Thermal Properties of Silk Proteins in Silkworms - American Chemical

Chapter 19 Thermal

Properties in 1

of

Silk

Proteins

Silkworms 2

3

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on September 14, 2015 | http://pubs.acs.org Publication Date: December 3, 1993 | doi: 10.1021/bk-1994-0544.ch019

Shigeo Nakamura , Jun Magoshi , and Yoshiko Magoshi 1

Department of Applied Chemistry, Faculty of Engineering, Kanagawa University, Kanagawa-ku, Yokohama 221, Japan National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305, Japan National Institute of Sericultural and Entomological Science, Tsukuba, Ibaraki 305, Japan 2

3

The thermal behavior of s i l k proteins in s i l k worms has been investigated in d e t a i l . As the temperature of the amorphous random-coil fi broin of Bombyx mori is r a i s e d , water i s evaporated off up to 100º C. Intra- and inter molecular hydrogen bonds are broken between 150 and 180° C. The glass t r a n s i t i o n is ob served at 175° C. The random-coil to β-form t r a n s i t i o n accompanied by reformation of hydrogen bonds occurs above 180° C. C r y s t a l l i zation to the β-form crystals starts at above 190° C. The α-form and β-form fibroins lose water up to 100° C on heating, and molecular motions in the crystalline regions starts at 175° C. The α-form to β - f o r m t r a n s i t i o n is induced thermally at 270° C. Silk sericin shows a glass transition at 170° C and c r y s t a l lizes at 250° C. The glass transition tempera ture of wild s i l k fibroins varies with species of silkworm ranging from 160 to 210º C. It has been observed that, when being heat-treated, s i l k filament starts to lose i t s weight gradually at 175° C, and the color changes from white to light yellow, brown and then to black at 250° C (1). Schwenker and Dusenbury (2) has f i r s t recorded DTA curves of s i l k filament. In the DTA curves of s i l k f i l a ment, a peak due to evaporation of water appears at 108°C and an endothermic peak of thermal degradation occurs at 326° C. Several studies have been reported on the thermal behavior of s i l k . Ishikawa et a l . (3) found that the DTA peak due to thermal degradation appears at 305, 348 and 0097-6156/94/0544-0211$06.00/0 © 1994 American Chemical Society In Silk Polymers; Kaplan, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

212

SILK POLYMERS: MATERIALS SCIENCE AND BIOTECHNOLOGY

2 3 4 ° C f o r d o m e s t i c s i l k , t u s s a h s i l k and s i l k f r o m anaphe infracta, respectively. However, s y s t e m a t i c r e s e a r c h on t h e t h e r m a l b e h a v i o r o f s i l k , e s p e c i a l l y f i b r o i n , t h e main component o f s i l k filament, has n o t been c a r r i e d out u n t i l the authors report d e t a i l e d i n v e s t i g a t i o n of thermal behavior of s i l k f i b r o i n (4).


