The Gelling Mechanism and Relationship to Molecular Structure of

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Downloaded by UNIV OF CALIFORNIA SAN DIEGO on April 12, 2016 | http://pubs.acs.org Publication Date: November 17, 1980 | doi: 10.1021/bk-1980-0141.ch025

The Gelling Mechanism and Relationship to Molecular Structure of Microbial Polysaccharide Curdlan W. S. FULTON and E. D. T. ATKINS H. H . Wills Physics Laboratory, University of Bristol, Bristol BS8 1TL U.K. The e x t r a c e l l u l a r microbial polysaccharide curdlan i s e s s e n t i a l l y a l i n e a r homopolymer o f 3 - ( 1 - 3 ) - D - g l u c a n as i l l u s t r a t e d i n Figure 1. I t i s produced from the mutant s t r a i n of the b a c t e r i a A l c a l i g e n i s faecal i s and was f i r s t i s o l a t e d and i n v e s t i g a t e d by Harada and coworkers (T_-_3) h ° coined the name curdlan to describe the g e l l i n g behaviour of the polysaccharide a t elevated temperatures. When an aqueous suspension (2%w/v) o f curdlan i s heated, a number of d i s t i n c t phases can be observed. F i r s t the suspension c l e a r s forming a s o l between 50-55°C. Then g e l a t i o n begins a t approximately 65°C forming a low s e t g e l . This i s a weak and e a s i l y disrupted g e l . I f the temperature i s r a i s e d f u r t h e r to 95°C, a t r a n s l u c e n t , r e s i l i e n t gel ( F i g . 2) i s produced, which has been termed the high s e t g e l . Both the low s e t and high set gels are thermally i r r e v e r s i b l e . As regards the morphology o f these two types o f g e l , only that o f the high s e t gel i s reported i n t h i s work. A number o f X-ray d i f f r a c t i o n patterns o f o r i e n t e d curdlan gels have been reported (2^4). They may be separated i n t o two families. The f i r s t group Fears a strong resemblance to the p r e v i o u s l y reported X-ray pattern f o r 3 - ( l - 3 ) - D - x y l a n and which was i n t e r p r e t e d i n terms o f a s i x - f o l d t r i p l e - s t r a n d e d molecule (5^6). S i m i l a r patterns have a l s o been obtained from 3 - ( 1 - 3 ) D-gTucan i n fungal t i s s u e by Jelsma and Kreger (7) and from l e n t i n a n by Bluhm and Sarko ( 8 ) . The second type o f X-ray p a t t e r n , which e x h i b i t s l a y e r l i n e s o f spacing 2.70nm, has been suggested by Takeda e t a K (2) to correspond to a m a j o r i t y component o f seven foTd singTe h e l i c e s . We have examined the X-ray r e s u l t s from various o r i e n t e d curdlan gels and report our f i n d i n g s and c a l c u l a t i o n s on a number o f models. In a d d i t i o n we have i n v e s t i g a t e d the behaviour of curdlan gels i n a s e r i e s o f deuterium exchange experiments monitored with i n f r a - r e d spectroscopy. w

0-8412-0589-2/80/47-141-385$06.50/0 © 1980 American Chemical Society French and Gardner; Fiber Diffraction Methods ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

FIBER

DIFFRACTION

METHODS


386

Figure 2.

Resilient gel obtained from curdlan at temperatures in excess of 95°C

French and Gardner; Fiber Diffraction Methods ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


25.

