e r a t i oo f n e g a t i v e l y c h a r g e d - t o - n e u t r a l s p e c i e s i s 1 : 2 a n dm o s t o f t h e n e g a t i v e l y c h a r g e d m o l e c u l e s w i ll b ec o n s t a n t l y i n v o l v e d i n h y d r o g e n b o n d i n g e v e n i ft h e se h y d r o g e n b o n d s a r e c o n t in u a l l y b e i n g b r o k e na n d r e f o r m e d . H o w e v e r , a t h i g h e r p H , t h i s r a t i oi n c r e a s e s a n d t h e p r o b a b i l i t y o f n o n - p a r t i c i p a t i o no f s o m e n e g a t i v e l y c h a r g e d l i p i d i n h y d r o g e nb o n d i n g a t a n y o n e m o m e n t i n c r e a s e s . T h i s n e g a -t i v e l y c h a r g e d l i p i d w i l l c a u s e c h a r g e r e p u l s i o n

a n d w i l l d e s t a b i l i z e t h e g e l p h a s e l o w e r i n g t h et r a n s i t i o n t e m p e r a t u r e . A s i m i l a r g r a d u a l d r o p o fa f e w d e g r e e s i n t h e t r a n s i t i o n t e m p e r a t u r e o f P Eo c c u r s a s t h e p H i s i n c r e a s e d f r o m l o w p H t o p H3 a t h i g h io n i c s t r e n g t h F i g . 4 ) , o r p H 1 1 a t l o wi on i c s t r eng t h F i g . 11 ). Th i s i l l u s t r a t e s t he ab i l i t yo f e v e n a s m a l l a m o u n t o f c h a r g e d l i p id , w h i c h i st e m p o r a r i l y n o t i n v o l v e d i n h y d r o g e n b o n d i n g , t od e s t a b i l i z e t h e g e l p h a s e a n d l o w e r t h e t r a n s i t i o nt e m p e r a t u r e . T h e m u c h l a r g e r d r o p i n t r a n s i t i o nt e m p e r a t u r e w h i c h o c c u r s f o r P S w h e n t h e c a r b o -x y l b e c o m e s d e p r o t o n a t e d F i g. 5 A ) r e f le c t s t h ep r e s e n c e o f o n e n e g a t i v e l y c h a r g e d g r o u p p e rm o l e c u l e w h i c h e i t h e r d o e s n o t p a r t i c i p a t e a t a l l inh y d r o g e n b o n d i n g o r e l s e s h a r e s t h e h y d r o g e nb o n d d o n o r s w i t h t he o t h e r n e g a t i v e ly c h a r g e dg r o u p . I n e i t h e r c a s e , t h e c h a r g e r e p u l s i o n i s g r e a t e ra n d d e s t a b i l i z e s t h e g e l p h a s e m o r e .T h e d i f f e r e n c e i n t r a n s it i o n t e m p e r a t u r e b e -t w e e n t h e p u t a t i v e h y d r o g e n b o n d i n g s t a t e s o f P E- + ), P A - ) , a n d P S - - + ) a n d th e i r r e p u l -

s iv e ly c h a r g e d s ta t es , P E - ) , P A - - ) a n d P S- - ) , i s 2 2 - 2 5 C d e g T a b l e I I I ) [ 7 ,4 2 , 44 , 54 , 67 ] .

T h e s e l a r g e d i f f e re n c e s c a n n o t b e e n t i r e lya c c o u n t e d f o r b y e l e c t r o s t a t i c r e p u l s i o n [ 4 , 8 ] .S c r e e n i n g o f th e c h a r g e a t h i g h i o n i c s t re n g t h d o e sn o t r a i se t h e te m p e r a t u r e t o th o s e o f P E - + ),P A - ) a n d P S - - + ) , c o n s i s t e n t w i t h t h e i d e at h a t t h e s e i o n i z a t i o n s t a t e s a r e i n v o l v e d i n 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 [4 2]. A l a r g e i n c r e a s ei n tr a n s it io n t e m p e r a t u r e o f P G a n d P M o f 1 7 - 2 0C d e g T a b l e I I I ) o n l o w e r i n g t h e p H t o 4 i n t h ec a s e o f P M [ 7,6 8] a n d e v e n l o w e r f o r P G [ 4 4 ,6 3 ,6 0 ],a l s o c a n n o t b e a c c o u n t e d f o r b y e l e c t r o s t a t i c e f -f ec t s [4 ,8 ,69 ], s ugges t i ng t h a t t he s e l i p i d s m u s t a l s ob e i n v o l v e d i n h y d r o g e n b o n d i n g a t lo w p H .

