Ordered Media in Chemical Separations - American Chemical Society

Chapter 9

Ordered Media in Chemical Separations Downloaded from pubs.acs.org by UNIV OF CALIFORNIA SAN DIEGO on 02/17/16. For personal use only.

Extraction of Proteins and Amino Acids Using Reversed Micelles T. Alan Hatton Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139

The solubilisation of proteins and amino acids in organic solvents by reversed micelles provides a new method for the selective recovery, separation and concentration of bioproducts using liquid-liquid extraction techniques. Selectivity is affected by electrostatic interactions between the charged residues or moieties of the solute and the surfactant headgroups. These interactions are mediated by electrostatic screening as affected by solution ionic strength. The more hydrophobic the amino acid residue, the more favourable is the solubilisation of this residue in the partially structured water pool of the reversed micelle relative to the bulk, unstructured water phase. The l a r g e s c a l e r e c o v e r y of p o l a r b i o l o g i c a l m o l e c u l e s s u c h as enzymes and o t h e r p r o t e i n s , n u c l e i c a c i d s , p o l y p e p t i d e s and amino a c i d s has been g i v e n new emphasis i n r e c e n t years as a r e s u l t o f t h e s i g n i f i c a n t advances made i n recombinant DNA t e c h n i q u e s and g e n e t i c e n g i n e e r i n g . These advances have opened up many new v i s t a s f o r t h e p r a c t i c a l u t i l i s a t i o n o f b i o t e c h n o l o g y on an unprecedented s c a l e . The problem s t i l l r e m a i n s , however, as how b e s t t o a c h i e v e t h e r e q u i r e d s e p a r a t i o n , c o n c e n t r a t i o n and p u r i f i c a t i o n o f t h e b i o l o g i c a l p r o d u c t s on a continuous b a s i s . Many t e c h n i q u e s a r e a v a i l a b l e f o r t h i s p u r p o s e , and each has i t s n i c h e i n t h e complex arena of b i o p r o d u c t s e p a r a t i o n s . I n some cases t h e r e may be more than one s e p a r a t i o n method t h a t would work, a l t h o u g h g e n e r a l l y t h e s e methods were developed f o r bench-^scale s e p a r a t i o n s and s u f f e r from l o s s of r e s o l u t i o n on s c a l e ^ u p . Moreover they do n o t o f f e r t h e advantages o f economy of s c a l e s o f r e q u e n t l y found w i t h s e p a r a t i o n o p e r a t i o n s i n o t h e r chemically-«related i n d u s t r i e s . A s e p a r a t i o n o p e r a t i o n t h a t has n o t r e c e i v e d t h e c o n s i d e r a t i o n i t d e s e r v e s f o r b i o p r o d u c t r e c o v e r i e s i s t h a t of l i q u i d - l i q u i d e x t r a c t i o n , w h i c h c a n o f f e r b o t h moderate t o h i g h s e l e c t i v i t y , and can be o p e r a t e d on a c o n t i n u o u s , l a r g e - s c a l e b a s i s . One o f t h e p r i m a r y reasons f o r t h i s n e g l e c t i s t h e l a c k of s u i t a b l e s o l v e n t systems h a v i n g the d e s i r e d s e l e c t i v i t y and c a p a c i t y f o r t h e p r o d u c t s of i n t e r e s t . Many of t h e p r o d u c t s a r e i o n i c i n c h a r a c t e r a t t h e pH 0097-6156/87/0342-0170$06.00/0 © 1987 American Chemical Society


