Interactions of Counteroins with Pectins Studied by Potentiometry and

6 Interactions of Counteroins with Pectins Studied by Potentiometry and Circular Dichroism 1

J. F. Thibault and M . Rinaudo

Downloaded by UNIV OF ARIZONA on January 6, 2013 | http://pubs.acs.org Publication Date: June 5, 1986 | doi: 10.1021/bk-1986-0310.ch006

Centre de Recherches sur les Macromolécules Végétales, B.P. 68, 38402 Saint Martin D'heres Cedex, France

The aim of this study was to investigate the binding of counterions in dilute salt-free solutions of pectins with different levels and patterns of esterification. The influence of the charge parameter and of the distribution pattern of carboxyl groups on the interaction polyanion-counterion (sodium or calcium) were studied by the determination of calcium and sodium activity coefficients which were compared to theoretical values. Very low values of calcium activity coefficients (0.085) were obtained for the most charged pectin-sample. These results were completed by circular dichroism spectroscopy in order to have local informations on the conformational changes during the neutralization of pectinic acids with sodium or calcium hydroxides. The results suggest that three different conformations exist for the low-methoxyl pectins in acidic form, sodium and calcium forms. The i n t e r a c t i o n s o f p e c t i n s w i t h mono- o r d i v a l e n t c o u n t e r i o n s a r e o f a g r e a t importance i n many f i e l d s . I t i s well-known t h a t t h e s e p o l y e l e c t r o l y t e s p l a y an i m p o r t a n t r o l e as n a t u r a l ion-exchangers i n p l a n t p h y s i o l o g y , p h y t o p a t h o l o g y , n u t r i t i o n (1-2) and t h e i r a b i l i t y t o form g e l s w i t h c a l c i u m i o n s i s w i d e l y used by f o o d t e c h n o l o gists (3). The strong cooperative (4) c a l c i u m b i n d i n g o f low-methoxyl p e c t i n s may be e x p l a i n e d by t h e s t e r e o c h e m i c a l f e a t u r e o f t h e 1,4l i n k e d monomeric u n i t s (5) l e a d i n g t o t h e f o r m a t i o n o f p o l a r c a v i t i e s which c a n be o c c u p i e d by c a l c i u m i o n s , a s w e l l a s by 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 polymer which c a n be i d e a l i z e d by t h e j u x t a p o s i t i o n o f " h a i r y r e g i o n s " where t h e n e u t r a l sugar s i d e - c h a i n s a r e c o n c e n t r a t e d and o f "smooth" r e g i o n s o f u n s u b s t i t u t e d homogalacturonans ( 6 , 7 ) . Rees and co-workers (5,8,9) s t u d i e d t h e c a l c i u m b i n d i n g m a i n l y by e q u i l i b r i u m d i a l y s i s and c i r c u l a r d i c h r o i s m on g e l s . The i n t e r 1 Current address: Laboratoire de Biochimie et Technologie des Glucides, Institut National de la Recherche Agronomique, rue de la Géraudière, 44072 Nantes, France. 0097-6156/86/0310-0061 $06.00/0 © 1986 American Chemical Society

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

62

CHEMISTRY AND

FUNCTION OF PECTINS

a c t i o n s between c a l c i u m c o u n t e r i o n s and c a r b o x y l groups were d e s c r i bed by the "egg-box" model and a two s t a g e s p r o c e s s was p o s t u l a t e d : an i n i t i a l d i m e r i z a t i o n and subsequent a g g r e g a t i o n o f t h e s e preformed "egg-boxes". The i n t e r m o l e c u l a r b i n d i n g was c o n f i r m e d by Kohn and Luknar (10) and by Ravanat and Rinaudo (11) by measuring c a l c i u m a c t i v i t y c o e f f i c i e n t s on c a l c i u m p e c t a t e s o l u t i o n s . Our aim was t o i n v e s t i g a t e the b i n d i n g o f c o u n t e r i o n s i n d i l u t e s a l t - f r e e s o l u t i o n s o f p e c t i n s w i t h d i f f e r e n t l e v e l s and p a t t e r n s o f e s t e r i f i c a t i o n . We used methods such as p o t e n t i o m e t r y and c i r c u l a r d i c h r o i s m i n o r d e r t o o b t a i n b o t h g e n e r a l and l o c a l i n f o r m a t i o n s on the c o n f o r m a t i o n a l s t a t e s o f the polymer depending on i t s i o n i c form.


