Science, Faculty of
Botany, Department of
Zoology, Department of
DSpace
UBCV
Wallace, Julia C.
Master of Science - MSc
University of British Columbia
Suspensions of sn-2 deuterated ²H-DPPC-d31 containing various concentrations of either lanosterol or cholesterol were compared using deuterium nuclear magnetic resonance spectroscopy (²H-NMR) and differential scanning calorimetry (DSC). The alpha face of cholesterol is smooth whereas a hydroxyl group protrudes from the alpha face of lanosterol. At the sterol concentrations studied, (0%, 5%, 10%, 15%), the phase behaviour of lanosterol and cholesterol was similar. M, profiles showed that the gel to liquid crystalline phase transition is broadened with the incorporation of both sterols and that mixed phase regions exist at comparable concentrations and temperatures. Both lanosterol and cholesterol increase the average order of the phospholipids above the transition temperature. Since both lanosterol and cholesterol produce this ordering, it is proposed that the significant structural feature producing this result is the rigidity of the cholestane ring. T[sub 2e] increases with the incorporation of both cholesterol and lanosterol. This could indicate the importance of the rigid ring structure which may modify slow motions such as the collective motions of the phospholipid chains.
A 2H-NMR STUDY OF THE COMPARATIVE EFFECTS OF LANOSTEROL AND B.Sc., U n i v e r s i t y o f New B r u n s w i c k , 1983 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIOLOGY We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA CHOLESTEROL ON MODEL MEMBRANES by J U L I A C. WALLACE OCTOBER 1985 © J u l i a C. W a l l a c e , 1985 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e The U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head of my Depa r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . DEPARTMENT OF BIOLOGY The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5 D a t e : JANUARY 1986 A b s t r a c t S u s p e n s i o n s of sn-2 d e u t e r a t e d 2H-DPPC-d31 c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s of e i t h e r l a n o s t e r o l o r c h o l e s t e r o l were compared u s i n g d e u t e r i u m n u c l e a r m a g n e t i c r e s o n a n c e s p e c t r o s c o p y ( 2H-NMR) and 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 (DSC). The a l p h a f a c e of c h o l e s t e r o l i s smooth whereas a h y d r o x y l g r o u p p r o t r u d e s f r o m t h e a l p h a f a c e of l a n o s t e r o l . A t t h e s t e r o l c o n c e n t r a t i o n s s t u d i e d , ( 0 % , 5%, 10%,15%), t h e phase b e h a v i o u r o f l a n o s t e r o l and c h o l e s t e r o l was s i m i l a r . M, p r o f i l e s showed t h a t 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 hase t r a n s i t i o n i s b r o a d e n e d w i t h t h e i n c o r p o r a t i o n o f b o t h s t e r o l s and t h a t m i x e d p h a s e r e g i o n s e x i s t a t c o m p a r a b l e c o n c e n t r a t i o n s and t e m p e r a t u r e s . B o t h l a n o s t e r o l and c h o l e s t e r o l i n c r e a s e t h e a v e r a g e o r d e r o f t h e p h o s p h o l i p i d s above t h e t r a n s i t i o n t e m p e r a t u r e . S i n c e b o t h l a n o s t e r o l and c h o l e s t e r o l p r o d u c e t h i s o r d e r i n g , i t i s p r o p o s e d t h a t t h e s i g n i f i c a n t s t r u c t u r a l f e a t u r e p r o d u c i n g t h i s r e s u l t i s t h e r i g i d i t y o f t h e c h o l e s t a n e r i n g . T^Q i n c r e a s e s w i t h t h e i n c o r p o r a t i o n o f b o t h c h o l e s t e r o l and l a n o s t e r o l . T h i s c o u l d i n d i c a t e t h e i m p o r t a n c e o f t h e r i g i d r i n g s t r u c t u r e w h i c h may m o d i f y s l o w m o t i o n s s u c h a s t h e c o l l e c t i v e m o t i o n s of t h e p h o s p h o l i p i d c h a i n s . i i T a b l e o f C o n t e n t s A b s t r a c t i i L i s t o f T a b l e s v L i s t of F i g u r e s v i A c knowledgements i x I . INTRODUCTION 1 A. B a c k g r o u n d 3 B. Phase B e h a v i o u r of P h o s p h l i p i d S y s t e m s -.1 C. C h o l e s t e r o l / P h o s p h o l i p i d Systems 10 D. F l u i d i t y 12 E . Aims o f R e s e a r c h 12 11 . NMR AND DSC THEORY 14 A. D e u t e r i u m N u c l e a r M a g n e t i c R e s o n a n c e 14 B. O r d e r P a r a m e t e r s 17 C. The 2H Powder P a t t e r n S p e c t r u m , D e p a k i n g 17 D. F i r s t Moment 18 E. R e l a x a t i o n 19 F. 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 e r y 20 I I I . MATERIALS AND METHODS 22 A. NMR Sample P r e p a r a t i o n 22 B. NMR Measurements 23 C. C a l o r i m e t r y 24 IV. RESULTS 26 A. I n t r o d u c t i o n 26 B. P u r e DPPC-d3l a t 44°C 26 C. L a n o s t e r o l : D P P C - d 3 l s y s t e m s 29 D. C h o l e s t e r o l : D P P C - d 3 1 Systems 38 E. 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 47 i i i V. DISCUSSION 52 A. O v e r v i e w 52 B. Moments 54 C. R e l a x a t i o n B e h a v i o u r .58 D. R e s o l u t i o n of t h e S p e c t r a l L i n e s h a p e s 59 V I . C o n c l u s i o n 60 REFERENCES 63 i v L i s t o f T a b l e s T a b l e Page 1. Q u a d r u p o l e s p l i t t i n g s (kHz) 29 2. T- R e l a x a t i o n T i mes a t 44°C 3 3 v L i s t of F i g u r e s F i g u r e Page 1. Molecular s t r u c t u r e s of c h o l e s t e r o l , l a n o s t e r o l and d i pa l m i t o y l p h o s p h a t i d y l c h o l i n e 2 2. L i p i d phase t r a n s i t i o n s . (A) Phase t r a n s i t i o n between g e l and l i q u i d c r y s t a l l i n e s t a t e s . (B) DSC t r a c e of the c a l o r i m e t r i c behaviour of DPPC~d3l 8 3. The temperature composition p l o t f o r D P P C - d 6 2 / c h o l e s t e r o l . L i s the l i q u i d c r y s t a l f l u i d phase. G i s the g e l phase. L + 0, G + 0 and L + G are two phase r e g i o n s , o 2H NMR data, n D i f f e r e n c e spectroscopy data, •• DSC data. Diagram reproduced from [6] 11 4. Quadrupole S p e c t r a . (A) Perturbed Zeeman energy l e v e l s . (B) A T y p i c a l Powder P a t t e r n AI>Q 16 5. 2H NMR S p e c t r a . (A)DPPC-d3l powder p a t t e r n taken at 44°C. (B) Depaked v e r s i o n of the above spectrum e x h i b i t i n g 8 r e s o l v e d peaks. The peak assignments i n d i c a t e the carbon p o s i t i o n the deuterons are bonded to 27 6. Comparison of the powder p a t t e r n s p e c t r a f o r pure DPPC-D31 measured over a range of temperatures 31 7. Comparison of the powder p a t t e r n s p e c t r a f o r 5% lanosterol/DPPC-d31 measured over a range of temperatures 32 8. Comparison of the powder p a t t e r n s p e c t r a f o r 10% v i lanosterol/DPPC-d31 measured over a range of temperatures 35 9. Comparison of the powder p a t t e r n s p e c t r a f o r 15% lansterol/DPPC-d31 measured over a range of temperatures 36 10. V a r i a t i o n of the f i r s t moment, M,, with temperature f o r 0, 5, 10 and 15 percent l a n o s t e r o l c o n c e n t r a t i o n s 37 11. Comparison of the powder p a t t e r n s p e c t r a f o r 5% cholesterol/DPPC-d31 measured over a range of temperatures 39 12. Comparison of the powder p a t t e r n s p e c t r a f o r 10% cholesterol/DPPC-d31 measured over-a range of temperatures 41 13. Comparison of the powder p a t t e r n s p e c t r a f o r 15% cholesterol/DPPC-d31 measured over a range of temperatures 42 14. V a r i a t i o n of the f i r s t moment, M,, with temperature f o r 0, 5,10 and 15 percent c h o l e s t e r o l c o n c e n t r a t i o n s 43 15. Comparison of the powder p a t t e r n s p e c t r a of a)the pure DPPC-d31 sample, b) the 5% l a n s o s t e r o l sample and c) the 5% c h o l e s t e r o l sample a l l measured at 44°C 45 16. Comparison of the depaked s p e c t r a of a)t h e pure DPPC-d31 sample, b) the 5% l a n s o s t e r o l sample and c) the 5% c h o l e s t e r o l sample a l l measured at 44°C. .46 v i i 17. DSC t r a c e s of lanosterol/DPPC systems measured at a scan r a t e of l5°/hr 48 18. DSC t r a c e s of cholesterol/DPPC systems measured at a scan r a t e of l5°/hr 49 19. V a r i a t i o n of the e n t h a l p i e s of t r a n s i t i o n with l a n o s t e r o l concent r a t ion . ... . 50 20. F r a c t i o n of DPPC~d31 i n the g e l phase f o r 0% and 5% s t e r o l c o n c e n t r a t i o n s as a f u n c t i o n of temperature 56 v i i i Acknowledgements I w o u l d l i k e t o t h ank P r o f . Myer Bloom f o r s u g g e s t i n g t h i s p r o j e c t and f o r t h e d i s c u s s i o n s and g u i d a n c e he p r o v i d e d t h r o u g h o u t t h i s work. I am a l s o g r a t e f u l and i n d e b t e d t o D r . A l e x MacKay f o r h i s i n v a l u a b l e h e l p and a d v i c e . S i n c e r e t h a n k s t o D r . P.R. C u l l i s f o r d i s c u s s i o n s c o n c e r n i n g t h e b i o c h e m i c a l p r o p e r t i e s of membrane l i p i d s and-t o a l l t h r e e f o r t h e i r c a r e f u l r e a d i n g and c r i t i c i s m s o f my t h e s i s . A s p e c i a l t h a n k s f o r t h e s u g g e s t i o n s and a s s i s t a n c e p r o v i d e d by members of t h e NMR g r o u p , e s p e c i a l l y t o D r . G i n a H o a t s o n and Ed S t e r n i n . A l s o t h e c o n t i n u e d s u p o r t and e n c o u r a g e m e n t g i v e n by my f a m i l y was much a p p r e c i a t e d . F i n a l l y , t h e f i n a n c i a l s u p p o r t o f t h e N a t i o n a l S c i e n c e s and E n g i n e e r i n g C o u n c i l of Canada i s g r a t e f u l l y a c k n o w l e d g e d . ix I . INTRODUCTION C h o l e s t e r o l i s a major component o f many b i o l o g i c a l membranes. T h e r e i s e v i d e n c e t o s u g g e s t i t p e r f o r m s some s p e c i a l i z e d r o l e ( s ) i n n a t u r a l membranes. F i r s t l y , i t i s s t r u c t u r a l l y v e r y d i f f e r e n t f r o m t h e o t h e r l i p i d components. S e c o n d l y , c h o l e s t e r o l i s s y n t h e s i z e d v i a a complex pathway and a s i g n i f i c a n t amount of m e t a b o l i c e n e r g y i s expended d u r i n g i t s f o r m a t i o n [ 1 ] , F i n a l l y , and most s i g n i f i c a n t l y , c h o l e s t e r o l i s p r e s e n t i n e q u i m o l a r c o n c e n t r a t i o n s w i t h t h e p h o s p h o l i p i d s i n most e u k a r y o t i c membranes but i s i n g e n e r a l a b s e n t f r o m p r o k a r y o t i c membranes [ 2 ] . I n v e s t i g a t i o n s w i t h model membranes have shown t h a t t h e p r e s e n c e o f c h o l e s t e r o l i n f l u e n c e s t h e p e r m e a b i l i t y [ 3 - 5 ] , p h ase b e h a v i o u r [6 and r e f e r e n c e s t h e r e i n ] and d e g r e e of o r d e r o f t h e l i p i d components i n t h e b i l a y e r [ 7 , 8 ] . T h e s e r e s u l t s have been i n t e r p r e t e d i n t e r m s o f two key s t r u c t u r a l f e a t u r e s of c h o l e s t e r o l ; t h e p l a n a r r i n g s t r u c t u r e and t h e s moothness o f t h e a l p h a f a c e [ 9 , 1 0 ] , ( f i g u r e 1 ) . L a n o s t e r o l , ( f i g u r e 1 ) , i s an i n t e r m e d i a t e i n c h o l e s t e r o l b i o s y n t h e s i s and u n d e r g o e s t h r e e s e q u e n t i a l o x i d a t i v e d e m e t h y l a t i o n s t e p s a t p o s i t i o n s 4, 4 and 14 t o f o r m c h o l e s t e r o l [ 1 ] . I t i s n o r m a l l y p r e s e n t i n t r a c e amounts [11] and d o e s n o t p e r f o r m any known p h y s i o l o g i c a l r o l e . The 14 a m e t h y l g r o u p w h i c h p r o j e c t s f r o m t h e a l p h a f a c e o f l a n o s t e r o l i s t h o u g h t t o be t h e most s i g n i f i c a n t s t r u c t u r a l d i f f e r e n c e between l a n o s t e r o l and c h o l e s t e r o l and c o u l d i n t e r f e r e w i t h t h e Van d e r Waal i n t e r a c t i o n s between 1 2 F i g u r e 1. M o l e c u l a r s t r u c t u r e s o f c h o l e s t e r o l , l a n o s t e r o l and d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e . 