Polyelectrolytic Behavior of Ionic Polysaccharides - American

23 Polyelectrolytic Behavior of Ionic Polysaccharides V. CRESCENZI, M. DENTINI, and R. RIZZO

Downloaded by UNIV OF SYDNEY on March 21, 2013 | http://pubs.acs.org Publication Date: April 21, 1981 | doi: 10.1021/bk-1981-0150.ch023

Istituto di Chimica-Fisica, Università di Roma, Roma, Italy

An important aim of physico-chemical studies on natural and synthetic ionic-polysaccharides is to help elucidate the correlations between chemical structure-conformational characteristics of such polymers and their equilibrium properties in solution. In this context, attention is being given in our laboratory to the thermodynamics of macroion-counterion interactions and of polyion-polyion interactions, including [soluble] complex formation between polysaccharide macroions and different polyampholites (e.g. proteins) in dilute aqueous solution. Our experimental approach is mainly based on the use of micro_ calorimetric, potentiometric, and chirooptical techniques. Polymers considered include: 1) sulfated polysaccharides (i-carrageenan, dextran sulfate); 2) glycosaminoglycans (heparins); 3) microbial polysaccharides (Xanthan, PS-10). Species listed above (sodium salts) have been characterized in water and/or aqueous NaCl solution in terms of Na counterion activity coefficient, heat of dilution, and heat of Cu2 ions binding. These experiments are part of a systematic investigation on the relationship between "charge-density" along polyelectrolyte chains and (metal) ion-binding, comparing experimental evidence with existing theories (1,2,3). Nicrocalorimetric and spectroscopic data have then been collected on the interaction of human serum albumin with dextran sulfate and heparin, respectively, in dilute aqueous solution. The purpose is to afford possible, original evidence on the energetics of complex formation between glycosaminoglycans and different proteins of the biological fluids. Finally, the enthalpy of protonation as well as the circular dichroism of Xanthan have been studied in a rather wide range of pH values in water and in aqueous NaCl. This was done in order further characterize from a "structural" and thermodynamic standpoint the dissociation behavior of such a conformationally pecu+

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0097-6156/81/0150-0331$05.00/0 © 1981 American Chemical Society In Solution Properties of Polysaccharides; Brant, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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l i a r p o l y c a r b o x y l i c a c i d (_4, 5_) . F o r c o m p a r i s o n p u r p o s e s , t h e same e x p e r i m e n t s have been done u s i n g a n o t h e r m i c r o b i a l p o l y s a c c h a r i d e , PS-10 (6_, 7), whose c h a i n s a r e supposed t o l a c k any c o n formational order. Work s i now i n p r o g r e s s t o i n v e s t i g a t e s o l u t i o n p r o p e r t i e s o f t h e K l e b s i e l l a e K-63 p o l y s a c c h a r i d e (J3). We w i s h t o b r i e f l y summarize h e r e a f e w o f t h e r e s u l t s o b t a i ned so f a r i n o u r l a b o r a t o r y f r o m work on t h e t h r e e main r e s e a r c h l i n e s e x e m p l i f i e d above. R e s u l t s and D i s c u s s i o n I) S u l f a t e d polysaccharides

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a) Heat o f d i l u t i o n and o f c o u n t e r i o n b i n d i n g ( N a , C u ) . The a c t i v i t y c o e f f i c i e n t o f N a c o u n t e r i o n s ( }>Na ) f o r i - c a r r a geenan, d e x t r a n s u l f a t e , and h e p a r i n i n w a t e r a t 25°C, a s e v a l u a t e d f r o m o u r p o t e n t i o m e t r i c d a t a , a r e r e p o r t e d i n F i g . 1. Although t h e c o n c e n t r a t i o n range i n v e s t i g a t e d i s r a t h e r l i m i t e d , one o b s e r v e s t h a t / N a * i s low, and q u i t e i n s e n s i t i v e t o d i l u t i o n , t h e more so t h e h i g h e r t h e c h a r g e - d e n s i t y a l o n g t h e c h a i n s . These r e s u l t s a r e i n l i n e w i t h a number o f s i m i l a r d a t a c o l l e c t e d by v a r i o u s a u t h o r s (_9, _1Q_) : t h e agreement i s however o n l y q u a l i t a tiven,when a comparison i s f e a s i b l e a t a l l , given the l a c k o f s i m i l a r i t y among d i f f e r e n t s a m p l e s used i n d i f f e r e n t l a b o r a t o r i e s . A more i m p o r t a n t p r o p e r t y u s e f u l f o r t h e t h e r m o d y n a m i c c h a r a c t e r i z a t i o n o f aqueous p o l y e l e c t r o l y t e s o l u t i o n s i s t h e e n thalpy o f d i l u t i o n . Our m i c r o c a l o r i m e t r i c r e s u l t s (25°C) a r e r e p o r t e d i n F i g . 2 f o r d e x t r a n s u l f a t e , and i n F i g . 3 f o r segmented i - c a r r a g e e n a n and f o r h e p a r i n , r e s p e c t i v e l y . I t i s seen t h a t , f o r a g i v e n d i l u t i o n r a n g e : 1) t h e i n t e g r a l heat o f d i l u t i o n ( A H Q : c a l / p o l y e l . e q u i v . ) i s higher the lower t h e charge d e n s i t y along t h e c h a i n s ; 2 ) d i l u t i o n i n t h e p r e s e n c e o f NaCl Cat a f i x e d N a C l m o l a r i t y ) l e a d s t o d i s t i n c t l y s m a l l e r heat e f f e c t s t h a n i n w a t e r . To o u r knowledge no c a l o r i m e t r i c d a t a o f c o m p a r a b l e a c c u r a c y on i o n i c p o l y s a c c h a r i d e s o l u t i o n s c a n be f o u n d i n t h e l i t e r a t u r e , w i t h t h e n o t a b l e e x c e p t i o n o f t h e r e s u l t s r e c e n t l y r e p o r t e d by R. L. C l e l a n d [V\J on t h e e n t h a l p y o f m i x i n g g l y c o s a m i n o g l y c a n s with aqueous N a C l . A d d i t i o n a l e v i d e n c e on t h e complex i n t e r p l a y o f f a c t o r s w h i c h govern p o l y s a c c h a r i d e - c o u n t e r i o n i n t e r a c t i o n s i s c l e a r y a f f o r d e d by t h e c a l o r i m e t r i c d a t a on t h e b i n d i n g o f C u ions reported i n F i g . 4. Here t h e " c h a r g e - d e n s i t y " o f t h e c h a i n s i s n o t t h e o n l y dominant p a r a m e t e r a s , q u i t e o b v i o u s l y , a l s o t h e n a t u r e and r e l a t i v e c o n f i g u r a t i o n o f f i x e d - c h a r g e s ( h e p a r i n ) and t h e p r o p e n s i t y o f t h e c h a i n s t o assume o r d e r e d c o n f o r m a t i o n s (i-carrageenan) c o n t r i b u t e i m p o r t a n t l y t o the observed o v e r a l l e n e r g e t i c e f f e c t s upon C u - b i n d i n g . I n t h e case o f i-carrageenan t h e p e c u l i a r shape o f t h e Q a g a i n s t [ C u ] /N p l o t ( s e e F i g . 4; Q B m i x i n g p o l y e l e c t r o l y t e a n d Cu(N03)2 s o l u t i o n s , c o r r e c t e d f o r d i l u t i o n e f f e c t s ; Ν = p o l y e l e c t r o l y t e concentration i n equiv./I) should +