M a t e r i a l and Methods Pectin

samples

The s t a r t i n g p e c t i n was a commercial a p p l e p e c t i n ( R a p i d - s e t p e c t i n , from U n i p e c t i n e , Redon, F r a n c e ) . I t was p u r i f i e d by p r e c i p i t a t i o n w i t h c u p r i c i o n s ( 1 2 ) . A p e c t i n sample w i t h a h i g h degree o f e s t e r i f i c a t i o n was a l s o used i n t h i s study ( 1 3 ) . Alkaline

deesterification

P r e p a r a t i o n s o f p e c t i n w i t h v a r i o u s degrees o f e s t e r i f i c a t i o n were o b t a i n e d by p a r t i a l a l k a l i n e s a p o n i f i c a t i o n . Aqueous s o l u t i o n s o f the i n i t i a l p e c t i n were c o o l e d a t 2°C i n an i c e - s a l t b a t h and the pH was a d j u s t e d t o 7 w i t h NaOH 0.1N. Then amounts o f sodium h y d r o x i d e c o r r e s p o n d i n g t o the amount o f methoxyl groups t h a t s h o u l d be s a p o n i f i e d were added. A f t e r two days a t 2°C, the pH o f t h e s o l u t i o n was brought t o 4.5 w i t h HC1 IN. P e c t i n s were then p r e c i p i t a t e d w i t h 80% e t h a n o l , e x t e n s i v e l y washed w i t h 60% e t h a n o l and d r i e d under vacuum a t a temperature l e s s than 40°C. Enzymic

deesterification

A p e c t i n e s t e r a s e (E.C.3.1.1.11) from orange p e e l was p u r c h a s e d from Sigma. P e c t i n was d i s s o l v e d i n a 4 χ 10~ M c i t r a t e - p h o s p h a t e b u f f e r at pH 6 c o n t a i n i n g 0.1M N a C l . The amount o f enzyme and the r e a c t i o n time, a t 30°C, were chosen i n o r d e r t o o b t a i n t h e d e s i r e d degree o f e s t e r i f i c a t i o n . The r e a c t i o n was s t o p p e d by b r i n g i n g the pH v a l u e down t o 4.5 and the enzyme was i n a c t i v a t e d by h e a t i n g a t 100°C the r e a c t i o n m i x t u r e f o r 10 min. The samples were r e c o v e r e d as above. C h a r a c t e r i z a t i o n o f the

samples

The g a l a c t u r o n i c a c i d c o n t e n t as w e l l as the degree o f e s t e r i f i c a t i o n (DE) were measured by c o n d u c t i m e t r y and the n e u t r a l s u g a r c o n t e n t was determined by gas chromatography, as d e s c r i b e d elsewhere (14,15). L i g h t s c a t t e r i n g experiments were c a r r i e d out f o r an a n g l e o f 90° at 25.0 ± 0.1°C on p e c t i n s o l u t i o n s a f t e r f i l t r a t i o n through 0.45μπι Millipore filters.


6.