FULTON

A N D ATKINS

Microbial Polysaccharide Curdlan

387

M a t e r i a l s and Methods (a) F i b r e Formation and X-ray D i f f r a c t i o n . The suspension of curdlan (2% w/v) was poured into a convoluted glass tube o f i n t e r n a l diameter 1-2mm. and heated i n a water bath to 95°C f o r 10 minutes to form a high s e t g e l . The tube was removed from the bath, broken and gel f i b r e s were p u l l e d out o f the tube and subsequently annealed under tension i n an autoclave a t 120°C f o r an hour before drying over s i l i c a g e l . A t y p i c a l X-ray d i f f r a c t ion pattern from t h i s gel f i b r e i s shown i n Figure 3. An a l t e r n a t i v e f i b r e forming technique involved the d i s s o l u t i o n o f curdlan i n 1M NaOH to give a 5%-7% w/v s o l u t i o n , which was c a s t onto a t e f l o n b l o c k . A f t e r d r y i n g , the r e s u l t i n g f i l m was washed i n water and simultaneously stretched to produce a t h i n gel f i b r e . This was examined by X-ray d i f f r a c t i o n under the f o l l o w i n g c o n d i t i o n s : i) a t 100% r e l a t i v e humidity ( F i g . 4a) ii) a f t e r drying over s i l i c a gel ( F i g . 4b) iii) a f t e r annealing i n an autoclave and drying over s i l i c a gel ( F i g . 4 c ) . X-ray d i f f r a c t i o n photographs were recorded on f l a t f i l m using a camera with pinhole c o l l i n a t i o n (250 um diam.) and N i - f i l t e r e d Cu Ka r a d i a t i o n . C a l c i t e ( c h a r a c t e r i s t i c spacing 0.3035 nm) was used as an i n t e r n a l d i f f r a c t i o n c a l i b r a t i o n . Interplanar spacings were measured o f f the o r i g i n a l photographs and the u n i t c e l l dimensions were r e f i n e d , where p o s s i b l e , by a l e a s t - s q u a r e s procedure. (b) Film Formation and I n f r a - r e d Spectroscopy. A l l deute r a t i o n exchange experiments and drying of deuterated f i l m s were undertaken i n a dry-box. Water vapour was excluded from the i n t e r n a l environment by molecular s i e v e and s i l i c a g e l , which functioned as a d e s s i c a n t and a l s o gave an i n d i c a t i o n o f the water content i n the box. L i q u i d phase deuteration was c a r r i e d out on the polysaccharide by completely immersing the clamped f i l m i n heavy water f o r a t l e a s t 24 hours. The f i l m was removed, d r i e d and placed i n an i n f r a - r e d c e l l with sodium c h l o r i d e windows and the spectrum recorded using the Unicam S . P . 100 i n f r a - r e d spectrometer. Gel F i l m . A gel f i l m was produced by p l a c i n g a suspension (2% w/v) o f curdlan i n water on a clean glass p l a t e . A ground glass r i n g was l a i d on the p l a t e and the suspension evenly d i s t r i b u t e d over the enclosed a r e a . The p l a t e was held over b o i l i n g water and heated to approximately 100°C f o r 10 minutes with minimal evaporation o f water from the sample. A f t e r the f i l m had g e l l e d i t was removed from the glass p l a t e , l a i d on a t e f l o n block and allowed to dry over the drying agent, phosphorous pentoxide. A l k a l i Gel F i l m . Curdlan was d i s s o l v e d i n 1M NaOH (515-715 w/v), spread i n t o a t h i n f i l m on a c l e a n glass p l a t e and allowed to d r y . The f i l m , when immersed i n d i s t i l l e d water, swelled and became transparent as the excess a l k a l i was washed o u t . The gel


FIBER


388

Figure 3.

DIFFRACTION

METHODS

X-ray fiber diffraction photograph obtained from a dry, annealedoriented fiber of curdlan gel (fiber axis vertical)



389


FULTON AND ATKINS

Figure 4. X-ray diffraction photographs of curdlanfibersobtained from alkali solution: (a) at 100% relative humidity; (b) after drying over silica gel; (c) after annealing and drying



