The Interaction of Sodium Dodecyl Sulfate, a Competing Ligand, with

33 The Interaction of Sodium Dodecyl Sulfate, a Competing Ligand, with Iodine Complexes of Amylose and Amylopectin SHOBHANA V. BHIDE, MEENA S. KARVE, and N. R. KALE

Downloaded by DICLE UNIV on November 14, 2014 | http://pubs.acs.org Publication Date: April 21, 1981 | doi: 10.1021/bk-1981-0150.ch033

Department of Chemistry, Division of Biochemistry, University of Poona, Poona-411 007, India

Amylose and a m y l o p e c t i n a r e t h e i s o t a c t i c homopolymers o f oc-D-glucose which i n t e r a c t w i t h i o d i n e (I5) i n aqueous systems t o g i v e the c h a r a c t e r i s t i c blue c o l o u r e d complexes. The h e l i c a l nature o f the amylose-iodine-complex, w i t h s i x D-glucose u n i t s i n the C - l c o n f i g u r a t i o n p e r t u r n o f t h e h e l i x and the i o d i n e m o l e c u l e s packed i n s i d e the lumen, p a r a l l e l t o t h e a x i s o f t h e h e l i x has been w e l l e s t a b l i s h e d by X-ray d i f f r a c t i o n s t u d i e s ( 1 j . E l e c t r o n m i c r o s c o p i c s t u d i e s of a m y l o s e - i o d i n e complex i n t h e form o f f i b r i l s have r e v e a l e d r o d l i k e s t r u c t u r e s w i t h 40 nm i n diameter and the l e n g t h depending on the degree o f p o l y m e r i z a t i o n o f the polymer c h a i n , and the h e l i c e s f o l d e d p a r a l l e l t o the l o n g a x i s ( 2 ) . The r e c e n t s t u d i e s o f t h i s complex by Raman resonance and i o d i n e - 1 2 9 Mossbauer s p e c t r o s c o p y have p r o v i d e d evidence f o r the presence o f I E s p e c i e s w i t h i n the amylose h e l i x ( I - I — I " — I - I ) ( 3 ) . U l t r a f i l t r a t i o n s t u d i e s have demonstrated t h a t a d d i t i o n o f excess o f i o d i n e t o amylose s o l u t i o n compl e t e l y p r e v e n t s the passage of amylose m o l e c u l e s t h r o u g h the f i l t e r . T h i s i s a t t r i b u t e d t o the i o d i n e induced c o i l — H a e l i x t r a n s i t i o n , forming r a t h e r t i g h t h e l i c e s i n the amylose-iodine-complex ( 4 ) . Amylose a l s o forms c r y s t a l l i n e h e l i c a l complexes w i t h a v a r i e t y o f o r g a n i c compounds such as 1-butanol, f a t t y a c i d s , d i m e t h y l s u l p h o x i d e e t c . (£,6,7). The h e l i c a l complexes o f amylose w i t h SDS, 1 - b u t a n o l and c y c l o h e x a n o l a r e found t o be r e s i s t a n t t o the a c t i o n of amylases ( 8 , £ ) . P h y s i c o c h e m i c a l methods employed f o r t h e study o f c o n f o r m a t i o n o f amylose i n the f r e e and complexed s t a t e have f a i l e d t o d e t e c t any s i g n i f i c a n t change i n c o n f o r m a t i o n o f amylose on complexing. The e x t e n s i v e work on hydrodynamic s t u d i e s ( 1 0 , 1 j j suggests t h a t 0097-6156/81/0150-0491$05.25/0 © 1981 American Chemical Society

In Solution Properties of Polysaccharides; Brant, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.


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amylose i n n e u t r a l aqueous s o l u t i o n e x i s t s as a f l e x i b l e random c o i l and i n t h e presence o f complexing agents forms compact h e l i c a l c o i l s w i t h l i g a n d molec u l e s caged i n s i d e the h e l i c e s . The h e l i c a l r e g i o n s h a v i n g 110-130 D-glucose u n i t s a r e i n t e r s p e r s e d by random c o i l r e g i o n s , which c o n t r i b u t e f l e x i b i l i t y t o the polymer c h a i n ( 1_2). P o t e n t i o m e t r i c s t u d i e s by Bates, F r e n c h and Rundle (1 3) have shown t h a t amylose combines w i t h i o d i n e i s o p o t e n t i a l l y and the p o t e n t i a l i s c h a r a c t e r i s t i c o f amylose f o r i t s degree o f p o l y m e r i z a t i o n and s u g g e s t s a h i g h c o o p e r a t i v i t y . However e q u i l i b r i u m (V4) and k i n e t i c (1J3) s t u d i e s i n d i c a t e moderate c o o p e r a t i v i t y of the r e a c t i o n . On the b a s i s o f e x p e r i m e n t a l evidence and computer model b u i l d i n g , Brant and h i s coworkers (L^tiZ) have concluded t h a t amylose i n aqueous s o l u t i o n behaves a s a s t a t i s t i c a l c o i l w i t h no h e l i c a l c h a r a c t e r and i n the presence o f complexing agent a c q u i r e s a h e l i c a l c o n f o r m a t i o n . A Monte C a r l o study of amylose c h a i n c o n f o r m a t i o n has shown t h a t an approp r i a t e m o l e c u l a r model o f amylose c h a i n can generate a randomly c o i l e d c h a i n w i t h p e r c e p t i b l e r e g i o n s o f l e f t handed p s e u d o h e l i c a l backbone t r a j e c t o r y ( 1 8 ) . The e x t i n c t i o n c o e f f i c i e n t o f the a m y l o s e - i o d i n e complex, the wavelength o f the maximum a b s o r p t i o n and the s t a b i l i t y i s i n f l u e n c e d by the degree o f p o l y m e r i z a t i o n o f t h e amylose c h a i n (]_£, 20,21_, 22). Formation or xhe amylose-iodine-complex i s a f f e c t e d by temperat u r e , pH, i o n i c s t r e n g t h , c o n c e n t r a t i o n o f i o d i d e i o n s and the n a t u r e o f amylose. The study o f t h e i n f l u e n c e o f s o l v e n t s on the f o r m a t i o n o f the s t a r c h - i o d i n e - c o m p l e x has r e v e a l e d t h a t the water r e q u i r e m e n t i s n o t r e l a t e d t o e i t h e r the d i p o l e moment o r 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 (2^5). M o u l i k and Gupta have shown t h a t s u r f a c t a n t s and c o s o l v e n t s , mainly d e s t a b i l i z e the amylosei o d i n e - c o m p l e x , and the o v e r a l l p o l a r i t y o f the mixed medium i s i n s u f f i c i e n t t o s y s t e m a t i z e the s o l v e n t e f f e c t (24). Ono e t a l have r e p o r t e d t h a t the blue c o l o u r o f t h e amylose-iodine-complex i s n o t formed i n dimethyl sulphoxide-water mixtures containing l e s s than 28 moles o f water p e r l i t r e (25). A l l these observations i n d i c a t e that c o i l — ^ h e l i x t r a n s i t i o n o f amylose i n the presence o f complexing agents i n v o l v e s only minor s t r u c t u r a l changes. The e n t h a l p y change o f t h i s complex f o r m a t i o n i s -11 t o -20 k c a l / m o l i o d i n e (26). Our e a r l i e r s t u d i e s on the l i g a n d i n d u c e d s t r u c t u r a l changes i n amylose p a r t i a l l y complexed w i t h i o d i n e have shown t h a t competing l i g a n d produces a



