HAL Id: jpa-00223460
https://hal.archives-ouvertes.fr/jpa-00223460
Submitted on 1 Jan 1983
HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
ELLIPSOMETRIC MEASUREMENT OF THE SPECIFIC VOLUMES OF PHOSPHOLIPIDS AND
PROTEINS AT SOLID-WATER INTERFACES
P. Cuypers, J. Corsel, J. Kop, M. Janssen, W. Hermens
To cite this version:
P. Cuypers, J. Corsel, J. Kop, M. Janssen, W. Hermens. ELLIPSOMETRIC MEASURE- MENT OF THE SPECIFIC VOLUMES OF PHOSPHOLIPIDS AND PROTEINS AT SOLID- WATER INTERFACES. Journal de Physique Colloques, 1983, 44 (C10), pp.C10-495-C10-498.
�10.1051/jphyscol:198310100�. �jpa-00223460�
JOURNAL DE PHYSIQUE
Colloque C10, suppl6ment au n012, Tome 44, d6cernbre 1983 page CI 0-495
ELLIPSOMETRIC MEASUREMENT OF THE S P E C I F I C VOLUMES OF PHOSPHOLIPIDS AND PROTEINS AT SOLID-WATER INTERFACES
P.A. Cuypers, J.W. Corsel, J.M.M. Kop, M.P. Janssen and W.Th. Hermens
Departments of Biophysics and Biochemistry, Biomedical Centre, University of Limburg, Maastricht, The Netherlands
~ b s u m s
-
L e s v o l u m e s s p 6 c i f i q u e s v d e p h o s p h o l i p i d e s dGposGs s u r d e s p l a q u e s d e chrome, s o n t d s t e r m i n s s d a n s un tampon d e s o l u t G p h y s i o l o g i q u e . L e s c h a n g e m e n t s d e v d u s 2 l ' h y d r a t a - t i o n e t 2 l a v a r i a t i o n d e t e m p e r a t u r e s o n t a u s s i m e s u r g s , a i n s i q u e l e v o l u m e s n 6 c i f i q u e d e l a p r o t h r o m b i n e a d s o r b 6 e s u r d e s mu1 t i c o u c h e s d e p h o s p h o l i p i d e s . Q u e l q u e s c o m p l i c a t i o n s Gventu- e l l e s d e c e t t e mGthode s o n t d i s c u t s e s .A b s t r a c t
-
The s p e c i f i c v o l u m e s v o f p h o s p h o l i p i d s d e p o s i t e d o n chromium s l i d e s , a r e m e a s u r e d i n b u f f e r e d p h y s i o l o g i c a l s a l i n e . C h a n g e s i n v d u e t o h y d r a t i o n and d u e t o v a r i a t i o n o f t e m p e r a - t u r e a r e a l s o m e a s u r e d , a s w e l l a s t h e s p e c i f i c volume o f p r o - t h r o m b i n a d s o r b e d t o p h o s p h o l i p i d m u l t i l a y e r s . Some p o s s i b l e c o m p l i c a t i o n s o f t h e method a r e d i s c u s s e d .D e t e r m i n a t i o n o f t h e p a r t i a l s p e c i f i c v o l u m e s v o f p h o s p h o l i p i d s d i s p e r s e d i n a q u e o u s s o l u t i o n s i s hampered by t h e f a c t t h a t t h e s e v a l u e s a r e v e r y close t o t h a t o f t h e a q u e o u s s o l v e n t , w h i c h may c a u s e c o n s i d e r e a b l e e r r o r i n t h e u s u a l d e n s i t o m e t r i c d e t e r m i n a t i o n s ( 1 ) . M o r e o v e r , p r e p a r a t i o n s o f p u r e p h o s p h o l i p i d s w e r e d i f f i c u l t t o o b t a i n u n t i l r e c e n t l y . F o r many p r o t e i n s , a c c u r a t e v a l u e s o f v a r e known f o r d i l u t e s o l u t i o n s . However i t r e m a i n s a m a t t e r o f d e b a t e w h e t h e r t h e s e v a l u e s r e m a i n v a l i d f o r p r o t e i n / w a t e r m i x t u r e s c o n t a i n i n g w e i g h t f r a c t i o n s o f more t h a n 30-40% o f p r o t e i n ( 2 , 3 ) , s u c h a s e x i s t f o r p r o t e i n s a d s o r b e d a t s o l i d - w a t e r i n t e r f a c e s . The p r e s e n t s t u d y demon- s t r a t e s t h a t s u c h v a l u e s o f v c a n b e d e t e r m i n e d e l l i p s o m e t r i c a l l y b y means o f a r e l a t i o n b a s e d o n t h e L o r e n t z - L o r e n z e q u a t i o n .