390

FIBER

DIFFRACTION

METHODS

f i l m was c a r e f u l l y removed from the glass p l a t e and allowed to dry on a t e f l o n b l o c k . Deuterated g e l . Dried curdlan powder was mixed into a 2% w/v suspension i n heavy water. This suspension was spread on a g l a s s p l a t e and placed i n an a i r t i g h t c o n t a i n e r , shown schemati c a l l y i n Figure 5. The container was heated to approximately 100°C, thus g e l l i n g the polymer. The f i l m was washed i n D2O, to stop the r a p i d l o s s of D2O from the hot f i l m , and then c a r e f u l l y removed and d r i e d . ( A l l operations except that of heating were c a r r i e d out i n the dry-box.) Deuterated a l k a l i g e l . NaOD was d i s s o l v e d i n D2O to form a 1M NaOD s o l u t i o n . Curdlan was d i s s o l v e d i n t h i s s o l u t i o n (5% w/v) and spread into a t h i n f i l m on a t e f l o n b l o c k . A f t e r d r y i n g , the f i l m was washed i n D2O to remove any excess heavy a l k a l i and then allowed to d r y . F i l m from Formic A c i d . A s o l u t i o n (1% w/v) of the curdlan i n formic acid was spread onto a t e f l o n block and allowed to dry, forming a t h i n transparent f i l m . (c) Molecular Model B u i l d i n g . Molecular models were generated by using a linked-atom procedure ( 9 ) . The p o s i t i o n s of the atoms were defined i n terms of the bond l e n g t h s , bond angles and t o r s i o n angles of the molecule. Stereochemical parameters f o r the 3-D-glucose rings i n the standard ^Ci c h a i r were based on the average set given by Arnott and Scott ( 1 0 ) . Bond lengths and bond angles were held constant, i n c l u d i n g the angles f o r the g l y c o s i d i c bridges which were assigned a value o f 116.5°. This defines the glucose residues as r i g i d bodies and leaves the g l y c o s i d i c t o r s i o n angles and a l s o the t o r s i o n angle that defines the o r i e n t a t i o n of the hydroxmethyl group as e x p l i c i t v a r i a b l e s . These parameters were v a r i e d to produce a model with the appropr i a t e h e l i c a l symmetry and p i t c h and with the minimal amount of non-bonded s t e r i c compression ( 1 1 , 1 2 ) . (d) C y l i n d r i c a l l y Averaged F o u r i e r Transform. The c y l i n d r i c a l l y averaged F o u r i e r transforms of the various s t r u c t u r e s were c a l c u l a t e d using the equations of F r a n k l i n and Klug (ljl) and atomic s c a t t e r i n g f a c t o r s computed using the a n a l y t i c a l expression given i n International Tables of Crystallography (J_4). These values were modified to approximate the e f f e c t of the disordered water i n the voids between chains ( 1 5 ) . X-Ray D i f f r a c t i o n The X-ray d i f f r a c t i o n pattern obtained from the d r y , annealed f i b r e (see M a t e r i a l s and Methods) i s shown i n Figure 3. The r e f l e c t i o n s index on a hexagonal u n i t c e l l with dimensions a = b = 1.438 nm, c ( f i b r e a x i s ) = 0.582 nm with meridional r e f l e c t i o n s o c c u r r i n g only on l a y e r l i n e s with A = 2 n, where n i s an i n t e g e r . These c o n s t r a i n t s on the M i l l e r i n d i c e s hkl are i n accordance with



FULTON AND ATKINS

Microbial Polysaccharide Curdlan-

391

suspension __airtight sear

Figure 5.

Diagrammatic representation of the equipment used to obtain curdlan gels in heavy water