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Figure 1. Activity coefficient of Na counterions (γ +) of ionic polysaccharides in water (25° C), Ν is the polyelectrolyte concentration in equivalents/liter: (A) isocarrageenan (ξ = 1.2); (+) heparin (£ = 1.3); ( A ) dextran sulfate. (M = 4 · 10 ; Να

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Figure 2. Enthalpy of dilution of dextran sulfate (M = 4 · 10 ) at 25°C: (+) in water; (Φ) in 1 · 10 M NaCl; (A) in 3 · 10' M NaCl; (O) in 5 · 10~ M NaCl; (A) in 1 · 10 M NaCl. 3

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F/gwre 3. Enthalpy of dilution of iso-carrageenan segments and of heparin at 25°C: (A) iso-carrageenan in water; (Φ) iso-carrageenan in 1 · 10 M NaCl; (+) iso-carrageenan in 5 · 10 M NaCl; (A) heparin in water.


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i n f a c t be t r a c e d back t o a d i s o r d e r order t r a n s i t i o n , as c l e a r l y s u p p o r t e d by o p t i c a l a c t i v i t y measurements (J_2) . C r i t i c a l c o m p a r i s o n o f o u r d a t a ( F i g s 1-4) w i t h c u r r e n t t h e o r i e s o f p o l y e l e c t r o l y t e s o l u t i o n s would e n t a i l a r a t h e r l e n g t h y p r e s e n t a t i o n and d i s c u s s i o n o f a numer o f e q u a t i o n s and u n d e r l y ing assumptions. F o r sake o f b r e v i t y l e t us s i m p l y p o i n t o u t here t h a t , c o n s i d e r i n g M a n n i n g ' s t h e o r y (13_, _1_4), we f i n d o n l y a q u a l i t a t i v e , r o u g h agreement w i t h o u r e x p e r i m e n t a l r e s u l t s . In p a r t i c u l a r , t h e h e a t o f d i l u t i o n d a t a i n aqueous N a C l ( F i g s 2 and 3) seem "anomalous" w i t h r e s p e c t t o t h e o r e t i c a l p r e d i c t i o n s ( Y\_). A d e t a i l e d d i s c u s s i o n on t h e s e p o i n t s w i t h t h e p r e s e n t a t i o n o f d a t a a l s o f o r d i f f e r e n t p o l y e l e c t r o l y t e s w i l l be g i v e n e l s e w h e r e (_1_5) . b) M i c r o c a l o r i m e t r i c and s p e c t r o s c o p i c d a t a on i o n i c p o l y s a c c h a r i d e - p r o t e i n i n t e r a c t i o n s . The a s s o c i a t i o n between d i f f e r e n t c h a r g e d p o l y m e r s i s a w e l l known phenomenon w i t h i m p o r t a n t i m p l i c a t i o n s i n t h e c a s e o f many b i o l o g i c a l p r o c e s s e s . For i n s t a n c e , the e x t e n s i v e a s s o c i a t i o n o f h e p a r i n w i t h c e r t a i n plasma p r o t e i n s i s c u r r e n t l y t h o u g h t t o be t h e b a s i s f o r t h e b i o l o g i c a l a c t i v i t y o f t h i s g l y c o s a m m i n o g l y c a n (1_6). The i n t e r a c t i o n between i o n i c p o l y s a c c h a r i d e s and a number o f ~ ~ p r o t e i n s l e a d i n g t o s o l u b l e complex f o r m a t i o n has been c h a r a c t e r i z e d i n v i t r o u s i n g d i f f e r e n t e x p e r i m e n t a l t e c h n i q u e s ( 1 7 ) , b u t much r e m a i n s t o be u n d e r s t o o d a b o u t e n e r g e t i c a s p e c t s a s w e l l a s s p e c i f i c mechanisms a t t h e molecular l e v e l . I n t h i s f i e l d , o u r r e s e a r c h program i n c l u d e s s t u d i e s o f t h e c o m p l e x a t i o n p r o c e s s e s between i o n i c - p o l y s a c c h a r i d e s , i n p a r t i c u l a r m u c o p o l y s a c c h a r i d e s , and s e l e c t e d p r o t e i n s i n aqueous s o l u t i o n by means o f m i c r o c a l o r i m e t r i c and s p e c t r o s c o p i c experiments. A few r e s u l t s o f the l a t t e r experiments concerning t h e d i l u t e aqueous s y s t e m s : d e x t r a n s u l f a t e - h u m a n serum a l b u m i n (NaDS-HSA), and h e p a r i n - h u m a n serum a l b u m i n (Hep-HSA), r e s p e c t i v e l y , a r e r e p o r t e d i n F i g s . 