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c o n d i t i o n s t y p i c a l of f e r m e n t a t i o n media and t e n d t o be i n s o l u b l e and/or l a b i l e i n the o r g a n i c s o l v e n t s t r a d i t i o n a l l y used i n extraction operations. As a consequence, o r g a n i c s o l v e n t s have not been c o n s i d e r e d l i k e l y c a n d i d a t e s f o r t h e s e s e p a r a t i o n s , and the f i e l d has not r e c e i v e d the a t t e n t i o n g i v e n t o o t h e r t e c h n i q u e s s u c h as chromatography and ion^exchange. I n t h i s paper, we c o n s i d e r a new c l a s s of e x t r a c t a n t s which do not have the i n h e r e n t l i m i t a t i o n s of c o n v e n t i o n a l s o l v e n t s i n the recovery of b i o l o g i c a l p r o d u c t s f r o m t h e i r n a t i v e soups. The concepts are based on t h e o b s e r v a t i o n t h a t c e r t a i n s u r f a c t a n t s a g g r e g a t e i n a p o l a r media t o form r e v e r s e d m i c e l l e s . These m i c e l l e s g e n e r a l l y c o n s i s t of a p o l a r c o r e w h i c h i n c l u d e s the s u r f a c t a n t headgroups, s o l u b i l i s e d w a t e r , and any o t h e r s o l u b i l i s e d p o l a r m a t e r i a l s , s u r r o u n d e d by h y d r o p h o b i c s u r f a c t a n t tails p r o t r u d i n g i n t o t h e c o n t i n u o u s o r g a n i c medium e n v e l o p i n g the micelle. Under the a p p r o p r i a t e conditions, these reversed m i c e l l e - c o n t a i n i n g o r g a n i c s o l u t i o n s can e x i s t i n e q u i l i b r i u m w i t h a b u l k aqueous phase, and can be used t o e x t r a c t p o l a r m a t e r i a l s from t h i s aqueous s o l u t i o n . The m a t e r i a l s s o l u b i l i s e d i n the i n n e r c o r e s of t h e s e r e v e r s e d m i c e l l e s are g e n e r a l l y water and salts, a l t h o u g h o t h e r p o l a r s p e c i e s , i n p a r t i c u l a r p r o t e i n s and amino a c i d s , can a l s o be t a k e n up by t h e s e m i c e l l e s and t h e r e b y s o l u b * i l i s e d i n an o t h e r w i s e i n h o s p i t a b l e o r g a n i c environment. The s o l u b i l i s a t i o n of a p r o t e i n i n a r e v e r s e d m i c e l l e i s i l l u s t r a t e d s c h e m a t i c a l l y i n F i g u r e 1. I n the development of these concepts f o r b i o p r o d u c t r e c o v e r y , i t i s i m p o r t a n t t o have a good u n d e r s t a n d i n g of t h e f a c t o r s i n f l u e n c i n g t h e s e l e c t i v i t y o f t h e e x t r a c t i o n p r o c e s s f o r the s o l u t e s of interest. T h i s i s t h e t o p i c of the o v e r v i e w p r e s e n t e d h e r e , where i t w i l l be apparent t h a t e l e c t r o s t a t i c i n t e r a c t i o n s are an i m p o r t a n t f a c t o r i n t h i s s e l e c t i v i t y , but a l s o t h a t h y d r o p h o b i c i n t e r a c t i o n s can p l a y a s i g n i f i c a n t r o l e i n d e t e r m i n i n g s o l u b i l i s a t i o n b e h a v i o u r . Protein Solubilisation The a b i l i t y of p r o t e i n s t o t r a n s f e r from an aqueous s o l u t i o n t o a r e v e r s e d m i c e l l e ^ c o n t a i n i n g o r g a n i c p h a s e , and be s u b s e q u e n t l y r e c o v e r e d i n a second aqueous phase, was f i r s t e s t a b l i s h e d by the group of L u i s i ( 2 , 3 ) . I t has s i n c e been suggested by van't R i e t and Dekker (4,5) and Goklen and H a t t o n (6-9) t h a t t h i s phenomenon be e x p l o i t e d i n the recovery, s e p a r a t i o n and c o n c e n t r a t i o n of b i o p r o d u c t s from complex aqueous m i x t u r e s . I n the p a s t t h r e e y e a r s , s i g n i f i c a n t p r o g r e s s has been made i n t h i s d i r e c t i o n , and i t has been e s t a b l i s h e d t h a t t h e s e s o l v e n t s can be s e l e c t i v e i n the s e p a r a t i o n of b i n a r y and t e r n a r y p r o t e i n m i x t u r e s (7f9_) and i n the r e c o v e r y of an e x t r a c e l l u l a r a l k a l i n e p r o t e a s e from a c l a r i f i e d f e r m e n t a t i o n b r o t h (1_0). I t has a l s o been demonstrated t h a t the p r o c e s s can be o p e r a t e d on a c o n t i n u o u s b a s i s ( 5 ) . The p h y s i c o - c h e m i c a l i n t e r a c t i o n s t h a t can be e x p l o i t e d i n t h e s e l e c t i o n and o p t i m i s a t i o n of these p r o c e s s e s are d i s c u s s e d below. We w i l l r e s e r v e f o r f u t u r e communications a d e t a i l e d d i s c u s s i o n of t h e t e c h n o l o g i c a l a s p e c t s and p o t e n t i a l p r o b l e m s i n the i m p l e m e n t a t i o n of r e v e r s e d m i c e l l a r e x t r a c t i o n of b i o p r o d u c t s in l a r g e - s c a l e c o n t i n u o u s o p e r a t i o n s , t h e s e t o p i c s b e i n g beyond the scope of t h i s o v e r v i e w .