392

FIBER DIFFRACTION METHODS

space group assignment P 6 3 . The e s s e n t i a l features o f the X-ray pattern are s i m i l a r to that obtained from 3 - ( l - 3 ) - D - x y l a n ( 6 ) . (The dimensions of the xylan u n i t c e l l are s l i g h t l y d i f f e r e n t : a = b = 1.340 nm, c ( f i b r e a x i s ) = 0.598 nm.) A t k i n s and Parker X§) were able to i n t e r p r e t such a d i f f r a c t i o n pattern i n terms o f a triple-stranded structure. Three c h a i n s , of the same p o l a r i t y , i n t e r t w i n e about a common axis to form a t r i p l e - s t r a n d molecular rope. The i n d i v i d u a l polysaccharide chains t r a c e out a h e l i x with s i x saccharide units per turn and are r e l a t e d to t h e i r neighbours by azimuthal r o t a t i o n s o f 2TT/3 and 4TT/3 r e s p e c t i v e l y , with zero r e l a t i v e t r a n s l a t i o n . A s i m i l a r model f o r curdlan i s i l l u s t r a t e d i n F i g u r e 6. Examinations o f t h i s model shows that each chain repeats at a d i s t a n c e 3 x 0.582 = 1.746 nm. Thus i f f o r any reason the p r e c i s e symmetrical arrangement between chains (or with t h e i r a s s o c i a t e d water o f c r y s t a l l i z a t i o n ) i s d i s r u p t e d , we would expect r e f l e c t i o n s to occur on l a y e r l i n e s which are orders of 1.746 nm. Indeed such a d d i t i o n a l r e f l e c t i o n s have been observed v i a patterns obtained from specimens a t d i f f e r e n t r e l a t i v e humidity (4) o f f e r i n g c o n f i r m a t i o n f o r the t r i p l e - s t r a n d ed model. Since the 5 - p o s i t i o n o f s u c c e s s i v e saccharide units occur on the o u t s i d e surface o f the t r i p l e - s t r a n d molecule i t was evident a f t e r the d i s c o v e r y of such a model f o r 3 - ( l - 3 ) - D - x y l a n t h a t a s i m i l a r s t r u c t u r e would be f e a s i b l e f o r 3 - ( l - 3 ) - D - g l u c a n . D e t a i l e d conformational analyses by Bluhm and Sarko (8) have supported a t r i p l e - s t r a n d right-handed s t r u c t u r e . It i s convenient, i n t h i s p a r t i c u l a r c o n t e x t , to index the observed Bragg r e f l e c t i o n s with respect to the longer 1.746 nm repeat f o r the £ axis i n a n t i c i p a t i o n o f the conformational changes envisaged, which w i l l be discussed l a t e r . Table I provides a l i s t o f the observed r e f l e c t i o n s together with the interplanar spacings. The measured d e n s i t y o f 1.52 g/cm i s compatible with one t r i p l e - s t r a n d e d molecule running through the u n i t eel 1. The X-ray d i f f r a c t i o n patterns obtained f o r the a l k a l i prepared gel (see M a t e r i a l s and Methods) i s shown i n Figure 4 a . I t i s s i m i l a r to the pattern reported by Takeda e t a K (J5) who indexed the r e f l e c t i o n s on an orthorhombic u n i t c e l l with dimensions a = 1.64 nm, b = 2.64 nm and c ( f i b r e a x i s ) = 2.265 nm. I t was proposed that the u n i t c e l l contained two s i n g l e - s t r a n d s e v e n - f o l d h e l i c e s together with an estimated 250 water molecules. The patterns we obtained show d i s c r e t e e q u a t o r i a l r e f l e c t ions and streaked l a y e r l i n e s i n d i c a t i n g l o n g i t u d i n a l d i s o r d e r i n the specimen. The e q u a t o r i a l r e f l e c t i o n s index on a hexagonal net with dimensions ai = ]D = 1.701 nm and the l a y e r l i n e spacing c ( f i b r e axis) = 2.270 nm. The a x i a l advance per saccharide residue i n the t r i p l e - s t r a n d model discussed p r e v i o u s l y ( F i g s . 3 and 6) i s 1.746/6 = 0.291 nm. I t would appear l o g i c a l t h e r e f o r e to a s s o c i a t e the l a r g e r l a y e r l i n e spacing o f 2.270 nm with seven or e i g h t saccharide units although there i s no 3


FULTON

AND

ATKINS


393


25.

Figure 6. Computer generator ORTEP projections of the triple-strand structure corresponding to the x-ray diffraction pattern shown in Figure 3. The pitch of 1.746 nm for each six-fold helix is reduced to 1.746/3 = 0.581 nm by the symmetry related chains, (a) and (b) are different projections normal to the molecular axis and (c) is a projection parallel to the molecular axis.


394

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Table I .

Miller

METHODS

Comparison o f c a l c u l a t e d and measured d-spacings f o r d r y , annealed curdlan f i b r e . The r e f l e c t i o n s are indexed on a hexagonal u n i t c e l l o f dimensions a = b = 1.438 nm, c ( f i b r e a x i s ) = 1.746 nm. indices

d(measured) nm

d(calculi

100, 010

1.241

1.245

110

0.719

0.719

200, 020

0.621

0.623

120, 210

0.464

0.471

300, 030

0.411

0.415

130, 310

0.336

0.345

140, 410

0.263

0.272

150, 510

0.220

0.224

103, 013

0.530

0.524

113

0.455

0.453

203, 023

0.424

0.425

123, 213

0.364

0.366

303, 033,

0.335

0.338

133, 313

0.297

0.297

403, 043

0.271

0.274

006

0.284

0.291

116

0.270

0.270

126, 216

0.251

0.248

306, 036

0.238

0.238



25.