THIBAULT AND

RINAUDO

Interactions of Counterions with Pectins

63

Potentiometry The calcium and sodium a c t i v i t y c o e f f i c i e n t s were d e t e r m i n e d a t 25.0 ± 0.1°C w i t h an O r i o n e l e c t r o d e (model 92-32) and a Radiometer e l e c t r o d e (model G502 Na), r e s p e c t i v e l y . A s a t u r a t e d c a l o m e l e l e c t r o de was used as the r e f e r e n c e . C a l i b r a t i o n c u r v e s were o b t a i n e d u s i n g CaCl o r NaCl s o l u t i o n s b e f o r e and a f t e r each measurement. The C a C l ^ and NaCl c o n c e n t r a t i o n s were measured by p o t e n t i o m e t r i c d e t e r m i n a t i o n s o f the c h l o r i d e s w i t h s i l v e r n i t r a t e and w i t h a s i l v e r e l e c trode.


Circular

dichroism

A Dichro V spectropolarimeter (Jobin-Yvon, F r a n c e ) was used a t 25°C. S o l u t i o n s o f p e c t i n s c o n t a i n i n g 1 mequiv o f c a r b o x y l groups/1 were put i n a 1 mm p a t h l e n g t h c e l l . An i n t e g r a t i o n time o f 0.2 sec was used and 10-15 scans (0.2 nm/sec, 185-250 nm) were accumulate J. The d i f f e r e n t i a l e x t i n c t i o n c o e f f i c i e n t (Δε) was e x p r e s s e d i n cm" . equiv .1. O t h e r methods The a c i d i c form o f the p e c t i n ( p e c t i n i c a c i d s ) were o b t a i n e d by p e r c o l a t i o n through a s t r o n g H exchanger column ( A m b e r l i t e IRN 77). S a l t s ( p e c t i n a t e s ) o f t h e s e p e c t i n i c a c i d s were o b t a i n e d by e x a c t n e u t r a l i z a t i o n w i t h the d e s i r e d c a r b o n a t e - f r e e hydroxide. +

R e s u l t s and

discussion

C h a r a c t e r i s t i c s o f the

samples

The i n i t i a l p e c t i n was p r e c i p i t a t e d by c u p r i c i o n s i n o r d e r t o remove the n e u t r a l p o l y s a c c h a r i d e s which a r e n o t l i n k e d t o the p e c t i c back bone. Ion-exchange chromatography on DEAE-Sepharose CL-6B showed t h a t o n l y 0.5% o f the i n i t i a l m a t e r i a l was n o t bound t o the g e l , i n d i c a t i n g a p u r i t y o f 99.5%. The p u r i f i e d p e c t i n s have a g a l a c t u r o nic a c i d c o n t e n t o f 78.5%, a n e u t r a l sugar c o n t e n t o f ^ 1 0 % and a degree o f e s t e r i f i c a t i o n o f 72.1%. Samples o f p e c t i n s w i t h v a r y i n g degrees o f e s t e r i f i c a t i o n (060%) were o b t a i n e d from t h i s p r e p a r a t i o n by d e e s t e r i f i c a t i o n w i t h a l k a l i and w i t h enzyme. The main c h a r a c t e r i s t i c s o f t h e s e samples a r e i n d i c a t e d i n T a b l e I . I t i s l i k e l y t h a t the a c t i o n o f a l k a l i l e a d s t o p e c t i c m o l e c u l e s i n which f r e e and methyl e s t e r i f i e d c a r b o x y l i c a c i d s a r e randomly d i s t r i b u t e d ( 1 6 ) . The temperature was chosen i n o r d e r t o m i n i m i z e c h a i n - c l e a v a g e by 3 - e l i m i n a t i o n (17) and i n t r i n sic v i s c o s i t y measurements o f the samples (sodium form i n 0.1 M NaCl) showed t h a t the d e g r a d a t i o n was minimal ( 1 5 ) . Other p e c t i n s w i t h degree o f e s t e r i f i c a t i o n o f about 10, 30 and 40% ( T a b l e I) were o b t a i n e d by enzymic d e e s t e r i f i c a t i o n o f the i n i t i a l p r e p a r a t i o n . An orange p e c t i n e s t e r a s e was used and the pH o f the r e a c t i o n was 6 i n o r d e r t o a v o i d c o n c u r r e n t base-catalyzed s a p o n i f i c a t i o n . The a c t i o n o f p l a n t p e c t i n e s t e r a s e i s known (16-18) to r e s u l t i n a blockwise arrangement o f f r e e c a r b o x y l groups i n the p e c t i c m o l e c u l e s . The enzymic p r e p a r a t i o n does n o t c o n t a i n depolymerase a c t i v i t i e s as shown by the c o n s t a n c y o f the intrinsic