33.

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drop i n absorbance a t 640 nm and i s accompanied by a blue s h i f t (640-580 nm) i n the a b s o r p t i o n spectrum. We had chosen an i o d i n e d e f f i c i e n t system i n o r d e r t o a v o i d the s t a b i l i z i n g i n f l u e n c e o f i o d i n e i o n s on the complex. Under these c o n d i t i o n s , i o d i d e i o n s g e n e r a t e d by h y d r o l y s i s o f i o d i n e are s u f f i c i e n t to produce a s t a b l e , blue c o l o u r e d amylose-iodine-complex, though the i n t e n s i t y i s low. However, t h i s s y s t e m was found to be v e r y s e n s i t i v e t o the g e n e r a t i o n o f i o d i d e i o n s , due to the o x i d a t i o n of t r a c e i m p u r i t i e s i n the l i g a n d . In the homologous s e r i e s of a l o c o h o l s , the b l u e s h i f t t h a t was observed was more pronounced as the l e n g t h o f the h y d r o c a r b o n c h a i n i n c r e a s e d , i m p l y i n g t h a t noncov a l e n t i n t e r a c t i o n s l i k e Η-bonds, Van der Waals f o r c e s , h y d r o p h o b i c i n t e r a c t i o n s and water p l a y an i m p o r t a n t r o l e i n s t a b i l i z i n g these h e l i c a l s t r u c t u r e s (27). H o l l o ' e t a l have a l s o r e p o r t e d s i m i l a r o b s e r v a t i o n s on the s t a b i l i t y of a r a y l o s e - a l c o h o l complexes (28). Kim and Robinson have shown t h a t the h e l i c a l c o n f o r mation o f amylose was the most i m p o r t a n t f a c t o r i n the b i n d i n g o f s u r f a c t a n t m o l e c u l e s i n the h e l i c a l c a v i t y of amylose c h a i n , r e s u l t i n g i n a s l i g h t decrease i n the i n t r i n s i c v i s c o s i t y , f a l l i n the p-amylolysis l i m i t and the i o d i n e b i n d i n g c a p a c i t y ( 2 9 ) A r e c e n t r e p o r t on the i n t e r a c t i o n of amylose w i t h a h y d r o p h o b i c f l u o r e s c e n t probe, has shown t h a t i t enhances the f l u o r e s c e n c e i n d i c a t i n g the presence of a h y d r o p h o b i c environment (30). In t h i s paper, we p r e s e n t some o f our o b s e r v a t i o n s on the n a t u r e o f the amylose-iodine-complex/amylop e c t i n - i o d i n e - c o m p l e x u s i n g SDS a s a complexing l i g a n d - p r o b e which might shed some l i g h t on the l i g a n d i n d u c e d s t r u c t u r a l changes i n amylose and a m y l o p e c t i n . M a t e r i a l s and

Methods f

f

A l l r e a g e n t s were o f A n a l a r grade, and water d i s t i l l e d i n a l l - g l a s s a p p a r a t u s was used. Sodium d o d e c y l s u l p h a t e (SDS), F l u k a ( S w i t z e r l a n d ) , 90-93$ was r e c r y s t a l l i z e d t h r i c e from e t h a n o l . S t e a r i c a c i d , p a l m i t i c a c i d and m y r i s t i c a c i d , a l l of e x t r a - p u r e q u a l i t y { 9 9 % ) were o b t a i n e d from SISCO R e s e a r c h Lab., Bombay. A h i g h m o l e c u l a r weight p o t a t o amylose was p r e p a r e d by the u r e a d i s p e r s i o n method and i t was p r e s e r v e d a s a wet p r e c i p i t a t e i n ethanol(6o%) a t +4°C (2L). P o t a t o a m y l o p e c t i n was i s o l a t e d from p o t a t o s t a r c h by the same method, and t r a c e q u a n t i t i e s of amylose from a m y l o p e c t i n f r a c t i o n were removed by a d s o r p t i o n on d e f a t t e d c e l l u l o s e ( £ 2 ) . Amylose and