FULTON AND ATKINS


395

p a r t i c u l a r need to have an i n t e g e r number o f saccharide residues per r e p e a t . I t i s s t r a i g h t f o r w a r d to envisage the s i x - f o l d t r i p l e - s t r a n d e d s t r u c t u r e untwisting s l i g h t l y , d e s t r o y i n g the p e r f e c t P63 symmetry r e l a t i o n s h i p , and t h e r e f o r e a l l o w i n g l a y e r l i n e s o f spacing ^2 nm to appear. The measured d e n s i t y i s 1.24 g/cm3 which would allow one t r i p l e - s t r a n d molecule per u n i t c e l l together with approximately 2 water molecules per glucose residue. When the sample i s dehydrated the X-ray d i f f r a c t i o n p a t t e r n obtained i s o f poorer q u a l i t y ( F i g . 4b) and i s s i m i l a r to that reported f o r l e n t i n a n ( 8 ) . We suggest that the removal o f water causes a t w i s t i n g o f the chains back toward the s i x - f o l d t r i p l e stranded model. On a n n e a l i n g , the sample completes t h i s t r a n s i t i o n ( F i g . 4c) by e x h i b i t i n g a pattern s i m i l a r to Figure 3. The r e f l e c t i o n s observed i n Figure 4c index on a hexagonal u n i t c e l l with dimensions a = b = 1.530 nm. c ( f i b r e a x i s ) = 1.76 nm and the measured densTty Ts 1.52 g/crrr. A model f o r the s e v e n - f o l d t r i p l e - s t r a n d e d s t r u c t u r e i s shown i n Figure 7. I n f r a Red Spectra A d e t a i l e d band assignment i s not presented i n t h i s work, but a summary i s given i n Table I I . Throughout a l l the d e u t e r a t i o n and rehydrogenation procedures there are three main features to note i n the s p e c t r a . i) The band a t 3300 c n H , due to the s t r e t c h i n g mode o f the hydrogen bonded hydroxl group, p e r s i s t s a f t e r d e u t e r a t i o n with only a small decrease i n i n t e n s i t y which i s estimated a t 20%. ii) The band a t 1645 c n H , due to the i n - p l a n e deformation of the water molecule. T h i s water i s the s t r o n g l y bonded water of c r y s t a l l i z a t i o n which i s i n a c c e s s i b l e to exchange as i t i s s t i l l present i n the deuterated f i l m s . iii) The band a t 2500 c m " l , due to 0D s t r e t c h on d e u t e r a t i o n which i s removed completely when the sample i s rehydrogenated. G e l - f i l m ( F i g . 8 ) . Deuteration ( B) shows that ROD-D2O ( c r y s t ) i s present with a s l i g h t r e d u c t i o n i n H2O ( c r y s t ) . Therefore only a small proportion o f the water o f c r y s t a l l i z a t i o n i s exchangeable. Differences i n the deuterated spectrum lead to the assumption that some o f the OH groups undergo an exchange r e a c t i o n to form 0D. Rehydrogenation proves that t h i s i s a completely r e v e r s i b l e r e a c t i o n and that the D2O ( c r y s t ) i s exchangeable, contrary to the " b u r i e d " H 0 ( c r y s t ) . F o u r i e r transform i n f r a - r e d techniques were performed on a block o f hydrated g e l , s i m i l a r i n form to that shown i n Figure 2. The r e s u l t i n g spectra are shown i n F i g u r e 9 . Spectrum B i s a f t e r the water background had been s u b t r a c t e d . A comparison o f t h i s spectrum with t h a t o f A i n Figure 8 i n d i c a t e s no real s t r u c t u r a l d i f f e r e n c e between the bulk gel and a d r i e d gel f i l m . 2



396

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DIFFRACTION

2-27 nm

3

s*

Figure 7. Proposed triple-strand structure incorporating seven-fold helical chains. Each chain related azimuthally by 2n/3 and 4tr/3, respectively, and with zero relative translation and similar polarity, (a) and (b) are different projections normal to the helix axis; (c) is a projection looking along the helix axis.