64

CHEMISTRY AND

FUNCTION OF PECTINS

v i s c o s i t y v a l u e s . S i n c e the s t a r t i n g sample was 72% esterified, i t i s l i k e l y t h a t a f t e r the enzymic d e e s t e r i f i c a t i o n , the samples c o n t a i n some amounts o f randomly d i s t r i b u t e d c a r b o x y l g r o u p s . They a r e c o n s i d e r e d n e v e r t h e l e s s as r e p r e s e n t a t i v e o f a b l o c k w i s e d i s t r i bution. The charge parameter λ i s a l s o i n d i c a t e d i n the T a b l e I . T h i s fundamental parameter (19) g o v e r n s the b e h a v i o r o f the p o l y e l e c t r o l y t e p o l y m e r s . I t s v a l u e i s g i v e n a t 25°C by : e


λ

2

= bDkT

X

( 1

D

"

E

/

1

0

0

)

=

1.61 (1 - DE/100)

(1)

where e i s the e l e c t r o n charge, kT theι Boltzmann term , b the l e n g t h o f the monomeric u n i t and 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 . The b v a l u e was taken as 4. 35 A ( 2 0 ) . The c a l c u l a t i o n o f λ n e g l e c t s the s m a l l c o n t e n t ( < 1%, w/w) o f L-Rhamnose which i s p r e s e n t i n the p o l y g a l a c t u r o n a t e backbone. Table

I . C h a r a c t e r i s t i c s o f the p e c t i n s

DE

Alkali-deesterified 1 2 3 4 5 6 7 8 9

a

AG

samples 91.4 90.7 84.6 83.6 80.7 81.3 79.1 78.5 75.4

1.578 1.454 1.265 1.170 0.997 0.832 0.673 0.451 0.274

samples

10 11 12

87.1 80.2 80.1

10.1 27.7 39.7

1.447 1.164 0.971

degree o f e s t e r i f i c a t i o n anhydrogalacturonic a c i d content s t r u c t u r a l charge parameter Counterion

activity

coefficients

The sodium and calcium a c t i v i t y c o e f f i c i e n t s were determined by s p e c i f i c e l e c t r o d e s , i n d i l u t e s a l t - f r e e s o l u t i o n s (c < 10~~ equiv/]) o f sodium and c a l c i u m p e c t i n a t e s . R e s u l t s were compared t o those c a l c u l a t e d from Manning's t h e o r y ( 2 1 ) . T h i s model i s p r o p o s e d f o r infinitely dilute s o l u t i o n s o f r o d l i k e p o l y e l e c t r o l y t e s and the a c t i v i t y c o e f f i c i e n t s a r e d i r e c t l y imposed by the charge parameter : γ

= e~

|

z

|

x

|

2

\z\\4

1

(2)


6.

THIBAULT A N D

RINAUDO


/2


γ = e~

(ΙζΙλΓ

1

|ζ|λ>

1

65

(3)

