Ordered Media in Chemical Separations - ACS Publications

Chapter 2

Membrane Mimetic Separations Janos H. Fendler

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 12, 2017 | http://pubs.acs.org Publication Date: June 30, 1987 | doi: 10.1021/bk-1987-0342.ch002

Department of Chemistry, Syracuse University, Syracuse, NY 13244-1200

Development of new separation techniques requires a fundamental understanding of the relationship between molecular structures and permeabilities. Initiation of interdisciplinary researches in biology, biophysics, polymer and colloid chemistry is proposed to provide the insight to membrane transport processes at the molecular level. Mother nature's most talented transporter - the biological membrane should inspire this endeavor. Following a survey of the properties of, and recognized transport mechanisms in, biomembranes, membrane mimetic chemistry is introduced to serve as a bridge between biological and polymeric membranes. Surfactant aggregates micelles, monolayers, organized multilayers (Langmuir-Blodgett films), bilayer lipid membranes (BLMs), vesicles and polymerized vesicles - are shown to be the media in membrane mimetic chemistry. Properties of these organized surfactant assemblies are summarized. Emphasis is placed on our current research on the potential use of BLMs to reconstitute active and transport systems and on the development of their simultaneous electrical and spectroscopic measurements. M i c e l l e s and o t h e r o r g a n i z e d s u r f a c t a n t a g g r e g a t e s a r e i n c r e a s i n g l y u t i l i z e d i n a n a l y t i c a l a p p l i c a t i o n s (I). They i n t e r a c t w i t h r e a g e n t s and a l t e r s p e c t r o s c o p i c and e l e c t r o c h e m i c a l p r o p e r t i e s which, i n t u r n , o f t e n r e s u l t s i n i n c r e a s e d s e n s i t i v i t i e s . O r g a n i z e d a s s e m b l i e s h a v e a l s o been employed i n s e p a r a t i o n p r o c e s s e s . Gas, l i q u i d and t h i n l a y e r m i c e l l a r c h r o m a t o g r a p h i c t e c h n i q u e s have been d e v e l o p e d (2) . R e a l i z i n g t h e f u l l p o t e n t i a l o f o r g a n i z e d assembly m e d i a t e d s e p a r a t i o n s n e c e s s i t a t e s , I b e l i e v e , w e l l c o n c e i v e d and w e l l executed i n t e r d i s c i p l i n a r y researches. The p u r p o s e o f t h i s p r e s e n t a t i o n i s t o s t i m u l a t e such i n t e r d i s c i p l i n a r y a p p r o a c h e s . Our

0097-6156/87/0342-0083$06.25/0 © 1987 American Chemical Society

Hinze and Armstrong; Ordered Media in Chemical Separations ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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O R D E R E D M E D I A IN C H E M I C A L SEPARATIONS


s t a r t i n g p o i n t w i l l be mother n a t u r e ' s most t a l e n t e d t r a n s p o r t e r t h e b i o l o g i c a l membrane. F o l l o w i n g a b r i e f d e s c r i p t i o n o f the b i o l o g i c a l membrane ( i n t h e s e c t i o n on B i o l o g i c a l Membranes), t h e r e c o g n i z e d t r a n s p o r t mechanisms w i l l be d e l i n e a t e d t h e r e i n ( s e c t i o n on R e c o g n i z e d T r a n s p o r t Mechanisms A c r o s s B i o l o g i c a l Membranes), The s e c t i o n on Membrane M i m e t i c C h e m i s t r y w i l l d i s c u s s t h e p h i l o s o p h y o f t h e membrane mimetic approach and t h e most f r e q u e n t l y used mimetic systems. The s e c t i o n on S i m u l t a n e o u s E l e c t r i c a l and S p e c t r o s c o p i c M e a s u r e m e n t s o f BLMs w i l l h i g h l i g h t o u r c u r r e n t r e s e a r c h e s on BLM s p e c t r o s c o p y . The t r e a t m e n t s w i l l be, o f c o u r s e , i l l u s t r a t i v e r a t h e r than comprehensive. B i o l o g i c a l Membranes B i o l o g i c a l membranes d e f i n e t h e v e r y e x i s t e n c e o f c e l l s . They p r o v i d e c o m p a r t m e n t s f o r t h e d i f f e r e n t components o f t h e l i v i n g s y s t e m ; i n t e r a c t w i t h , t r a n s p o r t and a r e permeable t o s u b s t r a t e s . T h e y a r e i n v o l v e d i n l i p i d a n d p r o t e i n s y n t h e s e s , energy t r a n s d u c t i o n , i o n a n d g r o u p t r a n s p o r t , i n f o r m a t i o n t r a n s m i s s i o n and m o l e c u l a r and c e l l u l a r r e c o g n i t i o n . These m u l t i t u d e o f a c t i v i t i e s a r e a c c o m p l i s h e d by t h e u n i q u e m o r p h o l o g y o f t h e b i o l o g i c a l membrane a n d by i t s a b i l i t y t o a f f e c t t h e t r a n s p o r t o f s p e c i e s by d i f f e r e n t mechanisms. C e l l membranes a r e composed o f 25-75% l i p i d s , 25-75% p r o t e i n s and l e s s t h a n 10% c a r b o h y d r a t e s . The o r g a n i z a t i o n o f t h e s e c o m p o n e n t s i n t h e membrane i s b e s t d e s c r i b e d i n t e r m s o f t h e bilayer-lipid g l o b u l a r - p r o t e i n " f l u i d m o s a i c " model ( 3 , 4 ) . As i l l u s t r a t e d i n F i g u r e 1, t h e l i p i d s ( p h o s p h o l i p i d s and/or g l y c o l i p i d s ) are arranged i n b i l a y e r s with t h e i r p o l a r headgroups e x p o s e d t o t h e e x t e r i o r s u r f a c e o f t h e membrane. Proteins are e i t h e r a p e r i p h e r a l o r i n t e g r a l p a r t o f t h e membrane. The f o r m e r , a t t a c h e d e l e c t r o s t a t i c a l l y , i s e a s i l y d i s s o c i a t e d from t h e membrane by c h a n g i n g t h e pH o r t h e i o n i c s t r e n g t h o f t h e s o l u t i o n . Integral p r o t e i n s p a r t i a l l y i n t e r c a l a t e t h e membrane o r f u l l y s p a n t h e b i l a y e r (transmembrane p r o t e i n ) . Globular proteins are p a r t i a l l y embedded i n t o one o r t h e o t h e r s i d e o f t h e membrane and form a m o s a i c p a t t e r n w i t h t h e l i p i d headgroups. The d e p t h o f i n c o r p o r a t i o n d e p e n d s upon t h e s i z e o f t h e g l o b u l a r p r o t e i n , i t s h y d r o p h o b i c i t y and charge d i s t r i b u t i o n . An i m p o r t a n t r e q u i r e m e n t o f t h e f l u i d mosaic model i s t h e dynamic n a t u r e o f t h e l i p i d - p r o t e i n i n t e r a c t i o n s i n t h e membrane. P r o t e i n s may r o t a t e around t h e i r axes, d i f f u s e l a t e r a l l y i n t h e p l a n e o f t h e membrane o r move a c r o s s the b i l a y e r . A d d i t i o n a l l y , t h e y may u n d e r g o v i b r a t i o n a l a n d c o n f o r m a t i o n a l changes. B e i n g l e s s than c a t e g o r i c a l i n d e s c r i b i n g p r o t e i n m o b i l i t i e s has been i n t e n t i o n a l . W h i l e most p r o t e i n s move a b o u t , some c a n n o t f r e e l y d i f f u s e i n t h e membrane under p h y s i o l o g i c a l conditions. The l i p i d s t h e m s e l v e s a r e h i g h l y m o b i l e . Steady s t a t e and t i m e r e s o l v e d s p e c t r o s c o p y ( a b s o r p t i o n , e m i s s i o n , i r , raman, nmr, epr) and a n i s o t r o p y measurements have r e v e a l e d r o t a t i o n a l , v i b r a t i o n a n d s e g m e n t a l m o t i o n s o f t h e headgroups and t h e h y d r o carbon t a i l s o f the l i p i d s . T r a n s l o c a t i o n o f a l i p i d from one h a l f o f t h e b i l a y e r t o t h e o t h e r , ( " f l i p - f l o p " ) as w e l l as intermembrane


2.