METHODS

FULTON


e II.

A N D ATKINS


The band assignment f o r the i n f r a - r e d spectrum o f a curdlan gel f i l m ( 1 A ) .

397

absorption

Band cm"^

Assignment

3370

OH s t r e t c h bonded)

2917 2894

CH, CH, s t r e t c h

1647

H-0-H bend

1460

C0-H i n - p l a n e bend (hydrogen bonded)

1423

CH£ deformation

1374

CH bend

1317

CH wag

1258

C-0H i n - p l a n e bend

1234

C-0H i n - p l a n e bend o r CH i n - p l a n e bend

1162

C-j-O-Cg antisymmetric

(hydrogen

2

stretch 1107

Antisymmetric in-phase r i n g s t r e t c h o r C-0 stretch

1082

C-0 s t r e t c h OH groups)

1050

C-0 s t r e t c h o r C-C stretch

1007

C-0 s t r e t c h

930

C-0 s t r e t c h alcohol)

894

C-, group frequency (common to linkage)

(secondary

(primary


FIBER

DIFFRACTION

METHODS


398

fttfMwr lea

Figure 8.

4

I

IR spectra obtained from curdlan sample gelled in water: (a) original film; (b) film after deuteration; (c) rehydrogenated film



399


FULTON AND ATKINS

Figure 9. FTIR spectra of: (a) the hydrated gel block (see Figure 2); (b) after water background was substracted


400

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A l k a l i Gel F i l m . The exchange r e a c t i o n i s only p a r t i a l i . e . KUH and H2U ( c r y s t ) bands remain on deuteration ( F i g . 1 0 ) . Apart from the ROD band there i s no apparent change i n the spectrum, implying no apparent s t r u c t u r a l change.


Gel i n D2O. G e l a t i o n i n heavy water s t i l l leaves a high proportion of the polysaccharide undeuterated ( F i g . 1 1 ) , both ROH and H2O ( c r y s t ) bands remaining. Heavy Alkali/Heavy Water Gel F i l m . This strong technique s t i l l cannot completely deuterate curdlan ( F i g . 12) i n d i c a t e d by the p e r s i s t e n t ROH and H2O ( c r y s t ) bands. Furthermore that part of the polymer which i s deuterated can be completely rehydrogenated. F i l m from Formic A c i d . D i s s o l u t i o n i n formic a c i d a l s o e s t e r i f i e s curdlan g i v i n g the formate bands i n the spectrum ( F i g . 13). 2848 cm

•1

— O H - C — Hi

0 1724 cm

1166 cm

-1

-1

-0

— C— H

f - 0 — C-f-H

Exchange f o r OD i s a small percentage, o c c u r r i n g with groups not already e s t e r i f i e d . In a l l the f i v e methods, complete d i s s o c i a t i o n o f the p o l y mer chains was expected and therefore complete d e u t e r i a t i o n except f o r the hydroxy1 groups buried i n the c e n t r a l core o f the t r i p l e - s t r a n d e d molecule. Complete d i s s o c i a t i o n was not o b t a i n ed even when using i n c r e a s i n g l y severe deuteration techniques. Discussion The X-ray evidence favours a model f o r the curdlan gel based on t r i p l e - s t r a n d e d molecules. This s t r u c t u r e i s present i n both the gel prepared from aqueous suspension and from a l k a l i s o l u t i o n . The a l k a l i gel contains the proposed seven-fold t r i p l e - s t r a n d e d model ( F i g s . 7 and 14a) which converts to the more symmetric s i x - f o l d t r i p l e h e l i x by annealing ( F i g s . 6 and 14b). This


FULTON AND ATKINS


401


25.