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E f f e c t of pH. The pH of the s o l u t i o n w i l l a f f e c t t h e s o l u b i l i s a t i o n c h a r a c t e r i s t i c s o f a p r o t e i n p r i m a r i l y i n t h e way i n which i t m o d i f i e s t h e charge d i s t r i b u t i o n over t h e p r o t e i n s u r f a c e . With i n c r e a s i n g pH, t h e p r o t e i n becomes l e s s p o s i t i v e l y charged u n t i l i t r e a c h e s i t s i s o e l e c t r i c p o i n t , or p o i n t of zero n e t c h a r g e , p i . A t pH*s above t h e p i the p r o t e i n w i l l take on a n e t n e g a t i v e c h a r g e . If electrostatic i n t e r a c t i o n s play a s i g n i f i c a n t r o l e i n the s o l u b i l i s a t i o n process, s o l u b i l i s a t i o n with anionic surfactants s h o u l d be p o s s i b l e o n l y a t p H s l e s s t h a n t h e p i of t h e p r o t e i n , w h e r e t h e p r o t e i n i s p o s i t i v e l y c h a r g e d and e l e c t r o s t a t i c a t t r a c t i o n s between t h e p r o t e i n and t h e s u r f a c t a n t headgroups are favourable. At pH s above t h e p i , e l e c t r o s t a t i c r e p u l s i o n s would i n h i b i t the p r o t e i n s o l u b i l i s a t i o n . The r e v e r s e t r e n d s would be a n t i c i p a t e d i n t h e case o f c a t i o n i c s u r f a c t a n t s . The e x p e c t e d t r e n d s a r e born o u t f o r t h e l o w m o l e c u l a r w e i g h t enzymes r i b o n u c l e a s e - a , cytochrome-c, and l y s o z y m e , as shown i n F i g u r e 2. These r e s u l t s a r e p r e s e n t e d as t h e p e r c e n t a g e of t h e p r o t e i n t r a n s f e r r e d from a 1 mg/ml aqueous p r o t e i n s o l u t i o n t o an e q u a l volume of i s o o c t a n e c o n t a i n i n g 50 mM of t h e a n i o n i c s u r f a c t a n t A e r o s o l 0T, o r AOT ( d i - 2 - e t h y l h e x y l sodium s u l f o s u c c i n a t e ) . As a n t i c i p a t e d , o n l y a t pH s l o w e r t h a n t h e p i was t h e r e any a p p r e c i a b l e s o l u b i l i s a t i o n of a g i v e n p r o t e i n , w h i l e above t h e p i the s o l u b i l i s a t i o n appears t o have been t o t a l l y s u p p r e s s e d . Note, however, t h a t as t h e pH was l o w e r e d even f u r t h e r , t h e r e was a drop i n t h e degree of s o l u b i l i s a t i o n of t h e p r o t e i n s . T h i s was a c c o m p a n i e d by t h e f o r m a t i o n of a p r e c i p i t a t e at t h e i n t e r f a c e between t h e two phases, a t t r i b u t e d t o a d e n a t u r a t i o n of t h e p r o t e i n . These r e s u l t s were not always found f o r l a r g e r p r o t e i n s a t t h e same l e v e l of s u r f a c t a n t l o a d i n g s . F o r the c l a s s of p r o t e i n s t r y p s i n , a l p h a - c h y m o t r y p s i n , e l a s t a s e and a l p h a - c h y m o t r y p s i n o g e n i t was f o u n d t h a t t h e pH had t o be r e d u c e d t o v a l u e s significantly below t h e p i f o r t h e r e t o be any a p p r e c i a b l e s o l u b i l i s a t i o n . Even t h e n t h e s o l u b i l i s a t i o n o c c u r r e d o n l y over a v e r y narrow pH range b e f o r e d e c r e a s i n g r a p i d l y a g a i n w i t h f u r t h e r decreases i n t h e pH of t h e a q u e o u s f e e d p h a s e , a c c o m p a n i e d by p r e c i p i t a t i o n a t t h e interface. With increased s u r f a c t a n t c o n c e n t r a t i o n s , the e a r l i e r b e h a v i o u r was r e c o v e r e d , i . e . , t h e p i a g a i n marked t h e p o i n t of t r a n s i t i o n between s i g n i f i c a n t s o l u b i l i s a t i o n and no s o l u b i l i s a t i o n of t h e p r o t e i n ( F i g u r e 3). These r e s u l t s a l l p o i n t t o t h e e l e c t r o s t a t i c i n t e r a c t i o n s between t h e s o l u t e p a r t i c l e s and t h e s u r f a c t a n t headgroups b e i n g a c o n t r o l l i n g f a c t o r i n the s o l u b i l i s a t i o n process. While t h i s i s undoubtedly t r u e , r e s u l t s p r e s e n t e d below on t h e e f f e c t s of i o n i c s t r e n g t h i n d i c a t e some more s u b t l e phenomena come i n t o p l a y , t o o . The q u e s t i o n t h a t a r i s e s i s p r e c i s e l y how i s t h e p r o t e i n p o s i t i o n e d w i t h i n the m i c e l l e . The presence of r e v e r s e d m i c e l l e s i n t h e s e studies i s undisputed. Measurements of water c o n t e n t s using K a r l - F i s h e r t i t r a t i o n , and m i c e l l e s i z e d e t e r m i n a t i o n s v i a dynamic l i g h t - s c a t t e r i n g (V1_) a n d s m a l l - a n g l e n e u t r o n s c a t t e r i n g (1_2) confirm t h e i r existence. T h i s of course does not g u a r a n t e e t h a t t h e p r o t e i n i s c o n t a i n e d w i t h i n t h e p o l a r c o r e of t h e m i c e l l e . R a t h e r , i t i s p o s s i b l e t h a t s o l u b i l i s a t i o n o c c u r s by s i m p l e i o n - p a i r i n g


f

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Fig. 2


Schematic R e p r e s e n t a t i o n of P r o t e i n Reversed M i c e l l e s .

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Solubilisation i n

E f f e c t of pH on S o l u b i l i s a t i o n of Cytochrome-c Lysozyme (D) , and R i bonuclease-a (A).

(O) ,


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b e t w e e n t h e p o s i t i v e l y charged s u r f a c e r e s i d u e s and t h e a n i o n i c s u r f a c t a n t headgroups. T h i s s c e n a r i o , however, i g n o r e s t h e f a c t t h a t over t h e pH range o f i n t e r e s t , many o f t h e s u r f a c e r e s i d u e s w i l l be n e g a t i v e l y charged, and would need t o be h y d r a t e d when t h e p r o t e i n i s p u l l e d i n t o t h e o r g a n i c phase. T h i s would p o i n t t o s o l u b i l i s a t i o n w i t h i n t h e water pool of t h e m i c e l l e . The r e s u l t s d i s c u s s e d above r e l a t e t o t h e a n i o n i c s u r f a c t a n t AOT. For c a t i o n i c s u r f a c t a n t s , a d i f f e r e n t t r e n d i s o b s e r v e d . V a n ' t R i e t ' s group have f o u n d t h a t w i t h t r i o c t y l m e t h y l ammonium c h l o r i d e as t h e s u r f a c t a n t , s i g n i f i c a n t s o l u b i l i s a t i o n of t h e enzyme a l p h a - a m y l a s e was observed over a narrow pH range o n l y , i n t h e v i c i n i t y o f 10 «10.5 (£). I n t h i s pH r a n g e , i t can be a n t i c i p a t e d t h a t a l l b a s i c r e s i d u e s w i l l be d e p r o t o n a t e d , t h e o n l y charged r e s i d u e s b e i n g t h e c a r b o x y l groups b e a r i n g a n e g a t i v e charge. These would be a v a i l a b l e f o r i o n - p a i r i n g , and indeed i t would appear t h a t t h i s i s t h e mechanism f o r t h e s o l u b i l i s a t i o n o f t h i s p r o t e i n . S i m i l a r r e s u l t s have been o b s e r v e d i n our l a b o r a t o r y , even f o r p r o t e i n s h a v i n g l o w p i ' s , s u c h as c a r b o n i c anhydrase and ribonuclease-na. Hinze has a l s o observed t h i s t r e n d (13) • Thus, i t can be argued t h a t depending on t h e t y p e of s u r f a c t a n t used, d i f f e r e n t s o l u b i l i s a t i o n mechanisms c o u l d be o p e r a t i v e . For the a n i o n i c s u r f a c t a n t AOT, i t appears t h a t mi c e l l a r s o l u b i l i s a t i o n i s o c c u r r i n g , w h i l e i o n - * p a i r i n g i s t h e mechanism f o r c a t i o n i c s u r f a c t a n t s . I t s h o u l d be emphasised, however, t h a t these g e n e r a l * i s a t i o n s a r e n o t always v a l i d . For i n s t a n c e , r e c e n t r e s u l t s on t h e s o l u b i l i s a t i o n of c a t a l a s e u s i n g t h e c a t i o n i c s u r f a c t a n t DTAB ( d o d e c y l t r i m e t h y l ammonium bromide) i n n-octane, w i t h hexanol as c o s u r f a c t a n t , i n d i c a t e d t h a t t h e s o l u b i l i s a t i o n was by r e v e r s e d m i c e l l e s and n o t by s t r i c t i o n - ^ p a i r i n g . W h i l e below t h e p i of about 5.3 t h e r e was no s o l u b i l i s a t i o n of t h i s enzyme, above t h i s pH v a l u e , s i g n i f i c a n t t r a n s f e r occurred. This r e s u l t , plus the observation t h a t t h e m i c e l l e s i z e i n c r e a s e d d r a m a t i c a l l y above t h e pH of 5.3, g i v e s t r o n g e v i d e n c e f o r t h e m i c e l l e s o l u b i l i s a t i o n mechanism b e i n g operative. From these o b s e r v a t i o n s , a c l e a r e r p i c t u r e o f t h e s o l u b i l i s a t i o n process i s b e g i n n i n g t o emerge, a l t h o u g h i t i s s t i l l not s u f f i c i e n t l y advanced t o be a p p l i e d i n a p r e d i c t i v e s e n s e .