where ζ i s the v a l e n c e o f the c o u n t e r i o n . Samples w i t h a random d i s t r i b u t i o n o f c a r b o x y l groups a r e c h a r a c t e r i z e d by e x p e r i m e n t a l v a l u e s which a r e d e c r e a s i n g w i t h d e c r e a s i n g DE, i . e . w i t h i n c r e a s i n g charge parameter (1^5). T h i s b e h a v i o r is s i m i l a r to t h a t observed f o r o t h e r p o l y e l e c t r o l y t e s (22,23). E x p e r i m e n t a l sodium a c t i v i t y c o e f f i c i e n t s are i n good agreement w i t h the t h e o r e t i c a l ones, e x c e p t f o r samples w i t h low DE which a r e c h a r a c t e r i z e d by h i g h e r v a l u e s than p r e d i c t e d ( 1 5 ) . V a l u e s o f c a l c i u m p e c t a t e (sample 1) and f o r c a l c i u m s a l t s o f samples 7 and 8 (DE = 58.2 and 72.1 r e s p e c t i v e l y ) a r e c l o s e t o those r e p o r t e d by Ravanat and Rinaudo (11) and by Rinaudo and M i l a s (22) f o r p e c t i n s w i t h s i m i l a r DE. A c o n t i n u o u s d e c r e a s e o f the c a l c i u m a c t i v i t y c o e f f i c i e n t s was o b t a i n e d whereas Kohn and Luknar (4) o b s e r v e d a sudden drop o f the c a l c i u m a c t i v i t y c o e f f i c i e n t as measured by a m e t a l i n d i c a t o r method f o r p e c t i n a t e s w i t h DE lower than 40%, a l t h o u g h b o t h s e t s o f e x p e r i mental v a l u e s a r e c l o s e t o each o t h e r . I f the r a t i o experimental/ t h e o r e t i c a l c a l c i u m a c t i v i t y c o e f f i c i e n t i s p l o t t e d v e r s u s the DE ( F i g u r e 1 ) , a t r a n s i t i o n i s e v i d e n c e d as t h i s r a t i o d e c r e a s e s below a DE o f 50%. Values o f calcium a c t i v i t y c o e f f i c i e n t obtained f o r the polymers w i t h l o w e s t DE a r e v e r y low. These v a l u e s s u g g e s t s p e c i fic i n t e r a c t i o n s o f the p e c t i c m o l e c u l e s with calcium ions. The f a c t t h a t the c a l c i u m a c t i v i t y c o e f f i c i e n t i s about h a l f o f the t h e o r e t i c a l v a l u e s u g g e s t s t h a t the c a l c i u m p e c t a t e behaves r o u g h l y as a p o l y e l e c t r o l y t e w i t h a doubled charge pjensity, and may be a s c r i bed t o an i n t e r m o l e c u l a r b i n d i n g o f the Ca i o n s t o c a r b o x y l groups o f two macromolecules l e a d i n g t o the f o r m a t i o n ο ξ dimers, even i n v e r y d i l u t e s o l u t i o n s (range t e s t e d 5.10"" < c < 10~ equiv/1).. The i n f l u e n c e o f the p a t t e r n o f e s t e r i % i c a t i o n on the b i n d i n g o f c a l c i u m i o n s i s c l e a r l y shown i n F i g u r e 1. The e n z y m e - d e e s t e r i f i e d pectins a r e c h a r a c t e r i z e d by low v a l u e s o f c a l c i u m a c t i v i t y c o e f f i c i e n t s c l o s e t o those o f c a l c i u m p e c t a t e (sample 1) due t o the p r e s e n ce i n these samples o f c o n t i g u o u s not m e t h y l a t e d g a l a c t u r o n i c a c i d s (9,10), i n agreement w i t h r e s u l t s r e p o r t e d by Kohn e t a l . ( 1 8 ) . The changes i n s c a t t e r e d l i g h t have been r e c o r d e d (15) d u r i n g the n e u t r a l i z a t i o n o f some samples. The n e u t r a l i z a t i o n o^ h i g h l y e s t e r i f i e d p e c t i n s by p o t a s s i u m or c a l c i u m h y d r o x i d e s does not induce any i m p o r t a n t change i n s c a t t e r e d l i g h t , as i s the case d u r i n g the n e u t r a l i z a t i o n by p o t a s s i u m h y d r o x i d e o f the most c h a r g e d samples. In c o n t r a s t , the n e u t r a l i z a t i o n w i t h c a l c i u m h y d r o x i d e o f the samples w i t h the h i g h e s t charge d e n s i t y l e a d s t o an i n c r e a s e i n s c a t t e r e d l i g h t . These changes may be e x p l a i n e d by an i n c r e a s e i n a p p a r e n t molecular weight, c o n f i r m i n g the h y p o t h e s i s o f an a s s o c i a t i o n o f c h a i n s i n d u c e d by c a l c i u m c o u n t e r i o n s . I n f l u e n c e o f the polymer c o n c e n t r a t i o n on c a l c i u m a c t i v i t y c o e f f i c i e n t In a d d i t i o n , the dependence o f c a l c i u m a c t i v i t y c o e f f i c i e n t s upon the c a l c i u m p e c t i n a t e c o n c e n t r a t i o n has been i n v e s t i g a t e d . S o l u t i o n s o f p e c t i n i c a c i d s o f d i f f e r e n t c o n c e n t r a t i o n s were p r e p a r e d , n e u t r a lized by c a l c i u m h y d r o x i d e and the r e s u l t i n g c a l c i u m s a l t s were a n a l y z e d by p o t e n t i o m e t r y f o r the c a l c i u m a c t i v i t y c o e f f i c i e n t .