3 t h e a l p h a f a c e and t h e a c y l c h a i n s [ 9 , 1 0 ] , G i v e n t h a t l a n o s t e r o l i s a p r e c u r s o r t o c h o l e s t e r o l and th e c h i e f d i f f e r e n c e i s t h e "bump" o r p r o t r u s i o n from t h e a l p h a f a c e , t h e c o m p a r a t i v e e f f e c t s o f t h e s e two s t e r o l s on membranes s h o u l d i n d i c a t e t h e i m p o r t a n c e o f h a v i n g t h e smooth f a c e . Any d i f f e r e n c e s s h o u l d a l s o p r o v i d e a c l u e t o why c h o l e s t e r o l i s f u n c t i o n a l l y s u p e r i o r . B e f o r e r e v i e w i n g some o f t h e work done w i t h t h e s e two s t e r o l s some n e c e s s a r y b a c k g r o u n d m a t e r i a l w i l l be i n t r o d u c e d . A. BACKGROUND B i o l o g i c a l membranes c o n t a i n a huge d i v e r s i t y o f components. The m a j o r i t y of t h e s e components c a n be s e p a r a t e d i n t o two g r o u p s ; p r o t e i n s and l i p i d s . Many s t u d i e s have been u n d e r t a k e n t o e s t a b l i s h t h e c o n t r i b u t i o n e a c h component makes t o t h e o v e r a l l f u n c t i o n i n g o f t h e t h e membrane. The p h y s i o l o g i c a l r o l e s o f p r o t e i n s i n c l u d e t h e t r a n s p o r t of s o l u t e s a c r o s s t h e membrane and e n z y m a t i c a c t i v i t i e s [ 1 2 ] , M e m b r a n e - a s s o c i a t e d p r o t e i n s c a n be of two g e n e r a l t y p e s . I f t h e y a r e d e e p l y embedded i n t h e membrane t h e y a r e known as i n t e g r a l p r o t e i n s whereas p e r i p h e r a l p r o t e i n s a r e a s s o c i a t e d w i t h t h e h y d r o p h i l i c r e g i o n s o f e i t h e r t h e l i p i d s o r t h e i n t e g r a l p r o t e i n s on t h e membrane s u r f a c e . L i p i d s p r o v i d e a m a t r i x f o r t h e p r o t e i n s [ 1 3 ] . The r e a s o n f o r s u c h a l a r g e number o f d i f f e r e n t l i p i d s i s not 4 f u l l y u n d e r s t o o d . S t u d i e s have i n d i c a t e d t h a t membrane p r o p e r t i e s s u c h as f l u i d i t y and p e r m e a b i l i t y a r e dependent on l i p i d c o m p o s i t i o n . A l s o , a v a r i e t y of l i p i d s may be n e c e s s a r y t o s a t i s f y p a c k i n g r e q u i r e m e n t s of' p r o t e i n s [ 1 4 , 1 5 ] . The r o l e s o f t h e i n t e g r a l p r o t e i n s a r e l i k e l y t o be i n f l u e n c e d by 1 i p i d / p r o t e i n i n t e r a c t i o n s . T h e s e i n t e r a c t i o n s ' have been d i s c u s s e d i n d e t a i l e l s e w h e r e [ 1 6 ] . I t w i l l be of i n t e r e s t t o compare t h e p r o t e i n / 1 i p i d i n t e r a c t i o n s t o l i p i d / l i p i d i n t e r a c t i o n s i n l i g h t o f t h e r e s u l t s of t h i s work. I n t e r a c t i o n s between l i p i d s depend on t h e e n v i r o n m e n t and t h e s t r u c t u r e s o f t h e l i p i d s i n v o l v e d . The r e s p o n s e of p h o s p h o l i p i d s t o c h a n g e s i n t h e e n v i r o n m e n t v a r y w i t h a c y l c h a i n l e n g t h , d e g r e e o f s a t u r a t i o n and t h e t y p e o f h e a d g r o u p [ 1 3 ] . T h i s makes t h e i n t e r p r e t a t i o n o f r e s u l t s f r o m b i o l o g i c a l membranes b o t h c o m p l i c a t e d and ambiguous. To remove some o f t h i s a m b i g u i t y , model membranes a r e o f t e n u s e d . The a c t u a l model c h o s e n depends on t h e n a t u r e of t h e s t u d y . A s i m p l e model may c o n s i s t o f a p a r t i c u l a r p h o s p h o l i p i d d i s p e r s e d i n w a t e r . More s o p h i s t i c a t e d models may i n c o r p o r a t e d i f f e r e n t l i p i d s a n d / o r p r o t e i n s . A t y p i c a l model i s f o r example an aqueous d i s p e r s i o n o f t h e p h o s p h o l i p i d DPPC ( f i g u r e 1 ) . B e c a u s e o f t h e a m p h i p a t h i c n a t u r e o f p h o s p h o l i p i d s ( i . e . t h e y p o s s e s s b o t h h y d r o p h i l i c and h y d r o p h o b i c r e g i o n s ) and, i n t h e c a s e o f DPPC, t h e b a s i c a l l y c y l i n d r i c a l shape o f t h e m o l e c u l e , m u l t i l a m e l l a r 5 v e s i c l e s (MLVs) a r e t h e most e n e r g e t i c a l l y f a v o r a b l e s t r u c t u r e s f o r m e d when t h e p h o s p h o l i p i d s a r e d i s p e r s e d i n water [ 1 3 ] . T h e s e MLVs c o n s i s t of c o n c e n t r i c b i l a y e r s of l i p i d s w h i c h r e s e m b l e i n s t r u c t u r e t h e s k i n s of an o n i o n . W i t h i n t h e v e s i c l e s , t h e l i p i d s a r e o r i e n t e d s u c h t h a t t h e c h o l i n e h e a d g r o u p s a r e p o s i t i o n e d a t t h e l i p i d / w a t e r i n t e r f a c e w h i l e t h e a c y l c h a i n s e x t e n d i n t o t h e c e n t r a l r e g i o n of t h e b i l a y e r . S t e r o l s a r e a l s o a m p h i p a t h i c , t h e h y d r o y l g r o u p r e p r e s e n t s t h e p o l a r m o i e t y and t h e c h o l e s t a n e r i n g and a c y l c h a i n t h e h y d r o p h o b i c p a r t . The i n c o r p o r a t i o n o f s t e r o l s i n t o p h o s p h o l i p i d model membranes d r a s t i c a l l y m o d i f i e s t h e i r p h y s i c a l p r o p e r t i e s , i n c l u d i n g p e r m e a b i l i t y and p h a s e b e h a v i o u r . T h i s must be b r o u g h t a b o u t by some i n t e r a c t i o n between t h e s t e r o l and t h e p h o s p h o l i p i d s . I t i s t h e r e f o r e i m p o r t a n t t o d e t e r m i n e t h e p o s i t i o n o f t h e c h o l e s t e r o l m o l e c u l e s w i t h r e s p e c t t o t h e b i l a y e r s . X - r a y and n e u t r o n d i f f r a c t i o n s t u d i e s [17-19] on e g g - P C / c h o l e s t e r o l . m i x t u r e s i n d i c a t e t h a t t h e 3 - 0 - h y d r o x y l g r o u p o f c h o l e s t e r o l i s p o s i t i o n e d a t t h e p h o s p h o l i p i d / w a t e r i n t e r f a c e i n t h e v i c i n i t y o f t h e a c y l - e s t e r l i n k a g e and t h e r i n g s t r u c t u r e i s o r i e n t e d 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 and s p ans t h e f i r s t 8-10 m e t h e l e n e g r o u p s [ 7 ] , S i n c e l a n o s t e r o l a l s o has a 3-/3 h y d r o x y l and i s p l a n a r i t i s r e a s o n a b l e t o assume t h a t i t i s o r i e n t e d i n l i p i d b i l a y e r s i n a s i m i l a r manner t o c h o l e s t e r o l . F u r t h e r 6 e v i d e n c e i s p r o v i d e d by 2H NMR s t u d i e s u s i n g c h o l e s t e r o l - 3 a d , and l a n o s t e r o l - 3 a d , i n PC v e s i c l e s [ 2 0 ] . I t i s r e a s o n a b l e t o ask whether t h e 14-a m e t h y l g r o u p of l a n o s t e r o l would p e r t u r b t h e a c y l c h a i n o r i e n t a t i o n a l o r d e r i n t h e v i c i n i t y of t h e number 9 t o 12 c a r b o n s i n a manner d i f f e r e n t from c h o l e s t e r o l . The a c t u a l p o s i t i o n c l o s e s t t o t h e 14-a m e t h y l g r o u p i s d e p e n d e n t on t h e t e m p e r a t u r e due t o t h e c hange i n l e n g t h o f t h e a c y l c h a i n s w i t h i n c r e a s i n g t e m p e r a t u r e . The r e s u l t s o f a s t u d y u s i n g f a t t y a c i d s p i n l a b e l s i n l e c i t h i n (PC) v e s i c l e s w i t h t h e two s t e r o l s showed t h a t a s i g n i f i c a n t p e r t u r b a t i o n i n t h e f a t t y a c i d c h a i n s o c c u r r e d i n t h e v i c i n i t y o f C 1 2 [ 2 0 ] . C h o l e s t e r o l e x h i b i t s a " c o n d e n s a t i o n e f f e c t " [2] w i t h some p h o s p h o l i p i d s . In o t h e r words, t h e m i x i n g o f c h o l e s t e r o l and t h e p h o s p h o l i p i d s i s n o t i d e a l , so t h a t t h e mean m o l e c u l a r a r e a of a f i l m of a c h o l e s t e r o l / p h o s p h o l i p i d m i x t u r e i s not a s t r a i g h t f o r w a r d sum of t h e a r e a s o f t h e i n d i v i d u a l c omponents. I t i s assumed t h a t t h e c r o s s s e c t i o n a l a r e a of c h o l e s t e r o l i s c o n s t a n t . The t e r m c o n d e n s a t i o n t h e r e f o r e r e f e r s t o t h e r e d u c t i o n i n c r o s s - s e c t i o n a l a r e a o f t h e p h o s p h o l i p i d p e r p o l a r h e a d g r o u p . 7 Two s t r u c t u r a l f e a t u r e s of t h e s t e r o l a r e e s s e n t i a l f o r c o n d e n s a t i o n t o o c c u r ; t h e 3/3-hydroxyl g r o u p and t h e p l a n a r c o n f o r m a t i o n of t h e r i n g , b o t h o f w h i c h l a n o s t e r o l p o s s e s s e s [ 2 2 , 2 3 ] . T h i s e f f e c t p r o b a b l y a r i s e s from Van -der Waals f o r c e s between t h e r i g i d t e t r a c y c l i c n u c l e u s and t h e a c y l c h a i n s . The p l a n a r i t y of t h e a l p h a f a c e p r o m o t e s maximal i n t e r a c t i o n . P h y s i c a l s t u d i e s w i t h model membranes i n d i c a t e t h a t l a n o s t e r o l d o e s not c o n d e n s e p h o s p h o l i p i d f a t t y c h a i n s [ 1 0 ] , B. PHASE BEHAVIOUR OF PHOSPHLIPID SYSTEMS In a p u r e l i p i d s y s t e m , t h e a c y l c h a i n s o f t h e l i p i d s u n d e r g o f r e e z i n g ( o r m e l t i n g ) a t a c h a r a c t e r i s t i c t r a n s i t i o n t e m p e r a t u r e . Below t h i s t e m p e r a t u r e t h e d i f f u s i o n r a t e of t h e l i p i d s i s < l O ~ 1 0 c m 2 / s and t h e c h a i n s a r e o r i e n t e d a t an a n g l e w i t h r e s p e c t t o t h e b i l a y e r ( f i g u r e 2) [ 1 3 ] . T h i s i s known a s t h e g e l - p h a s e o f t h e l i p i d s . The a c y l c h a i n s m e l t as t h e t e m p e r a t u r e i s r a i s e d t h r o u g h t h e t r a n s i t i o n . Above 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 h e l i p i d s a r e i n a l i q u i d c r y s t a l l i n e p hase i n w h i c h t h e o r i e n t a t i o n a l o r d e r o f t h e c h a i n s i s r e d u c e d c o n s i d e r a b l y and i n w h i c h t h e a x i s of symmetry f o r r e o r i e n t a t i o n a l m o t i o n s o f t h e c h a i n i s 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 . The l i p i d s d i f f u s e l a t e r a l l y ( i . e . i n t h e p l a n e of t h e b i l a y e r ) w i t h a d i f f u s i o n r a t e of =*1 0" 8 c m 2 / s . The change i n s t a t e w i t h i n c r e a s i n g t e m p e r a t u r e i n v o l v e s c h a n g e s i n thermodynamic q u a n t i t i e s s u c h a s e n t r o p y polar head group D " O a Q: y E Pretransit ion Temperature F i g u r e 2. L i p i d p hase t r a n s i t i o n s . (A) Phase t r a n s i t i o n between g e l and l i q u i d c r y s t a l l i n e s t a t e s . (B) DSC t r a c e of th e c a l o r i m e t r i c b e h a v i o u r o f DPPC-d31. 