M a t e r i a l s a n d m e t h o d s
P r e p a r a t i o n s o f p h o s p h a t i d y l s e r i n e ( P S )
,
p h o s p h a t i d y l c h o l i n e (PC),
t r i t i u m - l a b e l e d p r o t h r o m b i n a n d i o d i n e - l a b e l e d a l b u m i n w e r e o b t a i n e d a s d e s c r i b e d ( 4 ) . M u l t i l a y e r s o f PS were d e p o s i t e d o n c h r o m i u m - c o a t e d g l a s s s l i d e s b y u s i n g t h e L a n g m u i r - B l o d g e t t t e c h n i q u e s ( 4 ) . M u l t i - l a y e r s o f PC c a n n o t b e p r e p a r e d i n t h i s way b e c a u s e t h e n e c e s s a r y p h o s p h o l i p i d - c a l c i u m c o m p l e x e s a r e n o t f o r m e d . T h e r e f o r e a d i f f e r e n t t e c h n i q u e was a d o p t e d . A d r o p o f a c h l o r o f o r m s o l u t i o n c o n t a i n i n g 2 mg/ml o f PC i s p u t o n t h e chromium s l i d e a n d r a p i d l y s p r e a d w i t h t h e s t a n d a r d t e c h n i q u e f o r t h e p r e p a r a t i o n o f a b l o o d s m e a r , u s i n g a t e f l o n s l i d e . The chromium s l i d e s a r e p l a c e d i n a q u a r t z c u v e t t e f i l l e d w i t h 4 m l o f b u f f e r e d p h y s i o l o g i c a l s a l i n e . The b u f f e r i s m a g n e t i c a l l y s t i r r e d a n d k e p t a t t h e r e q u i r e d t e m p e r a t u r e b y m e a n s o f a P e l t i e r e l e m e n t . B u f f e r s u s e d i n t h e e x p e r i m e n t s were 0 . 0 5 M T r i s - HC1, pH = 7 . 5 , c o n t a i n i n g 0 . 1 M NaCl a n d 0 . 0 1 M CaC12 ( ~ r i s - b u f f e r ) a n d 0.05 M H e p e s , pH = 5.0 ( H e p e s - b u f f e r ) . E x p e r i m e n t s were p e r f o r m e d w i t h a n a u t o m a t e d e l l i p s o m e t e r , u s i n g a l a s e r ( A = 632.8 nm) a n d a n a n g l e o f i n c i d e n c e o f 6 8 d e g r e e s ( 4 , 5 ) . The p o s i t i o n s o f two p o l a r i z -
Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:198310100
C10-496 JOURNAL DE PHYSIQUE
i n g p r i s m s , t h e " p o l a r i z e r " P a n d t h e " a n a l y z e r " A , a r e d e t e r m i n e d e v e r y 3-5 s e c . The o p t i c a l c o n s t a n t s o f t h e chromium s l i d e , a n d t h e r e f r a c t i v e i n d e x n a n d t h i c k n e s s d o f a p h o s p h o l i p i d o r p r o t e i n f i l m a d s o r b e d t o t h e s l i d e , a r e c a l c u l a t e d f r o m t h e ( c h a n g e s i n ) p o s i t i o n s o f P a n d A. The L o r e n t z - L o r e n z r e l a t i o n f o r a s u b s t a n c e w i t h r e f r a c - t i v e i n d e x n c a n b e w r i t t e n as ( 6 ) :
w h e r e p O i s t h e m a s s d e n s i t y o f t h e p u r e s u b s t a n c e ( g / m l ) a n d r i s t h e s p e c i f i c r e f r a c t i v i t y ( m l / g ) w h i c h c a n b e c a l c u l a t e d f r o m t h e m o l e c u - l a r s t r u c t u r e ( 7 ) . F o r a l a r g e number o f o r g a n i c compounds t h e s e c a l c u l a t e d v a l u e s o f r d i f f e r l e s s t h a n 1% f r o m t h e v a l u e s o b t a i n e d b y i n s e r t i n g e x p e r i m e n t a l v a l u e s o f po a n d n i n t o e q u a t i o n ( 1 ) .