EXPERIMENTAL L i q u i d s i l k u s e d f o r e x p e r i m e n t s was t a k e n f r o m t h e p o s t e r i o r p a r t of the middle d i v i s i o n of the s i l k gland i n f u l l - g r o w n l a r v a e (one day b e f o r e s p i n n i n g ) o f s i l k w o r m . A f t e r t h e s e r i c i n e n v e l o p i n g f i b r o i n a s an o u t e r l a y e r was removed by w a s h i n g t h o r o u g h l y w i t h d e i o n i z e d w a t e r , the l i q u i d s i l k was d i l u t e d w i t h w a t e r . The aqueous s o l u t i o n o f s i l k f i b r o i n was d i a l y z e d a g a i n s t t h e d e i o n i z e d w a t e r and d i l u t e d t o a p r e d e t e r m i n e d c o n c e n t r a t i o n . Amorphous f i l m w i t h r a n d o m - c o i l c o n f o r m a t i o n was c a s t f r o m a 0.8% s o l u t i o n onto g l a s s p l a t e s . Differential s c a n n i n g c a l o r i m e t r y (DSC) c u r v e s were o b t a i n e d on a P e r k i n - E l m e r t y p e II DSC a p p a r a t u s u n d e r nitrogen. T h e r m a l e x p a n s i o n measurement was made u s i n g a hand-made a p p a r a t u s by r e c o r d i n g t h e change i n l e n g t h o f the f i l m specimen under a c o n s t a n t t e n s i o n o f l e s s than 250gcm" . The dynamic modulus E* and t h e l o s s t a n g e n t ( t a n δ) were measured w i t h R h e o v i b r o n model D D V - I I a t a f r e q u e n c y o f 110Hz. D i e l e c t r i c measurement was made w i t h a 4270A automatic capacitance bridge (Yokogawa-Hewlett Packard) e q u i p p e d w i t h a vacuum c e l l . A t h i n g o l d l a y e r was e v a p o r a t e d on t h e s u r f a c e o f t h e s p e c i m e n . X - r a y d i f f r a c t i o n p a t t e r n s were o b t a i n e d w i t h a R i g a k u D-3F x - r a y a p p a r a t u s . The t e m p e r a t u r e o f s p e c i m e n was c o n t r o l l e d t o _+0.5' C . I n f r a r e d s p e c t r a were m e a s u r e d on a JASCO model 402G i n f r a r e d s p e c t r o m e t e r . The s p e c i m e n was p l a c e d between two C a F o r N a C l p l a t e s . 2

2

RESULTS AND DISCUSSION Thermal Behavior o f Random-coil F i b r o i n .

Two e n d o t h e r m i c

p e a k s a p p e a r a t a b o u t 100 and 2 8 0 ° C i n t h e DSC c u r v e o f amorphous s i l k f i b r o i n o f d o m e s t i c s i l k w o r m Bombyx m o r i with random-coil conformation (Figure 1(a)) (5). An e x o t h e r m i c peak i s o b s e r v e d a t 2 1 2 ° C and an e n d o t h e r m i c s h i f t o f t h e b a s e l i n e o c c u r s a t 175* C . The p r o m i n e n t e n d o t h e r m i c p e a k a t a b o u t 2 8 0 ° C i s a t t r i b u t e d to the d e g r a d a t i o n of s i l k f i b r o i n . A weight l o s s s t a r t s a b r u p t l y a t a b o u t 2 5 0 ° C i n t h e TG c u r v e . The b r o a d e n d o t h e r m i c peak a t a b o u t 100" C i s due t o t h e e v a p o r a t i o n of water i n the specimen. T h i s peak becomes s m a l l e r by d r y i n g the s p e c i m e n p r i o r to measurement (Figure 1(b)). The e n d o t h e r m i c s h i f t s t a r t i n g a t 1 7 5 ° C i s the glass transition of silk fibroin, because it d i s a p p e a r s by h e a t - t r e a t i n g the specimen at 220° C to induce c r y s t a l l i z a t i o n (Figure 1 ( C ) ) .