9 and e n t h a l p y w h i c h c a n be i n t e r p r e t e d q u a n t i t a t i v e l y i n t e rms of t h e o b s e r v e d d e c r e a s e i n o r d e r o f • t h e c h a i n s . In t h e g e l - s t a t e t h e a c y l c h a i n s a r e i n a t i l t e d a l l - t r a n s c o n f i g u r a t i o n whereas i n t h e l i q u i d c r y s t a l l i n e p h a s e one o r more gauche c o n f o r m e r s may e x i s t , r e s u l t i n g i n i n c r e a s e d i n t e r c h a i n s p a c i n g . The c o n f o r m a t i o n o f t h e c h a i n d e t e r m i n e s t h e a n g l e t h e C-D bonds make w i t h n o r m a l t o t h e b i l a y e r . T h i s a n g l e t h e r e f o r e p r o v i d e s some i n f o r m a t i o n on c h a i n l e n g t h , t h i c k n e s s and p a c k i n g and c a n be d e t e r m i n e d from t h e o r d e r p a r a m e t e r s of t h e d e u t e r i u m s down t h e c h a i n [ 2 1 ] . A l s o a q u a n t i t a t i v e measure o f t h e e x t e n t of m o t i o n a l a v e r a g i n g i s p r o v i d e d by t h e o r d e r p a r a m e t e r s . F o r a C-D bond making an a n g l e 6(t) a t t i m e t w i t h t h e n o r m a l t o t h e b i l a y e r , t h e o r d e r p a r a m e t e r S C D i s d e f i n e d as [24] S C D = \ < 3 c o s 2 ( 0 ( t ) ) - 1 > 1.1 where t h e a v e r a g e i s t a k e n o v e r a l l m o l e c u l a r c o n f o r m a t i o n s and m o t i o n s . The m e t h y l e n e g r o u p s down t h e a c y l c h a i n a t t e m p e r a t u r e s below t h e t h e c h a i n m e l t i n g t e m p e r a t u r e e x h i b i t a more o r l e s s c o n s t a n t o r d e r p a r a m e t e r . Above 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 h e d e g r e e o f m o t i o n e x h i b i t e d by d e u t e r o n s on c a r b o n p o s i t i o n s 10-16+ i n c r e a s e s m o n o t o n i c a l l y w i t h c a r b o n number. The d i f f e r e n c e between t h e o r d e r p a r a m e t e r s o f d e u t e r o n s c l o s e r t o t h e h e a d g r o u p ( p o s i t i o n s 2-9) i s s m a l l w h i c h r e s u l t s i n t h e c h a r a c t e r i s t i c p l a t e a u r e g i o n i n t h e o r d e r p a r a m e t e r p r o f i l e s . 10 D i f f e r e n t i a l Scanning C a l o r i m e t r y can be used to fo l l o w the t r a n s i t i o n . The t r a c e shown i n f i g u r e 2 shows the heat absorbed by a pure p h o s p h o l i p i d system as a f u n c t i o n of temperature. The peak occurs at the t r a n s i t i o n temperature. For the pure l i p i d system the t r a n s i t i o n occurs over one degree as i n d i c a t e d by the width of the t r a n s i t i o n peak. The behaviour of complex m u l t i - l i p i d systems i s more r e p r e s e n t a t i v e of b i o l o g i c a l membranes. Because of the number of d i f f e r e n t l i p i d s the system does not undergo an isothermal phase t r a n s i t i o n . The system may e x i s t i n a mixed g e l / l i q u i d c r y s t a l l i n e phase over a wide range of temperature. The m o t i v a t i o n f o r studying phase behaviour i s not only to model the thermodynamic behaviour of b i o l o g i c a l membranes ( s i n c e at p h y s i o l o g i c a l temperatures membranes are in a l i q u i d c r y s t a l l i n e s t a t e ) but to e s t a b l i s h how the d i f f e r e n t l i p i d s are i n t e r a c t i n g . C. CHOLESTEROL/PHOSPHOLIPID SYSTEMS C h o l e s t e r o l i s thought to decrease the order of l i p i d s i n t h e i r gel-phase and order them i n t h e i r l i q u i d - c r y s t a l l i n e phase. The s t a t e of a model membrane c o n t a i n i n g DPPC and c h o l e s t e r o l t h e r e f o r e depends on both the s t e r o l c o n c e n t r a t i o n and the temperature. Even f o r t h i s t e r n a r y system t h i s r e s u l t s i n complex phase behaviour. The complexity i s r e f l e c t e d i n the phase diagram determined from DSC and Deuterium Nuclear Magnetic Resonance (2H-NMR) ( f i g u r e 3) [6], 11 F i g u r e 3. The temperature composition p l o t for D P P C - d 6 2 / c h o l e s t e r o l . L i s the l i q u i d c r y s t a l f l u i d phase. G i s the g e l phase. L + 0, G + 0 and L + G are two phase r e g i o n s , o 2H NMR data, n D i f f e r e n c e spectroscopy data, •• DSC data. Diagram reproduced from [ 6 ] . 1 2 DSC measurements i n d i c a t e t h a t t h e h e a t a b s o r b e d 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 d e c r e a s e s l i n e a r l y 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 [ 4 , 2 5 ] , whereas t h e w i d t h o f t h e t r a n s i t i o n peak b r o a d e n s . The c o n d e n s a t i o n e f f e c t d epends on t h e e x i s t e n c e of an i n t a c t s i d e c h a i n and a 3/3 h y d r o x y l g r o u p . I t i s t h e r e f o r e i n t e r e s t i n g t h a t t h e s e same r e q u i r e m e n t s a r e needed f o r t h e b r o a d e n i n g and p o s s i b l e e l i m i n a t i o n of 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 hase t r a n s i t i o n peak [ 2 , 4 , 2 6 ] . D. FLUIDITY The t e r m " f l u i d i t y " i s o f t e n u s e d t o d e s c r i b e t h e p h y s i c a l s t a t e of membranes. U n f o r t u n a t e l y i t i s a l o o s e l y d e f i n e d p a r a m e t e r and has been u s e d t o d e s c r i b e d i f f e r e n t membrane p r o p e r t i e s . F l u i d i t y i s u s e d i n some c a s e s t o d e s c r i b e t h e o r i e n t a t i o n a l o r d e r of t h e m o l e c u l e s i n a s y s t e m w h i l e i n o t h e r c a s e s t h e d e f i n i t i o n o f f l u i d i t y a s b e i n g i n v e r s e l y r e l a t e d t o m i c r o v i s c o s i t y i s u s e d . E. AIMS OF RESEARCH I n t e r a c t i o n s between c h o l e s t e r o l and DPPC m a n i f e s t t h e m s e l v e s i n many p h y s i c a l l y o b s e r v a b l e ways. D i f f e r e n t i a l e f f e c t s between l a n o s t e r o l and c h o l e s t e r o l have been o b s e r v e d f o r membrane p r o p e r t i e s s u c h a s m i c r o v i s c o s i t y , p e r m e a b i l i t y e t c . The a i m o f t h i s r e s e a r c h was t o compare t h e i n f l u e n c e o f l a n o s t e r o l on t h e c h a i n d y n a m i c s o f DPPC t o c h o l e s t e r o l . C a l o r i m e t r y was u s e d t o compare t h e 1 3 thermodynamic b e h a v i o u r of t h e s y s t e m s s t u d i e d . I n f o r m a t i o n a b o u t t h e o r i e n t a t i o n a l o r d e r was o b t a i n e d u s i n g 2H-NMR. DPPC m o l e c u l e s w i t h a p e r d e u t e r a t e d sn-2 c h a i n were u s e d w i t h t h e hope t h a t i f c e r t a i n r e g i o n s of t h e c h a i n were p e r t u r b e d by t h e b u l k y m e t h y l g r o u p of l a n o s t e r o l , t h e 2H s p e c t r a w o u l d r e f l e c t t h i s . F o r f u r t h e r c o m p a r i s o n between t h e s y s t e m s t h e NMR. r e l a x a t i o n t i m e T„ was a l s o m e a s u r e d . R e l a x a t i o n t i m e s a r e ze i n f l u e n c e d by t h e a m p l i t u d e s a n d t i m e s c a l e s of t h e i n t e r a c t i o n s between t h e d e u t e r o n s and t h e i r e n v i r o n m e n t . I I . NMR AND DSC THEORY A. DEUTERIUM NUCLEAR MAGNETIC RESONANCE A n u c l e u s w i t h s p i n I > 1/2 p o s s e s s e s an e l e c t r i c q u a d r u p o l e moment (eQ). E l e c t r o n s n e a r t h e s i t e of t h e n u c l e u s g i v e r i s e t o an e l e c t r i c f i e l d g r a d i e n t (EFG) w h i c h i n t e r a c t s w i t h t h e e l e c t r i c q u a d r u p o l e moment [ 2 7 ] , The EFG can be e x p r e s s e d a s a t r a c e l e s s , symmetric s e c o n d rank t e n s o r V ^ . By c h o o s i n g a s e t o f p r i n c i p a l a x e s i n r e l a t i o n t o w h i c h = 0 f o r a * o n l y two p a r a m e t e r s a r e needed t o d e s c r i b e V^^; t h e asymmetry p a r a m e t e r (rj) and t h e f i e l d q r a d i e n t (eq), where [27] eq = V 2 Z 2.1 v = V x x v y y , , Vz z In NMR i n h i g h m a g n e t i c f i e l d s , t h e d i r e c t i o n of t h e e x t e r n a l m a g n e t i c f i e l d i s a n a t u r a l c h o i c e f o r one o f t h e a x e s i n t h e l a b o r a t o r y f r a m e . The p r i n c i p a l a x i s o f t h e EFG does n o t n e c e s s a r i l y c o i n c i d e w i t h t h e l a b o r a t o r y f r a m e . T r a n s f o r m a t i o n i n t o t h e l a b o r a t o r y r e f e r e n c e frame c a n be a c c o m p l i s h e d by t h e Wigner R o t a t i o n M a t r i x D(aPy) o p e r a t i n g on t h e EFG t e n s o r when w r i t t e n i n terms o f a s p h e r i c a l b a s i s [ 2 8 ] , The a n g l e s a,0 and 7 a r e t h e E u l e r a n g l e s d e f i n i n g t h e t r a n s f o r m a t i o n . 14 1 5 The i n t e r a c t i o n between t h e EFG and t h e e l e c t r i c g u a d r u p o l e moment l i f t s t h e d e g e n e r a c y of t h e Zeeman e n e r g y l e v e l s . I n a l a r g e m a g n e t i c f i e l d , H 0, t h e e l e c t r i c q u a d r u p o l e i n t e r a c t i o n i s much s m a l l e r t h a n t h e Zeeman I n t e r a c t i o n and can be t r e a t e d a s a f i r s t o r d e r p e r t u r b a t i o n on t h e Zeeman I n t e r a c t i o n [ 2 7 ] . S i n c e d e u t e r i u m has s p i n 1=1, t h e r e a r e two a l l o w e d t r a n s i t i o n s (m=-1 t o m=0) and (m=0 t o m=l). As a r e s u l t o f t h e q u a d r u p o l e i n t e r a c t i o n t h e s e two t r a n s i t i o n s a r e c h a r a c t e r i z e d by d i f f e r e n t e n e r g i e s a s i l l u s t r a t e d i n f i g u r e 4. The s p e c t r u m from a s y s t e m of e q u i v a l e n t 2 H - n u c l e i w i l l be a d o u b l e t . The f r e q u e n c y s e p a r a t i o n o f t h e two s i g n a l s i s [24] A"Q = 1 ^ " h ^ [ ( 3 c o s 2 ) + r ? c o s ( 2 a ) s i n 2 (p) ] 2.3 where a, 0 a r e E u l e r a n g l e s s p e c i f y i n g t h e o r i e n t a t i o n o f t h e EFG t e n s o r w i t h r e s p e c t t o t h e m a g n e t i c f i e l d H 0 . The EFG t e n s o r of t h e p h o s p h o l i p i d s i s i n a m o l e c u l e f i x e d s y s t e m . M o t i o n s m o d u l a t e t h e o r i e n t a t i o n o f t h e m o l e c u l e s w i t h r e s p e c t t o H 0 and t h u s t h e o b s e r v e d q u a d r u p o l e s p l i t t i n g w i l l be LvQ = | ^ ~ h ^ < ( 3 c o s 2 ) + 7 7 C O s ( 2 a ) s i n 2 (£)> 2.4 w h i c h i s a v e r a g e d o v e r a l l m o t i o n s t h a t a r e f a s t compared t o t h e s t a t i c q u a d r u p o l e c o u p l i n g c o n s t a n t , e2qQ/h (=* 170 kHz f o r most C-D b o n d s ) . 16 Q u a d r u p o l a r B. F r e q u e n c y Figure 4. Q u a d r u p o l e S p e c t r a . (A) P e r t u r b e d Zeeman e n e r g y l e v e l s . (B)A T y p i c a l Powder P a t t e r n . 