E i b l a n d c o l l a b o r a t o r s [ 4 , 8 ] i n v e s t i g a t e d t h ee f f e c t o f lo w e r p H o n P M u s i n g l o w i o n ic s t r e n g t hs o l u t i o n s a n d f o u n d t h a t a f u r t h e r d e c r e a s e i n p Hb e l o w 4 c a u s e s t h e t r a n s i t i o n t e m p e r a t u r e t o d r o pb y 7 - 9 C d e g F i g. 1 1 ) s im i l a r to t h e e f f e c t o f lo wp H o n P A F i g. 9) . T h e y s u g g e st e d t h a t a t a p Hw h e r e t h e t r a n s i t i o n t e m p e r a t u r e o f P M i s a t am a x i m u m , t h e l i p i d i s i n a s t a t e o f d i s s o c i a t i o n o f0 .5 a n d 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 o c c u r sb e t w e e n t h e p r o t o n a t e d a n d i o n i z e d f o r m s o f t h el ip i d. W h e n t h e l ip i d is c o m p l e t e l y p r o t o n a t e d t h et r a n s i t i o n t e m p e r a t u r e f a l l s . T h u s , t h e d i f f e r e n c ei n t r a n s i t i o n t e m p e r a t u r e b e t w e e n t h e n e u t r a l ,


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80-~ ~ 0 - - 0 - . . - - 0 ~ O 0

~ 6 0 - **, t2-1-tH- 6PE_ o z . ~ \ 1 2 P P G P M e I

k o / I

4 0 I O/O'O~t2-MM-GPMel3 6 9 1 2P R O T O N C O N C E N T R A T I O Np H )

F i g . 1 1 . p H d e p e n d e n c e o f t h e g e l to l i q u i d - c r y s t a l li n e p h a s e -t r an s i ti o n t e m p e ra t u re o f D H P E ) , D P P M * ) a n d D M P MO ) d e t e r m i n e d b y E i b l [ 8 ] . R e p r o d u c e d f r o m R e f . 8 w i t h

p e r m i s s i o n f r o m t h e a u t h o r a n d A c a d e m i c P r e s s , I n c .

non-hydrogen bonding states and the repulsivelysingly charged states was concluded to be only8-12 Cdeg, a difference which can more easily beaccounted for by electrostatic repulsion [4,8].

However, when completely protonated PA andpossibly also PM are in a crystalline dehydratedstate in which hydrogen bonding may still occur.It may not be valid to compare the transitiontemperature of this crystalline state to that of themore hydrated state when partially ionized. Thusit is difficult to know what the transition tempera-ture of the neutral, non-hydrogen bonding specieswould be if it were in the same kind of bilayerphase as when ionized. Cevc et al. [69] showedthat screening of the charge of PG - ) at highionic strength raised the transition temperature byonly 5.5-6.5 Cdeg, indicating that the electrostaticcontribution is of this magnitude. If hydrogenbonding occurs in addition to elimination of elec-trostatic repulsion, it raises the transition tempera-

367

ture by an additional 8-16 Cdeg. Thus, mereelimination of repulsion does not stabilize the gelstate over the liquid-crystalline state as much asintroduction of hydrogen bonding interactions.