FENDLER

Membrane Mimeûc Separations

85


( o r i n t e r v e s i c u l a r ) l i p i d exchanges have a l s o been recognized. F i g u r e 2 i l l u s t r a t e s some o f t h e motions o f l i p i d s . P r o t e i n s a n d l i p i d s i n t e r a c t c o o p e r a t i v e l y i n t h e membrane. The t y p e ( s ) a n d s t a t e ( s ) o f l i p i d s i n f l u e n c e t h e m o b i l i t y and c o n f o r m a t i o n o f t h e p r o t e i n s i n t h e membrane m a t r i x . This, i n t u r n , may w e l l a l t e r t h e p r o p e r t i e s o f t h e membrane p r o t e i n s . S i m i l a r l y , p r o t e i n s a f f e c t t h e phase b e h a v i o r o f t h e l i p i d s and/or p r o m o t e d o m a i n f o r m a t i o n i n membranes c o n t a i n i n g m i x t u r e s o f l i p i d s . Morphological a l t e r a t i o n of the l i p i d a r c h i t e c t u r e leads t o changes i n t h e membrane p e r m e a b i l i t y . Phase t r a n s i t i o n i s an i m p o r t a n t p r o p e r t y o f membranes. 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 , l i p i d s a r e t i l t e d and h i g h l y ordered. They a r e i n t h e i r s o l i d or " g e l " s t a t e . Increasing the t e m p e r a t u r e l e a d s t o a p r e - t r a n s i t i o n , c h a r a c t e r i z e d by p e r i o d i c undulations and s t r a i g h t e n i n g of the hydrocarbon c h a i n . Further i n c r e a s e o f t h e t e m p e r a t u r e c a u s e s t h e main phase t r a n s i t i o n . Above t h e main p h a s e t r a n s i t i o n temperature, l i p i d s are f l u i d o r " l i q u i d c r y s t a l l i n e . " F i g u r e 3 shows t h e p h a s e d i a g r a m f o r t h e i n t e r a c t i o n o f water w i t h a l i p i d as w e l l as i t s i n f e r r e d a r r a n g e m e n t s i n a model membrane (5). Phase t r a n s i t i o n s i n membranes and membrane m o d e l s h a v e b e e n e x t e n s i v e l y s t u d i e d b y s p e c t r o s c o p i c t e c h n i q u e s and by 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 . M o s t membranes a r e o s m o t i c a l l y active. They s h r i n k i f e l e c t r o l y t e s a r e added e x t e r n a l l y . They s w e l l i f p l a c e d i n a s o l u t i o n w h i c h i s more d i l u t e t h a n t h e i r i n t e r n a l e l e c t r o l y t e concentrations. Most membranes a r e asymmetric w i t h r e s p e c t t o t h e d i s t r i b u t i o n of l i p i d s , c h a r g e s and p r o t e i n s b e t w e e n t h e i r e x t e r i o r s a n d interiors. Uneven d i s t r i b u t i o n o f i o n s between t h e o u t s i d e and t h e i n s i d e o f membranes i s r e s p o n s i b l e , a t l e a s t i n p a r t , f o r membrane potentials. The i n s i d e o f l i v i n g c e l l s ( c y t o p l a s m , f o r example) i s t y p i c a l l y more n e g a t i v e t h a n t h e e x t r a c e l l u l a r medium. This d i f f e r e n c e i n c h a r g e s i s r e f e r r e d t o a s t h e r e s t i n g o r membrane potential. T r a n s i e n t changes i n t h e membrane p o t e n t i a l , caused by r e v e r s i b l e charge r e d i s t r i b u t i o n s , a r e r e s p o n s i b l e f o r i n f o r m a t i o n and i m p u l s e t r a n s m i s s i o n i n n e r v e and m u s c l e f i b e r s . There i s a n o t h e r i m p o r t a n t a s y m m e t r y i n membranes: the segregation of c e r t a i n l i p i d s (phase s e p a r a t i o n ) g i v i n g r i s e t o domains. The p r e c i s e f u n c t i o n o f domains has n o t been e l u c i d a t e d . Emphasis i s p l a c e d h e r e on f e a t u r e s of the b i o l o g i c a l membranes w h i c h a r e i m p l i c a t e d i n s u b s t r a t e t r a n s p o r t . The l i p i d b i l a y e r i n t h e " g e l " s t a t e , i n the absence o f a d d i t i v e s , forms an e f f e c t i v e b a r r i e r a g a i n s t p o l a r i o n s and water s o l u b l e s u b s t r a t e s . C h a n g i n g t h e f l u i d i t y , by phase t r a n s i t i o n ( i n d u c e d by t e m p e r a t u r e c h a n g e s and/or by the a d d i t i o n o f f o r e i g n i o n s o r m o l e c u l e s ) o r by t h e i n c o r p o r a t i o n o f a d d i t i v e s ( c h o l e s t e r o l , f o r example), p r o foundly influences the structure and, hence, the t r a n s p o r t p r o p e r t i e s o f membranes. T h i s , and t h e p r e s e n c e o f c h a n n e l o r pore forming peptides o r p r o t e i n s , o p e n s t h e d o o r t o a number o f t r a n s p o r t m e c h a n i s m s w h i c h w i l l be s u m m a r i z e d i n t h e f o l l o w i n g section.



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ORDERED MEDIA IN C H E M I C A L SEPARATIONS

F i g u r e 1. A s c h e m a t i c r e p r e s e n t a t i o n o f the cross s e c t i o n o f t h e l i p i d - g l o b u l a r p r o t e i n mosaic model o f membrane s t r u c t u r e . The g l o b u l a r p r o t e i n s ( w i t h d a r k l i n e s d e n o t i n g t h e p o l y p e p t i d e c h a i n ) a r e a m p h i p a t h i c m o l e c u l e s w i t h t h e i r i o n i c and h i g h l y p o l a r g r o u p s exposed a t t h e e x t e r i o r s u r f a c e s o f t h e membranes; the degree t o which t h e s e m o l e c u l e s a r e embedded i n t h e membrane i s u n d e r t h e r m o d y n a m i c c o n t r o l . The b u l k o f t h e p h o s p h o l i p i d s ( w i t h f i l l e d c i r c l e s r e p r e s e n t i n g t h e i r p o l a r head groups and t h i n wavy l i n e s t h e i r f a t t y a c i d c h a i n s ) i s o r g a n i z e d a s a discontinuous b i l a y e r .