E f f e c t of I o n i c S t r e n g t h . The e f f e c t of i o n i c s t r e n g t h i s p r i m a r i l y t o m e d i a t e t h e e l e c t r o s t a t i c i n t e r a c t i o n s between t h e p r o t e i n surface and t h e s u r f a c t a n t headgroups. The w e l l - k n o w n Debye s c r e e n i n g determines t h e e l e c t r i c a l double l a y e r p r o p e r t i e s a d j a c e n t to any c h a r g e d s u r f a c e , a n d a f f e c t s t h e r a n g e o v e r w h i c h e l e c t r o s t a t i c i n t e r a c t i o n s can overcome t h e r m a l m o t i o n o f t h e s o l u t e molecules. The c h a r a c t e r i s t i c d i s t a n c e f o r t h e s e electrostatic i n t e r a c t i o n s i s t h e Debye l e n g t h , which i s i n v e r s e l y p r o p o r t i o n a l t o the s q u a r e r o o t of t h e i o n i c s t r e n g t h ( I j p . Thus, i n c r e a s e s i n t h e i o n i c s t r e n g t h w i l l d e c r e a s e t h i s i n t e r a c t i o n d i s t a n c e , and hence i n h i b i t t h e s o l u b i l i s a t i o n of the p r o t e i n . This decreased i n t e r a c t i o n has been n e a t l y c o n f i r m e d i n t h e case of AOT r e v e r s e d m i c e l l e s i n i s o o c t a n e i n e q u i l i b r i u m w i t h s a l t s o l u t i o n s . As t h e s a l t c o n c e n t r a t i o n i n c r e a s e s , t h e r e p u l s i v e headgroup i n t e r a c t i o n s between s u r f a c t a n t s w i l l be s u p p r e s s e d , p e r m i t t i n g t h e f o r m a t i o n o f