F o r a b i n a r y m i x t u r e o f b u f f e r ( b ) a n d s u b s t a n c e ( s ) t h e L o r e n t z - L o r e n z r e l a t i o n t a k e s t h e f o r m ( 6 , 4 ) : p b r b
+.
pgfS = ( n 2 - 1 ) ( n 2 + 2 ) . T h e v o l u m e f r a c t i o n o f b u f f e r i n s u c h a m i x t u r e is psvsr w h e r e v i s t h e p a r t i a l s p e c i f i c v o l u m e o f s u b s t a n c e s. Assuming a n i d e a l m i z t u r e we t h u s o b t a i n pb = p o b ( l - p s v ) w i t h p i t h e d e n s i t y o f p u r e b u f f e r . I n - s e r t i n g t h i s e x p r e s s i o n &r P b o n e o b t a i n e a f t e r r e a r r a n g e m e n t o f t e r m s a n d u s i n g e q u a t i o n ( 1 ) f o r p u r e b u f f e r :w h e r e
r =
p,.d i s t h e a d s o r b e d m a s s p e r u n i t s u r f a c e a r e a ( g / c m ) o f s u b - 2 s t a n c e s i f a l a y e r o f t h i c k n e s s d ( c m ) o f t h e b i n a r y m i x t u r e h a s a d s o r b e d t o t h e s l i d e . I f t h e v a l u e o fr
i s known, e q u a t i o n ( 2 ) a l l o w s c a l c u l a t i o n o f v f r o m e l l i p s o m e t r i c a l l y d e t e r m i n e d v a l u e s o f n a n d d . V a l u e s u s e d i n t R e s e c a l c u l a t i o n s w e r e ( 4 ) : n b = 1 , 3 3 5 ; r = 0 . 2 6 8 ml/g f o r 14:0/14:0 PS; r = 0 . 2 7 2 ml/g f o r 1 8 : 1 / 1 8 : 1 PS; r = 0 . 2 7 8 ml/g f o r 1 6 : 0 / 1 6 : 0 PC; r = 0.236 ml/g f o r p r o t h r o m b i n a n d r = 0 . 2 4 3 ml/g f o r a l b u m i n .R e s u l t s
T a b l e 1 s h o w s t h e r e s u l t s o f m e a s u r e m e n t s f o r s t a c k e d P S - m u l t i l a y e r s a n d f o r r a d i o l a b e l e d p r o t e i n s a d s o r b e d o n s l i d e s c o v e r e d w i t h PS.