F i g u r e 2. An o v e r s i m p l i f i e d r e p r e s e n t a t i o n o f m o l e c u l a r m o t i o n s i n liposome b i l a y e r s . I n d i v i d u a l l i p i d s can r o t a t e ( A ) , undergo s e q u e n t i a l m o t i o n ( B ) , f l i p - f l o p ( C ) , undergo l a t e r a l d i f f u s i o n ( D ) , o r i n t e r v e s i c l e exchange ( E ) .


Hinze and Armstrong; Ordered Media in Chemical Separations ACS Symposium Series; American Chemical Society: Washington, DC, 1987. 20 %

Water

Smectic θ

Smectic A

Multilamellar

99

rigid

fluid

{vesicles'

mmmitm

F i g u r e 3. S c h e m a t i c r e p r e s e n t a t i o n o f a p h o s p h o l i p i d - w a t e r p h a s e d i a g r a m . T h e t e m p e r a t u r e s c a l e i s a r b i t r a r y and v a r i e s f r o m l i p i d t o l i p i d . F o r t h e sake o f c l a r i t y phase s e p a r a t i o n s and o t h e r c o m p l e x i t i e s i n t h e 2 0 - 9 9 % w a t e r r e g i o n a r e n o t i n d i c a t e d . S t r u c t u r e s p r o p o s e d f o r the p h o s p h o l i p i d b i l a y e r s a t d i f f e r e n t t e m p e r a t u r e s a r e shown on the r i g h t - h a n d s i d e . A t low t e m p e r a t u r e , t h e l i p i d s are arranged i n t i l t e d one-dimensional lattices. A t t h e p r e - t r a n s i t i o n temperature, two-dimensional a r r a n g e m e n t s a r e f o r m e d w i t h p e r i o d i c u n d u l a t i o n s . Above t h e main phase, t r a n s i t i o n s l i p i d s r e v e r t t o o n e - d i m e n s i o n a l l a t t i c e a r r a n g e m e n t s , s e p a r a t e d somewhat f r o m e a c h o t h e r , and assume mobile l i q u i d - l i k e conformations.

10

Crystals

Liquid

Smectic Β

Crystals

Liquid

Smectic A

Lamellar

compartment

single


88


Recognized Transport

Mechanisms A c r o s s

Biological

Membranes


T r a n s p o r t a c r o s s b i o l o g i c a l membranes i s c l a s s i f i e d a c c o r d i n g t o t h e t h e r m o d y n a m i c s o f the p r o c e s s . P a s s i v e t r a n s p o r t i s a thermodynamically downhill process; the species move t o w a r d the equilibrium. The d r i v i n g f o r c e f o r the p a s s i v e t r a n s p o r t i s the p o t e n t i a l d i f f e r e n c e between the two s i d e s o f the membrane. Active t r a n s p o r t i s a thermodynamically u p h i l l process, i t i s coupled to a c h e m i c a l r e a c t i o n and i s d r i v e n by i t . The f o l l o w i n g t r a n s p o r t mechanisms have been r e c o g n i z e d : Passive Transport. T r a n s p o r t by s i m p l e d i f f u s i o n : T h i s mode o f transport i s a v a i l a b l e for apolar molecules. Permeation i s p r e d o m i n a n t l y governed by p a r t i t i o n i n g o f the s u b s t r a t e between the l i p i d and w a t e r . The membrane s i m p l y a c t s as a p e r m e a b i l i t y b a r r i e r ; s m a l l m o l e c u l e s p a s s more e a s i l y than l a r g e ones. The t r a n s p o r t i s e x p l a i n e d i n terms o f a s i m p l e d i f f u s i o n model i n v o l v ing three steps: p a s s a g e o f the s u b s t r a t e from the e x t e r i o r i n t o t h e membrane, d i f f u s i o n t h r o u g h the membrane, and passage out o f the membrane. T r a n s p o r t by f a c i l i t a t e d d i f f u s i o n : A l a r g e number o f molec u l e s and i o n s were shown t o permeate membranes c o n s i d e r a b l y f a s t e r than e x p e c t e d from t h e i r l i p i d - w a t e r p a r t i t i o n i n g behavior. This led to the r e c o g n i t i o n of a d d i t i o n a l t r a n s p o r t mechanisms. Systematic i n v e s t i g a t i o n s o f p e r m e a b i l i t y r a t e s i n membranes, r e c o n s t i t u t e d membranes, and membrane models as f u n c t i o n s o f the t e m p e r a t u r e ; o f t h e n a t u r e and c o n c e n t r a t i o n o f the permeant; i n t h e a b s e n c e and i n t h e p r e s e n c e o f a d d i t i v e s , s u g g e s t e d t h r e e d i f f e r e n t f a c i l i t a t e d p a s s i v e t r a n s p o r t mechanisms: 1) C a r r i e r m e d i a t e d t r a n s p o r t - s u b s t r a t e s are t r a n s p o r t e d a c r o s s t h e membrane by a d i f f u s a b l e c a r r i e r , t y p i c a l l y an enzyme. Once a g a i n , t h e r e a r e t h r e e s t e p s : c o m p l e x a t i o n o f the s u b s t r a t e w i t h t h e c a r r i e r on o n e - s i d e o f t h e membrane, d i f f u s i o n o f the s u b s t r a t e - c a r r i e r complex t o the o t h e r s i d e and d e c o m p l e x a t i o n : S +

I ES

Il

j

*

ES

I + S

II

(1)

The s u g a r - t r a n s p o r t system i s the most o f t e n c i t e d example f o r the c a r r i e r m e d i a t e d f a c i l i t a t e d t r a n s p o r t o f a c o v a l e n t molecule. T r a n s p o r t o f s u g a r s i n t o the r e d b l o o d c e l l s i s p a s s i v e ( i t o c c u r s o n l y i n the p r e s e n c e of a c o n c e n t r a t i o n g r a d i e n t ) , s e l e c t i v e (D-glucose i s transported, while L-glucose i s n o t ) , and the k i n e t i c s show a s a t u r a t i o n b e h a v i o r ( o b s e r v e d t y p i c a l l y f o r enzyme mediated i n t e r a c t i o n s ) . T h e s e o b s e r v a t i o n s are i n s u p p o r t o f a f a c i l i t a t e d p a s s i v e t r a n s p o r t mechanism which i n v o l v e s an enzyme as t h e c a r r i e r . V e r i f i c a t i o n must a w a i t t h e i s o l a t i o n and full c h a r a c t e r i z a t i o n o f the s p e c i f i c enzyme(s) i n v o l v e d i n the t r a n s port of a given molecule. Transport o f c a t i o n s by membrane d i f f u s a b l e m a c r o c y c l i c a n t i b i o t i c s (valinomycin, nigericin, for e x a m p l e ) a l s o b e l o n g s t o the c a t e g o r y o f c a r r i e r m e d i a t e d p a s s i v e



2.