5 a , and 5_b. S p e c t r a l d a t a were c o l l e c t e d f o r t h e above s y s t e m s ( a t pH = 5.0; i o n i c s t r e n g t h 0.1 N) u s i n g two d i f f e r e n t o p t i c a l p r o b e s , namely: f l u o r e s c e i n e and rhodamine-B. These d y e s do n o t i n t e r a c t ( f l u o r e s c e i n e ) o r i n t e r act o n l y very weakly (rhod-B) w i t h the i o n i c - p o l y s a c c h a r i d e s considered. For the p a r t i c u l a r experimental c o n d i t i o n s used, f l u o r e s c e i ne may be c o n s i d e r e d as e x t e n s i v e l y bound on a s i n g l e , p r i m a r y b i n d i n g s i t e o f HSA (JJ3, 1_9) i n t h e a b s e n c e o f e x t r a n e o u s p o l y electrolytes . A d d i t i o n o f NaDS (lï = 4 . 1 0 ) p e r t u r b s t h e f l u o r e s c e i n e s p e c t r u m i n t h e d i r e c t i o n o f an e x t e n s i v e d i s p l a c e m e n t o f dye m o l e c u l e s f r o m t h e p r o t e i n w i t h an a p p a r e n t " e q u i v a l e n c e - p o i n t " c o r r e s p o n d i n g t o a s t o i c h i o m e t r i c N / [ H S A ] r a t i o c l o s e t o 15, a c c o r d i n g t o a b s o r p t i o n a n d f l u o r e s c e n c e d a t a (see F i g . 5a_) . A t pH = 6.5, on t h e c o n t r a r y , any e f f e c t was b a r e l y d e t e c t a b l e . This i s i n l i n e w i t h e x t e n s i v e complex f o r m a t i o n between NaDS a n d HSA d r i v e n e s s e n t i a l l y by e l e c t r o s t a t i c f o r c e s , w h i c h w o u l d engage many o f t h e f i x e d p o s i t i v e c h a r g e s o f HSA, p r o b a b l y i n c l u d i n g most o f 4


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Figure 5b.

Δ Α data for the heparin-HSA system. Same experimental conditions as in Figure 5a: (+c)fluoresceine;(Φ) rhodamine-B.


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those a t t h e f l u o r e s c e i n e primary b i n d i n g - s i t e . Quite opposite i s t h e b e h a v i o r o f r h o d a m i n e - B . Data o f F i g . 5a show i n f a c t t h a t t h i s dye i s o n l y v e r y w e a k l y bound t o f r e e HSA m o l e c u l e s ( u n d e r o u r e x p e r i m e n t a l c o n d i t i o n s ) b u t t h a t a d d i t i o n o f NaDS w o u l d p r o mote i t s b i n d i n g . The " e q u i v a l e n c e - p o i n t " f o r NaDS o f Π = 4 . 1 0 i s i n t h i s c a s e f o u n d a t an N/ £HSA] r a t i o a p p r o x i m a t e l y 3 0 . I n v i e w o f t h e z w i t t e r i o n i c n a t u r e o f rhodamine-B a p o s s i b l e e x p l a n a t i o n o f t h i s e f f e c t may be t h a t i n t e r a c t i o n o f NaDS w i t h HSA b r i n g s p a i r s o f p o s i t i v e and n e g a t i v e f i x e d c h a r g e s i n t o s u f f i c i e n t l y c l o s e p r o x i m i t y w i t h t h e onset o f " b i n d i n d - s i t e s " f o r rhodamine. I t i si n t e r e s t i n g t o point out that the "equivalencep o i n t " f o r NaDS w i t h Π = Θ . 1 0 i s c e r t a i n l y l o w e r t h a n t h a t f o r t h e h i g h e r M s a m p l e , as shown i n F i g . 5 a . D a t a c o l l e c t e d u s i n g h e p a r i n i n s t e a d o f NaDS b u t f o r o t h e r w i s e i d e n t i c a l c o n d i t i o n a r e g i v e n i n F i g . 5b. The t r e n d o f t h e a b s o r p t i o n d a t a a r e q u i t e s i m i l a r t o t h o s e f o u n d w i t h NaDS: a p p a r e n t " e q u i v a l e n c e - p o i n t s " a r e i n t h i s c a s e d i s c e r n i b l e a t N/ [JHSA] v a l u e s a r o u n d 20 ( f l u o r e s c e i n e ) and 25 ( r h o d . B ) . These i n t e r e s t i n g f e a t u r e s d e s e r v e f u r t h e r i n v e s t i g a t i o n . Here we l i m i t o u r s e l v e s t o deduce f r o m t h e d a t a o f F i g s 5a and 5b t h a t f o r a N/ [HSA] r a t i o o f about 3 0 , w i t h [HSA] ca 1 0 ~ M, t h e m a j o r i t y o f HSA m o l e c u l e s s h o u l d be bound by NaDS o r by h e p a r i n c h a i n s . Ini t i a l l y , more t h a n one p r o t e i n m o l e c u l e w o u l d t h u s be bound p e r polysaccharide chain. I t has t o be p o i n t e d o u t however t h a t t h e s p e c t r o s c o p i c " e q u i v a l e n t - p o i n t s " c o r r e s p o n d t o N/JHSAJ v a l u e s m i n i m a l i n o r d e r t o n e a r l y c a n c e l ( f l u o r e s c e i n e ) o r t o f u l l y deve lop (rhodamine) s p e c t r a l p e r t u r b a t i o n s o f probe chromophores, f o r our working c o n d i t i o n s . 4