In Silk Polymers; Kaplan, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


19. NAKAMURA ET AL.

Thermal Properties of Silk Proteins in Silkworms

Further detailed i n v e s t i g a t i o n is undertaken to c l a r i f y t h e t h e r m a l b e h a v i o r o f s i l k f i b r o i n w i t h randomc o i l conformation. I n t h e l i n e a r t h e r m a l e x p a n s i o n c u r v e o f amorphous r a n d o m - c o i l f i b r o i n f i l m ( F i g u r e 2) ( 4 ) , t h e specimen c o n t r a c t s v e r y s l o w l y up t o 100" C . This contraction is a t t r i b u t e d to the e v a p o r a t i o n o f water, because the con t r a c t i o n becomes s m a l l e r w i t h p r i o r d r y i n g a t 100° C . The l e n g t h of specimen b e g i n s to i n c r e a s e a b r u p t l y at about 165'C. The t e m p e r a t u r e a t w h i c h t h e i n t e r s e c t i o n o f t h e s t r a i g h t l i n e at the lower temperature s i d e w i t h t h a t at t h e h i g h e r t e m p e r a t u r e s i d e i s 175 C, w h i c h a g r e e s w e l l w i t h t h e g l a s s t r a n s i t i o n t e m p e r a t u r e o b s e r v e d by DSC. The dynamic modulus ( Ε ' ) and t h e l o s s t a n g e n t ( t a n δ ) o f amorphous r a n d o m - c o i l f i b r o i n f i l m i s r e c o r d e d as a f u n c t i o n o f t e m p e r a t u r e ( F i g u r e 3) ( 4 ) . Figure 3 contains a l s o t h e r e s u l t s f o r f i l m s w i t h d i f f e r e n t β-form contents or d i f f e r e n t degrees o f c r y s t a l l i n i t y . These specimens a r e p r e p a r e d by i m m e r s i n g t h e r a n d o m - c o i l f i b r o i n f i l m i n m i x t u r e s o f CH3OH and H 0 w i t h v a r i o u s c o m p o s i t i o n s ( 6 , 7). A t lower t e m p e r a t u r e s the E o f r a n d o m - c o i l f i b r o i n h a r d l y d e c r e a s e s w i t h t e m p e r a t u r e and s t a r t s t o d e c r e a s e a b r u p t l y at about 170°C. I t r e a c h e s t h e minimum a t 200° C and t h e n i n c r e a s e s . A f t e r p a s s i n g t h r o u g h t h e maximum a t about 220°C, i t decreases a g a i n . The t a n δ b e g i n s t o i n c r e a s e a b r u p t l y a t a b o u t 170° C and r e a c h e s t h e maximum ( a ^ d i s p e r s i o n ) a t 195° C . Then i t t u r n s to d e c r e a s e a g a i n and r e a c h e s t h e minimum a t 230° C . The maximum o f E ' a t 220° C i n c r e a s e s i n h e i g h t w i t h i n c r e a s i n g £ - f o r m c o n t e n t or c r y s t a l l i n i t y , whereas the minimum a t 200° C becomes s m a l l e r . Only a shoulder is o b s e r v e d f o r t h e s p e c i m e n w i t h 25% c r y s t a l l i n i t y . F o r t h e s p e c i m e n s w i t h 27 and 30% c r y s t a l l i n i t y , b o t h t h e maximum and the minimum d i s a p p e a r . The t a n δ peak shifts s l i g h t l y and c o n t i n u o u s l y to h i g h e r t e m p e r a t u r e and d e c r e a s e s i n h e i g h t w i t h i n c r e a s i n g β-form content or crystallinity. O n l y a s h o u l d e r a p p e a r s f o r t h e 27 and 30% c r y s t a l l i n e specimens. A c c o r d i n g l y , t h e t a n δ peak a t 195° C i s r e l a t e d t o t h e m o l e c u l a r m o t i o n s i n t h e amorphous p h a s e , namely w h i c h i s i n d u c e d by t h e s e g m e n t a l m o t i o n s o f f i b r o i n m o l e c u l e s i n t h e amorphous r e g i o n . The i n c r e a s e i n modulus s t a r t i n g a t about 200° C i s a t t r i b u t e d t o t h e c r y s t a l l i z a t i o n o f amorphous f i b r o i n a c c o m p a n i e d by t h e r a n d o m - c o i l t o β-form conformational t r a n s i t i o n . The d i e l e c t r i c c o n s t a n t o f r e g e n e r a t e d amorphous fibroin films increases slowly with increasing temperature, h a s t h e maximum a t a b o u t 100° C a n d then s t a r t s t o i n c r e a s e a b r u p t l y a t a b o u t 150° C ( 8 ) . The peak a t 1 0 0 ° C i s due t o t h e e v a p o r a t i o n o f w a t e r i n t h e s p e c i m e n , b e c a u s e t h e peak c a n n o t be o b s e r v e d a f t e r the specimen i s h e a t - t r e a t e d . Two l o s s p e a k s a p p e a r i n t h e d i e l e c t r i c l o s s t a n g e n t measurement. The p r o m i n e n t peak a t 175° C i s i n d e p e n d e n t o f f r e q u e n c y . The m i n o r peak i s r e m a r k a b l y a f f e c t e d by the f r e q u e n c y and s h i f t s f r o m -40° C a t 1kHz t o 30°C a t 1MHz. The p r o m i n e n t l o s s peak does n o t e