The maximum transition temperatures reportedfor PG, PE, and PS at low pH may also reflecthydrogen bonding between phosphate acid andanion forms. A drop in transition temperature onlowering the pH further might be expected tooccur for these lipids as for PM and PA but hasnot yet been reported. It may be impossible tocompletely protonate PG, PE, and PS due to thelow intrinsic pK values of their phosphate groupsor to the decreased attraction of protons to thebilayer surface as the lipids become more proto-nated, and in the case of PE and PS, more posi-tively charged. Phosphate acid-anion hydrogenbonding for these lipids also may lower theirintrinsic pK values as it appears to do for fattycarboxylic acid-anion mixtures see subsection II-B). Alternatively, these lipids, or at least PG,which is uncharged in its completely protonatedstate, may be completely protonated at low pHbut continues to interact by intermolecular hydro-gen bonding. Thus, the transition temperature ofPG at low pH, which is similar to that of PE- + ) and PA - ) in their interactive states, may

reflect the completely protonated form which hy-drogen bonds intermolecularly via the glycerolOH and P-OH groups. In support of this, ad-dition of palmitic acid to DPPG at pH 1 does notcause phase separation of two populations ofDPPG as it does for DPPC, suggesting that onlyone population, the completely protonated form,is present [53].

Studies with neutral unionized glycolipids indi-cate that it is not necessary for the lipid to haveionizable groups in order to participate in inter-molecular hydrogen bonding. The high transitiontemperature of the glycerol-based glycolipidMGDG, which is similar to that of PE - + ) andPA - ) of identical hydrocarbon chain composi-tion Table I), indicates that intermolecular hydro-gen bonding occurs between the sugar hydroxyls[41,48,70,71]. Intermolecular hydrogen bondingbetween neighboring molecules of the neutral lipiddipalmitin may be responsible for its high transi-tion temperature also and for its formation of adehydrated crystalline phase in water Table I).


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368Single crystal analysis of 2,3-dilauroyl-D-glycerolshowed that it forms bilayers with intermolecularhydrogen bonds of length 2.8 ,~ between the hy-droxyl head group and the v-carbonyl oxygen ofthe ester linkage of adjacent molecules [72]. Thus,it is reasonable to suppose that PG may alsointeract by intermolecular hydrogen bonding in itscompletely protonated state.

The various lipids with different head groupsand in different ionization states, can be classifiedinto two categories on the basis of their phasetransition temperatures (Table III). There is nocorrelation between head group size or charge inthese categories but there is a correlation withtheir capacity for intermolecular hydrogen bond-ing interactions. Those which have a phase-transi-tion temperature of 41-45 C (also including thoselipid analogues listed in Table II) are dominatedby repulsive forces which stabilize the liquid crys-talline phase. Those which have a phase t ransitiontemperature of 61-73 C are dominated by inter-molecular hydrogen bonding interactions whichstabilize the gel phase.

Van Dijck et al. [73] noted that the large dif-ference in transition temperature between PE andPC disappears if there is a is unsaturated chainin both the 1 and 2 positions. This suggests thatthe large molecular volume taken up by the hydro-carbon chains may cause too much lateral sep-aration for hydrogen bonding to occur for PEeven in the gel phase. Branched chains may have asimilar effect. Diisopalmitoyl PE goes into twophases, one of which has a similar transit ion tem-perature as the corresponding type of PC [74]. Theother, in which interbilayer hydrogen bonding maytake place, since it occurs more when the lipid isconcentrated, has a transition temperature only 13Cdeg higher than PC. Thus, although isobranchedchains do not have a large disordering effect onthe gel phase, this result suggests that they mayinhibit lateral hydrogen bonding for PE. Morenatural, 1-saturated, 2-unsaturated forms of PEstill have transition temperatures 15-25 Cdeghigher than the corresponding PC, however, asdetermined on POPE and POPC, SOPE and SOPC,and on egg PC and the PE prepared from it bytransphosphatidylation [4,56,75,76].PE and some of the other hydrogen bondinglipids go into other phases in which intermolecular