E x p e r i m e n t a l s y s t e m - n d ( n m ) r ( y g / c m 2 ) v ( m l / g ) 1 2 l a y e r s 1 4 : 0 / 1 4 : 0 PS i n T r i s - 1 . 4 8 6 3 5 . 7 0 . 2 7 8 0 . 8 8 2 b u f f e r ; T = 2 0 ° C ( N = 1 0 )
-
+0.009 2 1 . 8-
C0.003 + 0 . 0 1 0 1 2 l a y e r 1 8 : 1 / 1 8 : 1 PS i n Hepes- 1 . 4 8 2 3 0 . 2 0 . 2 2 3 0 . 8 9 0 b u f f e r ; T = 2 0 ° C ( ~ = 1 0 )-
+ 0 . 0 0 8 s . 2-
+O. 0 0 3 +O. 0 1 3 3 ~ - p r o t h r o m b i n o n 8 l a y e r s 1 8 : 1 / 1 . 4 6 0 5.17 0 . 3 5 4 0 . 7 0 7 1 8 : l PS. T r i s - b u f f e r ; T=37OC ( N = 3 ) 2 0 . 0 2 4 5 . 1 0 2 0 , 0 2 2 t 0 . 0 1 9 3 ~ - p r o t h r o m b i n o n 8 l a y e r s 1 4 : 0 / 1 . 9 0 0 2.79 0 . 5 1 0 0 . 4 8 6 14:O PS. H e p e s - b u f f e r ; T=37 "C-
+ 0 . 2 0 5 0 . 7 7 t 0 . 0 3 0 2 0 . 0 5 4 (N=3 )1 2 5 ~ - a l b u m i n o n 8 l a y e r s 1 4 : 0 / 1 . 7 3 1 1 . 4 2 0 . 2 5 4 0 . 6 3 8 14:O PS. H e p e s - b u f f e r ; T=37OC - +0.056 5 0 . 1 4 t 0 . 0 1 1 5 0 . 0 2 6
(N=3 )
T a b l e 1. E x p e r i m e n t a l r e s u l t s . F i g u r e s i n d i c a t e mean
2
S D .V a l u e s o f T f o r t h e P S - m u l t i l a y e r s w e r e d e t e r m i n e d f r o m t h e m o n o l a y e r s u r f a c e a r e a d i s a p p e a r i n g f r o m t h e f i l m b a l a n c e d u r i n g s t a c k i n g . F o r
F i g . 1 a n d F i g . 2 show c h a n g e s i n v d u r i n g h y d r a t i o n a n d d u r i n g a t e m p e r a t u r e - d e p e n d e n t p h a s e t r a n s i t i o n . I n F i g . 1, t h e c a l c i u m b o n d s b e t w e e n t h e p h o s p h o l i p i d h e a d g r o u p s i n t h e s t a c k e d s y s t e m were b r o k e n b y a d d i n g EDTA, and a s u b s e q u e n t s w e l l i n g i s o b s e r v e d . The s w o l l e n s y s t e m s t i l l c o n t a i n s i n t a c t l a y e r s a s shown b y t h e o s m o t i c e f f e c t Of a d d i n g NaC1. F i g . 2 s h o w s t h e r e v e r s i b l e s w e l l i n g o f P C f i l m a s a f u n c t i o n o f t e m p e r a t u r e . A s t h e v a l u e o f
r
was unknown i n t h i s c a s e , t h e v a l u e o f v a t 50 OC was e s t i m a t e d i n d e p e n d e n t l y . A d r o p o f concen- t r a t e d egg-PC i n c h l o r o f o r m was e v a p o r a t e d o n t h e p r i s m o f a n Abbe r e f r a c t o m e t e r ( Z e i s s ) a n d h y d r a t e d by c o o l i n g t h e p r i s m t o 1 0 ° C f o r 1 h o u r , a l l o w i n g m o i s t e n i n g o f t h e PC f i l m . H e r e a f t e r , a v a l u e Of nsO=1 . 4 7 0 was f o u n d o r , u s i n g e q u a t i o n ( I ) , ~ ~ ~ = 0 . 9 9 6 ml/g. U s i n g t h l s v a l u e
r
was c a l c u l a t e d f r o m e q u a t i o n ( 2 ) . F i g . 2 a l s o c o n t a i n s a c u r v e c a l c u l a t e d f o r a v a l u e o f v = 0 . 9 7 3 ml/g a s f o u n d f r o m X-ray d i f f r a c - t i o n f o r a q u e o u s d i s p e r s i o n s 5 8 f 16:0/16:0 PC ( 8 ) .p r o t e i n s I' was d e t e r m i n e d by c h o s i n g e x p e r i m e n t a l c o n d i t i o n s s u c h t h a t d e s o r p t i o n o f p r o t e i n is n e g l i g e a b l e when t h e c u v e t t e is r i n s e d w i t h b u f f e r a f t e r t h e a d s o r p t i o n . T h e r e a f t e r , p r o t e i n s w e r e d e s o r b e d f o r m t h e s l i d e s w i t h 1 M HC1 and t h e amount o f r a d i o a c t i v i t y wa.s c o u n t e d .