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a m y l o p e c t i n s o l u t i o n s were p r e p a r e d by d i s p e r s i o n i n NaOH(1.0 M) a t room temperature(26-28°C) f o l l o w e d by n e u t r a l i z a t i o n w i t h HC1(0.5 M) t o pH 7.0, and c e n t r i f u g e d ( 2 , 4 l 8 g) b e f o r e use. F r e s h l y p r e p a r e d s o l u t i o n s were used and the p o l y s a c c h a r i d e c o n t e n t was d e t e r mined by t h e p h e n o l - s u l p h u r i c a c i d method ( 3 3 ) . TABLE I


C h a r a c t e r i z a t i o n o f p o t a t o amylose and a m y l o p e c t i n Iodine Binding Capacity (IBC)

Blue Value (BV)

(31)

•«j}-amylolysis I n t r i n s i c Limit(#) viscosity

io: .irU/ 2

Amylose

19.8

1 .42

96-98

3.53

Amylopectin

0.21

0.16

48-50

1.49

* E x p r e s s e d as p e r c e n t c o n v e r s i o n t o m a l t o s e Blue v a l u e r e a g e n t (BVR) - I o d i n e (7.87 χ 10"*M) + KI (1.21 χ 10" M) was used f o r the i o d i n e s o l u t i o n d j ) , The r e a t i o n s were c a r r i e d out i n g l a s s - s t o p p e r e d t e s t tubes m a i n t a i n e d a t 30°C ( + 0.05°) i n a thermo s t a t . The s p e c t r o p h o t o m e t r y r e c o r d s were made on SHIMADZU UV 300, a t 30°C ( + 0.05°) i n q u a r t z c e l l s (1.0 cm), u s i n g water as a~blank. I n f l u e n c e o f i o d i n e (BVR) on the f o r m a t i o n o f b l u e coloured amylose-iodine-complex(34): I t was s t u d i e d to determine the optimum c o n c e n t r a t i o n o f i o d i n e t h a t g i v e s maximum absorbance a t 640 nm, w i t h o u t p r e c i p i t a t i o n o f the complex. A system c o n t a i n i n g amylose (0.07 mg) - 0.2 ml + a c e t a t e b u f f e r ( p H 4.8, 0.1 M) 0.5 ml + i o d i n e ( B V R - 0.1—^0.8 ml) + water t o make t h e t o t a l volume t o 5.0 ml, was m a i n t a i n e d a t 30 C f o r 30 minutes to a t t a i n the e q u i l i b r i u m and t h e s p e c t r a were r e c o r d e d . The i o d i n e c o n c e n t r a t i o n (BVR-0.6 ml) g i v i n g the maximum absorbance a t 640 nm was chosen t o study the i n f l u e n c e o f the competing l i g a n d s l i k e SDS and fatty acids. I n f l u e n c e o f SDS on i o d i n e complexes o f amylose and a m y l o p e c t i n : ( F i g u r e 1) A system c o n t a i n i n g amylose(0.07 mg) - 0.2 ml/amylopectin(0.1 8 mg) - 0.3 ml + a c e t a t e b u f f e r ( p R 4.8, 0.1 M) - 0 . 5 m l + SDS (1.0 x 10~ M) - 0.2-V5.0 ml was e q u i l i b r a t e d a t 30°C f o r 30 minutes f o l l o w e d by t h e a d d i t i o n o f i o d i n e ( B V R ) - 0.6 m l and the t o t a l volume was a d j u s t e d t o 5.0 ml 2

4



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Amylose-SDS-iodine complex (Q); amylopectin-SDS-iodine (A); absorbance (ABS) (\ )-

complex

max

Figure 2. (A) Difference spectra: amylose-SDS-iodine complex minus amyloseiodine complex in acetate buffer, pH 4.8, for three different sequences (1, 2, and 3). (Β) Absorption spectra for amylose-SDS-iodine complex in acetate buffer, pH 4.8, for three different sequences (1,2, and 3).