FENDLER

Membrane Mimetic Separations

89

transport. S y n t h e t i c m a c r o c y c l i c compounds (crown e t h e r s , c r y p t ands, f o r example) are i n c r e a s i n g l y u t i l i z e d f o r o b t a i n i n g fundam e n t a l u n d e r s t a n d i n g o f c a r r i e r m e d i a t e d t r a n s p o r t mechanisms i n membrane models. 2) C h a n n e l m e d i a t e d t r a n s p o r t - c a t i o n s are mainly t r a n s p o r t e d by t h e i r p a s s i v e d i f f u s i o n t h r o u g h c h a n n e l s ( o r p o r e s ) i n the membranes. G r a m i c i d i n A i s t h e b e s t u n d e r s t o o d c h a n n e l f o r m i n g substance. I t i s a l i n e a r p o l y p e p t i d e c o n s t i t u t e d from 15 n e u t r a l amino a c i d s . Two m o l e c u l e s o f G r a m i c i d i n A r e v e r s i b l y a s s o c i a t e t o f o r m a h e a d t o h e a d d i m e r w h i c h s p a n s a p p r o x i m a t e l y 30 A, t h e t h i c k n e s s o f a t y p i c a l membrane, F i g u r e 4 ( 4 ) . C o n d u c t a n c e m e a s u r e m e n t s a c r o s s a G r a m i c i d i n A c o n t a i n i n g membrane ( a t a f i x e d p o t e n t i a l ) r e s u l t i n s m a l l p o s i t i v e c u r r e n t jumps o f c o n s t a n t a m p l i t u d e w h i c h c o r r e s p o n d t o the a s s o c i a t i o n and d i s s o c i a t i o n o f the d i m e r s and, h e n c e , t o t h e o p e n i n g and c l o s i n g o f t h e i o n channels. G r a m i c i d i n A ceases to f a c i l i t a t e the t r a n s p o r t of c a t i o n s i n membranes t h i c k e r than 30 Â. A p p a r e n t l y , t h e c h a n n e l f o r m i n g d i m e r s do n o t s p a n t h i c k membranes. Conversely, the a b i l i t y of valinomycin t o t r a n s p o r t c a t i o n s does n o t d i m i n i s h i n t h i c k membranes. These o b s e r v a t i o n s a r e i n a c c o r d w i t h G r a m i c i d i n A f o r m i n g c h a n n e l s o f d e f i n e d l e n g t h s and v a l i n o m y c i n a c t i n g as a d i f f u s a b l e c a r r i e r i n t h e membrane. 3) Gate m e d i a t e d t r a n s p o r t - anions are mainly transported by t h e i r f a c i l i t a t e d d i f f u s i o n t h r o u g h a s w i n g i n g gate formed by a t r a n s m e m b r a n e enzyme u n d e r g o i n g c o n f o r m a t i o n a l changes, F i g u r e 5. E x c h a n g e o f H C O 3 " f o r C l ~ t h r o u g h t h e e r y t h r o c i t e membrane d u r i n g the flow o f b l o o d i s b e l i e v e d t o o c c u r t h r o u g h t h i s mechanism. Transport by f l u x - c o u p l i n g (co-transport or symport): E n h a n c e d p e r m e a b i l i t y o f a m o l e c u l e i n t h e p r e s e n c e o f a n o t h e r has been o b s e r v e d . F o r e x a m p l e , i n some membranes t h e t r a n s p o r t o f D - g l u c o s e ( b u t n o t L - g l u c o s e ! ) i s s u b s t a n t i a l l y i n c r e a s e d by t h e p r e s e n c e o f sodium i o n s . The enhanced t r a n s p o r t i s t h e consequence o f h a v i n g more t h a n one r e c o g n i t i o n s i t e on a g i v e n t r a n s p o r t protein. Sodium i o n s b i n d complimentarily t o the glucose t r a n s p o r t i n g enzyme a n d , h e n c e , f a c i l i t a t e i t s p a s s a g e a c r o s s t h e membrane. Active Transport. By d e f i n i t i o n , a c t i v e t r a n s p o r t o c c u r s i n t h e a b s e n c e o f a n y e l e c t r o c h e m i c a l p o t e n t i a l o r i g i n a t i n g i n a concent r a t i o n g r a d i e n t (4,6) . A c t i v e t r a n s p o r t i s d r i v e n by a c o u p l e d c h e m i c a l r e a c t i o n . D i s t i n c t i o n i s made between p r i m a r y and secondary a c t i v e transport. Primary a c t i v e transport: Primary a c t i v e t r a n s p o r t i s q u i t e s i m p l y the c o u p l i n g o f a l o c a l c h e m i c a l r e a c t i o n (X >Y) t o p r o v i d e e n e r g y f o r an u p h i l l f a c i l i t a t e d (by E, which may be a c a r r i e r , a c h a n n e l o r a g a t e ) d i f f u s i o n o f a s p e c i e s S a c r o s s t h e membrane:



90


2.5- 3.0 nm

F i g u r e 4. P r o j e c t i o n of a t h r e e - d i m e n s i o n a l m o d e l o f an e l e c t r i c a l l y c o n d u c t i n g p o r e o f g r a m i c i d i n A. To span the f u l l t h i c k n e s s o f the l i p i d b i l a y e r membrane, two m o l e c u l e s , e n d - t o e n d , a r e r e q u i r e d . The s i d e c h a i n s o f t h e amino a c i d s a r e not shown. The m o d e l was o r i g i n a l l y p r o p o s e d by U r r y P r o c . Nat. A c a d . S c i . USA 6 8 , 672 ( 1 9 7 1 ) . Reproduced w i t h p e r m i s s i o n from Ref. 4. C o p y r i g h t 1983, Springer-Verlag.

F i g u r e 5. Diagram o f a s i m p l i f i e d model o f the mechanism o f C I " e x c h a n g e d i f f u s i o n t h r o u g h a n o n c o n d u c t i n g pore o f the e r y t h r o c y t e membrane. The g a t e m e c h a n i s m i s shown f u n c t i o n i n g i n c o m b i n a t i o n w i t h a c o n f o r m a t i o n a l change i n the pore w a l l . The b a s i c c o n c e p t i s t h a t the gate can o n l y f l i p o v e r from the c i s t o t h e t r a n s p o s i t i o n and b a c k i f a c h l o r i d e i o n i s bound. A c o n f o r m a t i o n a l c h a n g e t h e n t a k e s p l a c e nearby i n the p r o t e i n , which l e a d s t o a s c r e e n i n g o f the b i n d i n g s i t e from the c i s s i d e and an o p e n i n g t o w a r d s t h e t r a n s s i d e . F o r s i m p l i c i t y , the c o n f o r m a t i o n a l c h a n g e shown i n t h e d i a g r a m a f f e c t s the whole protein. R e p r o d u c e d w i t h p e r m i s s i o n f r o m Ref. 4. Copyright 1983, Springer-Verlag.


2.

91


FENDLER


Energy i s p r o v i d e d , f o r example, by ATP f o r pumping sodium i o n s out o f and potassium ions i n t o the c e l l . Another i m p o r t a n t example o f primary a c t i v e t r a n s p o r t i s the proton c o n c e n t r a t i o n gradient d r i v e n ATP s y n t h e s i s ( M i t c h e l l - h y p o t h e s i s ) . Secondary a c t i v e t r a n s p o r t : Secondary active transport i s more c o m p l e x . I t i n v o l v e s t h e p e r m e a t i o n o f two d i f f e r e n t sub s t a n c e s (A a n d B) a c r o s s t h e membrane. The t r a n s p o r t o f A i s a c t i v e - i t i s an u p h i l l p r o c e s s d r i v e n by t h e c h e m i c a l r e a c t i o n X—>Y. The t r a n s p o r t o f Β i s p a s s i v e , b u t f a c i l i t a t e d by a c a r r i e r C, w h i c h c o - t r a n s p o r t s A ( E q u a t i o n 3 ) . C o - t r a n s p o r t i s d e f i n e d above i n t h e s e c t i o n on p a s s i v e t r a n s p o r t .