hydrogen bonding is also undoubtedly involved. Ifwater is added to PE at a temperature below itsphase transition temperature it forms a crystalline,dehydrated or nearly dehydrated phase whosetransition to the liquid-crystal phase occurs at ahigher temperature and with a higher enthalpythan the gel to liquid-crystal phase transition[59,77-79]. Incubation of a previously heated gelphase sample at 2C for prolonged periods canallow the crystal phase to reform [80,81]. The lesshydrated phase of PS ( - + ) with the highesttransition temperature (Fig. 5A) may also be asimilar crystalline phase [82]. Saturated forms ofMGDG and DGDG in which the chains areester-linked also undergo a transition from ametastable phase to a stable gel phase which has ahigher phase-transition temperature and enthalpythan the metastable phase [48,83]. The stable phasemay be less hydrated than the metastable phase.Intermolecular hydrogen bonding must occur inthe less hydrated stable.phases of these lipids alsoand would be expected to be stronger than thatfor the more hydrated gel phase. Intermolecularhydrogen bonding in these less hydrated phasesmay be interlamellar rather than intralamellar.

The effect of modification of the head group ofPE on the transition temperatures of its gel andcrystalline phases, TG and TcR, respectively, hasbeen determined in a number of studies[80,81,84-87]. The head group was modified inways which affect its size, hydrophobicity, andability to hydrogen bond. Data from one of themost comprehensive of these studies [80] is givenin Table IV.Methylation of the amine decreases T~ but in anon-linear way with increasing number of methylgroups, suggesting that increasing head group sizeis not responsible. Data from a number of studiesshow that one methyl group decreases the temper-ature by 24-28 of the difference between PE andPC, while two methyl groups decreases it by61-76 [80,81,84-87]. The effect of an ethyl groupis between that of one and two methyls [80,81].Alkylation of the amine would be expected todecrease the probability of hydrogen bonding in-teractions due to steric hindrance, with one smallgroup having less effect than two groups or onelarger group. Interestingly, X-ray d iffraction anal-ysis of a single crystal of the dimethyl form of


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T A B L E I VE F F E C T O F M O D I F I C A T I O N S T O E T H A N O L A M I N ET Y P E H E A D G R O U P O N T R A N S I T IO N T E M P E R A -T U R E S a

C H 2 - O RR O - C H O

C H 2 - O - P - O - XO -

Group X TG b TCR bC) C)C H 2 - C H 2 - N H ~- D M P E ) 5 0.1 5 7.3C H 2 - C H 2 - N H 2 C H 3 ) + M e D M P E ) 4 2.7 cC H 2 - C H 2 - N H C H 3 ) ~- M e 2 D M P E ) 3 1.4 cC H E - C H E - N C H 3 ) ~- D M P C ) 23 d cC H 2 - C H 2 - N H 2 C H 2 - C H 3 ) + 37.7 59.3

C H 3C H 2 - C H - N H ~ 4 3.4 ) e 64.4 , 73 .2

C H 3C H 2 - C - N H ~ 3 7 .5 ) e 8 0, 8 1

CH3C H 2 - c a 3

C H 2 - C H - N H ~ 3 4.2 ) e 6 2.0

C H 2 ) 3 - N H ~ 4 1 .9 5 2.5C H 2 ) 4 - N H ~ 3 4.4 5 1.6

a R i s m y r i s t i c a c i d. C h a r g e o n h e a d g r o u p i s - + ) . C o m m o nn a m e i s g i v e n i f t h e r e i s o n e . A l l d a t a f r o m R e f . 8 0 e x c e p tw h e r e n o t e d .

b T ~ i s t e m p e r a t u r e o f g e l to l i q u i d c r y s t a l p h a s e t r a n s i t i o n ,T R i s t e m p e r a t u r e o f h i g h e n t h a l p y c r y s t a l l i n e t o l i q u i d -c r y s t a l p h a s e t r a n s i t io n .

c C r y s t a l li n e p h a s e d o e s n o t f o r m .d F r o m R e f . 5 5 .e V e r y l o w e n t h a l p y p e a k s , o f t e n p r e c e d e d o r f o l l o w e d b y

e x o t h e r m i c t r a n s i t i o n s s e e t e x t) .