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w i t h water. The system was m a i n t a i n e d a t 30°C f o r 30 minutes t o a t t a i n t h e e q u i l i b r i u m and t h e absorbance was r e c o r d e d a t the r e s p e c t i v e λ max. Sequence s t u d i e s : ( F i g u r e 2 & 3) The system was same a s d e s c r i b e d above. The r e a g e n t s were added i n the sequence g i v e n i n F i g u r e - 2 . I o d i n e s o l u t i o n ( B V R ) 0.6 ml and SDS s o l u t i o n (1.0 χ 10"^M) - 2.0 ml were used i n t h i s system. The b l a n k (b) was w i t h o u t SDS. Perturbation studies : Reduction of iodine with sodium t h i o s u l p h a t e ( F i g u r e 4 & 5) The system was same a s d e s c r i b e d f o r the sequence s t u d i e s . Two s e t s of experiments were r u n under i d e n t i c a l c o n d i t i o n s . I o d i n e was c o m p l e t e l y reduced by a d d i n g r e q u i r e d amount o f sodium t h i o s u l p h a t e ( 1 .0 χ 10""3M). One s e t was e q u i l i b r a t e d a t 30°C and t h e o t h e r a t 60 C f o r 30 minutes r e s p e c t i v e l y . The s e t m a i n t a i n e d a t 6o°C was c o o l e d t o 30°C and both the s e t s were r e i o d i n a t e d by a d d i n g i o d i n e ( B V R ) - 0.6 ml f o l l o w e d by e q u i l i b r a t i o n f o r 30 minutes a t 30°C. I n f l u e n c e o f u r e a on a m y l o s e - i o d i n e - c o m p l e x / amylose-SDS-iodine-complex The d e s t a b i l i z i n g i n f l u e n c e of u r e a on t h e absorbance o f amylose-iodine-complex was i n v e s t i g a t e d by r e c o r d i n g t h e absorbance a t 640 run i n the p r e s e n c e o f u r e a ( 1 . 6 — * 5 . 3 M). A system c o n t a i n i n g amylose(0.07 mg) - 0.2 ml + a c e t a t e b u f f e r ( p H 4 . 8 , 0.1 M) - 0 . 5 ml + i o d i n e ( B V B ) - 0.5 ml + u r e a (8 M) 1.0-*2·8 ml was a d j u s t e d t o 5.0 ml w i t h water. The system was e q u i l i b r a t e d a t 30°C f o r 30 minutes and absorbance was r e c o r d e d a t 640 nm ( F i g u r e 6 A ) . I n f l u e n c e o f urea ( 1 . 6 — * 5 . 3 M) on amylose-SDSi o d i n e - c o m p l e x was s t u d i e d by u s i n g e s s e n t i a l l y the same system a s d e s c r i b e d f o r t h e sequence s t u d i e s . The sequence-1 which gave maximum r e d u c t i o n i n a b s o r bance (640 nm) was chosen f o r t h i s experiment. The i n c r e a s e i n absorbance a t 640 nm was r e c o r d e d t o study the p e r t u r b a t i o n caused by u r e a ( 1 . 6 — * 5 . 3 M) t o o v e r come the i n h i b i t o r y i n f l u e n c e o f SDS ( F i g u r e 6 B ) . P e r t u r b a t i o n w i t h u r e a ( F i g u r e 7 ) : The system c o n t a i n s amylose(0.07 mg) - 0.2 ml, a c e t a t e b u f f e r (pH 4 . 8 , 0.1 M) - 0.5 ml, SDS(1.0 x 10~ M) - 1.0 ml, u r e a (8 M) - 2.8 m l . The r e a g e n t s were mixed i n the sequence ( 1 , 2 & 3) as g i v e n i n F i g u r e 7. The b l a n k (b) was w i t h o u t SDS. Two s e t s o f experiments were conduc t e d under i d e n t i c a l c o n d i t i o n s . One s e t was e q u i l i b r a t e d a t 30°C and the o t h e r a t 60 C f o r 30 minutes r e s p e c t i v e l y . The s e t m a i n t a i n e d a t 60°C was c o o l e d to 30°C and i o d i n e s o l u t i o n ( B V R ) - 0 . 5 ml was added to both t h e s e t s f o l l o w e d by e q u i l i b r a t i o n f o r 30 minutes a t 30°C. 4


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STUDIES

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AMYLOPECTIN-SDS-IODINE


-COMPLEX

WAVELENGTH ( n m )

Figure 3. (A) Difference spectra: amylopectin—SDS-iodine complex — amylopectin-iodine complex in acetate buffer, pH 4.8, for three different sequences (1, 2, and 3) as given in Figure 2. (B) Absorption spectra for amylopectin-SDS-iodine complex in acetate buffer, pH 4.8, for three different sequences (1,2, and 3) as given in Figure 2.


498

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STUDIES SODIUM

FOR

OF

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AMYLOSE-SDS-IODINE

THIOSULPHATE


-COMPLEX

PROPERTIES

WAVELENGTH ( n m )

Figure 4. Absorption spectra of amylose-SDS-iodine complex in acetate buffer, pH 4.8, for three different sequences (1,2, and 3). (A) The iodine was reduced with sodium thiosulfate, equilibrated at 60°C for 30 min, cooled to 30°C, and reiodinated. (B) The iodine was reduced with sodium thiosulfate, equilibrated at 30°C for 30 min, and reiodinated. (C) Control.


BHIDE

ET

AL.

499

SDS


PERTURBATION OF AMYLOPECTIN-SDS - IODINE COMPLEX BY SODIUM THIOSULPHATE

•Oj

200

I

300

1

A00

I

500 WAVELENGTH (nm)

I

600

1—

700

Figure 5. Absorption spectra of amylopectin-SDS-iodine complex in acetate buffer, pH 4.8, for three different sequences (1,2, and 3) as given in Figure 4. (A) The iodine was reduced with sodium thiosulfate, equilibrated at 60°C for 30 min, cooled to 30°C, and reiodinated. (B) The iodine was reduced with sodium thiosulfate, equilibrated at 30°C for 30 min, and reiodinated. (C) Control.


500

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POLYSACCHARIDES


=ii-o INFLUENCE OF UREA (16—~5·3 M) ON AMYLOSE-IODINE -COMPLEX AND AMYLOSE-SDS-IODINE -COMPLEX

WAVELENGTH (nm)

Figure 6. (A) The destabilizing influence of urea on the absorbance of the bluecolored amylose-iodine complex. (B) The inhibitory influence of SDS on the absorbance of amylose-iodine complex is overcome by urea; *, 4.5M urea. The arrow indicates the increasing concentration of urea (1.6-> 5.3M).

PERTURBATION STUDIES FOR AMYLOSE-SDS-IODINE -COMPLEX WITH UREA (4.5 M) b - AMYLOSE + UREA + BVR

i-04 200

1

300

L

ZÔÔ

_

,

,

500

600

,

I

7 0 0 8 0 0

WAVELENGTH (n m)

Figure 7. Absorption spectar of amylose-SDS-iodine complex in acetate buffer, pH 4.8, for three different sequences (1, 2, and 3) as given above. (A) Urea (4.5M) was added and the system was equilibrated at 60°C for 30 min, cooled to 30°C, followed by the addition of iodine. (B) Urea (4.5M) was added and the system was equilibrated at 30°C for 30 min, followed by the addition of iodine; b = blank without SDS.


33.