1 7 B. ORDER PARAMETERS L i p i d s i n t h e l i q u i d c r y s t a l l i n e s t a t e ( f i g u r e 2) e x h i b i t a h i g h d e g r e e o f a x i a l symmetry a r o u n d t h e n o r m a l t o t h e b i l a y e r ( w h i c h i s t a k e n t o be t h e z - d i r e c t i o n ) . T h i s l e a d s t o a z e r o a s y m e t r y p a r a m e t e r ( T? = 0 ) . R o t a t i o n s and f l e x i n g m o t i o n s of t h e l i q u i d c r y s t a l l i n e p h o s p h o l i p i d s can a l s o be c h a r a t e r i z e d by c y l i n d r i c a l symmetry [29] and t h e r e f o r e t h e a n g u l a r component c a n be s e p a r a t e d i n two, g i v i n g A v Q = 1 ^~\r ( 3 c o s 2 ( 6 ) - 1 ) S C D 2.5 where 6 i s t h e a n g l e between t h e m a g n e t i c f i e l d and t h e n o r m a l t o t h e b i l a y e r [ 2 4 ] , The t e r m s C D i s t h e o r d e r p a r a m e t e r f o r t h e C-D bond v e c t o r and i s d e f i n e d i n e q u a t i o n 1.1. C. THE 2H POWDER PATTERN SPECTRUM, DEPAKING Q u a d r u p o l e NMR s p e c t r a t h a t r e s u l t f r o m 2 H - l i p i d membranes a r e t y p i c a l o f t h e "powder p a t t e r n s " seen f o r r a n d o m l y o r i e n t e d s a m p l e s . The s p e c t r a a r e a s u p e r p o s i t i o n of d o u b l e t s w i t h q u a d r u p o l e s p l i t t i n g s f r o m e a c h o f t h e r a n d o m l y o r i e n t e d d o m a i n s . As a r e s u l t o f o r i e n t a t i o n d e p e n d e n t i n t e r a c t i o n s , t h e c o n t r i b u t i o n e a c h domain makes t o t h e l i n e s h a p e i s s c a l e d [ 3 2 ] . I f t h e w i d t h of t h e o r i e n t e d l i n e s h a p e .as w e l l as i t s s p l i t t i n g s c a l e as [ 3 c o s 2 8 - 1 ] / 2 , a d e p a k i n g p r o c e d u r e c a n be employed on t h e powder p a t t e r n s r e s u l t i n g i n t h e s p e c t r a t h a t would be 18 o b t a i n e d i f t h e sample had been o r i e n t e d w i t h t h e f i e l d [ 3 0 ] . The number of domains p e r p e n d i c u l a r t o H 0 ( i . e . 6 = 90 i n e q u a t i o n 2.5) f a r outnumber t h e domains p a r a l l e l t o t h e f i e l d [ 2 4 ] , t h e r e f o r e t h e two most i n t e n s e p eaks i n t h e powder p a t t e r n ( f i g u r e 4 ( b ) ) c o r r e s p o n d t o t h e 90° e d g e s . Powder p a t t e r n s a r e d e p a k e d t o g i v e t h e o r i e n t e d s p e c t r a w h i c h would be o b t a i n e d f o r domains o r i e n t e d a t 90° t o H 0 [ 3 0 ] . The e x p r e s s i o n f o r t h e s p l i t t i n g s o f t h e depaked l i n e s h a p e s r e d u c e s t o A " G = 1 ^ S C D 2.6 Thus t h e o r d e r p a r a m e t e r s c a n be e a s i l y d e t e r m i n e d f r o m t h e s p l i t t i n g s . B e c a u s e of t h e d e p e n d e n c e on bond a n g l e t h e s t a t e of t h e membrane can be d e s c r i b e d by t h e o r d e r p a r a m e t e r s . D. FIRST MOMENT In t h e c o n v e n t i o n a l d e f i n i t i o n o f t h e s p e c t r a l moments a l l odd moments o f a s y m m e t r i c s p e c t r u m a r e z e r o [ 3 1 ] , In t h e c a s e o f s p e c t r a d o m i n a t e d by q u a d r u p o l e i n t e r a c t i o n s i t i s c o n v e n i e n t t o c a l c u l a t e moments o f t h e h a l f s p e c t r u m . F o r example t h e f i r s t moment o f t h e h a l f s p e c t r u m as d e f i n e d below, i s s i m p l y h a l f 19 J f {CO0+OJ)CO6CO M,= ° 2.7 Oo / f (o) 0 +w) dcj o where w0 i s the Larmor frequency and f(w 0+cj) i s the l i n e s h a p e at a frequency co from u>0. In the powder p a t t e r n s that r e s u l t from p h o s p h o l i p i d s i n which a s i n g l e chain i s perdeuterated there i s a d i s t r i b u t i o n of quadrupolar s p l i t t i n g s ^VQ) °^UE T O T H E v a r i a t i o n , i n order along the c h a i n . The f i r s t moment gi v e s the average quadrupolar s p l i t t i n g ; M, = (4ir/3|/3)<6j;g> 2.8 Changes i n the degree of order r e s u l t i n g from temperature v a r i a t i o n or i n c r e a s i n g s t e r o l c o n c e n t r a t i o n can be f o l l o w e d by M,. I t i s t h e r e f o r e a p a r t i c u l a r l y u s e f u l parameter f o r examining the phase behaviour of model membranes. E. RELAXATION NMR r e l a x a t i o n times can g i v e i n f o r m a t i o n on the " s p e c t r a l d e n s i t y " of motions i n model membrane systems [24,32]. These motions cause f l u c t u a t i o n s i n the s p i n dependent i n t e r a c t i o n s . The quadrupole echo r e s u l t i n g from the quadrupole echo pul s e sequence (III.C) decays to zero as [ e x p ( ~ t / T 2 e ) J where T~ i s the s p i n - s p i n r e l a x a t i o n time. T~ i s s e n s i t i v e to 20 s l o w m o t i o n s s u c h as t r a n s l a t i o n a l d i f f u s i o n and c h a i n m o b i l i t y ( r > l 0 " 7 s ) . S p i n l a t t i c e r e l a x a t i o n measurements (T,) p r o v i d e i n f o r m a t i o n on m o t i o n s h a v i n g c o r r e l a t i o n t i m e s between 1 0 " 9 S t o 1 0 " b S a p p r o x i m a t e l y [16,32] i . e . o f t h e o r d e r of t h e l a r m o r f r e q u e n c y . I t has been p r o p o s e d [32] t h a t c o l l e c t i v e m o t i o n s of-h y d o c a r b o n c h a i n s g i v e r i s e t o a d i s t r i b u t i o n o f c o r r e l a t i o n t i m e s e x t e n d i n g t o q u i t e l o n g v a l u e s . Such a d i s t r i b u t i o n o f c o r r e l a t i o n t i m e s would l e a d t o s h o r t T_ v a l u e s . Ze P r e v i o u s s t u d i e s w i t h p e r d e u t e r a t e d p h o s p h o l i p i d s y s t e m s have f o u n d t h a t r e l a x a t i o n c u r v e s v a r y e x p o n e n t i a l l y w i t h s h o r t T v a l u e s where T i s t h e t i m e s e p a r a t i n g t h e two 9 0 ° p u l s e s ( I I I . C ) [ 3 3 ] , The i n i t i a l d e c a y r a t e p r o v i d e s an e s t i m a t e o f t h e a v e r a g e T 2 e f o r t h e many s p i n s i n t h e s y s t e m . F. DIFFERENTIAL SCANNING CALORIMETERY 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 e r y m e asures t h e d i f f e r e n c e i n h e a t needed t o m a i n t a i n a v e r y s m a l l but d e f i n i t e t e m p e r a t u r e d i f f e r e n c e between two s a m p l e s as t h e t e m p e r a t u r e i s v a r i e d [ 3 4 ] , One sample i s u s u a l l y i n e r t ( i . e . d o e s n o t u n d e r g o a phase t r a n s i t i o n i n t h e t e m p e r a t u r e r a n g e o f i n t e r e s t ) and i s u s e d a s a r e f e r e n c e . The DSC t r a c e shown p r e v i o u s l y ( f i g u r e 2) shows a v a r i a t i o n i n t h e e x c e s s s p e c i f i c h e a t (C ) w i t h ex t e m p e r a t u r e . The e n t h a l p y f o r t h e t r a n s i t i o n between g e l 21 (T < 39°C) and l i q u i d c r y s t a l l i n e (T > 39°C) i s [34] T AHcal = M L C e x dT 2.9 T i where M 2 i s the molecular weight of the l i p i d DPPC-d3l and T, and T 2 are the endpoints of the t r a n s i t i o n peak. This peak can a l s o be c h a r a c t e r i z e d by i t s h a l f w i d t h which i n d i c a t e s the temperature range of the t r a n s i t i o n . As the chains melt from the g e l c o n f i g u r a t i o n there i s a change i n the f r e e energy, AG. The enthalpy change f o r the t r a n s i t i o n i s _ d(AG/T) m ~ 8<1/T) 2 , 1 0 from AG=AH-TAS ( f o r constant pressure) where AH can be i d e n t i f i e d with the i n t e g r a t e d enthalpy of the DSC t r a c e . The enthalpy per mole of DPPC, AH/n,, v a r i e s l i n e a r l y with the c o n c e n t r a t i o n of c h o l e s t e r o l i n c o r p o r a t e d . The slope of a p l o t of AH/n, vs % c h o l e s t e r o l has been i n t e r p r e t e d as i n d i c a t i n g the f r a c t i o n of l i p i d not p a r t i c i p a t i n g i n the t r a n s i t i o n [35]. I I I . MATERIALS AND METHODS A. NMR SAMPLE PREPARATION The p h o s p h o l i p i d sn-2 [ 2 H 3 1 ] d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e (DPPC-d31) was k i n d l y p r o v i d e d by R. C u s h l e y . The s y n t h e s i s of t h i s l i p i d i s o u t l i n e d i n [ 3 6 ] . U s i n g TLC, t h e p u r i t y was f o u n d t o be g r e a t e r t h a n 98%. DPPC (99+% p u r i t y ) , c h o l e s t e r o l (98%+ p u r i t y ) , and l a n o s t e r o l ( 8 0 % p u r i t y ) were o b t a i n e d f r o m Sigma C h e m i c a l Co. S t o c k s o l u t i o n s o f t h e two s t e r o l s were p r e p a r e d i n c h l o r o f o r m and a p p r o p r i a t e v o l u m e s were added t o t h e p h o s p h o l i p i d samples t o r e a c h t h e d e s i r e d s t e r o l c o n c e n t r a t i o n . T y p i c a l l y , t h e d r y w e i g h t o f t h e samples was between 50-60 mg. The sample was g e n t l y r o t a t e d i n a warm b a t h w h i l e t h e s o l v e n t was pumped o f f , l e a v i n g a t h i n homogeneous f i l m on t h e f l a s k . To remove t h e r e m a i n i n g s o l v e n t t h e samples were d r i e d u nder h i g h vacuum f o r a p e r i o d o f a t l e a s t 6 h o u r s . Aqueous d i s p e r s i o n of t h e l i p i d s were formed by a d d i n g e x c e s s b u f f e r (50mM p o t a s s i u m p h o s p h a t e pH 7) t o t h e f i l m s . T h e s e d i s p e r s i o n s were s u b j e c t e d t o v i g o r o u s m i x i n g ( v o r t e x i n g ) t o f o r m m u l t i l a m e l l a r v e s i c l e s . F r e e z e / t h a w c y c l e s were r e p e a t e d a t l e a s t t h r e e t i m e s t o d i s r u p t t h e v e s i c l e s , c a u s i n g w ater t o be d i s t r i b u t e d e v e n l y t h r o u g h o u t t h e v e s i c l e s i n t o v e s i c l e s o f s i m i l a r s i z e s t h e r e b y e l i m i n a t i n g some o f t h e sample i n h o m o g e n e i t y . 22 23 The d i s p e r s i o n s were c e n t r i f u g e d and t h e r e s u l t i n g p e l l e t s were s e p a r a t e d f r o m t h e s u p e r n a t a n t and p l a c e d i n t o 6mm wide, 1.5cm l o n g t e s t t u b e s . D e u t e r i u m d e p l e t e d b u f f e r ( a g a i n 50mM p o t a s s i u m p h o s p h a t e ) was added i n e x c e s s , u s u a l l y a b o u t 60-100M1. A g a i n t h e f r e e z e / t h a w / v o r t e x i n g c y c l e s were r e p e a t e d . A l l sam p l e s were u s e d w i t h i n 2 d a y s o f p r e p a r a t i o n and a l l NMR e x p e r i m e n t s were c o m p l e t e d w i t h i n 4 d a y s . TLC was p e r f o r m e d b e f o r e and a f t e r t h e e x p e r i m e n t s t o e n s u r e no d e g r a d a t i o n had o c c u r r e d d u r i n g t h e e x p e r i m e n t . None was o b s e r v e d . The e l u t i n g s o l v e n t f o r t h e TLC was CHCl 3:MeOH:25%NH 3:H 20 i n t h e r a t i o 900:540:57:53. B. NMR MEASUREMENTS A l l NMR measurements were p e r f o r m e d on a l o c a l l y c o n s t r u c t e d 35 MHz d e u t e r i u m s p e c t r o m e t e r o p e r a t i n g on r e s o n a n c e . Q u a d r u p o l e e c h o s were o b t a i n e d by r e p e a t i n g t h e p u l s e c y c l e c o n s i s t i n g o f f o u r p a i r s o f p u l s e s [ 9 0 x ~ T - 9 0 ] , [ 9 0 _ x - T - 9 0 ^ ] [90 - r - 9 0 1, [90 - T-90 1 where t h e e c h o s r e s u l t i n g from x y x y e a c h p a i r o f p u l s e s were a l t e r n a t e l y added t o and s u b t r a c t e d f r o m t h e computer memory. The l e n g t h of t h e 90 d e g r e e p u l s e was =* 3jisec and T=40/usec. T h i s s e q u e n c e was r e p e a t e d a t a r a t e o f 2.5 s " 1 . S i n c e t h e T, o f t h e s e s a m p l e s was on t h e o r d e r o f 35 msec t h i s r e c y c l e t i m e was s u f f i c i e n t l y l o n g t o a l l o w t h e d e u t e r o n s t o e q u i l i b r a t e a l o n g t h e f i e l d ( w i t h t h e e x c e p t i o n of t h e m e t h y l d e u t e r o n s w h i c h have