3

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F u r t h e r a d d i t i o n o f p o l y s a c c h a r i d e s may i n f a c t l e a d t o f u r t h e r " i n t e r a c t i o n s " w i t h HSA no l o n g e r d e t e c t a b l e by t h e dyes e m p l o y e d . T h i s , among o t h e r t h i n g s , i s b o r n o u t by t h e c a l o r i m e t r i c e x p e r i m e n t s w h i c h i n d i c a t e t h a t a t pH = 6 t h e r e i s l i t t l e p o l y s a c c h a r i d e - H S A i n t e r a c t i o n ( i n agreement w i t h s p e c t r a l o b s e r v a t i o n ) w h i l e a t pH = 5.0 t h e A H ( i n K c a l p e r mole o f HSA, c o r r e c ted f o r d i l u t i o n e f f e c t s ) values p r o g r e s s i v e l y increase with i n c r e a s i n g p o l y s a c c h a r i d e c o n c e n t r a t i o n and a p p e a r t o f i n a l l y r e a c h a plateau (Fig. 6). Evidently, 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 mainly r e s p o n s i b l e f o r the observed e f f e c t s . I n t h e c a s e o f NaDS w i t h Π = 4 . 1 0 one c a n e s t i m a t e f r o m t h e c a l o r i m e t r i c d a t a an a p p a r e n t " e q u i v a l e n c e - p o i n t " i n f a i r a g r e e ment w i t h t h e s p e c t r o s c o p i c r e s u l t s ( N / [JHSA] c a . 3 0 ) . The i n f l u e n c e o f p o l y s a c c h a r i d e average molecular weight i s a l s o evident f r o m d a t a o f F i g . 6, a l o w e r Ν p r o d u c i n g a l o w e r i n g and s m e a r i n g out o f t h e AHC v a l u e s . W i t h NaDS M = 8 . 1 0 we f i n d i n f a c t , a t h i g h N/ [HSA] v a l u e s , a HC p r a c t i c a l l y c o i n c i d e n t w i t h t h a t f o r h e p a r i n b u t n e a r l y h a l f o f t h a t f o r NaDS w i t h M = 4 . 1 0 ( b u t a l ways w i t h DS = 2 ) . M o r e o v e r f o r theNaDS w i t h M = Θ . 1 0 and f o r h e p a r i n no e q u i v a l e n c e - p o i n t may be e s t i m a t e d f r o m F i g . 6. The p o l y d i s p e r s i t y o f o u r NaDS s a m p l e s , o f unknown m o l e c u l a r weight d i s t r i b u t i o n s , precludes q u a n t i t a t i v e i n t e r p r e t a t i o n o f C