0 A + A ·

, A + A

β

t

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(3)

ABC 4. Β + Β

Β + Β *

C

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C

I s o t o n i c w a t e r r e s o r p t i o n i n the e p h i t e l i u m i s an example f o r the secondary a c t i v e t r a n s p o r t . Water and sodium i o n s a r e symported from the b l o o d i s o t o n i c a l l y ( i . e . , a g a i n s t t h e i r concentration g r a d i e n t s ) a n d t h e r e i s no t r a n s p o r t o f e i t h e r i n t h e absence o f the o t h e r . E q u a t i o n s have been r e c e n t l y d e r i v e d f o r a g e n e r a l i z e d scheme encompassing p r i m a r y and secondary a c t i v e t r a n s p o r t systems ( ! ) . Membrane M i m e t i c

Chemistry

Membrane m i m e t i c c h e m i s t r y i s a r a p i d l y emerging d i s c i p l i n e con c e r n e d w i t h the development o f p r o c e s s e s which a r e i n s p i r e d by t h e b i o l o g i c a l membrane ( 8 ) . S u r f a c t a n t a g g r e g a t e s - m i c e l l e s , mono l a y e r s , o r g a n i z e d m u l t i l a y e r s (Langmuir-Blodgett f i l m s ) , b i l a y e r l i p i d membranes (BLMs), v e s i c l e s and p o l y m e r i z e d v e s i c l e s have been u s e d a s media i n membrane mimetic c h e m i s t r y . D i f f e r e n t aggregates formed from s u r f a c t a n t s a r e i l l u s t r a t e d i n F i g u r e 6. A q u e o u s m i c e l l e s a r e 40-80 A d i a m e t e r s p h e r i c a l a g g r e g a t e s which are d y n a m i c a l l y f o r m e d f r o m s u r f a c t a n t s i n water above a characteristic concentration, t h e CMC ( 9 ) · D e p e n d i n g on t h e c h e m i c a l s t r u c t u r e o f t h e i r h y d r o p h i l i c headgroups, s u r f a c t a n t s can be n e u t r a l o r charged ( p o s i t i v e l y o r n e g a t i v e l y ) . The a l k y l c h a i n o f t h e s u r f a c t a n t s t y p i c a l l y c o n t a i n s between 5-20 c a r b o n atoms. M i c e l l e s r a p i d l y b r e a k up and r e f o r m by two known p r o c e s s e s . The f i r s t p r o c e s s o c c u r s on t h e m i c r o s e c o n d time s c a l e and i s due t o the r e l e a s e and subsequent r e i n c o r p o r a t i o n o f a s i n g l e s u r f a c t a n t from and b a c k t o t h e m i c e l l e . The s e c o n d p r o c e s s o c c u r s on t h e m i l l i s e c o n d time s c a l e and i s a s c r i b e d t o the d i s s o l u t i o n o f the



92


Figure 6. An o v e r s i m p l i f i e d r e p r e s e n t a t i o n a g g r e g a t e s formed from s u r f a c t a n t s .

of

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2.

FENDLER


93

m i c e l l e a n d t o t h e s u b s e q u e n t r e a s s o c i a t i o n o f t h e monomers. S u b s t r a t e i n t e r a c t i o n w i t h t h e m i c e l l e i s a l s o dynamic. M o n o l a y e r s ( m o n o m o l e c u l a r l a y e r s ) a r e f o r m e d by s p r e a d i n g n a t u r a l l y occurring l i p i d s or synthetic surfactants, dissolved i n a v o l a t i l e s o l v e n t , o v e r water i n a Langmuir t r o u g h (10). The p o l a r headgroups of the s u r f a c t a n t s a r e i n contact with water, the s u b p h a s e , w h i l e t h e i r h y d r o c a r b o n t a i l s p r o t r u d e above i t . Monol a y e r s a r e c h a r a c t e r i z e d by s u r f a c e a r e a - s u r f a c e p r e s s u r e c u r v e s , s u r f a c e p o t e n t i a l s , and s u r f a c e v i s c o s i t i e s . In t h e gaseous s t a t e , s u r f a c t a n t s f l o a t f r e e l y , m o s t l y l y i n g f l a t , on t h e s u r f a c e w i t h o u t e x e r t i n g much f o r c e on e a c h o t h e r . M o n o l a y e r s i n t h e i r gaseous s t a t e may be i n f i n i t e l y e x p a n d e d w i t h o u t any phase change. Comp r e s s i n g t h e gaseous monolayers r e s u l t s i n a t r a n s i t i o n t o a f l u i d state. A t l e a s t two f l u i d s u b p h a s e s have been r e c o g n i z e d . The i n i t i a l t r a n s i t i o n on d e c r e a s i n g the s u r f a c e area o f gaseous m o n o l a y e r s r e s u l t s from a g r a d u a l r e o r g a n i z a t i o n o f m o l e c u l e s t o a p o s i t i o n more o r l e s s p e r p e n d i c u l a r t o t h e subphase s u r f a c e . I n t h i s s t a t e , t h e a v e r a g e i n t e r m o l e c u l a r d i s t a n c e s a r e much g r e a t e r than that i n bulk l i q u i d s . On f u r t h e r c o m p r e s s i o n , t h e d i s t a n c e b e t w e e n t h e s u r f a c t a n t headgroups d e c r e a s e s and t h e system assumes t h e l i q u i d c o n d e n s e d f l u i d phase. I n t h e s o l i d phase, s u r f a c t a n t s i n t h e m o n o l a y e r a r e p a c k e d as c l o s e l y as p o s s i b l e ; t h e y a l l a r e perpendicular t o t h e s u b p h a s e o r a r e t i l t e d a t an a n g l e . Monol a y e r s i n t h e i r s o l i d phase show low c o m p r e s s i b i l i t y as i n d i c a t e d by t h e v e r t i c a l s u r f a c e p r e s s u r e - s u r f a c e a r e a i s o t h e r m ( F i g u r e 7 ) . U l t i m a t e l y , compression leads t o a break or i n f l e c t i o n i n the i s o t h e r m w h i c h c o r r e s p o n d s t o t h e c o l l a p s e o f t h e monolayer i n t o b i l a y e r s and m u l t i l a y e r s . T e c h n i q u e s have been d e v e l o p e d f o r t r a n s f e r r i n g t h e monolayer o n t o a s o l i d s u p p o r t a n d f o r b u i l d i n g up o r g a n i z e d multilayer a s s e m b l i e s i n c o n t r o l l e d t o p o l o g i c a l arrangements ( F i g u r e 8) ( 1 1 ) . D e p e n d i n g on t h e m o n o l a y e r f o r m i n g m a t e r i a l a n d on t h e mode o f d e p o s i t i o n , t h r e e s t r u c t u r a l l y d i f f e r e n t m u l t i l a y e r s are recogn i z e d . The X - t y p e m u l t i l a y e r s ( p l a t e - s u r f a c t a n t t a i l - s u r f a c t a n t head-tail-head, e t c . ) a r e formed by t h e s e q u e n t i a l hydrophobic a t t a c h m e n t s o f monolayers onto t h e p l a t e upon immersion o n l y . The Y - t y p e m u l t i l a y e r s ( p l a t e - s u r f a c t a n t t a i l - s u r f a c t a n t head-headt a i l - t a i l , e t c . ) a r e b u i l t up b o t h by d i p p i n g and by w i t h d r a w i n g t h e p l a t e t h r o u g h t h e f l o a t i n g monolayer. The Z-type m u l t i l a y e r s ( p l a t e - s u r f a c t a n t head-surfactant t a i l , head-tail-head, etc.) are t h e r e s u l t o f s e q u e n t i a l h y d r o p h i l i c a t t a c h m e n t s o f t h e monolayers o n t o t h e p l a t e upon w i t h d r a w a l o n l y . A b s o l u t e and s c r u p u l o u s c l e a n l i n e s s i s a must i n a l l m o n o l a y e r a n d m u l t i l a y e r s t u d i e s . M o n o l a y e r s a n d m u l t i l a y e r s have been s t a b i l i z e d by p o l y m e r i z a t i o n (12-14). B i l a y e r ( b l a c k ) l i p i d membranes, BLMs, a r e formed by b r u s h i n g an o r g a n i c s o l u t i o n o f a s u r f a c t a n t ( o r l i p i d ) a c r o s s a p i n h o l e ( 2 - 4 mm d i a m e t e r ) s e p a r a t i n g two aqueous phases (15,16) . A l t e r n a t i v e l y , BLMs can be formed from monolayers by t h e M o n t a l - M u e l l e r method ( 1 7 , 1 8 ) . I n t h i s method, t h e s u r f a c t a n t , d i s s o l v e d i n an a p o l a r s o l v e n t , i s s p r e a d on t h e water s u r f a c e t o form a monolayer b e l o w t h e t e f l o n p a r t i t i o n i n g which c o n t a i n s t h e p i n h o l e (0.1-0.5 mm d i a m e t e r ) . C a r e f u l i n j e c t i o n o f an a p p r o p r i a t e e l e c t r o l y t e s o l u t i o n below t h e s u r f a c e r a i s e s t h e water l e v e l above t h e p i n h o l e