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s m a l l e r m i c e l l e s , a t r e n d w h i c h i s e v i d e n t i n the r e s u l t s shown i n F i g u r e M. The s t r o n g l i n e a r dependence of m i c e l l e s i z e on the r e c i p r o c a l square r o o t of the i o n i c s t r e n g t h argues i n f a v o u r of the i m p o r t a n c e of Debye s c r e e n i n g i n t h e s e systems. The r e s u l t s shown i n F i g u r e 5 f u r t h e r c o n f i r m the importance of i o n i c s t r e n g t h e f f e c t s on p r o t e i n s o l u b i l s a t i o n i n r e v e r s e d m i c e l l e s . W i t h i n c r e a s i n g i o n i c s t r e n g t h , t h e r e i s a f a i r l y abrupt change i n t h e s o l u b i l i s a t i o n , o c c u r i n g a t d i f f e r e n t salt c o n c e n t r a t i o n s f o r the d i f f e r e n t p r o t e i n s . I t i s of i n t e r e s t t o note t h a t t h e o r d e r of appearance of t h e s e c u r v e s i n t h i s f i g u r e i s d i f f e r e n t f r o m t h a t o b s e r v e d f o r t h e same p r o t e i n s i n the pH v a r i a t i o n case. T h i s d e g r e e of d i s c r i m i n a t i o n b e t w e e n the s i m i l a r l y - ^ s i z e d p r o t e i n s was u n e x p e c t e d , and p o i n t s t o t h e s e n s i t i v i t y of the m i c e l l e e x t r a c t i o n process t o t h e i n d i v i d u a l s t r u c t u r a l f e a t u r e s of each p r o t e i n . At t h i s stage no d e f i n i t e c o n c l u s i o n s can be drawn as t o the p r e c i s e phenomena o p e r a t i v e i n t h i s p r o c e s s , a l t h o u g h i t can be s p e c u l a t e d t h a t i t r e l a t e s t o the s u r f a c e t o p o l o g y of t h e p r o t e i n , and i n p a r t i c u l a r t o the d i s t r i b u t i o n of charged r e s i d u e s and h y d r o p h o b i c patches over the s u r f a c e s of the p r o t e i n . The s a l t t y p e w i l l a l s o a f f e c t t h e s o l u b i l i s a t i o n of the p r o t e i n s (1J_,1_5). G o k l e n and H a t t o n (9) o b s e r v e d t h a t t h e i m p o r t a n c e of the s a l t e f f e c t s i n d e t e r m i n i n g w a t e r s o l u b i l i s a t i o n c a p a c i t i e s i n r e v e r s e d m i c e l l e s f o l l o w e d the n o w - c l a s s i c l y o t r o p i c , o r H o f m e i s t e r s e r i e s w i t h i n any g i v e n v a l e n c y group. T h i s p o i n t s t o t h e i m p o r t a n c e o f the i o n s o l v a t i o n e f f e c t s , and p o s s i b l y a l s o s p e c i f i c a d s o r p t i o n phenomena i n the S t e r n l a y e r s of t h e m i c e l l e w a l l and t h e p r o t e i n s u r f a c e (1_4) i n t h e s e s y s t e m s , a g a i n i l l u s t r a t i n g the dominance of e l e c t r o s t a t i c i n t e r a c t i o n s i n t h e s o l u b i l i s a t i o n process. I n a d d i t i o n , i t was f o u n d t h a t w i t h CaCl2 t h e range of pH's over w h i c h s i g n i f i c a n t s o l u b i l i s a t i o n occured extended, beyond the p i of the p r o t e i n , i n d i c a t i n g some s p e c i f c i o n b i n d i n g of the d i v a l e n t C a c a t i o n t o the p r o t e i n thus m o d i f y i n g i t s charge c h a r a c t e r i s t i c s and s h i f t i n g the e f f e c t i v e p i of t h e p r o t e i n to higher values. The e f f e c t of i o n i c s t r e n g t h i s f u r t h e r e v i d e n t i n t h e r e s u l t s shown i n F i g u r e 6, where the degree of c y t o c h r o m e s s o l u b i l i s a t i o n as a f u n c t i o n of pH i s g i v e n f o r two d i f f e r e n t KC1 c o n c e n t r a t i o n s . I n c r e a s i n g t h e s a l t c o n c e n t r a t i o n r e s u l t s i n a n a r r o w i n g of t h e s o l u b i l i s a t i o n peak t o the e x t e n t t h a t s o l u b i l i s a t i o n o c c u r s o n l y a t p H s s i g n i f i c a n t l y below t h e nominal p i of the p r o t e i n . T h i s c o u l d be a t t r i b u t e d t o e i t h e r a s i m p l e D e b y e s c r e e n i n g o f the e l e c t r o s t a t i c i n t e r a c t i o n s between the p r o t e i n and the s u r f a c t a n t headgroups, which i s overcome by the i n c r e a s e d net p o s i t i v e s u r f a c e c h a r g e of t h e p r o t e i n a t t h e l o w e r pH's, o r t o s p e c i f i c or n o n - s p e c i f i c c h l o r i d e i o n b i n d i n g t o the p r o t e i n , r e n d e r i n g i t more n e g a t i v e l y charged t h a n would be i n d i c a t e d by i t s p i . Whatever the r e a s o n f o r the e f f e c t of i o n i c s t r e n g t h on the s o l u b i l i s t i o n c h a r a c t e r i s t i c s f o r these p r o t e i n s , i t i s a p p a r e n t t h a t m a n i p u l a t i o n of i o n i c s t r e n g t h can be employed t o v a r y t h e pH range over w h i c h s o l u b i l i s a t i o n o c c u r s , a l l o w i n g g r e a t e r s e l e c t i v i t y i n the e x t r a c t i o n p r o c e s s . + +

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e x p l o r e d i s the p o s s i b i l i t y of enhancing s e l e c t i v i t y i n r e v e r s e d mi c e l l a r b i o s e p a r a t i o n s t h r o u g h s u i t a b l e s y n t h e s i s of s u r f a c t a n t s endowed w i t h m o l e c u l a r r e c o g n i t i o n c a p a b i l i t i e s (1_6). For i n s t a n c e , the a f f i n i t y l i g a n d s c o n v e n t i o n a l l y used i n a f f i n i t y chromatography can be m o d i f i e d t h r o u g h the attachment of a l o n g a l k y l t a i l t o the a c t i v e r e c o g n i t i o n group ( f r e q u e n t l y a s u b s t r a t e or i n h i b i t o r f o r the enzyme). The t a i l l e n g t h s h o u l d be s e l e c t e d t o ensure the a f f i n i t y s u r f a c t a n t s t r a d d l e s the reversed m i c e l l e s u r f a c t a n t s h e l l , p e r m i t t i n g the head group t o p r o t r u d e i n t o the p o l a r core where i t can i n t e r a c t s e l e c t i v e l y w i t h the p r o t e i n s t o be r e c o v e r e d , w h i l e at the same t i m e e n s u r i n g t h a t the a f f i n i t y group i s anchored w i t h i n the e x t r a c t a n t phase and i s not s t r i p p e d i n t o the aqueous phase. The b a s i c concepts are i l l u s t r a t e d s c h e m a t i c a l l y i n F i g u r e 7 . Note t h a t f o r t h i s concept t o work, t h e r e i s a need f o r o n l y one a f f i n i t y s u r f a c t a n t per m i c e l l e , on average, s i n c e i t i s r e a s o n a b l e t o assume s i n g l e occupancy f o r t h e s e p r o t e i n * i m i c e l l e complexes, and t h u s low c o n c e n t r a t i o n s of t h e s e s u r f a c t a n t s w i l l be r e q u i r e d r e l a t i v e t o t h o s e used f o r f o r m i n g the r e v e r s e d m i c e l l e s . S o l u b i l i s a t i o n of Amino A c i d s An important c l a s s of b i o l o g i c a l s i s the amino a c i d group, not o n l y because they are v a l u a b l e i n t h e i r own r i g h t as f e e d supplements, e t c . , but a l s o because t h e y a r e the s u b s t r a t e s f o r the s y n t h e s i s of di*t and o l i g o p e p t i d e s u s e d , f o r e x a m p l e , as t h e r a p e u t i c and a n a l g e s i c drugs and a r t i f i c i a l s w e e t e n e r s , and a r e the b u i l d i n g blocks f o r a l l proteins. In f a c t , i t i s t h i s l a t t e r point that makes the s t u d y of amino a c i d s o l u b i l i s a t i o n i n r e v e r s e d m i c e l l e s p a r t i c u l a r l y i n t e r e s t i n g . A q u a n t i f i c a t i o n of t h e i n t e r a c t i o n s , b o t h e l e c t r o s t a t i c and h y d r o p h o b i c , between the w e l l - c h a r a c t e r i s e d amino a c i d s and t h e r e v e r s e d m i c e l l e p o l a r c o r e s s h o u l d provide a d d i t i o n a l i n s i g h t i n t o the importance of t h e s e phenomena i n p r o t e i n s o l u b i l i s a t i o n experiments. Because of t h e i r m o l e c u l a r s t r u c t u r e and i o n i s a b l e groups, a t p h y s i o l o g i c a l pH c o n d i t i o n s they a r e not r e a d i l y e x t r a c t e d i n t o o r g a n i c s o l v e n t s u s i n g c o n v e n t i o n a l ion**** p a i r i n g or c h e m i c a l c o m p l e x a t i o n t e c h n i q u e s as f o u n d i n e.g. the hydrometallurgical industries. The g e n e r a l m o l e c u l a r s t r u c t u r e of amino a c i d s i s i l l u s t r a t e d schematically i n Figure 8. I t i s the presence of b o t h the amino g r o u p ( - N H 2 ) and t h e c a r b o x y l g r o u p (->COOH) a t t a c h e d t o the alpha-icarbon t h a t imparts t o t h e amino a c i d s many of their i n t e r e s t i n g c h a r a c t e r i s t i c s . I n a d d i t i o n t o t h e s e g r o u p s , the r e s i d u e , o r " R e g r o u p , " a t t a c h e d t o t h e a l p h a ^ c a r b o n i s what d i s t i n g u i s h e s one amino a c i d from a n o t h e r , and i s r e s p o n s i b l e f o r the unique p h y s i c a l p r o p e r t i e s of each s p e c i e s . These m o i e t i e s can be e i t h e r c a t i o n i c o r a n i o n i c , o r can be n e u t r a l l y p o l a r or n o n p o l a r , d e p e n d i n g on t h e s p e c i e s c o n s i d e r e d (1_7). I t i s of i n t e r e s t t o note t h a t under p h y s i o l o g i c a l c o n d i t i o n s , b o t h the amino and t h e c a r b o x y l groups w i l l be i o n i s e d , so t h a t , f o r n o n i o n i c r e s i d u e s , the amino a c i d s w i l l be e l e c t r i c a l l y n e u t r a l . I t i s t h i s z w i t t e r i o n i c c h a r a c t e r t h a t makes f o r d i f f i c u l t s e p a r a t i o n s by e x t r a c t i o n methods. For the charged r e s i d u e s the problem becomes more a c u t e . S o l u b i l i z a t i o n of amino a c i d s i n AOT/isooctane r e v e r s e d m i c e l l e