FrtquMcy

( cm I

(li)-lflirmt mn* tttaMtetf ]

Figure 10. IR spectra obtained from curdlan after gelling from alkali: (a) original film; (b) after deuteration; (c) rehydrogenated film



FIBER

Frequency

DIFFRACTION

METHODS

( cm" )

I (i)- Reference beam attenuated ]

Figure 11.

1R spectrum obtained from gelling curdlan in D O


t


403


FULTON AND ATKINS

Frequency

( cm" )

Figure 12. IR spectra of curdlan after gelling in NaOD and D 0: (a) deuterated film; (b) rehydrogenated film 2



404

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METHODS

s

' ' "00

1

1

3000

2S00

if

Frtqvfucy

1

1

1

1 1S00

1

1

1

1

1

r

1100

I cm"' I

Id) - Rfttrmctfetam•tlfwtltO

Figure 13. IR spectra obtained after dissolving curdlan in formic acid: (a) original film; (b) deuterated film; (c) rehydrogenated film



25.

FULTON

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405

Figure 14. Comparison of triple-stranded models involve: (a) seven-fold helices; and (b) six-fold helices


406

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Triple

METHODS

7-j

»7>

Figure 15. Cylindrically averaged transforms of the two models illustrated in Figures 6 and 7: (a) intensity distribution on successive layer lines for the triplestranded 7 helix; (b) intensity distribution on successive layer lines for the triplestranded 6j helix. Note layer lines with index 1 = 1,2,4, and 5 are absent. t



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Triple Helix

Intermolecularly Bound Water of Crystallization

Figure 16. Cross section of an associated curdlan micelle showing the triple helices hydrogen bonded to the inaccessible water of crystallization

Micelles Containing Triple Helices

3D Gel network (proposed for Curdlan)

Figure 17. The 3D gel network proposed for curdlan. The micelles are associated at particular points forming the junction zones of the gel.



408

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METHODS

t w i s t i n g of the h e l i c e s i s stereochemically f e a s i b l e and does not i n v o l v e d i s r u p t i o n of the i n t e r n a l stack of i n t e r c h a i n hydrogen bonded t r i a d s which s t a b i l i z e the s t r u c t u r e ( 1 6 , 1 7 ) . This t r a n s i t i o n i s i l l u s t r a t e d s c h e m a t i c a l l y i n Figure 14. The conversion from s e v e n - f o l d to s i x - f o l d r e s u l t s i n only a small r e d u c t i o n i n the a x i a l advance per saccharide from 0.324 nm to 0.291 nm. The c y l i n d r i c a l l y averaged F o u r i e r transform of the sevenf o l d and s i x - f o l d t r i p l e - s t r a n d e d s t r u c t u r e s are shown i n Figure 15. The F o u r i e r transform of the s i x - f o l d t r i p l e - s t r a n d e d model i l l u s t r a t e s the symmetry of the system by the t o t a l absence of i n t e n s i t y on l a y e r l i n e s with index I 3 n, where n i s an integer. The F o u r i e r transform of the s e v e n - f o l d t r i p l e - s t r a n d ed s t r u c t u r e shows that i n destroying t h i s p r e c i s e symmetry r e l a t i o n s h i p i n t e n s i t y occurs on a l l l a y e r l i n e s which are orders of the 2.27 nm s p a c i n g . This r e i n f o r c e s the concept of an indigenous t r i p l e - s t r a n d e d s t r u c t u r e which i s perturbed s l i g h t l y by the i n t e r a c t i o n of s o l v e n t . The i n f r a - r e d r e s u l t s show that a high proportion of the various gels are i n a c c e s s i b l e to exchange with deuterium atoms. This phenomenon p e r s i s t s throughout a l l the g e l a t i o n and d i s s o l u t i o n processes. Consequently, the t r i p l e - s t r a n d e d molecules must be bound by hydrogen bonding to the i n t e r s t i t i a l water of c r y s t a l l i z a t i o n , thus forming a m i c e l l a r domain. A cross s e c t i o n of the proposed model i s shown i n Figure 16. Thus we envisage the g e l l i n g mechanism of curdlan to involve the i n t e r a c t i o n of these m i c e l l e s and not the untwining and retwining o f s i n g l e h e l i c e s i n t o t r i p l e - s t r a r i H e d j u n c t i o n zones. Therefore the g e l a t i o n mechanism would i n v o l v e the p a r t i a l d i s s o l u t i o n of the m i c e l l e s , which i n turn would allow t h e i r a s s o c i a t i o n to form the j u n c t i o n zones of the 3-dimensional gel network (Figure 17). Acknowledgements We thank Mr. C.G. Cannon, f o r his help and guidance i n the i n f r a - r e d a n a l y s i s , and Professor T. Harada f o r a generous supply of m a t e r i a l . We are g r a t e f u l to Dr. G. Fraser f o r taking the F o u r i e r transform i n f r a - r e d spectra ( F i g . 9 ) . We thank the Science Research Council f o r f i n a n c i a l support.