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these i n t e r e s t i n g o b s e r v a t i o n s . A p o s s i b l e q u a l i t a t i v e explana t i o n may however be t h a t a t v e r y low N/ £ H S A ] v a l u e s t h e p r o t e i n b i n d i n g p r o c e s s t a k e s p l a c e w i t h p r a c t i c a l l y i d e n t i c a l mechanisms, i n d e p e n d e n t o f NaDS m o l e c u l a r w e i g h t . T h i s i s i n agreement w i t h b o t h s p e c t r o s c o p i c and c a l o r i m e t r i c e x p e r i m e n t a l e v i d e n c e . F o r h i g h e r N/ [HSA] v a l u e s , i . e . n e a r t o 20 ( s e e F i g . 5a_), d i f f e r e n c e s i n t h e " b i n d i n g - s i t e s " f o r HSA a l o n g s h o r t c h a i n s and l o n g e r p o l y s a c c h a r i d e c h a i n s become e v i d e n t . One may assume t h a t w i t h t h e l o n g e r c h a i n s more f i x e d c h a r g e s may be engaged i n t h e b i n d i n g o f e a c h a l b u m i n g l o b u l a r m o l e c u l e ( a t h i g h N/ [jHSA] v a l u e s ) so t h a t more heat i s e v o l v e d ( F i g . 6) and more o p p o r t u n i t y i s g i v e n t o rhodamine t o p a r t i c i p a t e i n t h e c o m p l e x a t i o n ( F i g . 5a_) . The same q u a l i t a t i v e r e a s o n i n g m i g h t a p p l y a s w e l l t o t h e Hep-HSA c a s e (PI o f h e p a r i n c a . 104 ) w h i c h , t o a f i r s t a p p r o x i m a t i o n , may r e s e m b l e t h e HSA-NaDS ΙΊ = 8 . 1 0 c a s e . I t has t o be r e c a l l e d t h a t t h e c a l o r i m e t r i c e x p e r i m e n t s were p e r f o r m e d u s i n g HSA s o l u t i o n s n e a r l y t e n t i m e s more c o n c e n t r a t e d t h a n i n t h e s p e c t r o scopic experiments. F i n a l l y , c o n c e r n i n g t h e i n f l u e n c e of_NaDS mo l e c u l a r w e i g h t , i t must be s a i d t h a t u s i n g a sample w i t h Π = 5 . 1 0 3

5

(DS

= 2) p r o t e i n p r e c i p i t a t i o n o c c u r r e d . F i n d i n g s r e p o r t e d here deserve f u r t h e r study inasmuch as t h e s y s t e m s c o n s i d e r e d and t h e o r i g i n a l e x p e r i m e n t a l a p p r o a c h e s used may y i e l d v a l u a b l e " r e f e r e n c e " i n f o r m a t i o n f o r s i m i l a r i n v e s t i g a t i o n s on p o l y s a c c h a r i d e - p r o t e i n s s y s t e m s i n w h i c h s p e c i f i c i n t e r a c t i o n s do p l a y an i m p o r t a n t r o l e ( e . g . h e p a r i n - a n t i t h r o m b i n I I I ) . II) Natural carboxylated

polysaccharides

a) D i s s o c i a t i o n e q u i l i b r i a o f X a n t h a n and PS-10. P r i o r t o a d i s c u s s i o n o f d a t a c o n c e r n i n g t h e p r o t o n a t i o n e q u i l i b r i a , i t may be o f i n t e r e s t t o r e p o r t b r i e f l y a few r e s u l t s on t h e e n t h a l p y o f d i l u t i o n and on t h e e n t h a l p y o f C u i o n b i n d i n g f o r Xanthan and PS-10 i n d i l u t e aqueous s o l u t i o n . F i r s t o f a l l i t must be p o i n t e d out t h a t w h i l e f o r Xanthan t h e c h e m i c a l s t r u c t u r e and c o n f o r m a t i o n a l f e a t u r e s have been a l r e a d y e l u c i d a t e d a n d / o r t h r o u g h l y i n v e s t i g a t e d , _no s i m i l a r i n f o r m a t i o n i s a v a i l a b l e f o r PS-10. F o r t h e l a t t e r e x o c e l l u l a r p o l y s a c c h a r i d e , i n f a c t , i t a p p e a r s t h a t , so f a r , c h e m i c a l c o m p o s i t i o n (_6, 7) o n l y i s known ( g l u e : g a l a c t : g l u c A : f u c o s e = 6:4:3:2, and an 0 - a c e t y l c o n t e n t o f 4 . 5 % ) . Never t h e l e s s , we d e c i d e d t o use PS-10 i n o u r s t u d i e s e s s e n t i a l l y a s a r e f e r e n c e p o l y s a c c h a r i d e compound d e v o i d o f c h a i n c o n f o r m a t i o n a l o r d e r a t any pH o r i o n i c s t r e n g t h , a s opposed t o t h e c a s e o f Xan than . F o r t h e s e two n a t u r a l c a r b o x y l a t e d p o l y s a c c h a r i d e s w h i c h have e q u i v a l e n t w e i g h t s : 633 ( X a n t a h n ) and 854 ( P S - 1 0 ) , t h e heat o f d i l u t i o n d a t a a r e g i v e n i n F i g . 7. I t once more a p p e a r s t h a t Δ Η ρ i s a l i n e a r f u n c t i o n o f logN, a t l e a s t i n t h e l i m i t e d range o f Ν values s t u d i e d , as experienced w i t h the s u l f a t e d p o l y s a c c h a r i d e s ( F i g s 2 and 3: l o w Ν v a l u e s ) . H e r e , however, c o r r e l a t i o n o f t h i s f e a t u r e w i t h t h e o r i e s based on l i n e - c h a r g e models i s o u t o f t h e 2 +


SOLUTION PROPERTIES O F POLYSACCHARIDES


340

Figure 6. Calorimetric data on the interaction of HSA with dextran sulfate (*,+) and with heparin (A) in aqueous solution (25°C), pH = 5.04, acetate buffer (7 · 10 M), albumin concentration (constant) = 1.45 · 10 mol/L. (jf) dextran sul fate, M = 4 · 10 ; (%) dextran sulfate, Ή = 8 · 10 ; (A) heparin. 2

4

4

3

Figure 7. Enthalpy of dilution of xanthan and PS-10 (25°C): (A) PS-10 in water; (ic) xanthan in 5 · 10 M NaCl; (A) PS-10 in 1 · 10' M NaCl; (O) PS-10 in 5 · 10- M NaCl. 2