-- --
th&$;
-
=tR6sSk
D i s c u s s i o n
V a l y e s o f v f o r p h o s p h a t i d y l s e r i n e a r e v e r y s c a r c e and t h e v a l u e s p r e s e n t e d i n T a b l e 1 f o r 14:0/14:0 PS a n d 18:1/18:1 PS c a n n o t b e compared w i t h d a t a from t h e l i t e r a t u r e . The same i s t r u e f o r t h e c h a n g e i n v o f PS d u r i n g h y d r a t i o n . I t i s known t h a t c a l c i u m - m e d i a t e d c o m p l e x e s o f p u r e PS a r e a n h y d r o u s ( 9 ) and 45-Ca i s n o t r e l e a s e d f r o m t h e s t a c k e d s y s t e m s i n b u f f e r ( J . M . M . Kop, u n p u b l i s h e d r e s u l t s ) . The s w o l l e n s y s t e m i n F i g . 1 t h e r e f o r e p r o b a b l y r e p r e s e n t s t h e s i t u a t i o n f o r i s o l a t e d PS membranes i n b u f f e r a n d v a l u e s o f v f o r s u c h s y s t e m s w i l l b e a p p r o x i m a t e l y 7 % h i g h e r t h a n t h o s e q u o t e d i n T a b l e 1. The
(nm) t
150.0- O 0 5 M
N ~ C I
100.0-
50.0-
(nm) 40.0-
30.0-
20.0-
0.94
10.0-
1 .OO-
v
0.96 -
I I I
z
0.90
0.88-
0.5 I.'o timets.1031 1.5 i 5
40
4'5 & e m ~ . ( ~ c iFig.1. Changes i n t h i c k n e s s and s p e c i - F i g . 2 . Changes i n t h i c k n e s s and s p e c i - f i c volume of 14 l a y e r s 14:0/14:0 PS f i c volume of 16:0/16:0 PC a s a func-
during h y d r a t i o n . t i o n of temperature.
0.92-
'
/
0.88-0ClO-498 JOURNAL DE PHYSIQUE
i n c r e a s e i n v d u r i n g t h e t r a n s i t i o n f r o m t h e c o n d e n s e d t o t h e e x p a n d e d s t a t e i n F l g . 2 , i s somewhat h i g h e r t h a n t h e 4-68 i n c r e a s e r e p o r t e d i n t h e l i t e r a t u r e f o r 1 6 : 0 / 1 6 : 0 PC ( 8 , 1 0 , 1 1 ) .
The v a l u e o f v f o r p r o t h r o m b i n i n T a b l e 1, d e t e r m i n e d f r o m a d s o r p t i o n s o n 1 8 : 1 / 1 8 : 1 P S I i s i n g o o d a g r e e m e n t w i t h t h e v a l u e o f v 2 0 = 0 . 7 1 0 ml/g q u o t e d i n t h e l i t e r a t u r e ( 1 2 ) . However, t h e v a l u e s o f v f r o m t h e a d s o r p t i o n s o f p r o t h r o m b i n a n d a l b u m i n o n 14:0/14:0 PS s e e m t o i n d i - c a t e a v e r y d e n s e p a c k i n g o f p r o t e i n m o l e c u l e s , w h i c h is h i g h l y i m - p r o b a b l e b e c a u s e t h e s p e c i f i c v o l u m e o f p r o t e i n s c a n b e e s t i m a t e d f r o m t h e i r a m i n o a c i d c o m p o s i t i o n a n d i s h a r d l y c h a n g e d i n 8 M u r e a . An a l t e r n a t i v e e x p l a n a t i o n i s t h e f o l l o w i n g .