2 3-di lauroyl - rac-glycero- l -phospho -N N-d i rne th -y l e thano lamine showed tha t i n t e rmolecu la r hydro -g e n b o n d i n g o c c u r s b e t w e e n t h e p h o s p h a t e o x y g e nof one li p id and the s ing le N - H o f ano the r [88 ].H o w e v e r , t h e h e a d g r o u p i s e x t e n d e d p e r p e n d i c u -

369

l a r t o t he b i l aye r p l ane and i s i n t e rd ig i t a t ed i n tothe head g roup l aye r o f t he ad j acen t b i l aye r . Thus ,t h e p h o s p h a t e o f a m o l e c u l e in o n e b i l ay e r h y d r o -g e n b o n d s w i t h t h e a m i n e o f a m o l e c u l e i n t h ead jacen t b i l aye r . In con t ras t , PC, PE and PG havethe i r head g roups o r i en t ed pa ra l l e l t o t he b i l aye rp l ane in bo th t he c rys t a l and the hydra t ed ge lphase [16,19,61,62]. The fact that Me2PE is d i ffer-en t sugges t s t ha t a pe rpend icu l a r o r i en t a t i on s ig -n i f i can t ly i nc reases t he p robab i l i t y o f i t s hydrogenbond ing wi th ano the r mo lecu le . Thus , t he me thy l -a t e d f o r m s o f P E c a n h y d r o g e n b o n d i n te r m o l e c u -l a r ly i f a su i t ab l e o r i en t a t i on o f t he head g roupscan be ach i eved ; however , a pa ra l l e l o r i en t a t i onmay no t be ve ry su i t ab l e , pa r t i cu l a r ly fo r t hed imethy la t ed fo rm.

However , i t i s un l ike ly t ha t a pe rpend icu l a ro r i e n t a t i o n o c c u r s f o r M e 2 P E i n t h e p r e s e n c e o fw a t e r . I n d e e d , b o t h m o n o - a n d d i - N - m e t h y l a t i o nappear t o p reven t fo rmat ion o f t he c rys t a l l i nephase , i n wh ich in t e rb i l aye r hydrogen bond ingprobab ly occu rs [80 ,81 ,87 ] . In con t ras t , monoe th -y l a t i o n o f P E d o e s n o t p r e v e n t f o r m a t i o n o f t h ec rys t a l l i ne phase . In fac t i t ra i ses t he t empera tu reo f it s t rans i ti on to t he l iqu id -c rys t a l phase Tab leIV) indicat ing a s tabi l iz ing effect on the crysta l l inephase . Pe rhaps , because o f i t s g rea t e r hydro -phob ic i t y o r s t e r i c fac to rs , t he monoe thy l headgroup i s more l i ke ly t o be ex t ended pe rpend icu l a rto t he b i l aye r i n t he p resence o f wa te r , so t ha t i tcan hydrogen bond wi th l i p id i n t he appos ingb i l a y e r a n d b e c o m e d e h y d r a t e d . T h e l e s s h y d r o -p h o b i c a n d s m a l l e r m o n o m e t h y l f o r m m a y h a v ethe more usua l pa ra l l e l o r i en t a t i on and hydrogenbond on ly l a t e ra l l y w i th molecu les i n t he sameb i l aye r . La t e ra l hydrogen bond ing o f t he d ime thy lf o r m m u s t b e g r e a tl y w e a k e n e d b y t h e s u b o p t i m a lorienta t ion and s ter ic effects .

A n i n c r e a s i n g n u m b e r o f m e t h y l e n e g r o u p s b e -tween the phospha te and amine a l so dec reases t het rans i ti on t empera tu re , a l t hough wi th fou r me thy l -enes i t is st i l l higher than that of PC [80,81,85].The fac t t ha t t he c rys t a l l i ne phase s t i l l fo rmssuggests that these head groups can s t i l l par-t i c ipa t e i n i n t e rb i l aye r hydrogen bond ing . Themos t l i ke ly reason fo r t he dec rease i n TG is thatt h e s e l o n g er h e a d g r o u p s c a n h y d r o g e n b o n d l a te r -a l l y w i th ne ighbor ing l i p ids nea r ly a s we l l i n t hel iquid-crysta l phase as in the gel phase and, thus,


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370do not stabilize the gel phase as much asethanolamine does.