2

2


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q u e s t i o n i n view o f the complex, branched s t r u c t u r e o f Xanthan. I n o t h e r words, t h e l i n e a r i t y o f c a l o r i m e t r i c p l o t s s u c h a s t h o s e o f F i g . 7 ( d a t a i n w a t e r and i n aqueous N a C l , a t 25 C) i s n o t , i n our o p i n i o n , a m a n i f e s t a t i o n o f t h e " p o l y e l e c t r o l y t i c " n a t u r e o f species considered. On t h e o t h e r hand d a t a r e p o r t e d i n F i g . 8 i n d i c a t e t h a t t h e h i g h e r c h a r g e - d e n s i t y o f X a n t h a n compared t o PS-10 shows up i n a h i g h e r heat o f C u i o n b i n d i n g and i n a l o w e r a c t i v i t y c o e f f i cient o f Na counterions. From t h e i n i t i a l s l o p e o f t h e Q a g a i n s t [ C u ] /N p l o t s o f F i g . 8 one e s t i m a t e s f o r X a n t h a n i n water a d i f f e r e n t i a l enthalpy o f C u b i n d i n g o f about 1 K c a l per mole o f c o p p e r bound. I n c o n c l u s i o n , a s f o u n d w i t h t h e s u l f a t e d p o l y s a c c h a r i d e s ( F i g . 4) a s w e l l a s w i t h d i f f e r e n t s y n t h e t i c p o l y c a r b o x y l a t e s (2Π, c h e l a t i o n o f C u i o n s by p o l y a n i o n s i s a p r o c e s s s y s t e m a t i c a l l y d r i v e n by t h e e n t r o p y . 2 +

+

B

2 4

2 +


2 +

Passing t o the p r o t o n a t i o n e q u i l i b r i u m experiments, our c a l o r i m e t r i c r e s u l t s a r e r e p o r t e d i n F i g . 9. I t i s e v i d e n t t h a t i n t h e case o f Xanthan i n water the e n t h a l p y o f p r o t o n a t i o n i s r a t h e r anomalous. L e a v i n g a s i d e p o s s i b l e a g g r e g a t i o n Τ—» disaggregation phenomena w h i c h , i n o u r e x p e r i e n c e , may r e n d e r t r o u b l e s o m e a l l p h y s i u o - c h e m i c a l measurements on aqueous X a n t h a n but w h i c h s h o u l d be m i n i m i z e d f o r o u r t h e r m a l l y t r e a t e d s o l u t i o n s (2£) (see a l s o E x p e r i m e n t a l P a r t ) , we p r o p o s e t h a t t h e anomalous e n t h a l p y t r e n d may be a s c r i b e d t o a c o n f o r m a t i o n a l , i s o t h e r m a l change o f X a n t h a n chains. This hypothesis appears t o g a i n support from the c i r c u l a r d i c h r o i s m d a t a i l l u s t r a t e d i n F i g s 10 and 11 f o r PS-10 and X a n t h a n respectively. F o r PS-10 t h e change o f t h e CD s p e c t r u m w i t h pH e s s e n t i a l l y r e f l e c t s t h e u n d i s s o c i a t e d T"*" d i s s o c i a t e d e q u i l i b r i u m o f c a r b o x y l g r o u p s o f g l u c A r e s i d u e s (21_, _22) . F o r X a n t h a n t h e dependence o f t h e CD s p e c t r a on pH i s more c o m p l i c a t e d b e c a u s e d i f f e r e n t chromophores (23) p a r t i c i p a t e i n t h e above s a i d e q u i l i b r i u m : one c a n , n e v e r t h e l e s s , d i s t i n g u i s h peculiar v s . pH f e a t u r e s i n c o r r e s p o n d e n c e w i t h pH v a l u e s f o r which anomalies are a l s o d e t e c t a b l e i n the c a l o r i m e t r i c p l o t s (Fig. 8]. I n 0.1 N a C l (25°) a l l a n o m a l i e s a r e c a n c e l l e d o u t , and t h e o r d e r e d c o n f o r m a t i o n assumed by X a n t h a n i n t h i s m i l i e u (_4, 5_) would t h u s r e s i s t any pH p e r t u r b a t i o n . Experimental The segmented i - c a r r a g e e n a n sample was a K i n d g i f t o f Dr. S. Reid, U n i l e v e r Research Laboratory (England). The i n t a c t i - c a r r a _ geenan sample was a SIGNA-4 (USA) p r o d u c t , p u r i f i e d and c h a r a c t e r i z e d i n t h e CERNAV l a b o r a t o r y o f t h e U n i v e r s i t y o f G r e n o b l e (France). Dextran s u l f a t e o f Π = 5.10 , 4.10 and 8.10 were PHARMACIA (Sweden) s a m p l e s , w h i c h have been p u r i f i e d by d i a l y s i s . E l e m e n t a l a n a l y s i s o f t h e above p o l y s a c c h a r i d e s (Na s a l t s ) and p o t e n t i o m e t r i c t i t r a t i o n s c a r r i e d out a f t e r c o n v e r s i o n t o t h e 5