V a l u e s o f n a n d dp, f o r p r o t e i n a d s o r p t i o n s i n T a b l e 1 were c a l c u - l a t e d b y as&?gming t h a t t h e v a l u e s n p h a n d dph o f t h e u n d e r l y i n g p h o s - p h o l i p i d f i l m were n o t i n f l u e n c e d b y t h e p r o t e i n a d s o r p t i o n . I f t h e m e a s u r e m e n t s a r e p e r f o r m e d a t t w o d i f f e r e n t a n g l e s o f i n c i d e n c e , t h i s a s s u m p t i o n c a n b e c h e c k e d . I n t h a t c a s e two d i f f e r e n t s e t s P I , A1 a n d P2 A a r e o b t a i n e d a n d , f o r c h o s e n v a l u e s o f nph
,
dph,
t w o s e t s o f n p r r d p r a r e c a l c u l a t e d f r o m r e s p e c t i v e l y P I ,A1 a n d P 2 , A2. O n l y f o r t h e c o r r e c t v a l u e s o f n F h ,,
b o t h s e t s o f n p r , d p r w i l l c o i n c i d e . T h i s p r o c e d u r e is shown i n T%?e 2.T a b l e 2 D o u b l e - a n g l e e l l i p s o m e t r y o n p r o t h r o m b i n a d s o r b e d o n 1 4 : 0 / 1 4 : 0 PS. F i g u r e s i n d i c a t e w e i g h t e d d i f f e r e n c e s b e t w e e n t w o s e t s o f np,, d p r
( s e e t e x t ) . B a r s i n d i c a t e t h a t no s o l u t i o n w a s o b t a i n e d .
I n i t i a l v a l u e s i n t h i s p a r t i c u l a r e x p e r i m e n t w e r e nph= 1 . 4 6 a n d dph = 29 nm b u t , u s i n g t h e s e v a l u e s , n p r = 1 . 6 6 , d p r = 4.2 nm a n d npr = 1 . 4 3 , d r = 1 2 nm w e r e f o u n d a t a n g l e s o f i n c i d e n c e o f respectively 6 2 . 3 a n % 74.4 d e g r e e s . From T a b l e 2 i t f o l l o w s t h a t d u r i n g a d s o r p t i o n o f p r o t h r o m b i n t h e v a l u e s o f n p h a n d d p h c h a n g e d t o n p h = 1 . 5 0 a n d d ph = 3 3 nm. T h i s i m p l i e s t h a t t h e m a s s o f t h e p h o s p h o l i p i d f i l m h a s i n c r e a s e d a n d s o m e p r o t e i n m u s t h a v e p e n e t r a t e d i n t o t h i s l a y e r . U s i n g t h e s e c h a n g e d v a l u e s o f n
,
d p h , v a l u e s o f n p r = 1 . 3 4 a n d d = 1 4 3 nm w e r e f o u n d . From t h e s e P $ a t a we c o n c l u d e t h a t e r r o n e o u s h i g % v a l u e s o f n p r r a n d c o r r e s p o n d i n g l o w v a l u e s o f v p r , may b e f o u n d d u e t o ad- s o r p t i o n - i n d uced c h a n g e s i n t h e u n d e r l y i n g p h o s p h o l i p i d l a y e r .R e f e r e n c e s
1. Huang C-H., J . B i o l . Chem.
246
( 1 9 7 1 ) 2555.2. B e r n h a r d t J . , J . P h y s . Chem.
84
( 1 9 8 0 ) 1 5 8 . 3. De F e y t e r J . A . , B i o p o l y r n e r s17
( 1 9 7 8 ) 1 7 5 9 . 4 . C u y p e r s P.A., J . B i o l . Chem.258
( 1 9 8 3 ) 2 4 2 6 . 5. C u y p e r s P.A., A n a l . Biochem.84
( 1 9 7 8 ) 56.6 . B o r n M.: P r i n c i p l e s o f o p t i c s . P e r g a m o n P r e s s , O x f o r d , 1 9 6 4 . 7. V o g e l A., J. P h y s . Chem. 5 8 ( 1 9 5 4 ) 1 7 4 .
8 . A l b o n N., J . C o l l . ~ n t e r f r ~ c i .
92
( 1 9 8 3 ) 5 4 5 .9. E k e r d t R . , P r o c . N a t l . Acad. S C ~ . USA
2
( 1 9 8 2 ) 2273.1 0 . S h e e t z M.P., B i o c h e m i s t r y