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213

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280

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f/°C F i g u r e 1. DSC c u r v e s o f amorphous s i l k f i b r o i n i n the r a n d o m - c o i l c o n f o r m a t i o n : (a) untreated, (b) d r i e d p r i o r to measurement, (c) h e a t - t r e a t e d at 200° C . Heating rate: 8Kmin . Reproduced with p e r m i s s i o n from R e f e r e n c e 5. _ 1

t/°C F i g u r e 2. Thermal e x p a n s i o n of amorphous silk f i b r o i n i n the r a n d o m - c o i l c o n f o r m a t i o n . Heating rate: 3Kmin . Reproduced with permission from Reference 4. _ 1



19.

NAKAMURA ET AL.


F i g u r e 3. Temperature dependence of the dynamic modulus E (a) a n d t h e d y n a m i c l o s s t a n g e n t t a n δ (b) o f s i l k f i b r o i n w i t h d i f f e r e n t β-form content or different c r y s t a l l i n i t y . Reproduced w i t h p e r m i s s i o n from R e f e r e n c e 4 . T


215

216


appear f o r the h e a t - t r e a t e d specimen. T h i s peak i s c a u s e d by t h e c o n f o r m a t i o n a l t r a n s i t i o n t o t h e β - f o r m w h i c h has occurred during heat-treatment. The m i n o r p e a k is a t t r i b u t e d t o t h e l o c a l m o t i o n ( β-dispersion) of the amorphous f i b r o i n w i t h a b s o r b e d w a t e r s i n c e t h e s p e c i m e n d r i e d a t 100 C i n vacuum shows a peak a t a b o u t 10 C (0.3Hz). T h e r e f o r e , t h e peak i s s u p p o s e d t o be due t o t h e H 0-coupled l o c a l mode m o t i o n o f f i b r o i n i n t h e randomc o i l conformation. I t i s a l s o o b s e r v e d from t h e t e m p e r a t u r e dependence o f t h e l i n e w i d t h o f b r o a d - l i n e NMR t h a t t h e m i c r o B r o w n i a n m o t i o n i n t h e a m o r p h o u s r e g i o n b e c o m e s t o be v i g o r o u s a t a b o u t 170' C ( 9 ) . X - r a y d i f f r a c t i o n p a t t e r n s a r e r e c o r d e d f o r amorphous f i b r o i n f i l m during stepwise increase i n temperature ( F i g u r e 4) ( 4 ) . No c r y s t a l l i n e p e a k i s o b s e r v e d b e l o w 190° C . The a m o r p h o u s h a l o s l i g h t l y s h i f t s t o s m a l l e r a n g l e s owing to the t h e r m a l e x p a n s i o n o f the specimen. When t h e t e m p e r a t u r e i s r a i s e d t o 2 0 0 C , t h e c h a r a c t e r i s t i c x - r a y d i f f r a c t i o n p a t t e r n o f the β -form crystals appears. T h e r e f o r e , t h e e x o t h e r m i c peak o f DSC a t 212° C i s a t t r i b u t e d t o t h e c r y s t a l l i z a t i o n o f amorphous randomc o i l f i b r o i n to the β -form c r y s t a l s . Infrared spectra of random-coil s i l k f i b r o i n are r e c o r d e d i n t h e range 4 0 0 - 1 7 0 0 c m w i t h s t e p w i s e i n c r e a s ing temperature (4). The s p e c t r u m a t room t e m p e r a t u r e shows bands 1 6 6 0 , 1 5 4 0 , 1235 and 6 5 0 c m . These bands a r e a s s i g n e d t o t h e amide I , amide I I , amide I I I and amide V bands o f t h e r a n d o m - c o i l c o n f o r m a t i o n . A t 180° C, bands c a u s e d by t h e β-form appear at 1630, 1535, 1265, and 700cm . However, even a t 2 1 0 ° C i n f r a r e d s p e c t r a do n o t c o r r e s p o n d e x c l u s i v e l y t o t h e ^ - f o r m , and bands c a u s e d by the r a n d o m - c o i l conformation p e r s i s t . In the i n f r a r e d s p e c t r a o f the r a n d o m - c o i l f i b r o i n t h e a b s o r b a n c e o f t h e amide I , I I , and I I I bands d e c r e a s e s l i n e a r l y w i t h i n c r e a s i n g t e m p e r a t u r e and an a b r u p t change i n t h e s l o p e o c c u r s a t 180' C ( F i g u r e 5) ( 4 ) . The a b s o r bance o f t h e amide I band o f t h e β - f o r m a t 1 6 3 0 c m " d e c r e a s e s l i n e a r l y and s t a r t s t o i n c r e a s e a b r u p t l y a t 190"C (Figure 6). On c o o l i n g , t h e a b s o r b a n c e o f t h e s e b a n d s increases l i n e a r l y with decreasing temperature a n d no a b r u p t change i n the s l o p e i s o b s e r v e d as seen i n the b r o k e n l i n e s i n F i g u r e s 5 and 6. T h i s means t h a t t h e r a n d o m - c o i l t o β - f o r m t r a n s i t i o n o c c u r s i r r e v e r s i b l y on heating. The a b s o r b a n c e o f t h e 3 4 5 0 c m band due t o f r e e ( n o t hydrogen-bonded) OH s t r e t c h i n g v i b r a t i o n d e c r e a s e s w i t h t e m p e r a t u r e up t o 150' C, t h e n i n c r e a s e s up t o 180" C and t h e n a g a i n d e c r e a s e s , whereas t h a t o f t h e 3 2 7 0 c m " bands c a u s e d by t h e h y d r o g e n - b o n d e d NH s t r e t c h i n g d e c r e a s e s up t o 1 9 0 ° C and t h e n i n c r e a s e s . The change o f t h e a b s o r b a n c e o f t h e s e bands w i t h h e a t i n g i s a l s o i r r e v e r s i b l e . On cooling, the absorbance of these bands increases monotonously w i t h d e c r e a s i n g temperature. The i n c r e a s e i n a b s o r b a n c e o f t h e 3 4 5 0 c m " band on h e a t i n g i s i n d u c e d by t h e b r e a k i n g o f i n t r a - and i n t e r m o l e c u l a r hydrojgen bonds a n d t h e d e c r e a s e i n a b s o r b a n c e o f t h e 3450cm band i s e