4

3


SOLUTION

PROPERTIES

OF

POLYSACCHARIDES


342

2+

Figure 8. Calorimetric data on Cu binding by xanthan and PS-10 in aqueous solutions (25°C): (A) xanthan in 1 · 10' M NaCl; (+) PS-10 in water; (Φ) PS-10 in 1 - 10 M NaCl. In the insert y + for (+) PS-10 (in water) and (A) xanthan (in water). 2

2

Na



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Figure 9. Enthalpy of protonation data for xanthan and PS-10 (25 °C). Polymer concentration = 5 · 10' equiv/L. (O) PS-10 in water; (A) PS-10 in 0.1M NaCl; (if) xanthan in 0.1M NaCl; (Φ, A) xanthan (degraded, undegraded) in water. 3

Figure 10. CD spectra of PS-10 in aque ous solution at different pHs. Polymer concentration 3 . 10 monomol/L. The [®] in deg cm /dmol (mole of repeating units, assuming 2562 as the average unit's weight): (a) acid form; (b) sodium salt. In the insert: pH dependence of molecu lar ellipticity of PS-10 at 206 nm in water (O)andinO.lMNaCl (V). 3

2

200

210

220

230 240

(nm)


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PROPERTIES

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Polysaccharides

f r e e a c i d s by i o n exchange have y i e l d e d t h e f o l l o w i n g e q u i v a l e n t w e i g h t d a t a : 1) i - c a r r a g e e n a n segments, 270; 2) i n t a c t i - c a r r a g e e nan, 270; 3 ] d e x t r a n s u l f a t e , 219, t a k i n g o f c o u r s e i n t o a c c o u n t the d i f f e r e n t w a t e r c o n t e n t s o f t h e s a m p l e s . These v a l u e s c o r r e s p o n d t o a d e g r e e o f s u b s t i t u t i o n o f 2.0 f o r d e x t r a n s u l f a t e (number o f s u l f a t e g r o u p s p e r g l u c o s e r e s i d u e ) and o f 1.7 f o r i - c a r r a g e e n a n segments ( a v e r a g e number o f s u l f a t e g r o u p s p e r two s u g a r r e s i d u e s i n t h e c h a i n ) . The h e p a r i n used was a p u r i f i e d sample f r o m P r o f . B. Casu l a boratory ( M i l a n ) , with the f o l l o w i n g c h a r a c t e r i s t i c s : b i o l o g i c a l a c t i v i t y = 157 u.; e q u i v a l e n t w e i g h t = 168; r a t i o [ C O O H ] t_S0 ]= = 1/2.3. F o r t h e so c a l l e d c h a r g e d e n s i t y p a r a m e t e r ^ = e / D k T b where b i s t h e d i s t a n c e between n e i g h b o r i n g c h a r g e s on t h e macroi o n s , e the e l e c t r o n i c charge, D the d i e l e c t r i c c o n s t a n t o f the s o l v e n t , and k i s t h e B o l t z m a n n ' s c o n s t a n t , one can t h e r e f o r e c a l c u l a t e t h e f o l l o w i n g v a l u e s : 1.2 ( i - c a r r a g e e n a n ) ; 1 . 3 ( h e p a r i n ) ; 2.8 ( d e x t r a n s u l f a t e ) . Two X a n t h a n s a m p l e s have been u s e d ; one was a p u r i f i e d , c e l l u l a s e - d e g r a d e d , (lï = 2 . 1 0 ) sample f r o m P r o f . R i n a u d o l a b o r a t o r y ( G r e n o b l e ) ; t h e o t h e r was a K e l c o p r o d u c t , p u r i f i e d a c c o r d i n g t o the l i t e r a t u r e ( 2 4 ) . F o r b o t h samples t h e e q u i v a l e n t w e i g h t (by NaGH t i t r a t i o n o f t h e a c i d f o r m s ) was 633. S o l u t i o n s o f Xanthan were h e a t e d a t 80 f o r 10-15 h r s p r i o r t o measurements: s u c h a t r e a t m e n t l e a v e s unchanged t h e e q u i v . w e i g h t but e f f i c i e n t l y r e d u ces pplymer a g g r e g a t i o n . PS-10 was a K e l c o p r o d u c t . I t was s o n i c a t e d t o r e d u c e t h e v i s c o s i t y , p u r i f i e d by c e n t r i f u g a t i o n and p r e c i p i t a t i o n f r o m aqueous NaCl-EDTA s o l u t i o n w i t h i s o p r o p y l a l c o h o l . The p o l y m e r was r e d i s s o l v e d i n w a t e r and d i a l y z e d e x h a u s t i v e l y a g a i n s t r e d i s t i l l e d w a t e r a t 5 C, l y o p h i l i z e d and r e d i s s o l v e d a s r e q u i r e d . The e q u i v a l e n t w e i g h t (854) was d e t e r m i n e d by NaOH t i t r a t i o n o f the a c i d f o r m a s w e l l a s by f l a m e ( N a ) p h o t o m e t r y . Human serum a l b u m i n was a Sigma p r o d u c t ( l o t No. A - 9 5 1 1 ) . F l u o r e s c e i n e ( s o d i u m s a l t ) was p u r c h a s e d f r o m C. E r b a ( I t a l y ) and Rhodamine-B f r o m N e r c k (W. Germany): b o t h were used w i t h o u t further ourification. C o p p e r n i t r a t e s o l u t i o n s were p r e p a r e d u s i n g a p u r e Cu ( N O g ^ 3 H 0 (C. E r b a ) s a m p l e : t h e t i t r e o f t h e s o l u t i o n s was c o n t r o l l e d by t i t r a t i o n o f H N O 3 l i b e r a t e d upon p a s s a g e t h r o u g h an i o n - e x c h a n ge c o l u m n . A l l s o l u t i o n s were p r e p a r e d u s i n g d e i o n i z e d , t w i c e - d i s t i l l e d water. S p e c t r a l measurements were c a r r i e d o u t w i t h a C a r y - 2 1 9 s p e c t r o p h o t o m e t e r ; c i r c u l a r d i c h r o i s m measurements w i t h a Cary-61 dichrograph. The t e m p e r a t u r e o f t h e c e l l s was i n a l l c a s e s c o n t r o l l e d (+_ 0.1°C) by c i r c u l a t i n g w a t e r t h r o u g h an u l t r a t h e r m o s t a t (Haake). C a l o r i m e t r i c measurements were p e r f o r m e d u s i n g a LKB 10070b a t c h m i c r o c a l o r i m e t e r a t 25°C f o l l o w i n g a p r o c e d u r e s i m i l a r t o t h a t a l r e a d y d e s c r i b e d e l s e w h e r e ( 3 ). The r e s u l t s o f t h e d i l u t i o n e x p e r i m e n t s a r e r e p o r t e d i n F i g s . 2, 3 , and 7 a s i n t e g r a l