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s o l u t i o n s has been i n v e s t i g a t e d i n d e t a i l by T h i e n et a l . ( 1 8 ) . E x t r a c t i o n c u r v e s are shown i n F i g u r e 9 f o r a c a t i o n i c and a n e u t r a l a r o m a t i c amino a c i d . Superimposed on the graphs are the t i t r a t i o n curves c a l c u l a t e d f o r t h e i n d i v i d u a l s p e c i e s . It is readily apparent from t h e s e d a t a t h a t the s o l u b i l i s a t i o n i s p o s s i b l e o n l y when the amino a c i d s bear a net p o s i t i v e c h a r g e , and t h a t the degree of s o l u b i l i s a t i o n i s governed by the value of t h i s net p o s i t i v e charge. For n e g a t i v e l y charged s p e c i e s , s o l u b i l i s a t i o n i s suppressed. These r e s u l t s s u g g e s t t h a t the s o l u b i l i s a t i o n i s governed p r i m a r i l y by e l e c t r o s t a t i c i n t e r a c t i o n s between the a n i o n i c s u r f a c t a n t h e a d g r o u p s and t h e p o s i t i v e l y c h a r g e d amino a c i d moieties. I t i s of i n t e r e s t t o note t h a t a t low pH c o n d i t i o n s the p r o t o n a t e d amino group p a r t i c i p a t e s i n the electrostatic i n t e r a c t i o n s , but t h a t at h i g h e r pH's, where the c a r b o x y l group i s d e p r o t o n a t e d and b e a r s a n e g a t i v e charge, t h i s i n t e r a c t i o n i s n e u t r a l i s e d by the z w i t t e r i o n i c n a t u r e of the a l p h a ^ c a r b o n group. T h i e n et a l . ( 1 j 8 ) a l s o showed t h a t the degree of s o l u b i l i s a t i o n d e p e n d s on t h e r e l a t i v e h y d r o p h o b i c i t i e s of t h e amino a c i d residues. A measure of t h i s e f f e c t i s t h e s l o p e of the s o l u b i l i s a t i o n c u r v e when r e p l o t t e d as a f u n c t i o n of the net s o l u t e c h a r g e , as shown i n F i g u r e 1 0 f o r a r g i n i n e . I t was f o u n d t h a t t h i s s l o p e c o r r e l a t e d w e l l w i t h the h y d r o p h o b i c i t y s c a l e proposed by B u l l and Breese ( 1 _ 9 ) , as i s e v i d e n t from F i g u r e 1 1 . I t i s i n t r i g u i n g to n o t e t h a t t h e more h y d r o p h o b i c t h e r e s i d u e t h e g r e a t e r the s o l u b i l i s a t i o n of the amino a c i d . T h i s c o u l d be due t o one of t h r e e e f f e c t s , as d i s c u s s e d below. The f i r s t p o s s i b i l i t y i s t h a t the s o l u b i l i s a t i o n o c c u r s by s i m p l e i o n ^ p a i r i n g between the p r o t o n a t e d amino g r o u p s and the s u r f a c t a n t s . W h i l e t h i s cannot be r u l e d out at low pH, i t i s not t h e o p e r a t i v e mechanism at t h e i n t e r m e d i a t e pH's, where the d e p r o t o n a t e d c a r b o x y l groups f o r the charged p o l a r amino a c i d s must be h y d r a t e d and c o n s e q u e n t l y t h e s o l u t e must be s o l u b i l i s e d w i t h i n the p o l a r c o r e of the r e v e r s e d m i c e l l e i t s e l f . I n a d d i t i o n , dynamic l i g h t s c a t t e r i n g s t u d i e s and K a r l f l F i s c h e r water t i t r a t i o n s do not i n d i c a t e the s i g n i f i c a n t decrease i n water s o l u b i l i s a t i o n t h a t would be e x p e c t e d i f t h e s u r f a c t a n t were t i e d up i n i o n p a i r f o r m a t i o n and were no l o n g e r a v a i l a b l e f o r m i c e l l e f o r m a t i o n . A second s c e n a r i o i s t h a t t h e n o n p o l a r r e s i d u e of the amino a c i d i s l o c a t e d w i t h i n t h e s u r f a c t a n t s h e l l , p e r m i t t i n g the s t r o n g i n t e r a c t i o n between the p r o t o n a t e d amino group and t h e s u r f a c t a n t head r e q u i r e d f o r the s o l u b i l i s a t i o n of the s o l u t e . Again, t h i s c o u l d not be the case f o r the c h a r g e d p o l a r amino a c i d s . In a d d i t i o n , such an e f f e c t i s not c o n s i s t e n t w i t h the K a r l - F i s c h e r t i t r a t i o n s and l i g h t s c a t t e r i n g e x p e r i m e n t s , w h i c h i n d i c a t e an unchanged apparent s u r f a c t a n t head coverage on i n c o r p o r a t i o n of the s o l u t e w i t h i n the m i c e l l e s . An i n c r e a s e i n t h i s v a l u e would be e x p e c t e d i f the s o l u t e were t o occupy p a r t of the i n t e r f a c i a l s h e l l of t h e r e v e r s e d m i c e l l e . Moreover, t h e r e i s o t h e r e v i d e n c e t h a t i n normal m i c e l l e s , a r o m a t i c s o l u t e s such as benzene and n a p h t h a l e n e a r e not t a k e n up i n the a p o l a r h y d r o c a r b o n c o r e , but a r e r a t h e r l o c a t e d i n t h e aqueous p h a s e n e a r t h e s u r f a c t a n t h e a d g r o u p s (20,21_). T h i s s u p p o r t s the c o n t e n t i o n t h a t the n e u t r a l a r o m a t i c amino a c i d s such as p h e n y l a l a n i n e and t r y p t o p h a n are s o l u b i l i s e d t o t a l l y w i t h i n t h e water pools of t h e r e v e r s e d m i c e l l e s .