94


F i g u r e 7. Schematic r e p r e s e n t a t i o n s u r f a c e area isotherm f o r monolayers.

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95


Z-type

F i g u r e 8. Types o f monolayer no rearrangement o c c u r s .

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system i f



96


and b r i n g s t h e m o n o l a y e r i n t o a p p o s i t i o n t o f o r m t h e BLM. An a d v a n t a g e o f t h e M o n t a l - M u e l 1 e r method i s t h a t i t p e r m i t s t h e formation of disymmetrical BLMs. The i n i t i a l l y formed f i l m i s r a t h e r t h i c k and r e f l e c t s w h i t e l i g h t w i t h a grey c o l o r . W i t h i n a few m i n u t e s t h e f i l m t h i n s a n d t h e r e f l e c t e d l i g h t e x h i b i t s i n t e r f e r e n c e c o l o r s that u l t i m a t e l y turn black. At that point the f i l m i s considered t o be b i m o l e c u l a r (40-60 A, t h i c k n e s s ) . BLMs have been e x t e n s i v e l y u t i l i z e d i n t h e e l u c i d a t i o n of t r a n s p o r t mechanisms by e l e c t r i c a l measurements. V e s i c l e s a r e s m e c t i c mesophases o f s u r f a c t a n t s c o n t a i n i n g w a t e r b e t w e e n t h e i r b i l a y e r s ( 1_9) . P r e p a r e d by s o n i c a t i o n from s u c h s i m p l e s u r f a c t a n t s a s d i o c t a d e c y l d i m e t h y l a m m o n i u m bromide (DODAB) o r d i h e x a d e c y l p h o s p h a t e ( D H P ) , t h e y a r e s i n g l e b i l a y e r s p h e r i c a l a g g r e g a t e s w i t h d i a m e t e r s o f 500-1000 Â and b i l a y e r t h i c k n e s s o f c a . 50 Â. Once formed, v e s i c l e s , u n l i k e m i c e l l e s , do n o t b r e a k down on d i l u t i o n . N e v e r t h e l e s s , t h e y a r e dynamic s t r u c tures. T h e y u n d e r g o p h a s e t r a n s i t i o n , f u s e , and a r e o s m o t i c a l l y active. M o l e c u l a r m o t i o n s o f t h e i n d i v i d u a l s u r f a c t a n t s i n the v e s i c l e s i n v o l v e r o t a t i o n s , k i n k f o r m a t i o n , l a t e r a l d i f f u s i o n on t h e v e s i c l e p l a n e , and t r a n s f e r from one i n t e r f a c e o f t h e b i l a y e r to the other ( f l i p - f l o p ) . V e s i c l e s a r e capable o f o r g a n i z i n g a l a r g e number o f m o l e c u l e s i n t h e i r c o m p a r t m e n t s . H y d r o p h o b i c m o l e c u l e s c a n be d i s t r i b u t e d among t h e h y d r o c a r b o n b i l a y e r s o f vesicles. P o l a r m o l e c u l e s may move a b o u t r e l a t i v e l y f r e e l y i n v e s i c l e - e n t r a p p e d w a t e r p o o l s , p a r t i c u l a r l y i f they are e l e c t r o s t a t i c a l l y r e p e l l e d from t h e i n n e r s u r f a c e . S m a l l charged i o n s can be e l e c t r o s t a t i c a l l y a t t a c h e d t o t h e o p p o s i t e l y c h a r g e d v e s i c l e s u r f a c e s . Species having charges i d e n t i c a l with those of the v e s i c l e s c a n be a n c h o r e d o n t o t h e v e s i c l e s u r f a c e by a l o n g hydrocarbon t a i l . The n e e d f o r i n c r e a s e d s t a b i l i t i e s , c o n t r o l l a b l e s i z e s , and p e r m e a b i l i t i e s l e d t o t h e development o f polymerized surfactant vesicles (12-14,20). Vesicle-forming s u r f a c t a n t s have been f u n c t i o n a l i z e d by v i n y l , m e t h a c r y l a t e , d i a c e t y l e n e , i s o c y a n o , and s t y r e n e groups i n t h e i r h y d r o c a r b o n c h a i n s o r a t t h e i r headgroups. Accordingly, s u r f a c t a n t v e s i c l e s c o u l d be p o l y m e r i z e d i n t h e i r b i l a y e r s o r a c r o s s t h e i r headgroups. In the l a t t e r c a s e , e i t h e r t h e o u t e r o r t h e i n n e r v e s i c l e s u r f a c e s c o u l d be l i n k e d s e p a r a t e l y ( F i g u r e 9 ) . A l l p o l y m e r i z e d v e s i c l e s show a p p r e c i a b l e s t a b i l i t i e s compared w i t h their unpolymerized counterparts. They have e x t e n s i v e s h e l f l i v e s and remain u n a f f e c t e d by t h e a d d i t i o n o f up to 30% methanol. S u b s t r a t e o r g a n i z a t i o n i n membrane m i m e t i c systems l e a d s t o a l t e r e d s o l v a t i o n , i o n i z a t i o n and r e d u c t i o n p o t e n t i a l s and, hence, to a l t e r e d r e a c t i o n r a t e s , p a t h s and s t e r e o c h e m i s t r i e s . These p r o p e r t i e s have been a d v a n t a g e o u s l y e x p l o i t e d , i n turn, f o r r e a c t i v i t y c o n t r o l , c a t a l y s i s , drug d e l i v e r y and a r t i f i c i a l p h o t o s y n t h e s i s (8). There a r e o n l y l i m i t e d examples o f t h e u t i l i z a t i o n o f membrane m i m e t i c systems i n p e r m e a b i l i t y c o n t r o l . In order to g a i n i n s i g h t i n t o t h i s i m p o r t a n t a r e a , we have i n i t i a t e d a r e s e a r c h p r o g r a m i n BLMs. A s t a t u s r e p o r t o f our a c t i v i t i e s i n t h i s area w i l l be summarized i n t h e next s e c t i o n .