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501

SDS

D e t e r m i n a t i o n of Κρ,5: The c o n c e n t r a t i o n of i o d i n e C) r e q u i r e d to reduce the absorbance (640nm) t o a v a l u e , h a l f t h a t o f the s a t u r a t i o n l e v e l i s found to be v e r y c h a r a c t e r i s t i c f o r a g i v e n p o l y s a c c h a r i d e . For these experiments the system d e s c r i b e d above f o r the d e t e r m i n a t i o n of optimum c o n c e n t r a t i o n of i o d i n e f o r the f o r m a t i o n of b l u e c o l o u r e d amylosei o d i n e - c o m p l e x was used. The p o l y s a c c h a r i d e concen t r a t i o n was k e p t c o n s t a n t and the i o d i n e c o n c e n t r a t i o n was v a r i e d ( BVR - 0.1->0.8 m l ) . The c o n c e n t r a t i o n of p o l y s a c c h a r i d e s used f o r the d e t e r m i n a t i o n o f t h e i r KO.5> i n t h i s system were, amylose(0.07-0.09 mg), amylopectin(0.18-0.20 mg), p o t a t o starch(0.10-0.12 mg) and s o l u b l e starch(0.10-0.12 mg). The c o n c e n t r a t i o n o f the competing l i g a n d ( K Q R ) r e q u i r e d t o reduce the absorbance of amylose-SDSi o d i n e - c o m p l e x (Amax) to h a l f , as compared w i t h the blank(640 nm) was determined by u s i n g the sequence-1, which gave maximum r e d u c t i o n i n the absorbance. For these experiments the system d e s c r i b e d f o r the study of i n f l u e n c e o f SDS on the amylose-iodine-complex was used. The c o n c e n t r a t i o n of amylose(0.07 mg) and iodine(BVR - 0.6 ml) was k e p t c o n s t a n t and the concen t r a t i o n of the competing l i g a n d s such as SDS-(1.0 χ 1 0 " " % ) , sodium s t e a r a t e - ( 5 . 0 χ 10"5 M ) , sodium c m y r i s t a t e - ( 4 . 0 χ 10-5M), sodium p a l m i t a t e - ( 4 . 0 χ 10 M) was v a r i e d (0.2->3.0 m l ) . The absorbance was r e c o r d e d a t the r e s p e c t i v e A max. S t u d i e s on the i n h i b i t o r y I n f l u e n c e of SDS on amylose-iodine-complex ; ( F i g u r e 10 & 11) The i n f l u ence o f the c o n c e n t r a t i o n of i o d i n e ( I 3 ) (BVR - 0.1—> 0.8 ml) on the f o r m a t i o n o f amylose-iodine-complex was s t u d i e d by r e c o r d i n g the absorbance a t 640 nm (blank) and i n the presence of SDS f o r the sequences 1,2 & 3, a t the c o r r e s p o n d i n g A max. T h i s system con t a i n e d amylose(0.09 mg;- 0.2 ml and SDS(1.0 χ 10"*Μ) 1.0 ml.


(KQ

#

R e s u l t s and

Discussion

The competing l i g a n d l i k e SDS, appears to i n t e r a c t w i t h the l o o s e h e l i c a l r e g i o n s i n amylose and amylo p e c t i n t o form the c o r r e s p o n d i n g compact h e l i c a l complexes w i t h SDS m o l e c u l e s t r a p p e d i n s i d e the h e l i c e s . When i o d i n e ( I 3 ) i s added to t h i s system, a p a r t o f the SDS, which i s l o o s e l y a t t a c h e d i s d i s p l a c e d by iodine(Ι3). The a b s o r p t i o n s p e c t r a of the r e s u l t i n g amylose-SDS-iodine-ccmplex/amylopectin-SDSi o d i n e - c o m p l e x , when compared w i t h t h e i r r e s p e c t i v e s p e c t r a i n absence of SDS, show a r e d u c t i o n i n


502

SOLUTION


DETERMINATION

Figure 8.

OF

Kg

FOR

5

PROPERTIES

OF

POLYSACCHARIDES

AMYLOSE

(A) Lineweaver Burk plot; (B) direct linear plot; ABS = absorbance (640 nm); BVR = blue value reagent (I ~). 3

DETERMINATION 8·0ι

OF

Kg

5

FOR

SDS

1

Figure 9. (A) Lineweaver Burk plot; (B) direct linear plot; (A) ABS—ABS(blank640 nm) — ABS(Xmax) (ABS = absorbance (X x). The reagents were added as per Sequence 1 (Amylose-SDS-BVR). ma


BHIDE

ET

AL.

503

SDS

INHIBITORY -COMPLEX

INFLUENCE

OF

SDS

ON

(I)

AMYLOSE - I O D I N E

60 Δ

®

Δ

l< ·

^

—

20

1

1——


"07

1

j 0-5

\

Γ

^-""Ι

ι

1

03~f

I

IODINE (1·575χ10~ mM) 2

Figure 10. (A) Lineweaver Burk plot for three sequences (1,2, and 3); (B) direct linear plot for Sequence 3 (BVR - j - SDS + amylose) (ABS = absorbance (X ); b = blank without SDS). max

INHIBITORY -COMPLEX

INFLUENCE (Π)

OF

SDS

ON

AMYLOSE-IODINE °- , 0.73-H 0-658

®

Figure 11. (A) Direct linear plot for Sequence 2 (amylose + BVR + SDS); (B) direct linear plot for Sequence 1 (amylose + SDS + BVR) (ABS = absorb ance (Xmax); b = blank without SDS).