66

CHEMISTRY AND

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Samples w i t h a DE h i g h e r t h a n 50% have c o n s t a n t c a l c i u m a c t i v i t y c o e f f i c i e n t s over t h e c o n c e n t r a t i o n range t e s t e d (0.8 - 3 χ 10 e q u i v / 1 ) . In c o n t r a s t , p e c t i n s w i t h lower DE a r e c h a r a c t e r i z e d by a c a l c i u m a c t i v i t y c o e f f i c i e n t which i s d e c r e a s i n g above a c o n c e n t r a t i o n o f 10~ e q u i v / 1 . F u r t h e r m o r e , the c o n c e n t r a t i o n i n c a l c i u m p e c t i n a t e s has a p r o f o u n d i n f l u e n c e on the c a l c i u m a c t i v i t y c o e f f i cient f o r samples w i t h the l o w e s t DE o r f o r e n z y m e - d e e s t e r i f i e d p e c t i n s s i n c e the c a l c i u m a c t i v i t y c o e f f i c i e n t d e c r e a s e s t o z e r o . With h i g h e r c o n c e n t r a t i o n i n p e c t i n s , (c > 3 χ 10 equiv/1), a g e l a t i o n p r o c e s s can be o b s e r v e d .


P

These r e s u l t s show t h a t the c a l c i u m i o n s seem t o i n t e r a c t l a r g e l y w i t h the polymer and can be e x p l a i n e d by a m u l t i c h a i n aggregate f o r m a t i o n i . e . an a s s o c i a t i o n o f dimers l e a d i n g t o an i n c r e a s e o f the a p p a r e n t charge parameter. These r e s u l t s a r e i n disagreement w i t h t h o s e o b t a i n e d by Κσητί-, and Luknar (4) who o b s e r v e d a c o n s t a n t c a l c i u m a c t i v i t y c o e f f i c i e n t (as determined by t h e i r m e t a l l o c h r o m i c method) whatever i s the p e c t i n c o n c e n t r a t i o n , but they p r e p a r e d the s o l u t i o n s by d i l u t i o n s o f the most c o n c e n t r a t e d s o l u t i o n . C i r c u l a r d i c h r o i s m measurements In o r d e r t o complete the r e s u l t s o b t a i n e d by p o t e n t i o m e t r y , c i r c u l a r d i c h r o i s m measurements were c a r r i e d o u t . P e c t i c m o l e c u l e s exhibit a p o s i t i v e Cotton effect corresponding to a η IT * t r a n s i t i o n o f the c a r b o x y l groups ( 5 ) . The o p t i c a l a c t i v i t y o f the c a r b o x y l chromophore i s determined by the environment and hence i t s h o u l d be a f f e c t e d by c o n f o r m a t i o n a l changes due t o i n t r a - o r i n t e r m o l e c u l a r i n t e r a c t i o n s . The e f f e c t o f l e v e l and p a t t e r n o f e s t e r i f i c a t i o n , o f the degree o f n e u t r a l i z a t i o n and o f the n a t u r e o f the c o u n t e r i o n , on the c i r c u l a r ^ d i c h r o i s m c u r v e s were s t u d i e d on d i l u t e s o l u t i o n c