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ABSTRACT

The molecular structure and gelling mechanism of the bacter ial polysaccharide curdlan was investigated using X-ray diffract ion and infra-red spectroscopy. Gels were obtained from heating an aqueous suspension to 95°C and also from treatment of the alkaline solution. X-ray diffraction patterns of the oriented gel, obtained from aqueous suspension were indexed on a hexagonal unit cell with dimensions a = b = 1.438 nm and c (fibre axis) = 1.746 nm and interpreted in terms of a triple-stranded structure with space group symmetry P63, by analogy with the previously published structure for β-(1-3)-D-xylan. The X-ray patterns of the gel formed from alkaline solution gave equatorial reflections which indexed on a hexagonal lattice a = b = 1.701 nm and a layer line spacing c (fibre axis) = 2.270 nm. This is interpreted as a perturbed triple-stranded structure which undergoes a transition on annealing to form the six-fold triple helix. Infra-red spect roscopy and deuteration of the polysaccharide gel films indicated the presence of inexchangeable hydroxyl groups and the interstit ial water of crystallization. This interstitial water forms a hydrogen bonded network with the triple helices, binding them into a micellar structure; it is the association of these micelles which form the gel network.


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Literature Cited 1.

Harada, T. in "Extracellular Microbial Polysaccharides, ed. Sanford and Laskin, ACS Symposium Series 45, 1977, 265-283.

2.

Takeda, H.; Yasuoka, N.; Kasai, N.; Harada, T. Polymer J., 1978, 10, 365-368.

3.

Harada, T. in "Polysaccharides in Food", ed. Blanshard and Mitchell, Butterworths, London, 1978, 18-68.

4.

Marchessault, R.H.; Deslandes, Y.; Ogawa, K.; Sundarajan, P. R. Can. J. Chem., 1977, 55, 300-303.

5.

Atkins, E.D.T.; Parker, K.D.; Preston, R.D. Proc. Roy. Soc. B, 1969, 173, 205-221.

6.

Atkins, E.D.T; Parker, K.D., J. Poly. S c i . , 1969, C28, 69-81.

7.

Jelsma, J ; Kreger, D.R. Carbonhyd. Res. 1975, 43, 200-203.

8.

Bluhm, T.L; Sarko, A. Can. J. Chem., 1977, 55, 293-300.

9.

Arnott, S.; Wonacott, J.A. Polymer, 1966, 7, 157-166.

10. Arnott, S.; Scott, W.E. J. Chem. Soc., (Perkin Transactions II), 1972, 324-335. 11. Gardner, K.H.; Magill, J.M.; Atkins, E.D.T. Polymer, 1978 19, 370-378. 12. Smith, P.J.C.; Arnott, S. Acta Cryst. A, 1978, 34, 3-11. 13. Franklin, R.E; Klug, A. Acta Crystallogr., 1955, 8, 777-780. 14. International Tables for X-ray Crystallography, Kynoch Press, Birmingham, 1974. 15. Fraser, R.D.B.; MacRae, T.D.; Suzuki, E. J. Appl. Cryst., 1978, 11, 693-694. 16. Atkins, E.D.T.; Parker, K.D. Nature, 1968, 220, 784-785. 17.

Fulton, W.S.; Atkins, E.D.T.; Cannon, C.G. (in preparation).

RECEIVED

February

19,

1980.


The Gelling Mechanism and Relationship to Molecular Structure of

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