a much l o n g e r T,) [ 3 0 ] . Q u a d r a t u r e d e t e c t i o n was u s e d and t h e echo s i g n a l b o t h i n and o u t o f phase w i t h t h e a p p l i e d r . f . p u l s e were 24 c o l l e c t e d . The p h a s e s were a d j u s t e d so t h a t t h e s i g n a l i n t h e out of phase c h a n n e l was e f f e c t i v e l y z e r o . Above 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 t h e p h o s p h o l i p i d , a d w e l l t i m e of 5/isec was u s e d whereas a d w e l l t i m e o f 2usec was u s e d f o r l i p i d s below t h e t r a n s i t i o n t e m p e r a t u r e . E a c h s p e c t r u m was o b t a i n e d from t h e a c c u m u l a t i o n of 40,000 s c a n s . When n e c e s s a r y , t h e s i g n a l was s h i f t e d u s i n g an i n t e r p o l a t i o n t e c h n i q u e [37] t o e n s u r e t h a t t h e echo was s y m m e t r i c a l a b o u t t=2r. The s i g n a l was t r a n s f o r m e d i n t o t h e f r e q u e n c y domain u s i n g a f a s t F o u r i e r T r a n s f o r m . S p e c t r a l moments were c a l c u l a t e d f r o m t h e s p e c t r a i n t h e f r e q u e n c y domain and o r i e n t e d s p e c t r a were o b t a i n e d by d e p a k i n g t h e powder p a t t e r n s . The a v e r a g e T 2 e o f t h e d e u t e r o n s on t h e sn-2 c h a i n was measured u s i n g t h e q u a d r u p o l e echo s e q u e n c e w i t h v a r y i n g r v a l u e s . The t e m p e r a t u r e was r e g u l a t e d by a B r u k e r T e m p e r a t u r e C o n t r o l l e r . The h e a t i n g d e v i c e c o n s i s t e d o f an oven and t h e t e m p e r a t u r e was measured u s i n g a t h e r m o c o u p l e . A t e a c h t e m p e r a t u r e change t h e s y s t e m was a l l o w e d t o e q u i l i b r a t e f o r an h o u r . C. CALORIMETRY 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 (DSC) measurements were p e r f o r m e d on an MC-1 U l t r a S e n s i t i v e S c a n n i n g C a l o r i m e t e r M i c r o C a l I n c . The samples were p r e p a r e d i n a s i m i l a r manner t o t h e NMR samples e x c e p t DPPC n o t DPPC-d31 was u s e d . DSC 25 t r a c e s of DPPC-d3l were shown t o e x h i b i t s i m i l a r c a l o r i m e t r i c b e h a v i o u r t o DPPC, w i t h t h e o n l y o b s e r v a b l e d i f f e r e n c e b e i n g t h e 2 d e g r e e r e d u c t i o n i n t r a n s i t i o n t e m p e r a t u r e . E a c h sample c o n t a i n e d 5 mg o f DPPC and t h e a p p r o p r i a t e amounts of s t e r o l t o make 0,5,10,15,20 and 25 m o l a r p e r c e n t c o n c e n t r a t i o n s . The l i p i d s were d i s p e r s e d i n .7 ml o f b u f f e r ( a g a i n 50mM p o t a s s i u m p h o s p h a t e ) and t h e c a l o r i m e t r i c b e h a v i o u r o f t h e s e s a m p l e s was compared t o a r e f e r e n c e c e l l c o n t a i n i n g .7 ml o f b u f f e r . A l l DSC t r a c e s were run a t a s c a n r a t e of 14 d e g r e e s / h o u r . The e n t h a l p y o f t r a n s i t i o n was c a l c u l a t e d f r o m t h e a r e a under e a c h t r a c e . IV. RESULTS A. INTRODUCTION Aqueous d i s p e r s i o n s of sn-2 s u b s t i t u t e d DPPC-d31 w i t h v a r i o u s c o n c e n t r a t i o n s o f e i t h e r l a n o s t e r o l o r c h o l e s t e r o l were s t u d i e d u s i n g 2H-NMR t e c h n i q u e s . 2H-NMR i s s e n s i t i v e t o the b e h a v i o u r of t h e d e u t e r a t e d c h a i n s . Thus t h e s e c h a i n s ' can be v i e w e d as p r o b e s i n t o t h e i n t e r i o r o f t h e membranes, p r o v i d i n g i n f o r m a t i o n a b o u t t h e o r d e r and d y n a m i c s o f t h e b i l a y e r . B. PURE DPPC-D31 AT 44°C A t y p i c a l s p e c t r u m f o r t h e s e s y s t e m s i s shown i n f i g u r e 5 ( a ) . The s y s t e m c o n t a i n e d p u r e DPPC-d3l v e s i c l e s i n e x c e s s b u f f e r and had been a l l o w e d t o e q u i l i b r a t e a t 44°C f o r an h o u r . The c e n t r a l peak, ( a t co=0) , i s due t o t h e n a t u r a l a b undance of 2H i n wa t e r and was m i n i m i z e d by u s i n g a d e u t e r i u m d e p l e t e d b u f f e r . The c a r b o n p o s i t i o n s from t h e p h o s p h o l i p i d head down t o th e t e r m i n a l m e t h y l g r o u p e x h i b i t s u c c e s s i v e l y more m o t i o n a l a v e r a g i n g . Thus t h e pe a k s c o r r e s p o n d i n g t o t h e t e r m i n a l m e t h y l g r o u p s have t h e s m a l l e s t s p l i t t i n g and a r e c e n t r e d a r o u n d ±3 kHz, w h i l e t h e d e u t e r o n s p o s i t i o n e d c l o s e r t o t h e l i p i d / w a t e r i n t e r f a c e ( w i t h t h e e x c e p t i o n of t h e d e u t e r o n s on t h e number two c a r b o n ) have i n c r e a s i n g l y l a r g e r s p l i t t i n g s . T a k i n g i n t o a c c o u n t t h i s " f l e x i b i l i t y g r a d i e n t " , e a c h r e s o l v e d peak c a n be a s s i g n e d a c a r b o n p o s i t i o n ( o r 26 27 -40 •20 0 20 frequency (kHz) 40 WKJW _L J - _L - 4 0 - 2 0 0 2 0 F r e q u e n c y (kHz) 4 0 F i g u r e 5. 2H NMR S p e c t r a . (A) DPPC-d31 powder p a t t e r n taken at 44°C. (B) Depaked v e r s i o n of the above spectrum e x h i b i t i n g 8 r e s o l v e d peaks. The peak assignments i n d i c a t e the carbon p o s i t i o n the deuterons are bonded t o . 28 c a r b o n p o s i t i o n s ) on t h e c h a i n . T h i s c a n be a c h i e v e d a t l e a s t a p p r o x i m a t e l y by i n t e g r a t i n g t h e a r e a under t h e d e p a k e d s p e c t r u m ( f i g u r e 5 ( b ) ) . C o n t r i b u t i o n s t o t h e c o m p o s i t e l i n e s h a p e a r e made by t h e m e t h y l e n e d e u t e r o n s on c a r b o n p o s i t i o n s 2-15 and t h e m e t h y l p e a k s on p o s i t i o n 16. The t o t a l a r e a a t f r e q u e n c y s p l i t t i n g s g r e a t e r t h a n t h a t o f t h e m e t h y l ,peak ( l a b e l e d 16 i n f i g u r e 5 ( b ) ) can be p a r t i t i o n e d among t h e 28 m e t h y l e n e d e u t e r o n s . The a r e a under e a c h peak p r o v i d e s an e s t i m a t e o f t h e number o f d e u t e r o n s c o n t r i b u t i n g t o t h a t p e a k . Thus e a c h peak c a n be a s s i g n e d a c a r b o n p o s i t i o n a s shown i n f i g u r e 5 ( b ) . The r e c y c l e t i m e o f t h e e x p e r i m e n t was s h o r t e r t h a n t h e T, o f t h e t e r m i n a l m e t h y l g r o u p so t h e a r e a u n d e r t h e m e t h y l peak does n o t s c a l e w i t h t h e number o f d e u t e r o n s [ 3 0 ] . I t s h o u l d a l s o be m e n t i o n e d t h a t t h e d e u t e r o n s on t h e a l p h a p o s i t i o n c a r b o n a r e i n e q u i v a l e n t due t o a bend i n t h e c h a i n a t t h i s p o i n t [ 3 8 ] . The s m a l l p e a k s w i t h s p l i t t i n g s of a r o u n d 11 kHz and 17 kHz a r e p r o b a b l y a t t r i b u t a b l e t o t h e s e a l p h a p o s i t i o n d e u t e r o n s b u t t h e s i g n a l t o n o i s e r a t i o i s n o t good enough t o make any f i r m c o n c l u s i o n s . A s i m i l a r p r o c e d u r e was f o l l o w e d by P a u l s et al [33] i n t h e i r a n a l y s i s o f DPPC-d62 but t h e d i s p l a c e m e n t of t h e 2 H NMR s p l i t t i n g s a s s o c i a t e d w i t h t h e sn-1 and sn-2 c h a i n s r e l a t i v e t o e a c h o t h e r c a u s e d t h e s p l i t t i n g s i n t h e DPPC-d62 s p e c t r u m t o be l e s s d e f i n i t i v e t h a n t h o s e made f o r DPPC-d31.These 29 s p l i t t i n g s c a n be f o u n d i n f i g u r e 13 and t a b l e 1. T a b l e 1. Q u a d r u p o l e S p l i t t i n g s (kHz. ) t C a r b o n No. - DPPC-d31 5% C h o i . 3-9 26.2 31.0 10 23.5 31 .0 1 1 22.5 25.0 1 2 20.3 23.0 13 18.6 19.3 1 4 15.4 15.2 15 12.1 12.9 1 6 2.9 3.9 f A l l e r r o r s f o r t h e s p l i t t i n g s a r e ± 0.1 kHz. Two comments can be made on t h e a s s i g n m e n t of t h e p e a k s i n t h e d e p a k e d s p e c t r u m . F i r s t l y , t h e d i f f e r e n c e i n t h e s p l i t t i n g s f o r p o s i t i o n s 3-9 were n o t r e s o l v a b l e . T h e s e l i n e s h a p e s a r e s u p e r i m p o s e d , f o r m i n g what i s known as t h e " p l a t e a u " r e g i o n o f t h e s p e c t r u m . S e c o n d l y , s i n c e t h e a s s i g n m e n t o f t h e s e p e a k s i s s u b j e c t t o some a m b i g u i t y , f u r t h e r work w i t h s p e c i f i c a l l y l a b e l l e d l i p i d s w o u l d be n e c e s s a r y t o e s t a b l i s h u n q u a l i f i e d a s s i g n m e n t s . C. LANOSTEROL:DPPC-D31 SYSTEMS In o r d e r t o compare how t h e i n c o r p o r a t i o n o f l a n o s t e r o l and c h o l e s t e r o l i n t o DPPC-d3l b i l a y e r s i n f l u e n c e s t h e DPPC-d31 2 H NMR s p e c t r u m , s p e c t r a were o b t a i n e d f o r aqueous 30 d i s p e r s i o n s of DPPC-d31 samples c o n t a i n i n g 0%, 5%, 10% and 15% m o l a r p e r c e n t l a n o s t e r o l and 5%, 10% and 15% molar p e r c e n t c h o l e s t e r o l a s a f u n c t i o n of t e m p e r a t u r e . The r e l a x a t i o n t i m e T^e was a l s o measured t o p r o v i d e f u r t h e r i n s i g h t i n t o t h e d y n a m i c s of t h e l i p i d s . The s p e c t r a r e s u l t i n g f r o m p u r e DPPC-d3l v e s i c l e s can be seen i n f i g u r e 6 . As has a l r e a d y been d i s c u s s e d , t h i s s y s t e m e x h i b i t s a x i a l l y s y m m e t r i c s p e c t r a w i t h 8 c l e a r l y r e s o l v a b l e p e a k s above t h e phase t r a n s i t i o n . I t c a n a l s o be seen t h a t t h e phase t r a n s i t i o n i s s h a r p , i . e . i t o c c u r s o v e r a t e m p e r a t u r e r a n g e of l e s s t h a n two d e g r e e s and t h e r e i s no o b s e r v a b l e o v e r l a p p i n g of p h a s e s a t t e m p e r a t u r e s e i t h e r s i d e o f t h e t r a n s i t i o n ( 3 6 C C , 3 8 ° C ) . Below t h e phase t r a n s i t i o n t e m p e r a t u r e , T , a b r o a d g e l - t y p e s p e c t r u m i s o b t a i n e d . The shape i s c h a r a c t e r i s t i c o f t h e a s y m m e t r i c s p e c t r a o b t a i n e d f o r s p e c i f i c a l l y d e u t e r a t e d l i p i d s i n t h e g e l p h a s e . E x c e p t f o r t h e m e t h y l p e a k s i t i s e s s e n t i a l l y f e a t u r e l e s s . As t h e t e m p e r a t u r e i s i n c r e a s e d , t h e w i d t h o f t h e powder p a t t e r n d e c r e a s e s , i n d i c a t i n g t h a t t h e l i p i d s a r e becoming more m o b i l e . T h e s e o b s e r v a t i o n s w i l l be q u a n t i f i e d t o some e x t e n t when t h e f i r s t moments a r e p r e s e n t e d . The a d d i t i o n o f 5 m o l a r p e r c e n t l a n o s t e r o l r e d u c e s t h e r e s o l u t i o n o f t h e q u a d r u p o l a r p e a k s above T , and i n c r e a s e s t h e q u a d r u p o l e s p l i t t i n g s of t h e p e a k s t h a t r e m a i n r e s o l v e d , ( f i g u r e 7 ) . O n l y 5 p e a k s a r e c l e a r l y r e s o l v e d i n t h e de p a k e d s p e c t r a ( f i g u r e 16) so i t was not p o s s i b l e t o make 31 - 1 0 0 - 5 0 0 5 0 100 F r e q u e n c y ( k H z ) Figure 6. Comparison of the powder pattern spectra for pure DPPC-D31 measured over a range of temperatures. 