g

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Simultaneous E l e c t r i c a l

and S p e c t r o s c o p i c Measurements o f BLMs

BLMs p r e p a r e d f r o m p h o s p h o l i p i d s have been f r u i t f u l l y u t i l i z e d i n the p a s t s e v e r a l y e a r s i n e l e c t r i c a l measurements both i n the absence and i n t h e p r e s e n c e o f i o n o p h o r e s (2_1 ) . Holding the b i l a y e r membrane a t a p r e d e t e r m i n e d p o t e n t i a l and m e a s u r i n g t h e c o r r e s p o n d i n g c u r r e n t f l o w , i . e . , v o l t a g e c l a m p i n g , has c o n t r i b u t e d much t o t h e p r e s e n t d a y u n d e r s t a n d i n g o f i o n c h a n n e l s and impulse t r a n s m i s s i o n (22). I n v e s t i g a t i o n s o f BLMs s u f f e r f r o m two m a j o r d r a w b a c k s . F i r s t , BLMs a r e n o t o r i o u s l y u n s t a b l e . V e r y r a r e l y do they s u r v i v e l o n g e r t h a n a c o u p l e o f h o u r s . Second, v o l t a g e c l a m p i n g p r o v i d e s i n f o r m a t i o n o n l y on t h e t r a n s i t i o n from an open s t a t e t o a c l o s e d state i n i o n channels. C u r r e n t r e s e a r c h i n our l a b o r a t o r i e s i s d i r e c t e d t o o v e r c o m i n g t h e s e d i s a d v a n t a g e s by s t a b i l i z i n g BLMs by p o l y m e r i z a t i o n o r by p o l y m e r c o a t i n g , a n d by d e v e l o p i n g s i m u l t a n e o u s i_n s i t u s p e c t r o s c o p i c a n d e l e c t r i c a l t e c h n i q u e s f o r m o n i t o r i n g f u n c t i o n i n g BLMs. D i r e c t s p e c t r o s c o p i c measurements o f a b s o r p t i o n s c o u l d p r o v i d e s u b s t a n t i a l a n d m u c h - n e e d e d c o m p l i m e n t a r y i n f o r m a t i o n on t h e p r o p e r t i e s o f BLMs. D i f f i c u l t i e s of spectroscopic techniques l i e i n t h e e x t r e m e t h i n n e s s o f t h e BLM; a b s o r b a n c e s o f r e l a t i v e l y few m o l e c u l e s need t o be d e t e r m i n e d . We have overcome t h i s d i f f i c u l t y by I n t r a c a v i t y L a s e r A b s o r p t i o n S p e c t r o s c o p i c (ICLAS) measurements. A b s o r b a n c e s i n I C L A S a r e d e t e r m i n e d as i n t r a c a v i t y o p t i c a l l o s s e s ( 2J3) . S e n s i t i v i t y enhancements o r i g i n a t e i n the m u l t i p a s s , t h r e s h o l d and mode c o m p e t i t i o n e f f e c t s . Enhancement f a c t o r as h i g h as 10^ h a s b e e n r e p o r t e d f o r s p e c i e s whose a b s o r b a n c e s a r e narrow c o m p a r e d t o s p e c t r a l p r o f i l e o f t h e l a s e r (J_0) The enhancement f a c t o r f o r broad-band a b s o r b e r s , used i n o u r work, i s much s m a l l e r . Thus, f o r BLM-incorporated c h l o r o p h y l l - a , we o b s e r v e d an enhance ment f a c t o r o f 10^ a n d r e p o r t e d s e n s i t i v i t i e s f o r a b s o r b a n c e s i n the o r d e r o f 1 0 " ( 2 4 ) . F i g u r e 10 shows t h e s c h e m a t i c s o f t h e e x p e r i m e n t a l setup u s e d f o r i n t r a c a v i t y l a s e r a b s o r p t i o n s p e c t r o s c o p y (ICLAS) o f b i l a y e r l i p i d membranes (BLMs). S i m u l t a n e o u s e l e c t r i c a l and ICLAS measure m e n t s w e r e c a r r i e d out i n a two-compartment c o n t a i n e r c o n s t r u c t e d from two 1 cm p a t h l e n g t h s q u a r t z c e l l s ( F i g u r e 1 1 ) . I C L A S o f f e r e d a c o n v e n i e n t m o n i t o r i n g o f BLM f o r m a t i o n . The u p p e r p a r t o f F i g u r e 12 shows t h e t i m e d e p e n d e n t change o f t h e r e l a t i v e l a s e r i n t e n s i t y p a r a l l e l i n g BLM f o r m a t i o n i n t h e c a v i t y . B L M - f o r m i n g s o l u t i o n was b r u s h e d a c r o s s t h e t e f l o n a p e r t u r e a t t 0. Due t o t h e s c a t t e r i n g o f t h e v e r y t h i c k f i l m , initially p r e s e n t , as w e l l as t o n o n - u n i f o r m , l a r g e l o s s e s i n t h e c a v i t y , no l a s i n g was o b s e r v e d . A f t e r some time, i n d i c a t e d by A i n t h e upper p a r t o f F i g u r e 12 ( t y p i c a l l y 3-4 m i n u t e s ) , t h e f i l m s u f f i c i e n t l y t h i n n e d , a n d l a s i n g was o b s e r v e d . F u r t h e r t h i n n i n g r e s u l t e d i n a gradual i n c r e a s e of the t r a n s m i t t e d l i g h t i n t e n s i t y u n t i l i t reached a p l a t e a u v a l u e ( i n d i c a t e d by Β i n t h e upper p a r t o f F i g u r e 1 2 ) . A t t h i s p l a t e a u , t r u e b i m o l e c u l a r t h i c k membranes (BLMs) were p r e s e n t . The p l a t e a u v a l u e remained c o n s t a n t u n t i l t h e membrane was broken ( i n d i c a t e d by C i n t h e upper p a r t o f F i g u r e 1 2 ) . BLM f o r m a t i o n was s i m u l t a n e o u s l y o b s e r v e d b y e l e c t r i c a l measurements ( s e e lower p a r t o f o f F i g u r e 1 2 ) . A t r i a n g u l a r 6

s



FENDLER


Figure 10. Schematics o f t h e e x p e r i m e n t a l s e t u p f o r i n t r a c a v i t y l a s e r a b s o r p t i o n spectroscopy (ICLAS). CD » chopper d r i v e r ; PM power m e t e r ; H\, M 2 , M 3 , M4 * s p h e r i c a l h i g h r e f l e c t i o n m i r r o r s ; Mp = pump m i r r o r ; MN » m o n o c h r o m a t o r ; PMT * p h o t o m u l t i p l i e r ; SP « s i l i c o n p h o t o c e l l ; PC « P o c k e l s c e l l ; WF » wedged f i l t e r ; L I A * l o c k - i n a m p l i f i e r ; R - r e c o r d e r ; MS • m i c r o s c o p e ; OF * o p t i c a l f i b e r ; S * sample ( s o l u t i o n on BLM) ; IEM • i n s t r u m e n t s f o r e l e c t r i c a l measurements ( s e e F i g u r e 2 ) . a


99



100



Figure 12. (Left): P l o t of l a s e r i n t e n s i t y as a f u n c t i o n of time ( i n a r b i t r a r y u n i t s ) d u r i n g f i l m f o r m a t i o n ( 0 — * A ) , t h i n n i n g ( A — > Β) , p r e s e n c e ( B — > C ) and b r e a k i n g o f BLM (C) . d l i s the d i f f e r e n c e i n the i n t e n s i t y o f the l a s e r p r i o r and subsequent t o the b r e a k i n g o f the BLM.