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T h e s e o b s e r v a t i o n s beg t h e q u e s t i o n : why do t h e h y d r o p h o b i c s o l u t e s f a v o u r t h e p o l a r cores of t h e r e v e r s e d m i c e l l e s more t h a n do the p o l a r amino a c i d s ? T h i s e f f e c t would seem t o be due t o t h e unusual p r o p e r t i e s of t h e w a t e r w i t h i n t h e r e v e r s e d m i c e l l e s , w h i c h i s known t o be more s t r u c t u r e d t h a n water i n t h e b u l k aqueous phase (22) . There i s thus a d i s t i n c t e n t r o p i c advantage i n removing t h e h y d r o p h o b i c r e s i d u e from t h e b u l k aqueous phase, where i t induces e n t r o p i c a l l y d i s f a v o u r e d s t r u c t u r a l changes i n t h i s aqueous phase, and p o s i t i o n i n g i t w i t h i n t h e a l r e a d y p a r t i a l l y - s t r u c t u r e d water w i t h i n t h e m i c e l l e p o o l . The g a i n i n e n t r o p y i n such cases would be g r e a t e r f o r t h e nonpolar r e s i d u e s than f o r t h e p o l a r m o i e t i e s , w i t h t h e r e s u l t t h a t t h e former would be s o l u b i l i s e d t o a g r e a t e r e x t e n t t h a n t h e l a t t e r , as has been o b s e r v e d e x p e r i m e n t a l l y . This i s e s s e n t i a l l y a m a n i f e s t a t i o n of t h e well-known h y d r o p h o b i c e f f e c t (23) . These r e s u l t s on amino a c i d s o l u b i l i s a t i o n i n r e v e r s e d m i c e l l e s o l u t i o n s have i n d i c a t e d c l e a r l y t h a t s u c h systems c o u l d be u s e f u l f o r t h e r e c o v e r y , s e p a r a t i o n and c o n c e n t r a t i o n o f s m a l l , charged b i o l o g i c a l m o l e c u l e s from aqueous media. F u r t h e r m o r e , t h e y have shed some l i g h t on t h e r o l e t h a t h y d r o p h o b i c i n t e r a c t i o n s w i l l p l a y i n t h e s o l u b i l i s a t i o n of more complex m o l e c u l e s s u c h as p r o t e i n s , which have a d i s t r i b u t i o n o f p o l a r and nonpolar amino a c i d r e s i d u e s over t h e i r s u r f a c e s . Conclusion The use of r e v e r s e d m i c e l l e s i n t h e s e l e c t i v e r e c o v e r y and c o n c e n t r a t i o n o f low and h i g h m o l e c u l a r weight b i o p r o d u c t s f r o m d i l u t e aqueous streams appears t o be a p r o m i s i n g new avenue f o r i n n o v a t i v e r e s e a r c h and a p p l i c a t i o n s . To d a t e , i t has been shown t h a t e l e c t r o s t a t i c i n t e r a c t i o n s between t h e charged s o l u t e r e s i d u e s and t h e s u r f a c t a n t headgroups, as w e l l as h y d r o p h o b i c e f f e c t s , can p l a y a s i g n i f i c a n t r o l e i n determining the s e l e c t i v i t y of t h i s p r o c e s s f o r one p r o t e i n over a n o t h e r . Moreover, t h e r e appears t o be some l a t i t u d e i n t h e s e l e c t i o n o f s u r f a c t a n t s and cos u r f act ants t h a t enables enhancements i n s e l e c t i v i t y t o be made over and above t h o s e already inherent i n the process. W h i l e some u n d e r s t a n d i n g of the f a c t o r s r e s p o n s i b l e f o r t h e s e l e c t i v i t y of t h e e x t r a c t i o n process has been g a i n e d , t h e f i e l d i s s t i l l i n i t s i n f a n c y , and t h e c h a l l e n g e i s t o d e l v e f u r t h e r i n t o t h e s u b t l e t i e s of m i c e l l a r s o l u b i l i s a t i o n t o o b t a i n a more c o m p l e t e m o l e c u l a r l e v e l d e s c r i p t i o n o f the e l e c t r o s t a t i c and h y d r o p h o b i c interactions leading to the cooperative formation of t h e s o l u t e ^ s o l u b i l i s a t e complexes. I n t h i s c o n t e x t , an a r e a t h a t has r e c e i v e d no a t t e n t i o n t o d a t e i s t h e m e c h a n i s t i c d e s c r i p t i o n of t h e i n t e r f a c i a l t r a n s p o r t of t h e s o l u t e s , and o n l y f r a g m e n t a r y d a t a a r e a v a i l a b l e on mass t r a n s f e r r a t e s i n t h e s e systems. Such i n f o r m a t i o n w i l l c e r t a i n l y be r e q u i r e d as t h e t e c h n i q u e p r o g r e s s e s from t h e exploratory stage t o f i n a l a p p l i c a t i o n s i n l a r g e s c a l e production operations. I n c o n c l u s i o n , i t can be s t a t e d t h a t r e v e r s e d m i c e l l e s p r e s e n t an a t t r a c t i v e a l t e r n a t i v e t o c o n v e n t i o n a l b i o s e p a r a t i o n methods, but t h a t much fundamental and a p p l i e d r e s e a r c h i s r e q u i r e d b e f o r e t h i s p o t e n t i a l can be f u l l y r e a l i z e d . The i n t e l l e c t u a l , and p o s s i b l y