o n t a i n i n g 10 e q u i v / 1 o f f r e e c a r b o x y l groups i n o r d e r t o a v o i d g e l a t i o n i n presence o f calcium i o n s . V a l u e s ^of f o r the p e c t i n samples i n t h r e e i o n i c forms (Η , Na , Ca ) a r e shown i n F i g u r e 2. The a c i d i c forms a r e c h a r a c t e r i z e d by a c o n s t a n t λ v a l u e ( a t 210 nm), whatever i s the DE and the d i s t r i b u t i o n p a t t e r n o f the f r e e c a r b o x y l g r o u p s . F o r the sodium form o f the polymers, the λ v a l u e d e c r e a s e s from 208 nm t o 203 nm as the charge d e n s i t y o f the p o l y e l e c t r o l y t e i n c r e a s e s w i t h a t r a n s i t i o n f o r a DE about 40% w i t h no i n f l u e n c e o f the d i s t r i b u t i o n p a t t e r n o f the f r e e c a r b o x y l groups. The v a l u e s o f λ f o r calcium pectina t e s d e c r e a s e from 205 nm w i t h a broad t r a n s i t i o n and a c o n s t a n t v a l u e o f 193 nm i s o b t a i n e d when t h e DE i s l e s s t h a n 20%. The i n f l u ence o f the p a t t e r n o f e s t e r i f i c a t i o n i s c l e a r l y shown by the f a c t t h a t the i n c r e a s e i n λ v a l u e o c c u r s f o r h i g h e r DE f o r enzymed e e s t e r i f i e d p e c t i n s . These r e s u l t s a r e n o t i n agreement w i t h t h o s e o b t a i n e d by Kohn and S t i c z a y (23) who o b s e r v e d t h a t the p o s i t i o n o f the a b s o r p t i o n band was s h i f t e d o n l y w i t h i n a 2 nm range i f the i o n i c form changes from p o t a s s i u m t o c a l c i u m . x

+

m

a

x

I t can be c o n c l u d e d from t h e s e r e s u l t s t h a t a l l the p e c t i n s i n a c i d i c form have o n l y one c o n f o r m a t i o n a l s t a t e whatever a r e the l e v e l and p a t t e r n o f e s t e r i f i c a t i o n . In c o n t r a s t , the sodium and c a l c i u m s a l t s o f p e c t i n i c a c i d s a r e c h a r a c t e r i z e d by a c o n f o r m a t i o n a l t r a n s i t i o n which i s s t a b i l i z e d i f t h e DE i s lower t h a n 20-30%.


6. THIBAULT AND RINAUDO


67

alkali Na

enzyme


alkali

/

u

Ca α enzyme

20

40

60

80

DEGREE OF ESTERIFICATION

F i g u r e 1. V a r i a t i o n s o f t h e r a t i o e x p e r i m e n t a l / t h e o r e t i c a l v a l u e s o f c a l c i u m ( • , • ) and sodium ( · , Ο ) a c t i v i t y c o e f f i c i e n t s w i t h the degree o f e s t e r i f i c a t i o n o f a l k a l i - ( •• ) and enzymedeesterified ( ) pectins.

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Interactions of Counteroins with Pectins Studied by Potentiometry and

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