504

SOLUTION

PROPERTIES

OF

POLYSACCHARIDES

absorbance a t 640 nm accompanied by a blue s h i f t ( 6 4 0 570 nm) ( F i g u r e 1B & 1 A ) . T h i s change i s more pronoun ced i n the case of amylose ( l i n e a r ) than a m y l o p e c t i n (branched), as seen from t h e i r r e s p e c t i v e d i f f e r e n c e s p e c t r a ( F i g u r e 2A à 3 A ) . The c o n c e n t r a t i o n of SDS used i n t h e s e experiments i s below the c r i t i c a l micel l a r c o n c e n t r a t i o n (CMC) and SDS has no i n t e r a c t i o n w i t h i o d i n e ( I j ) . The optimum c o n c e n t r a t i o n of i o d i n e t h a t gave maximum absorbance a t 6^-0 nm f o r the i o d i n e complexes o f amylose/amylopectin, w i t h o u t p r e c i p i t a t i o n , was used. Under t h e s e c o n d i t i o n s most of the i o d i n e i s bound i n s i d e the h e l i c a l c a v i t y and a smal] amount of i o d i n e may be adsorbed on the s u r f a c e of the complexed polymer c h a i n ( 2 0 ) . The p l o t of 1/ABS vs SDS, i n case of amylose-SDSiodine-ccmplex gives a t y p i c a l rectangular hyperbola type c u r v e , i m p l y i n g t h a t a f t e r r e a c h i n g the s a t u r a t i o n l e v e l , t h e r e i s no f u r t h e r drop i n absorbance of the amylose-SDS-iodine-complex. Whereas i n case of amylopectin-SDS-iodine-complex, t h e r e i s a l i n e a r drop i n the absorbance and the s a t u r a t i o n l e v e l i s not r e a c h e d . T h i s i s p r o b a b l y due to low a f f i n i t y of SDS/ i o d i n e f o r the s h o r t o u t e r l i n e a r c h a i n s on a m y l o p e c t i n ( F i g u r e 1Β · Sequence s t u d i e s : ( F i g u r e 2B & 3B) To our g r e a t s u r p r i s e , i x was observed t h a t the r e d u c t i o n i n a b s o r bance a t 640 nm and the b l u e s h i f t (640-570 nm) caused by SDS was dependent on the sequence o f a d d i t i o n o f r e a g e n t s , even though the system was e q u i l i b r a t e d a t 30°C f o r 30 minutes, a f t e r the a d d i t i o n of SDS and i o d i n e , r e s p e c t i v e l y . T h i s time i s s u f f i c i e n t to a t t a i n an e q u i l i b r i u m c o n d i t i o n . The i n t e r a c t i o n o f i o d i n e ( I j ) w i t h the l o o s e h e l i c a l r e g i o n s o f amylose to produce blue c o l o u r e d compact h e l i c a l domains w i t h i o d i n e ( 1 ^ ) m o l e c u l e s t r a p p e d i n s i d e the h e l i c e s , i s c l o s e to t h a t o f a d i f f u s i o n c o n t r o l l e d p r o c e s s ( 7 x 10° M~*l s e c " ) and i s independent of temperature (1025°C) (^5). F u r t h e r , i t was n o t i c e d t h a t once the r e a g e n t s were mixed i n a g i v e n sequence, t h e r e was no change i n the a b s o r p t i o n spectrum of the amylose-SDSi o d i n e complex/amylopectin-SDS-iodine-complex, even a f t e r 16-18 hours of i n c u b a t i o n a t 30°C i n g l a s s s t o p p e r e d t e s t t u b e s , p r o t e c t e d from l i g h t . I t appears t h a t i o d i n e ( I ^ ) / S D S m o l e c u l e s once entrapped i n the h e l i x cannot d i f f u s e out f r e e l y i n the s u r r o u n d i n g aqueous environment, due t o the t i g h t or s t i f f n a t u r e of the h e l i x . T h i s i s more v a l i d f o r the h e l i c a l domains i n s i d e (endo)the polymer c h a i n than a t the end p o r t i o n ( e x o ) . The i n f l u e n c e o f SDS was more prominent i n sequence-1, i n t e r m e d i a t e i n sequence-3 and minimum i n sequence-2 as compared w i t h the b l a n k ( F i g u r e 2 & 3) a

1


33.

BHIDE

ET

AL.