TIME t

V

Id]

(b)

(a|

(Right): P l o t of v o l t a g e clamped (a) and c u r r e n t (b, c, d) waveforms i n the absence ο f i l m ( b ) , i n the p r e s e n c e o f BLM (c) and sub sequent t o the breakage o f the BLM ( d ) .

2.5

mv


102



v o l t a g e c l a m p e d w a v e f o r m ( a i n t h e l o w e r p a r t o f F i g u r e 12.) was applied across the f i l m . The o b s e r v e d c u r r e n t waveform changed w i t h t h e f o r m a t i o n o f a t h i c k f i l m subsequent t o t h e b r u s h i n g o f t h e membrane-forming s o l u t i o n a c r o s s t h e t e f l o n a p e r t u r e , w i t h t h e t h i n n i n g o f t h e f i l m t o BLM, a n d w i t h t h e b r e a k i n g o f t h e BLM. These e l e c t r i c a l changes c o r r e s p o n d e d t o changes o b s e r v e d by ICLAS. T h u s , no c u r r e n t p a s s e d a c r o s s t h e e l e c t r o d e s p r i o r t o a p p r e c i a b l e t h i n n i n g o f t h e membrane. The o b s e r v e d t r a c e b i n t h e lower p a r t o f F i g u r e 12 c o r r e s p o n d e d t o t h e 0 »A time domain ( s e e upper p a r t o f F i g u r e 12) o b s e r v e d b y I C L A S . I n c r e a s e i n t h e transmembrane c u r r e n t c o r r e s p o n d e d t o t h e t h i n n i n g o f t h e f i l m t o BLM ( s e e c i n t h e l o w e r p a r t a n d A - B i n t h e u p p e r p a r t o f F i g u r e 1 2 ) . The c u r r e n t waveform remained s t a b l e and u n a l t e r e d d u r i n g t h e p r e s e n c e o f t h e BLM ( s e e Β »C i n t h e upper p a r t o f F i g u r e 1 2 ) . B r e a k i n g o f t h e BLM was s i g n a l l e d b y t h e a p p e a r a n c e o f p e r f e c t square waves c o r r e s p o n d i n g t o t h e s a t u r a t i o n o f t h e a m p l i f i e r by l a r g e e l e c t r o d e c u r r e n t s ( s e e d i n t h e lower p a r t and C i n t h e upper p a r t o f F i g u r e 12). T h i n n i n g o f t h e f i l m was a l s o o b s e r v e d by m i c r o s c o p y . The i n i t i a l l y w h i t e f i l m g r a d u a l l y changed c o l o r and showed a v a r i e t y o f i n t e r f e r e n c e f r i n g e s (between p o i n t s A and Β i n Figure 12), which u l t i m a t e l y turned black ( a t p o i n t B ) . G e n e r a l l y , BLM f o r m a t i o n was c o m p l e t e w i t h i n 20 m i n u t e s . T y p i c a l l y , BLMs l a s t e d f o r 1-3 h o u r s . Microscopic observations afforded the c a l c u l a t i o n ofthe p h y s i c a l a r e a o f BLM, w h i c h , i n c o m b i n a t i o n w i t h electrical m e a s u r e m e n t s , l e d t o v a l u e s o f BLM c a p a c i t a n c e s p e r u n i t a r e a . T y p i c a l BLMs p r e p a r e d from DODAC ( b o t h i n t h e p r e s e n c e and i n t h e a b s e n c e o f c h l o r o p h y 1 1 - a ) h a d a r e a s o f 5.7 χ 1 0 " ^ cm^ a n d 0.7 c a p a c i t a n c e s . These v a l u e s a g r e e d w e l l w i t h t h o s e d e t e r m i n e d f o r BLMs p r e p a r e d from p h o s p h o l i p i d s ( c a p a c i t a n c e * 0.7-1.3 \i¥/cm^) and from s i n g l e - c h a i n s u r f a c t a n t s ( c a p a c i t a n c e = 0.3-0.6 ]i¥/cm^). Thickness a s s e s s e d from:

of the i n s u l a t i n g

. d

l a y e r , d , i n DODAC BLMs can be

ε εΑ ο m =

/ , \

(4)

where ε^ i s t h e d i e l e c t r i c c o n s t a n t i n vacuum, and t a k e n t o be 8.85 χ 10""1* CV~1 m~l , ε i s t h e d i e l e c t r i c c o n s t a n t o f t h e h y d r o c a r b o n and i s assumed t o be 2.1 ( 1 5 ) . A i s t h e a r e a o f membrane, d e t e r m i n e d h e r e t o be 5.7 χ 10"^ cm^ a n d C i s t h e c a p a c i t a n c e o f t h e BLM, d e t e r m i n e d h e r e t o be 4.0 nF. S u b s t i t u t i n g t h e s e v a l u e s i n t o E q u a t i o n 11 g a v e d « 26.5 Â f o r t h e t h i c k n e s s o f t h e i n s u l a t i n g l a y e r i n DODAC BLMs. T h i s v a l u e i s i n v e r y g o o d agreement w i t h t h o s e c a l c u l a t e d f o r p h o s p h o l i p i d b i l a y e r membranes (23-26 A) (25) making t h e same assumptions as used h e r e . tn

We h a v e a l s o p r e p a r e d BLMs from p o l y m e r i z a b l e s u r f a c t a n t s and p o l y m e r i z e d them i n s i t u (_26). E x t e n t s o f p o l y m e r i z a t i o n have been f o l l o w e d by nanosecond, t i m e - r e s o l v e d f l u o r e s c e n c e s p e c t r o s c o p y and a n i s o t r o p i c measurements (26) . E x p e r i m e n t s have been i n i t i a t e d f o r r e a l i z i n g t h e d i f f e r e n t b i o l o g i c a l t r a n s p o r t mechanisms i n p o l y m e r i z e d a n d p a r t i a l l y - p o l y m e r i z e d BLMs and f o r s t u d y i n g t h e i r mechanisms by s i m u l t a n e o u s e l e c t r i c a l and s p e c t r o s c o p i c measurements.


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Acknowledgments I thank my co-workers, whose names appear i n t h e r e f e r e n c e s l i s t e d , f o r t h e i r e n t h u s i a s t i c , d e d i c a t e d , and s k i l l f u l work. The N a t i o n a l S c i e n c e F o u n d a t i o n , Department o f Energy, and Army R e s e a r c h O f f i c e p r o v i d e d f i n a n c i a l s u p p o r t f o r d i f f e r e n t a s p e c t s o f our r e s e a r c h e s .

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15. 16. 17. 18.

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