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economic, rewards t o be g a i n e d i n t h e p u r s u i t of t h e s e g o a l s a r e adequate j u s t i f i c a t i o n f o r a c o n c e r t e d e f f o r t i n t h i s a r e a .


Acknowledgments I am i n d e b t e d t o p a s t and present graduate s t u d e n t s Kent Goklen and Mike T h i e n , r e s p e c t i v e l y , and u n d e r g r a d u a t e s K a r e n L e e a n d K i m Thompson, f o r p r o d u c i n g most of the r e s u l t s d i s c u s s e d i n t h i s o v e r v i e w . I have a l s o drawn on some u n p u b l i s h e d r e s u l t s by graduate s t u d e n t s Andy Bommarius and Reza Rahaman, and I thank them f o r t h e i r contributions. T h i s work was s u p p o r t e d by t h e NSF B i o t e c h n o l o g y P r o c e s s E n g i n e e r i n g C e n t r e a t MIT, t h e W.R.Grace Company, A l f a L a v a l , and a N e s t l e / W e s t r e c o F e l l o w s h i p G r a n t .

Literature Cited 1. Fendler, J. H. "Membrane Mimetic Chemistry"; Wiley: New York, 1982. 2. Luisi, P. L.; Bonner, F. J.; Pelligrini, A.; Wiget, P.; Wolf R. Helv. Chim. Acta 1979, 62, 740-53. 3. Wolf, R. Ph.D. Thesis ETH#7027, Swiss Fed. Inst. Tech., Zurich, 1982. 4. Van't Riet, K . ; Dekker, M. Proc. 3rd Eur.Cong. Biotechn., Vol. III, 1984, p.541. 5. Dekker, M.; Van't Riet, K.; Weijers, S. R.; Baltussen, J. W. A.; Bijsterbosch, B.; Laane, C. Biochem. Eng. J. 1987, in press. 6. Goklen, K. E.; Hatton, T. A. Biotech. Prog. 1985, 1, 69-74. 7. Goklen, K. E.; Hatton, T. A. Proc. ISEC '86, Vol. III, 1986, p.587. 8. Goklen, K. E.; Hatton, T. A. Sep. Sci. Technol. 1987, in press. 9. Goklen, K. E.; Hatton, T. A. Biotech. Bioeng. 1987, submitted. 10. Rahaman, R. S.; Cabral, J . ; Hatton, T.A. 1987, in preparation. 11. Goklen, K. E. Ph.D. Thesis, M.I.T., Cambridge MA, 1986. 12. Sheu, E . ; G o k l e n , K. E.; H a t t o n , T . A . ; Chen, S.-H. Biotech. Prog. 1986, 2, 175-86. 13. Hinze, W. L., personal communication. 14. Hiemenz, P. C. "Principles of Colloid and Surface Chemistry"; Marcel Dekker: New York, 1977; p.369. 15. Meier, P.; Imre, E.; Fleschar, M.; Luisi, P. L. In "Surfactants in Solution"; Mittal, K. L.; Lindman, B., Eds.; Plenum Press: New York, 1984. 16. Woll, J.; Hatton, T. A., work in progress. 17. Rawn, J. D. "Biochemistry"; Harper and Row: New York, 1983. 18. Thien, M. P.; Lee, K. K.; Thompson, K.; Hatton, T. A. 1987, in preparation. 19. Bull, H. B.; Breese, K. Acta Biochem and Biophysics 1974, 161, 665-70. 20. Eriksen, J. C. Acta Chem. 1963, 17. 21. Murkherjee, P . ; C a r d i n a l , J . R.; Desai, N. R. In "Micellization, Solubilization and Microemulsions"; Mittal, K. L., Ed.; Plenum Press: New York, 1976. 22. Wong, M.; Thomas, J. K.; Gratzel, M. JACS 1976, 98, 2391-7. 23. Tanford, C. R. "The Hydrophobic Effect"; Wiley: New York, 1980. RECEIVED May 11, 1987

Ordered Media in Chemical Separations - American Chemical Society

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