32 - 8 0 - 4 0 0 4 0 8 0 F r e q u e n c y ( k H z ) Figure 7. Comparison of the powder p a t t e r n s p e c t r a f o r 5% lanosterol/DPPC-d31 measured'over a range of temperatures. 33 unambiguous peak a s s i g n m e n t s . The i n c r e a s e d q u a d r u p o l e s p l i t t i n g s o f t h e s p e c t r a i s q u a l i t a t i v e l y c o n s i s t e n t w i t h t h e b e h a v i o u r r e p o r t e d f o r c h o l e s t e r o l [ 6 , 7 ] . At 44°C, t h e T 2 e r e l a x a t i o n t i m e ( t a b l e 2) i s s i m i l a r t o t h e c o r r e s p o n d i n g T 2 g f o r p u r e DPPC-d31 a t t h i s t e m p e r a t u r e . T a b l e 2. T_ R e l a x a t i o n T i mes a t 44°C. ^ e S t e r o l C o n c e n t r a t i o n T 2 e ( u s e c ) 0% s t e r o l 400 5% l a n o s t e r o l 450 10% l a n o s t e r o l 700 15% l a n o s t e r o l 600 5% c h o l e s t e r o l 800 10% c h o l e s t e r o l 850 15% c h o l e s t e r o l 600 Thus t h e d e c r e a s e d r e s o l u t i o n c a n n o t be e x p l a i n e d by homogeneous l i n e b r o a d e n i n g . F u r t h e r , t h e t r a n s i t i o n o c c u r s o v e r a r a n g e o f t e m p e r a t u r e s and between 34°C and 41°C t h e s p e c t r a c o n t a i n c o n t r i b u t i o n s f r o m b o t h t h e g e l and t h e l i q u i d c r y s t a l l i n e p h a s e s . L i t t l e a t t e m p t w i l l be made t o d e s c r i b e t h i s mixed phase r e g i o n o t h e r t h a n by making t h e o b s e r v a t i o n t h a t i t e x i s t s and l a t e r ( i n t h e d i s c u s s i o n ) by e s t i m a t i n g t h e f r a c t i o n s o f g e l and l i q u i d c r y s t a l l i n e p h a s e s u s i n g t h e f i r s t moments. 34 W i t h t h e l a n o s t e r o l c o n c e n t r a t i o n i n c r e a s e d t o 10%, t h e r e s o l u t i o n o f t h e s p e c t r a above t h e phase t r a n s i t i o n i s f u r t h e r r e d u c e d ( f i g u r e 8 ) . O n l y 4 peaks were r e s o l v e d i n the d e p a k e d s p e c t r a . The q u a d r u p o l e s p l i t t i n g s i n c r e a s e and th e p h a s e t r a n s i t i o n now e x t e n d s o v e r more t h a n 10 d e g r e e s . S i m i l a r l y , t h e a d d i t i o n o f 15% l a n o s t e r o l c o n t i n u e s t h e t r e n d s t a r t e d by t h e a d d i t i o n of t h e s m a l l e r c o n c e n t r a t i o n s ( f i g u r e 9 ) . Over t h e e n t i r e t e m p e r a t u r e r a n g e i t becomes d i f f i c u l t t o say whether t h e s y s t e m i s i n a s i n g l e o r mixed p h a s e . The s p e c t r a r e m a i n a x i a l l y s y m m e t r i c t o below 2 7 ° C . T h i s m i x i n g o f t h e p h a s e s c a n be f u r t h e r c h a r a c t e r i z e d by t h e f i r s t moment. As was d i s c u s s e d i n t h e t h e o r y s e c t i o n , M, g i v e s an e s t i m a t e o f t h e a v e r a g e o r d e r p a r a m e t e r s o r t h e a v e r a g e s p l i t t i n g s . A l t h o u g h t h e d e f i n i t i o n of M, g i v e n i n the t h e o r y s e c t i o n i n v o l v e d an i n t e g r a l , i n p r a c t i c e t h e sum ^m , L t(co0+co)co u=0 4 . 1 wm L f(o) 0+cj) co=0 i s e v a l u a t e d , where £(co) i s t h e i n t e n s i t y a t a f r e q u e n c y co from t h e l a r m o r f r e q u e n c y and u>m i s t h e f r e q u e n c y c h o s e n t o be i n t h e b a s e l i n e o f t h e s p e c t r u m . The f i r s t moments o f t h e s p e c t r a p r e s e n t e d t h u s f a r a r e shown i n f i g u r e 10. An i n i t i a l comment on t h e t r a n s i t i o n c a n be made. F o r p u r e DPPC-d31 t h e d i f f e r e n c e between M 1 above t h e phase t r a n s i t i o n (44°C) and below t h e pha s e t r a n s i t i o n (23°C) i s 3 5 - 8 0 - 4 0 0 4 0 8 0 Frequency (kHz) F i g u r e 8 . C o m p a r i s o n of t h e powder p a t t e r n s p e c t r a f o r 10% l a n s t e r o l / D P P C - d 3 1 measured o v e r a r a n g e o f t e m p e r a t u r e s . 1 1 1 " ' I I I I I I I M . • • , , | -100 -50 0 50 100 Frequency (kHz) F i g u r e 9. C o m p a r i s o n o f t h e powder p a t t e r n s p e c t r a f o r 1 l a n s t e r o l / D P P C - d 3 l measured o v e r a r a n g e o f t e m p e r a t u r e s . 3 7 14 X 15 20 25 30 35 40 4 5 T e m p e r a t u r e ( ° C ) F i g u r e 10. V a r i a t i o n o f t h e f i r s t moment, M,, w i t h t e m p e r a t u r e f o r 0, 5, 10 and 15 p e r c e n t l a n o s t e r o l c o n c e n t r a t i o n s . 38 7 . 5 * 1 0 + 4 s - 1 . However, f o r 5% l a n o s t e r o l t h i s d e c r e a s e s t o 6.4*10 +" s " 1 f u r t h e r d e c r e a s e s t o 5 . 8 * 1 0 + 4 s " 1 and 4.8*10 +" s ~ 1 w i t h t h e a d d i t i o n of more l a n o s t e r o l t o c o n c e n t r a t i o n s o f 10% and 15%. In f a c t a t a g i v e n t e m p e r a t u r e above T m , M, i n c r e a s e s w i t h s t e r o l c o n c e n t r a t i o n w h i l e below T , M, m 1 d e c r e a s e s w i t h s t e r o l c o n c e n t r a t i o n . The b e h a v i o u r of t h e moments s u p p o r t s t h e c o n c l u s i o n s a l r e a d y r e a c h e d a b o u t the' p h a s e t r a n s i t i o n . W i t h o u t t h e i n c o r p o r a t i o n o f s t e r o l , t h e t r a n s i t i o n f r o m g e l t o l i q u i d c r y s a l l i n e was s h a r p w h i l e w i t h t h e a d d i t i o n of 15 m o l a r p e r c e n t l a n o s t e r o l , a v e r y g r a d u a l change i n t h e moments o c c u r r e d . D. CHOLESTEROL;DPPC-D31 SYSTEMS The powder p a t t e r n s r e s u l t i n g f r o m t h e a d d i t i o n of 5% c h o l e s t e r o l a r e s i m i l a r t o t h e c o r r e s p o n d i n g 5% l a n o s t e r o l s p e c t r a ( f i g u r e 11). The q u a d r u p o l e s p l i t t i n g s a r e i n c r e a s e d t o a s l i g h t l y g r e a t e r e x t e n t t h a n t h e l a n o s t e r o l s p e c t r a (as w i l l be seen from t h e p l o t s o f M,) ( f i g u r e 1 6 ) . Seven p e a k s a r e c l e a r l y r e s o l v e d and can be a s s i g n e d c a r b o n p o s i t i o n s on t h e c h a i n . P o s i t i o n 10 i s no l o n g e r d i s t i n g u i s h a b l e f r o m t h e p l a t e a u peak. The q u a d r u p o l a r r e l a x a t i o n t i m e T 2 e has i n c r e a s e d d r a m a t i c a l l y ( t a b l e 2) from 400 Msec ( p u r e DPPC-d31) t o 800Msec ( 5 % c h o l e s t e r o l ) . S i n c e t h e w i d t h a t h a l f maximum f o r a p u r e homogeneously b r o a d e n e d s y s t e m i s 1 / , r T 2 e a c o m p a r i s o n o f t h e w i d t h s o f t h e p eaks i n t h e d e paked s p e c t r u m t o 1 / T T T - shows t h e r e i s an inhomogeneous Figure 11. Comparison of the powder pattern spectra for 5% cholesterol/DPPC-d3l measured over a range of temperatures. 40 c o n t r i b u t i o n t o t h e l i n e w i d t h . F o r DPPC-d31, 1/'"' T2 e = - 7 k H z whereas t h e mean h a l f w i d t h o f t h e p e a k s i s a r o u n d 1 kHz. In t h e p r e s e n c e of 5% c h o l e s t e r o l , 1 / 7 r T 2 e * s * ^ k H z w ^ ^ l e t h e a v e r a g e h a l f w i d t h of t h e p e a k s i s 1.3 kHz., t h i r t y p e r c e n t l a r g e r t h a n t h e a v e r a g e DPPC-d3l s p e c t r a l peak w i d t h . The t r a n s i t i o n f r o m 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 s o c c u r s o v e r a w i d e r range o f t e m p e r a t u r e s t h a n t h e p u r e DPPC~d31 t r a n s i t i o n as has p r e v i o u s l y been o b s e r v e d by o t h e r s [6] and a t 36.5°C t h e s p e c t r u m e x h i b i t s what has been d e s c r i b e d as t h e " l i q u i d g e l p h a s e " [ 3 9 ] . A t 28°C t h e s y s t e m a p p e a r s t o be i n t h e g e l p h a s e . The r e s u l t s f o u n d f o r t h e 10% c h o l e s t e r o l sample were c o n s i s t e n t w i t h t h e 5% c h o l e s t e r o l d a t a t h o u g h t h e peak r e s o l u t i o n had d i m i n i s h e d somewhat ( f i g u r e 1 2 ) . I n c r e a s i n g t h e c h o l e s t e r o l c o n c e n t r a t i o n t o 15% l e d t o a s i g n i f i c a n t d e c r e a s e i n t h e peak r e s o l u t i o n ( f i g u r e 1 3 ) . A g a i n t h e s e p a r a t i o n s of t h e q u a d r u p o l e s p l i t t i n g s have i n c r e a s e d . O n l y 4 p e a k s a r e r e s o l v e d i n t h e c e n t r a l r e g i o n . The b e h a v i o u r o f M, f o r 0%, 5%, 10% and 15% c h o l e s t e r o l i s shown i n f i g u r e 14. V e r y l i t t l e d i f f e r e n c e e x i s t s between t h e f i r s t moments o f c h o l e s t e r o l and l a n o s t e r o l a t c o m p a r a b l e c o n c e n t r a t i o n s and t e m p e r a t u r e s . In b o t h c a s e s t h e t r a n s i t i o n r e g i o n b r o a d e n s w i t h i n c r e a s i n g s t e r o l c o n c e n t r a t i o n . W i t h i n t h i s t r a n s i t i o n r e g i o n t h e l i p i d s e x i s t i n a mixed p h a s e . A l t h o u g h t h e moments p r o v i d e i n f o r m a t i o n a b o u t t h e a v e r a g e s p l i t t i n g s , i t would be o f i n t e r e s t t o see how t h e I I I I I I - 1 0 0 5 0 0 5 0 F R E Q U E N C Y ( k H z ) 4 4.0°C 39.0°C 36.5°C 34.5°C 23.0°C 100 F i g u r e 12. C o m p a r i s o n o f t h e powder p a t t e r n s p e c t r a f o r 1 c h o l e s t e r o l / D P P C - d 3 1 measured o v e r a range o f t e m p e r a t u r e s 42 - 8 0 - 4 0 0 4 0 8 0 F r e q u e n c y ( k H z ) F i g u r e 13. Comparison of the powder p a t t e r n s p e c t r a f o r l 5 % cholesterol/DPPC-d31 measured over a range of temperatures. 4 3 14 r~ • , 20 25 30 35 40 4 5 T e m p e r a t u r e (°C) F i g u r e 14. V a r i a t i o n o f t h e f i r s t moment, M,, w i t h t e m p e r a t u r e f o r 0, 5, 10 and 15 p e r c e n t c h o l e s t e r o l c o n c e n t r a t i o n s . 44 s p e c i f i c p e a k s a r e i n f l u e n c e d by t h e i n c o r p o r a t i o n of t h e two s t e r o l s . U n f o r t u n a t e l y , peak a s s i g n m e n t s c o u l d o n l y be made f o r t h e p u r e DPPC and 5% c h o l e s t e r o l s y s t e m s a t 44°C and t h e s p l i t t i n g s f o r t h e s e r e s o l v e d p e aks a r e compared i n t a b l e 1. D i r e c t c o m p a r i s o n can be made between t h e s p e c t r a of p u r e D P P C - d 3 l , 5% l a n o s t e r o l and 5% c h o l e s t e r o l a t 44°G shown i n f i g u r e 15. The q u a d r u p o l e s p l i t t i n g s f o r t h e two 5% s t e r o l s y s t e m s d i f f e r by o n l y a s m a l l amount. However 7 pe a k s a r e c l e a r l y r e s o l v e d f o r t h e 5% c h o l e s t e r o l s p e c t r a w h i l e o n l y 5 a r e r e s o l v e d f o r t h e c o r r e s p o n d i n g l a n o s t e r o l s p e c t r a . The de p a k e d v e r s i o n of f i g u r e 15 i s p r e s e n t e d i n f i g u r e 16. R e l a x a t i o n t i m e s have been p r e s e n t e d d u r i n g d i s c u s s i o n s of t h e s p e c t r a l l i n e w i d t h s . T 2 g i s s e n s i t i v e t o slow m o t i o n s and t h u s f u r t h e r c o m p a r i s o n s c a n be made between t h e i n f l u e n c e s of t h e s e two s t e r o l on m o t i o n s . The r e l a x a t i o n t i m e T 2 e f o r t h e d i f f e r e n t s a m p l e s a t 44°C can be f o u n d i n t a b l e 2. I t i s i n t e r e s t i n g t h a t b o t h s t e r o l s r e s u l t i n an i n c r e a s e i n T 2 e , and t h e i n c r e a s e i s g r e a t e r f o r c h o l e s t e r o l t h a n f o r l a n o s t e r o l . The f a c t o r o f two i n c r e a s e i n T_ w i t h 2e t h e a d d i t i o n o f 5% and 10% c h o l e s t e r o l h as a l s o been o b s e r v e d w i t h t h e a d d i t i o n of c e r t a i n a l c o h o l s [ 3 6 ] . Frequency (kHz) Figure 15. Comparison' of t h e powder p a t t e r n s p e c t r a of a ) t h p u r e DPPC~d31 sample, b) t h e 5% l a n s o s t e r o l sample and c t h e 5% c h o l e s t e r o l sample a l l measured a t 44° C . _ J I I I I I I ! 