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19. NAKAMURA ET AL.


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F i g u r e 4. X - r a y d i f f r a c t i o n p a t t e r n s o f amorphous r a n d o m - c o i l f i b r o i n i n the course o f s t e p - w i s e i n crease i n temperature.

0

100

200

f/°C F i g u r e 5. A b s o r b a n c e s o f the amide I , I I and I I I bands o f t h e r a n d o m - c o i l c o n f o r m a t i o n as a f u n c t i o n of temperature: heating ( ); c o o l i n g ( ). R e p r o d u c e d w i t h p e r m i s s i o n from R e f e r e n c e 4 .


217

218


c a u s e d by the r e f o r m a t i o n o f the h y d r o g e n bonds. T h e r e f o r e , decrease i n absorbance of the 3 4 5 0 c m and t h e i n c r e a s e i n a b s o r b a n c e o f t h e 3 2 7 0 c m " band a t above 180° C r e v e a l t h e r e f o r m a t i o n o f h y d r o g e n bonds once b r o k e n by heating. From t h e s e r e s u l t s , t h e t h e r m a l b e h a v i o r o f amorphous s i l k f i b r o i n w i t h r a n d o m - c o i l c o n f o r m a t i o n i s summarized as f o l l o w s . As the t e m p e r a t u r e i s i n c r e a s e d , water e x i s t i n g i n t h e s p e c i m e n i s e v a p o r a t e d o f f up t o a b o u t 100° C . The i n t r a - and i n t e r m o l e c u l a r h y d r o g e n bonds a r e b r o k e n i n t h e t e m p e r a t u r e r a n g e 1 5 0 - 1 8 0 ° C, r e s u l t i n g i n the i n c r e a s e i n m o l e c u l a r m o t i o n s . The g l a s s t r a n s i t i o n occurs at 175°C. The i r r e v e r s i b l e r a n d o m - c o i l t o β-form conformational transition takes place above 180° C accompanying the r e f o r m a t i o n of hydrogen bonds. Thermally induced c r y s t a l l i z a t i o n to β -form c r y s t a l s s t a r t s at a b o u t 190° C . - 1