/

3


2

5

+

2


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heats o f d i l u t i o n Δ Η Ρ ( c a l o r i e s per equivalent o f p o l y e l e c t r o l y t e ) a g a i n s t logN p l o t s , where Ν i s t h e p o l y e l e c t r o l y t e c o n c e n t r a t i o n in equiv/1. The r e s u l t s o f t h e C u i o n b i n d i n g e x p e r i m e n t s a r e c o l l e c t e d i n F i g s 4 and 8 where t h e measured h e a t s Qg ( c a l o r i e s per e q u i v a l e n t o f p o l y e l e c t r o l y t e , a f t e r c o r r e c t i o n f o r heats o f d i l u t i o n ) a r e p l o t t e d a g a i n s t t h e [ C U ] /N r a t i o . P o t e n t i o m e t r i c measurements f o r t h e e v a l u a t i o n o f N a a c t i v i t y c o e f f i c i e n t s were c a r r i e d o u t u s i n g a O r i o n 801 A n a l y s e r w i t h a Orion Na-electrode i n conjunction with a reference calomel elec t r o d e and a w a t e r j a c k e t e d t i t r a t i o n c e l l . 2+

2 +


+

Acknowledgements : T h i s work h a s been c a r r i e d o u t w i t h f i n a n c i a l s u p p o r t o f t h e I t a l i a n C.N.R. The a s s i s t a n c e o f D r . G. P a r a d o s s i and o f N r . L. P i e t r e l l i i n p e r f o r m i n g a number o f e x p e r i m e n t s i s acknowledged.

Literature Cited 1. Crescenzi V . ; "Thermochemistry of Polyelectrolyte Solutions", Charged and Reactive Polymers, Ed. E. Selegny, D. Reidel Publ. Co., Holland-USA 1974, pgs. 115-135. 2. Crescenzi V . , Delben F . , Quadrifoglio F . , Dolar D., J . Phys. Chem., 1973, 77, 539. 3. Paoletti S., Delben F . , Crescenzi V. J . Phys Chem., 1976, 2564. 4. Rinaudo M., Milas M. Biopolymers, 1978,

17,

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5. Southwick J . G . , McDonnell M . E . , Jamieson Α.M., and Blackwell J . Macromolecules, 1979, 12, 305. 6. Kang K . S . , Veeder G . T . , Richey D.D. "Extracellular Microbial Polysaccharides", Am. Chem. Soc. Symp. 45. Eds. P.A. Sandford, A. Laskin, Am. Chem. Soc., Washington, D . C . , 1977, p.211-2119. 7. Sandford P . Α . : "Exocellular, Microbial Polysaccharides" Adv. Carb. Chem. Biochem., 1979, 36, 297. 8. Joseleau J . P . , Marais M.F. Carb. Res, 1979, 77, 9. Milas M., Rinaudo M. Carb. Res., 1979,

76,

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189.

10. Pass G . , Phillips G.O., Wadlock D . J . , Morley R.G. "Interaction of Sulfated Polysaccharides with counterions". chaot. 17 of: Carbohydrate Sulfates, R.G. Schweiger E d . , Am. Chem. Soc. Symp. 1878, 77.


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11. Cleland R.L. Biopolymers, 1979, 18, 2673. 12. Crescenzi V . , Dentini M., Paradossi G . , and Rizzo R. Polymers B u l l . , 1979, 1, 777. 13. Manning G.S.; J. Chem. Phys., 1969,

51,

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14. Boyd G . E . , Wilson D.P. J . Phys. Chem. 1976, 80, 805 15. Delben F., Paoletti S., and Crescenzi V . : in preparation.


16. Casu B.; Pharm. Res Commun., 1979,

11, 1.

17. Ledward D.A.: "Protein-polysaccharide Interactions" Polysac charides in Food. J.M.V. Blandshard, and J.R. Mitchell Eds. Butterworths, 1979, p. 205-217. 18. Manzini G . , Ciana Α., and Crescenzi V. Biophys. Chem., 1979, 10, 389. 19. Manzini G . , Crescenzi V. Biophys. Chem., 1979,

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