1_ -40 -20 0 20 AO Frequency ( k H z ) Figure 16. C o m p a r i s o n o f t h e d e p a k e d s p e c t r a o f a ) t h e pure D P P C - d 3 l sample, b) t h e 5% l a n s o s t e r o l sample and c) t h e 5% c h o l e s t e r o l sample a l l measured a t 4 4 ° C . 47 E. DIFFERENTIAL SCANNING CALORIMETRY 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 (DSC) was p e r f o r m e d on b o t h c h o l e s t e r o l - D P P C and l a n o s t e r o l - D P P C s y s t e m s . The t r a n s i t i o n t e m p e r a t u r e f o u n d f o r DPPC-d3l u s i n g t h i s method was 39°C w h i c h i s midway between t h a t o f p u r e DPPC (41°C) and DPPC-d62 (37°C [ 3 0 , 4 0 ] . S i n c e t h e c a l o r i m e t r i c b e h a v i o u r of DPPC-d31 was f o u n d t o be q u a l i t a t i v e l y t h e same as t h a t o f DPPC, D P P C / s t e r o l s y s t e m s were compared. The DSC t r a c e s f o r 0,5,T5,20 and 25% l a n o s t e r o l : D P P C a r e shown i n f i g u r e 17. V e r y s i m i l a r b e h a v i o u r i s o b s e r v e d f o r c h o l e s t e r o l ( f i g u r e 18) and l a n o s t e r o l . W i t h t h e a d d i t i o n o f 5% c h o l e s t e r o l , t h e t r a n s i t i o n t e m p e r a t u r e i s r e d u c e d by 1.4°C, whereas t h e r e d u c t i o n i s 1.25°C f o r t h e l a n o s t e r o l t r a n s i t i o n t e m p e r a t u r e . A l s o a t t h i s c o n c e n t r a t i o n t h e h e i g h t o f t h e t r a n s i t i o n d e c r e a s e s w h i l e t h e w i d t h i n c r e a s e s . No s u b t r a n s i t i o n was n o t i c e a b l e when l a n o s t e r o l was i n t r o d u c e d . W i t h t h e a d d i t i o n of i n c r e a s i n g c o n c e n t r a t i o n s of l a n o s t e r o l , t h e h a l f w i d t h s i n c r e a s e d w h i l e t h e h e i g h t s f u r t h e r d e c r e a s e d . A c o n s t a n t amount o f DPPC was u s e d i n e a c h sample and t h e same s c a l e was us e d t h r o u g h o u t , t h u s t h e s e t r a c e s c a n be compared d i r e c t l y . The e n t h a l p y o f t r a n s t i t i o n i s p r o p o r t i o n a l t o t h e a r e a under t h e DSC c u r v e s . I t was f o u n d t h a t w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f l a n o s t e r o l , t h e e n t h a l p i e s were r e d u c e d . The v a r i a t i o n of t h e e n t h a l p y of t r a n s i t i o n w i t h l a n o s t e r o l c o n c e n t r a t i o n c a n be seen i n f i g u r e 19. A l i n e a r d e c r e a s e i n 4 8 Mole % Lanosterol 0 5 15 20 25 J • i . . . . i . . . . J i • . . . i ,. I 10 20 30 40 50 T C C ) Figure 17. DSC t r a c e s of lanosterol/DPPC systems measured at a scan r a t e of !5°/hr. 49 m o l e * C H O L . F i g u r e 18. DSC t r a c e s of cholesterol/DPPC s y s t e m s measured a t a s c a n r a t e o f 15°/hr, Jj/J 50 _ A — A A — A — i I 1 1 1 ! I 5 10 15 20 Molar Concentration of Lanosterol 25 F i g u r e 19. V a r i a t i o n o f t h e e n t h a l p i e s o f t r a n s i t i o n w i t h s t e r o l c o n c e n t r a t i o n . 51 e n t h a l p y w i t h c h o l e s t e r o l c o n c e n t r a t i o n has been o b s e r v e d p r e v i o u s l y [ 4 , 2 5 ] . Due t o t h e p r e s e n c e of a b r o a d component t h e e n t h a l p y measurements c o n t a i n a l a r g e u n c e r t a i n t y . F u r t h e r , a d i s t o r t i o n i n t h e s l o p e o f t h e c u r v e s r e s u l t s from t h e f i n i t e s c a n r a t e . V. DISCUSSION A c o m p a r i s o n between l a n o s t e r o l and c h o l e s t e r o l w i l l be made i n terms of t h e p h y s i c a l p r o p e r t i e s d e t e r m i n e d by 2H-NMR and DSC. In a d d i t i o n , t h e d e c a y t i m e of t h e q u a d r u p o l e e c h o , T 2 g , w i l l a l s o be d i s c u s s e d i n r e l a t i o n t o t h e e f f e c t of c h o l e s t e r o l on slow m o t i o n s . A. OVERVIEW W i t h t h e i n t r o d u c t i o n of oxygen i n t o t h e a t m o s p h e r e , t h e d e v e l o p m e n t o f membranes i n v o l v e d t h e i n c o r p o r a t i o n of c h o l e s t e r o l . L a n o s t e r o l was p r e s e n t o n l y as a t r a n s i e n t s t e p on t h e e v o l u t i o n a r y pathway t o c h o l e s t e r o l [ 1 ] . The i m p l i c a t i o n o f t h i s i s t h a t c h o l e s t e r o l b e s t o w s s u p e r i o r p h y s i c a l p r o p e r t i e s on t h e membranes. I t was h y p o t h e s i z e d t h a t i f t h e s u p e r i o r i t y of c h o l e s t e r o l r e s u l t e d from t h e smoothness of t h e a l p h a - f a c e , any "bump" on t h i s f a c e ( i n p a r t i c u l a r t h e 14 a m e t h y l g r o u p of l a n o s t e r o l ) would s e r i o u s l y i n h i b i t t h e f u n c t i o n i n g of t h e s t e r o l . I t was hoped t h a t i f t h i s were t h e c a s e i t would be r e f l e c t e d by c h a n g e s i n t h e NMR s p e c t r a . C a r b o n p o s i t i o n s 9 t o 12 a r e i n t h e r e g i o n t h a t i s l i k e l y t o be p e r t u r b e d by t h e 14-a m e t h y l g r o u p s . I t i s u n f o r t u n a t e t h a t p o s i t i o n s 9 and 10 were n o t r e s o l v a b l e from t h e p l a t e a u r e g i o n . However, c h a n g e s i n t h e s p l i t t i n g s of t h e p l a t e a u r e g i o n i n d u c e d by l a n o s t e r o l were c o m p a r a b l e t o t h e c h a n g e s o b s e r v e d f o r c h o l e s t e r o l . In a d d i t i o n t h e s p l i t t i n g s o f t h e p e a k s t h a t do r e m a i n r e s o l v e d i n c r e a s e 52 53 w i t h t h e i n c o r p o r a t i o n of h i g h e r c o n c e n t r a t i o n s of b o t h s t e r o l s . A s t u d y was p e r f o r m e d w i t h d i o l e o y l p h o s p h a t i d y l c h o l i n e (DOPC) s p e c i f i c a l l y l a b e l l e d a t p o s i t i o n s 9 and 10 [ 4 1 ] . I t was o b s e r v e d t h a t l a n o s t e r o l and c h o l e s t e r o l had d i f f e r e n t e f f e c t s on t h e c h a i n o r d e r i n g o f DOPC m o l e c u l e s . The i n c o r p o r a t i o n of c h o l e s t e r o l was f o u n d t o i n c r e a s e t h e o r d e r p a r a m e t e r s f o r m e t h y l e n e d e u t e r o n s on C 9 but had no e f f e c t on t h e o r d e r a t p o s i t i o n C 1 0 . By c o n t r a s t , l a n o s t e r o l d i d n o t e f f e c t t h e s p l i t t i n g s a t e i t h e r p o s i t i o n . Some c a u t i o n i s n eeded i n c o m p a r i n g t h e s e r e s u l t s w i t h t h o s e p r e s e n t e d i n t h i s work s i n c e t h e s p l i t t i n g s g i v e n i n r e f e r e n c e [41] were t a k e n f r om s p e c t r a t h a t were p o o r l y r e s o l v e d . I t s h o u l d a l s o be m e n t i o n e d t h a t o t h e r s t u d i e s [6] show t h a t t h e i n c o r p o r a t i o n of c h o l e s t e r o l i n c r e a s e s t h e q u a d r u p o l e s p l i t t i n g s a t a l l p o s i t i o n s down t h e c h a i n . A s e c o n d s t u d y u s i n g f a t t y a c i d s p i n l a b e l s i n egg-PC s y s t e m s a l s o f o u n d t h a t l a n o s t e r o l had l e s s e f f e c t on t h e o r d e r i n g o f m e t h y l e n e s c l o s e t o t h e h y d r o p h i l i c heads t h a n c h o l e s t e r o l and had no e f f e c t a t p o s i t i o n s C 1 2 and C t 6 [ 2 0 ] , The s y s t e m s u s e d i n t h e s e s t u d i e s [20,42] c o n t a i n e d u n s a t u r a t e d PC l i p i d s w h i c h e x h i b i t a marked c o n d e n s a t i o n e f f e c t . Medium l e n g t h f a t t y c h a i n l e c i t h i n s s u c h a s DPPC do n o t show s u c h a s i g n i f i c a n t r e d u c t i o n i n t h e c r o s s s e c t i o n a l a r e a w i t h t h e i n c o r p o r a t i o n o f c h o l e s t e r o l [ 4 2 , 4 3 ] , Below 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 t h e p u r e l i p i d , t h e i n t r o d u c t i o n o f c h o l e s t e r o l (<10 mole %) i n t o DPPC membranes 54 removes t h e t i l t and i n c r e a s e s t h e m o b i l i t y and a m p l i t u d e of th e m o t i o n s of t h e f a t t y a c i d c h a i n s [ 4 4 ] . Above T , by c o n t r a s t , t h e s y s t e m i s more o r d e r e d t h a n t h e p u r e p h o s p h o l i p i d s y s t e m . I t has been p r o p o s e d t h a t t h e c o n d e n s a t i o n e f f e c t a r i s e s f r o m Van d e r Waals i n t e r a c t i o n s b e c a u s e of t h e dependence on c h a i n l e n g t h . G i v e n t h a t DPPC m o n o l a y e r s do not e x h i b i t a marked c o n d e n s a t i o n e f f e c t i t i s p o s s i b l e t h a t t h e b e h a v i o u r o b s e r v e d when c h o l e s t e r o l i s i n t r o d u c e d i n t o t h e DPPC l i p o s o m e s i s due t o t h e r i g i d i t y o f t h e c h o l e s t a n e r i n g s w h i c h a r e o r i e n t e d 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 s and r e s t r i c t t h e a n g l e s t h e c h a i n s c a n make. Below T m t h e s y s t e m i s a l r e a d y o r d e r e d and t h e i n t r o d u c t i o n o f c h o l e s t e r o l d i s r u p t s t h e i n t e r a c t i o n s between t h e a c y l c h a i n s . I t i s t h e r e f o r e n o t s u r p r i s i n g t h a t l a n o s t e r o l e x h i b i t e d s i m i l a r b e h a v i o u r . B. MOMENTS As a r e s u l t o f t h e d e c r e a s e d r e s o l u t i o n of t h e l a n o s t e r o l / D P P C s p e c t r a , i t was n o t p o s s i b l e t o compare t h e s p l i t t i n g s o f many i n d i v i d u a l p e a k s . However a q u a n t i t a t i v e c o m p a r i s o n c o u l d be made u s i n g t h e f i r s t moments. M, i s e s p e c i a l l y u s e f u l when d i s c u s s i n g t h e g e l and m i x e d phase r e g i o n s i n w h i c h o n l y t h e m e t h y l p e a k s a r e r e s o l v e d . L a n o s t e r o l and c h o l e s t e r o l s y s t e m s have v e r y s i m i l a r moment p r o f i l e s between 20°C and 45°C a t t h e c o n c e n t r a t i o n s e x a m i n e d . Above t h e phase t r a n s i t i o n , M, i n c r e a s e s w i t h 55 s t e r o l c o n c e n t r a t i o n f o r b o t h s y s t e m s . T h i s c a n be i n t e r p r e t e d a s t h e r i g i d i f y i n g o f t h e a c y l c h a i n s . One may e x p e c t l a n o s t e r o l t o be s l i g h t l y l e s s e f f e c t i v e i n r i g i d i f y i n g t h e c h a i n s b e c a u s e of t h e p e r t u r b i n g e f f e c t s of t h e e x t r a m e t h y l g r o u p s . The o b s e r v a t i o n s , w h i c h do not a g r e e w i t h t h i s e x p e c t a t i o n , i n d i c a t e t h a t t h e r i g i d i t y of t h e c h o l e s t a n e r i n g of e a c h o f t h e two m o l e c u l e s i s more i m p o r t a n t t h a n t h e s m a l l p r o t r u s i o n . Below t 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 t h e r e v e r s e s i t u a t i o n o c c u r s . B o t h s t e r o l s a p p e a r t o d i s o r d e r t h e membranes. In t h e two phase r e g i o n , t h e f r a c t i o n o f t h e membrane i n t h e g e l p h a s e can be c a l c u l a t e d f rom; M, = f M , G + ( 1 - f ) M , L 5.1 where f i s t h e f r a c t i o n of l i p i d s i n t h e g e l phase and M, and M, L a r e t h e f i r s t moments o f t h e g e l and l i q u i d c r y s t a l l i n e components of t h e s p e c t r u m r e s p e c t i v e l y [45,46] d e t e r m i n e d a t t e m p e r a t u r e s w e l l away from 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 h i s i n t e r p r e t a t i o n o f M, as a l i n e a r c o m b i n a t i o n o f p h a s e s i s o n l y v a l i d i f t h e e x c h a n g e r a t e between t h e p h a s e s i s slow compared t o t h e s p e c t r a l w i d t h . The f r a c t i o n a l c o m p o s i t i o n of t h e m i x e d r e g i o n s can be seen i n f i g u r e 20. The mixed p h a s e r e g i o n o f t h e p u r e DPPC e x t e n d s o v e r l e s s t h a n 3 d e g r e e s whereas w i t h t h e a d d i t i o n o f 5% s t e r o l , t h e m i x e d r e g i o n e x t e n d s o v e r 10 d e g r e e s . 