1

Thermal Behavior o f C r y s t a l l i n e β-Form F i b r o i n . For the c r y s t a l l i n e β - f o r m f i b r o i n , t h r e e e n d o t h e r m i c DTA p e a k s a r e o b s e r v e d a t a b o u t 1 0 0 , 252 and 317° C a t a h e a t i n g r a t e of l K m i n . A b r o a d peak a t 1 0 0 ° C i s due t o t h e e v a p o r a t i o n of water. Two e n d o t h e r m i c p e a k s a t h i g h e r t e m p e r a t u r e s are a t t r i b u t e d to the d e g r a d a t i o n o f the β - f o r m silk fibroin. I n t h e DTA c u r v e o f t h e β - f o r m f i b r o i n , a w e i g h t l o s s o c c u r s i n two s t e p s . These r e s u l t s indicate t h a t the d e g r a d a t i o n o f the β -form s i l k f i b r o i n proceeds i n two s t e p s . I n t h e DTA c u r v e s o f the β - f o r m f i b r o i n a t d i f f e r e n t draw r a t i o s , a b r o a d e n d o t h e r m i c peak a t a b o u t 1 0 0 ° C i s due t o e v a p o r a t i o n o f w a t e r a n d an e n d o t h e r m i c p e a k a t h i g h e r temperature i s a t t r i b u t e d to the d e g r a d a t i o n o f the £-form f i b r o i n ( F i g u r e 7) ( 1 0 ) . The l a t t e r peak s h i f t s t o h i g h e r t e m p e r a t u r e s w i t h i n c r e a s i n g draw r a t i o . The dynamic modulus E ' o f t h e β - f o r m f i b r o i n f i l m i n c r e a s e s w i t h draw r a t i o , b u t t h e t a n δ d e c r e a s e s ( 1 0 ) . The d e c r e a s e i n modulus a t a b o u t 100° C i s n o t a f f e c t e d by draw r a t i o and i s a t t r i b u t e d t o e v a p o r a t i o n o f w a t e r . The d e c r e a s e i n modulus and t h e i n c r e a s e i n t a n δ s t a r t i n g a t 175° C a r e due t o t h e o n s e t o f m o l e c u l a r m o t i o n s i n t h e c r y s t a l l i n e r e g i o n b e c a u s e an a b r u p t i n c r e a s e i n s p a c i n g i s o b s e r v e d i n x - r a y d i f f r a c t i o n p a t t e r n s a t 175° C . - 1

Thermal B e h a v i o r o f C r y s t a l l i n e α-Form F i b r o i n . The o r i e n t e d α - f o r m f i b r o i n c a n n o t be o b t a i n e d b e c a u s e t h e α - f o r m t o β-form c o n f o r m a t i o n a l t r a n s i t i o n o c c u r s by drawing (11). The E and the t a n δ o f t h e drawn α - f o r m f i b r o i n f i l m s behave v e r y s i m i l a r l y t o t h o s e o f t h e drawn β - f o r m f i l m s ( F i g u r e 8 ) , because the α - f o r m to β-form t r a n s i t i o n i n d u c e d by d r a w i n g ( 1 0 ) . A t l o w e r draw r a t i o s a s m a l l e n d o t h e r m i c peak a p p e a r s a t 270° C i n DTA c u r v e s and i s a t t r i b u t e d t o t h e t h e r m a l l y i n d u c e d α - f o r m t o β-form t r a n s i t i o n , w h i c h i s supported by x - r a y d i f f r a c t i o n p a t t e r n s . f


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Thermal Properties of Silk Proteins in Silkworms - American Chemical

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