SDS

505

P e r t u r b a t i o n s t u d i e s ; R e d u c t i o n o f i o d i n e (I3) w i t h sodium t h i o s u l p h a t e - I t was o f g r e a t i n t e r e s t to know how the system behaves when one of the l i g a n d s such as i o d i n e ( 1 3 ) was removed by r e d u c t i o n w i t h sodium t h i o s u l p h a t e under e x t r e m e l y m i l d c o n d i t i o n s ( 3 0 ° C ) , i n the presence o f SDS. One would n o r m a l l y e x p e c t t h a t the SDS m o l e c u l e s from the s o l u t i o n would d i f f u s e i n the h e l i c a l c a v i t y t o occupy t h e v a c a n t spaces o f i o d i n e ( I ) . On r e i o d i n a t i o n the a b s o r p t i o n s p e c t r a of the systems g e n e r a t e d by the sequences (2 & 3) s h o u l d be i d e n t i c a l t o the a b s o r p t i o n spectrum o f the system g e n e r a t e d by s e q u e n c e - 1 . However, the r e s u l t s i n d i c a t e t h a t a t 3 0 ° C the e n t r y o f SDS molec u l e s i n the empty h e l i c a l r e g i o n s o f the polymer c h a i n i s r a t h e r a slow p r o c e s s ( F i g u r e 4 B & 5 B ) . T h i s p r o c e s s can be a c c e l e r a t e d by i n c r e a s i n g the temperature t o 6 0 ° C ( F i g u r e 4 A & 5 A ) . I t appears t h a t the h e l i c a l domains of the polymer c h a i n d e v o i d o f i o d i n e m o l e c u l e s r e t a i n t h e i r c o n f o n r a t i o n a l 'memory over a s h o r t p e r i o d . T h i s i s more prominent i n case o f amylose than amylopectin (Figure 4 & 5, sequence-2). I n f l u e n c e o f u r e a on a m y l o s e - i o d i n e - c c m p l e x / amylose-SDS-iodine-complex : Urea i s known to have a d e s t a b i l i z i n g i n f l u e n c e on the a m y l o s e - i o d i n e - c o m p l e x , r e s u l t i n g i n a r e d u c t i o n i n the absorbance a t 640 nm. T h i s i s e v i d e n t from the study o f i n f l u e n c e o f u r e a on a m y l o s e - i o d i n e - c o m p l e x ( F i g u r e 6 A ) . Urea a l s o i n c r e a s e s the f l e x i b i l i t y o f the polymer c h a i n by a f f e c t i n g the i n t r a m o l e c u l a r H-bonds ( 3 6 ) . The i n h i b i t o r y i n f l u e n c e of SDS on the absorbance o f amylose-iodine-complex a t 640 nm was r a p i d l y overcome a t 3 0 ° C when u r e a ( 4 . 5 M) was added to amylose-SDS-iodine-complex. T h i s i s c l e a r l y e v i d e n t from the study o f i n f l u e n c e o f u r e a on amylose-SDS-iodine-complex (sequence-1 : F i g u r e 6 B ) . The sequence o f a d d i t i o n o f r e a g e n t s had a l i t t l e i n f l u e n c e ( F i g u r e 7A) on the system e q u i l i b r a t e d a t 3 0 ° C and 6o°C ( F i g u r e Ik * 7 B ) . F u r t h e r , a d d i t i o n o f u r e a t o aniylcse-SDS-complex b e f o r e or a f t e r the a d d i t i o n o f i o d i n e ( I 3 ) made no d i f f e r e n c e . These r e s u l t s i m p l y 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 and R-bonds p l a y an i m p o r t a n t r o l e i n the f o r m a t i o n o f amylose-SDSi o d i n e -complex/amylopectin-SDS-iodine-complex. D e t e r m i n a t i o n o f K p R and Kj : A p l o t o f concen tration (I3) vs absorbance (640 nm) i n the case o f the a m y l o s e - i o d i n e - c o m p l e x / a m y l o p e c t i n iodine-complex gives a t y p i c a l rectangular hyperbola curve ( 3 4 ) , most o f t e n encountered i n the f i e l d o f enzyme k i n e t i c s . S i m i l a r l y , the b e h a v i o u r o f many p h y s i c a l and b i o l o g i c a l systems can be d e s c r i b e d i n


3

1

R


506

SOLUTION

PROPERTIES

OF

POLYSACCHARIDES

terms of a h y p e r b o l i c r e l a t i o n s h i p between a measured response and a c o n t r o l l e d v a r i a b l e . Hence M i c h a e l i s Menten e q u a t i o n w i t h s u i t a b l e s u b s t i t u t i o n s can be used to c a l c u l a t e Kg . 5 from Lineweaver Burk p l o t and i n p a r t i c u l a r the * d i r e c t l i n e a r p l o t developed by E i s e n t h a l and Cornish-Bowden (J5j[,J8) ( T a b l e I I ) . TABLE I I Michael!s-Menten Equation V(s) ν = ABS Km ( ) Downloaded by DICLE UNIV on November 14, 2014 | http://pubs.acs.org Publication Date: April 21, 1981 | doi: 10.1021/bk-1981-0150.ch033

+

A B S

max

(Iodine)

-

g

K

0.5

+

U °

d

i

n

e

)

Lineweaver Burk P l o t

I _I

*m

ν

V

V

#

1

1

(s)

ABS

Ko.5 A B S

A B S

max

(Iodine)

max

E i s e n t h a l and Cornish-Bowden Direct Linear Plot m

ax

= 1

(s) Competitive

Inhibition K

TABLE

= 1

(Iodine)

ABS

0.5

=

K

0.5

(

1

~ >

+

III

Polysaccharide

Iodine v

0.5

Amylose

7.1

x 10

Amylopectin

7.4

χ 10

Potato

3.8

χ 10

4,3

x 10

(mM)

-3 -2

starch

Soluble

starch

The c o n c e n t r a t i o n o f i o d i n e K the absorbance (64ο mr.) to a

Q

c r e q u i r e d to reduce v a l u e , h a l f t h a t of

0


33.

BHIDE

ET

AL.