56 Temperature ( ° C ) F i g u r e 20. F r a c t i o n of DPPC-d31 i n the g e l phase f o r the 0% and 5% s t e r o l c o n c e n t r a t i o n s as a f u n c t i o n of temperature. 57 The p h a s e d i a g r a m f o r c h o l e s t e r o l ( f i g u r e 3) shows t h a t a t 44°C 5% c h o l e s t e r o l / D P P C membranes a r e i n t h e l i q u i d c r y s t a l l i n e p h a s e . However a t t h e same t e m p e r a t u r e t h e 15% c h o l e s t e r o l / D P P C sample i s i n a m i x e d L + |3 phase [ 6 ] , T h i s has been d e s c r i b e d as an a v e r a g e of two p h a s e s , l i q u i d c r y s t a l l i n e and a s e c o n d phase w h i c h i s t h o u g h t t o have a h i g h e r c o n c e n t r a t i o n o f c h o l e s t e r o l t h a n t h e G and L p h a s e s . In t h i s r e g i o n i t was f o u n d t h a t t h e moments (and t h e r e f o r e t h e s p e c t r a ) c o u l d not be t r e a t e d as a l i n e a r c o n b i n a t i o n o f t h e two e n d p o i n t p h a s e s . The r e a s o n g i v e n f o r t h i s i s t h a t t h e r e i s f a s t e xchange between t h e L and 0 p h a s e s . E v i d e n c e f o r t h e /3 phase i s f o u n d i n t h e DSC t r a c e s w h i c h i n d i c a t e t h a t a t c o n c e n t r a t i o n s above 10% , t h e r e e x i s t s an u n d e r l y i n g b r o a d component f o r b o t h l a n o s t e r o l and c h o l e s t e r o l / D P P C s y s t e m s . At s m a l l s t e r o l c o n c e n t r a t i o n s , t h e r e i s a r e d u c t i o n 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 w h i c h i s c o n s i s t e n t w i t h t h e p r o p o s a l t h a t t h e r e i s a s m a l l m i x e d phase r e g i o n n e a r 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 t h e p u r e l i p i d [ 6 ] ( f i g u r e 3 ) . One r e a s o n f o r s t u d y i n g t h e p h a s e b e h a v i o u r i s t o g e t some i n s i g h t i n t o t h e i n t e r a c t i o n s between t h e components o f t h e s y s t e m . S i n c e t h e phase b e h a v i o u r o f l a n o s t e r o l a t t h e two c o n c e n t r a t i o n s measured i s v e r y s i m i l a r t o c h o l e s t e r o l , i t i s n e c e s s a r y t o examine t h e i n t e r a c t i o n s f r o m a n o t h e r a n g l e . 58 C. RELAXATION BEHAVIOUR The v a r i a t i o n of t h e d e c a y o f t h e q u a d r u p o l e echo d e t e r m i n e d f o r a l l t h e samples was e x p o n e n t i a l f o r s h o r t v a l u e s o f T. In t h e g e l p h a s e , T 2 e was of t h e o r d e r of lOOMsec. Above T^ t h e r e was a s u b s t a n t i a l i n c r e a s e i n T 2 e * T 2 e f u r t h e r i n c r e a s e d w i t h t h e a d d i t i o n of c h o l e s t e r o l ( t o 800 usee) and t o a s m a l l e r e x t e n t w i t h l a n o s t e r o l (700 u s e e ) . I t h a s a l r e a d y been m e n t i o n e d t h a t T 2 & i s s e n s i t i v e t o slow m o t i o n s . The dominant T 2 g r e l a x a t i o n p r o c e s s of p h o s p h o l i p i d membranes i s t h o u g h t t o be a s s o c i a t e d w i t h slow m o t i o n s o f t h e c h a i n s w h i c h m o d u l a t e t h e q u a d r u p o l e i n t e r a c t i o n s [ 3 0 , 4 0 ] . The a d d i t i o n o f s t e r o l s i n t o t h e membranes may i n some way a f f e c t t h e slow m o t i o n s . A more d e f i n i t i v e i n t e r p r e t a t i o n of t h e s e r e s u l t s c o u l d be made i f a r e l a x a t i o n s t u d y were p e r f o r m e d w h i c h l o o k e d a t t h e r e l a x a t i o n o f i n d i v i d u a l p e a k s t o d e t e r m i n e whether o r n o t th e slow m o t i o n s i n v o l v e c o l l e c t i v e m o t i o n s o f t h e e n t i r e c h a i n . The s i g n i f i c a n c e o f t h e d i f f e r e n c e i n r e l a x a t i o n t i m e s i s n ot known. I f i t i s p o s t u l a t e d t h a t t h e p r o t r u d i n g m e t h y l g r o u p i s i m p o r t a n t i n m o d u l a t i n g t h e r e l a x a t i o n b e h a v i o u r , t h e n t h i s "bump" may be i n t e r f e r i n g w i t h t h e m o t i o n s o f t h e p h o s p h o l i p i d s as t h e y p a s s t h e a l p h a f a c e . S i n c e t h e T 2 e o f c h o l e s t e r o l i s l o n g e r , t h i s i n t e r p r e t a t i o n i m p l i e s t h a t t h e smooth a l p h a i s i m p o r t a n t f o r t h e s e m o t i o n s . 59 D. RESOLUTION OF THE SPECTRAL LINESHAPES The d i f f e r e n c e i n T. v a l u e s of t h e 5% l a n o s t e r o l and 2e c h o l e s t e r o l c o u l d a c c o u n t f o r a s m a l l amount o f homogeneous b r o a d e n i n g o f t h e s p e c t r a l l i n e s of t h e l a n o s t e r o l sample. The b r o a d e n i n g o f t h e s e l i n e s i s t o o g r e a t however t o be e n t i r e l y due t o r e l a x a t i o n e f f e c t s , i . e . some inhomogeneous b r o a d e n i n g i s e x h i b i t e d . The d i f f e r e n c e i n r e s o l u t i o n may be due t o t h e s m a l l amount of d i h y d r o l a n o s t e r o l p r e s e n t i n t h e l a n o s t e r o l s a m p l e s . T h i s l i p i d i s p r e s e n t i n c e l l s t h a t c o n t a i n l a n o s t e r o l . V I . CONCLUSION P r e l i m i n a r y s t u d i e s w i t h s y s t e m s c o n t a i n i n g 5%, 10% and 15% s t e r o l / D P P C - d 3 1 i n d i c a t e t h a t t h e ph a s e b e h a v i o u r of l a n o s t e r o l i s c o n s i s t e n t w i t h t h e phase b e h a v i o u r o b s e r v e d f o r c h o l e s t e r o l a t c o m p a r a b l e c o n c e n t r a t i o n s and t e m p e r a t u r e s ( f i g u r e 3 ) . The p h a s e s were a s s i g n e d u s i n g 2H-NMR s p e c t r a , t h e i r c o r r e s p o n d i n g f i r s t moments and from DSC t r a c e s . The w i d t h o f t h e t r a n s i t i o n between t h e p*hases a t e a c h c o n c e n t r a t i o n c o u l d be d e t e r m i n e d f r o m t h e f i r s t moments. W i t h t h e a d d i t i o n of b o t h l a n o s t e r o l and c h o l e s t e r o l a t a c o n c e n t r a t i o n of 5%, t h e t r a n s i t i o n t e m p e r a t u r e was r e d u c e d by 1 d e g r e e and t h e w i d t h o f t h e t r a n s i t i o n i n c r e a s e d t o 4 d e g r e e s . T h i s b r o a d e n i n g c o n t i n u e d a t t h e h i g h e r c o n c e n t r a t i o n o f b o t h s t e r o l s . O t h e r a u t h o r s [6] i n t e r p r e t e d t h e r e d u c t i o n i n T as t h e e x i s t e n c e o f a s m a l l mixed phase m r e g i o n n e a r t h e t e m p e r a t u r e t r a n s i t i o n o f t h e p u r e l i p i d . I f t h i s i s t h e c a s e , b o t h c h o l e s t e r o l and l a n o s t e r o l e x h i b i t t h i s p h a s e . The b r o a d component i n t h e DSC t r a c e s a t s t e r o l c o n c e n t r a t i o n s above 10% has been i n t e r p r e t e d by t h e same a u t h o r s [6] a s a "j3" p h a s e . B o t h s t e r o l s a p p e a r t o e f f e c t t h i s p h a s e . F u r t h e r , t h e f i r s t moment was u s e d t o d e t e r m i n e t h e f r a c t i o n o f p h o s p h o l i p i d i n t h e g e l ph a s e a t t h e 5% c o n c e n t r a t i o n . T h i s r e s u l t i n d i c a t e d t h a t a t t h e c o n c e n t r a t i o n s examined, l a n o s t e r o l and c h o l e s t e r o l have t h e 60 61 same c o m p o s i t i o n of g e l phase l i p i d s . Above t h e ph a s e t r a n s i t i o n o f t h e p u r e p h o s p o l i p i d , s amples c o n t a i n i n g e i t h e r l a n o s t e r o l o r c h o l e s t e r o l a r e more o r d e r e d t h a n t h e p u r e p h o s p h o l i p i d s y s t e m s a t t h e same t e m p e r a t u r e . T h e r e f o r e t h e p r e s e n c e o f e i t h e r s t e r o l r e s t r i c t s t h e f o r m a t i o n of gauche bonds, " c o n d e n s i n g " t h e p h o s p h o l i p i d s . T h i s d i s a g r e e s w i t h s t a t e m e n t s by D a h l et al [20] The p r o t r u s i o n f r o m t h e a l p h a f a c e of l a n o s t e r o l does not i m p a i r t h e a b i l i t y o f t h e s t e r o l t o o r d e r p h o s p h o l i p i d s nor does i t a l t e r t h e p h a s e b e h a v i o u r . T h e s e o b s e r v a t i o n s may be a t t r i b u t a b l e t o t h e r i g i d n e s s of t h e r i n g s t r u c t u r e w h i c h i s p o s s e s s e d by b o t h s t e r o l s . The T 2 e r e l a x a t i o n t i m e s a r e s l i g h t l y d i f f e r e n t f o r l a n o s t e r o l and c h o l e s t e r o l . A t low c o n c e n t r a t i o n s o f c h o l e s t e r o l T 2 e v a l u e s i n c r e a s e d by a f a c t o r of two o v e r p u r e DPPC. T h i s s u g g e s t s c h o l e s t e r o l i s s p e e d i n g up t h e s l o w e s t m o t i o n o f t h e p h o s p h o l i p i d . The smoothness of t h e a - f a c e o f c h o l e s t e r o l may a l l o w p h o s p h o l i p i d s t o s l i d e by, unimpeded by any p r o t r u s i o n . I f t h i s were t h e c a s e t h e a - m e t h y l on l a n o s t e r o l would h i n d e r t h e movement o f t h e p h o s p h o l i p i d s . To d e t e r m i n e more c l e a r l y t h e n a t u r e o f t h e slow m o t i o n s m o d u l a t i n g T 2 e a more e x t e n s i v e r e l a x a t i o n s t u d y s h o u l d be p e r f o r m e d , i n c l u d i n g t h e o b s e r v a t i o n o f how t h e i n d i v i d u a l s p e c t r a l l i n e s r e l a x . 6 2 The d i f f e r e n c e i n r e s o l u t i o n of t h e two s t e r o l s c o u l d n o t be e x p l a i n e d c o m p l e t e l y i n t erms o f c h a n g e s i n T 2 e « One p o s s i b l e r e a s o n f o r t h e d e c r e a s e d r e s o l u t i o n may be t h a t t h e s m a l l amounts o f d i h y d r o l a n o s t e r o l i n t h e s a m p l e s c o n t r i b u t e t o t h e inhomogeneous l i n e b r o a d e n i n g . REFERENCES 1. B l o c h , K., (1965) S c i e n c e (Wash.,D.C.) 150, 19-28 2. Demel,R.A. and De K r u y f f , B . , ( 1 9 7 6 ) , B i o c h i m . B i o p y s . A c t a , 457, 109-132. 3. Demel, R.A., B r u c k d o r f e r , K . 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Biology
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A ²H-NMR study of the comparative effects of lanosterol and cholesterol on model membranes
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