Addition of more than one sugar to the headgroup of glycolipids may have a similar effect.DSDGDG has a transition temperature 20-30Cdeg below that of the corresponding MGDGand lower than DSPC [48,70]. Although the sugarhydroxyls of DGDG should also be able to par-ticipate in intermolecular hydrogen bonding, thishydrogen bonding may be able to occur equallywell in the liquid-crystal phase, because of thelarger size of the head group, and, hence, may notstabilize one phase relative to the other. However,two synthetic diglycosyldihexadecylglyceridescon-raining maltose and cellobiose have transitiontemperatures which are 11-15 Cdeg higher thanthe similar chain length DPPC, suggestive of hy-drogen bonding which does stabilize the gel phasein these cases [89].C-2 alkylation of the ethanolamine head groupwas found to decrease TG and increase TCR, some-times greatly as for C-2 dimethylation [80,81] (Ta-ble IV). This led to the conclusion that head groupsize and hydrophobicity are more important indetermining the transition temperatures than hy-drogen bonding. However, the enthalpies of thegel to liquid-crystal phase transitions observed forC-2 alkylated lipids are very small and are usuallypreceded or followed by exothermic transitionsand, at a higher temperature, by the large enthalpycrystalline to liquid-crystal phase transition. It isnot necessary to incubate these samples at lowtemperatures to convert them to the crystallinephase. Raman spectroscopy showed that duringthese exothermic transitions a decrease in thenumber of gauche conformers and an increase inchain packing density occurs [81]. Thus, the smalllower temperature endothermic and exothermictransitions observed for these lipids involve transi-tions to the crystalline phase, either directly fromthe gel phase or from the liquid-crystal phase; thelatter exists only transiently. The very rapid con-version of the liquid-crystal phase to the crystal-line phase suggests that the liquid-crystal phase ofthe C-2 alkylated PE s may be less hydra ted andmore involved in hydrogen bonding than that ofunmodified PE, due to the greater hydrophobicityof the C-2 alkylated forms. Thus, the liquid-crystalphase is more stable relative to the gel phase than

is the case for unmodified PE and the transitiontemperature is lower. The combined factors ofintermolecular hydrogen bonding and decreasedhydration forces caused by the greater by_drophobicity of the head groups also stabilize thecrystalline phase relative to the liquid-crystal phaseof the C-2 alkylated PE s, thus raising TCR. Thus,hydrogen bonding interactions must be one of thedetermining factors in the behavior of the C-2alkylated forms of PE, even though they have alower TG than unmodified PE.H C2. Oth er properties

Other properties of glycerolipids which can becorrelated with the repulsive or attractive forcesbetween their head groups are their molecularareas in monolayers, bilayer permeability, theirdegree of hydration, the change in enthalpy andentropy during the gel to liquid-crystalline phasetransition, and their molecular motion and confor-mation determined by use of spectroscopic tech-niques.Lack of reactivity of the amino group of purePE and PS with 2,4,6-trinitrobenzenesulfonic acidand increased reactivity of PE when mixed withPC led to the early suggestion that the amine isinvolved in inter- or intramolecular hydrogenbonding [90]. The permeability of PE vesicles isless than that of PC and does not depend on acylchain length, in contrast to PC, indicating that thereduced permeability of PE is controlled by itshead group interactions [91]. The permeability ofPE vesicles and hydrolysis of PE by phospholipaseA 2 do not increase at the phase-transition temper-ature, in contrast to PC [91,92], suggesting thatintermolecular hydrogen bonding of PE may per-sist in the liquid-crystalline state.The hydrogen bonding lipids PE (- + ) and PA( - ) pack more closely in monolayers than PC[93,94]. MGDG forms a more condensed mono-layer than DGDG [95-98]. However, the collapsepressure is higher for the distearoyl form of DGD Gwhich was attributed to its greater number ofhydrogen bonding groups [97]. Hydrogen bondingby DGDG may occur but does not cause it toform a closely packed monolayer because of thelarger si

1987_boggs_lipid intermolecular hydrogen bonding influence on structural organization and membrane...

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