507

SDS

the s a t u r a t i o n l e v e l i s very c h a r a c t e r i s t i c f o r a m y l o s e ( l i n e a r ) and a m y l o p e c t i n ( branched), and can be determined a c c u r a t e l y (Table I I I ) . S i m i l a r l y a p l o t o f 1/ABS vs SDS i n case of amylose i n sequence-1 g i v e s a t y p i c a l r e c t a n g u l a r h y p e r b o l a curve ( F i g u r e 1B). Hence Κ ς v a l u e s f o r d i f f e r e n t competing l i g a n d s can be '"Obtained s i m i l a r to t h a t of SDS ( F i g u r e 9) by u s i n g sequence-1. Here the fc' - value g i v e s the c o n c e n t r a t i o n of the complexing ligand. r e q u i r e d to reduce the absorbance of amylose-SDS-iodine-complex (Amax) to h a l f , as compared w i t h the blank (640 nm) under s t a n d a r d con d i t i o n s ( F i g u r e 9 ) . I t i s i n t e r e s t i n g to know t h a t 0.5 £° sodium stéarate h a v i n g the * same a l i p h a t i c hydrocarbon c h a i n are i d e n t i c a l and they decrease as the c h a i n l e n g t h i n c r e a s e s ^ (Table I V ) . The d i s s o c i a t i o n c o n s t a n t (K^) can be c a l c u l a t e d from t h i s data f o r SDS (Table I I & V). The v a l u e s of and a r e very c h a r a c t e r i s t i c of the s p e c i e s of *'the l i g a n d ( s ) - p o l y m e r complexes and they d i f f e r a c c o r d i n g to the sequence of a d d i t i c n of the r e a g e n t s . Thus i n sequence-1 the polymer c h a i n i s predominantly complexed w i t h SDS and i n the sequence-2, w i t h i o d i n e U ^ ) . In the sequence-3, the i o d i n e and SDS m o l e c u l e s a r e randomly d i s t r i b u t e d i n the h e l i c a l domains. Hence the v a l u e s of K , and K-$ f o r the sequence-3 are the mean v a l u e s of the sequence (1) &r\à (2) (Table V). S i n c e the r e s u l t a n t parameters K Q ^ and depend on the m i x i n g sequence, they cannot be*^ understood as e q u i l i b r i u m c o n s t a n t s i n the u s u a l sense. I t a l s o suggests t h a t the h e l i c a l domains of the polymer c h a i n c o n t a i n i n g the l i g a n d s are i d e n t i c a l . T h i s i s expected i n case of amylose, a homopolymer made o f D-glucose u n i t s l i n k e d through oc -D(1—*4) l i n k a g e s . The K Q ^ v a l u e s o b t a i n e d by Lineweaver Burk p l o t s and d i r e c v l i n e a r p l o t s were found t o be s i m i l a r ( F i g u r e 8,9,10,11). Mechanism o f i n t e r a c t i o n of SDS w i t h amyloseiodine-complex : The i n h i b i t o r y i n f l u e n c e of SDS on the f o r m a t i o n o f amylose-iodine-complex can be i n v e s t i gated by s t u d y i n g the i n f l u e n c e of i o d i n e c o n c e n t r a t i o n ( I j ) on the absorbance of amylose-iodine-complex i n the presence (Amax) and i n the absence of SDS (640 nm). I t i s i n t e r e s t i n g to note t h a t the mechanism of t h i s i n h i b i t o r y p r o c e s s i s governed by the sequence of a d d i t i o n of the r e a g e n t s . The sequence-3 shows a t y p i c a l c o m p e t i t i v e mechanism, wherein i o d i n e ( I ~ ) and SDS m o l e c u l e s i n t e r a c t i n a random way w i t h t h e ^ l o o s e h e l i c a l r e g i o n s of the amylose c h a i n to form compact h e l i c a l domains c o n t a i n i n g SDS-I^ m o l e c u l e s . The 0 #

% J


K

v

a

l

u

e

s

Γ

a n ( i

Q

c


SOLUTION

508

PROPERTIES

O F POLYSACCHARIDES

TABLE IV I n f l u e n c e o f L i g a n d s on t h e Absorbance of A m y l o s e - i o d i n e - c c m p l e x Ligand

K

0.5


x 10-3mM SDS

CH

Sodium Stéarate Sodium Myristate

· 0S0 Na

2.0

CHj . ( C H ) n

. 000Na

2.0

CH

3

. (0Ε )n 2

3

2

Sodium PaLoaitate

. (CH )

1 2

· COONa

0.8

CHj . ( C H )

1 4

. COONa

0.2

3

2

2

TABLE V 5

K0

and

Sequence 5 2

i

K

d

(b)

(1)

(2)

(3)

Mean (1)&(2)

0·70

6.1

2.8

3.8

3.75 (1)+(2)/2-(b)

-

2.6

6.9

4.6

4.79

0.65

0.58

0.73

0.65

0.65

x

10-3mM SUS ABS

f o r Amylose-SDS-iodine -complex

x

10" mM Iodine K

Κi

max

- Amylose-iodine-complex = 7.87 x 10-3 mM,

Iodine


33.

BHIDE

ET

AL.

509

SDS

outstanding feature of competitive i n h i b i t i o n i s that i t i s overcome by i n c r e a s i n g the c o n c e n t r a t i o n o f the competing l i g a n d ( s u b s t r a t e i n case o f enzyme). T h i s p r e c i s e l y happens when i o d i n e c o n c e n t r a t i o n i s i n c r e a s e d ( F i g u r e 1 0 , Table V - A B S ^ ^ v a l u e s ) . The sequence 1 and 2 showed a mixed type of i n h i b i t o r y mechanism.(Figure 1 1 ) . Acknowledgements


Financial assistance

i n support o f t h i s work from :

1. U n i v e r s i t y G r a n t s Commission - New D e l h i 2. C o u n c i l o f S c i e n t i f i c and I n d u s t r i a l R e s e a r c h New D e l h i 3. Youth S e r v i c e Dept. ( S c i e n c e & Technology C e l l ) Govt, o f M a h a r a s h t r a - Bombay 4 . S. H. K e l k a r C h a r i t y T r u s t - Bombay 5 . N a t i o n a l Science Academy - New D e l h i 6. Department o f S c i e n c e & Technology - New D e l h i , i s g r a t e f u l l y acknowledged.

Abstract Amylose i s a unique polysaccharide which forms a h e l i c a l blue-coloured complex with iodine (I 3). It also forms h e l i c a l complexes with a v a r i e t y of organic compounds such as 1-butanol, 1-pentanol, cyclohexanol, SDS etc. The i n t e r a c t i o n of SDS, a competing ligand with iodine (I 3) complexes of amylose and amylopectin i s studied spectrophotometrically. I t i s observed that the reduction i n absorbance at 640 nm accompanied by the blue s h i f t (640-570 nm) i n the absorption spectrum i s governed by the sequence of addition of the reagents, implying that this i n t e r a c t i o n i s closely associated with the coil-->helix t r a n s i t i o n of the polymer chain. Perturbation of this complex with sodium thiosulphate and urea has revealed that the t r a n s i t i o n from h e l i x -->coil i s rather sluggish and hydrophobic interactions play an important role i n the s t a b i l i t y of t h i s complex. -

-

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