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Structure and Molecular Conformation of Tussah Silk Fibroin Films: Effect of Methanol MASUHIRO TSUKADA,'. GlULlANO FREDDI,' PATRlZlA MONTI,' ALESSANDRO BERTOLUZZA,' and NOBUTAMI KASAI' 'National Institute of Sericultural and Entomological Science, Tsukuba City, lbaraki 305, Japan; 'Stazione sperimentale per la Seta, via G. Colombo, 81, 201 33 Milano, Italy; 'Dipartimento di Biochimica, Centro di Studi sulla Spettroscopia Raman, Universiti di Bologna, via Belrneloro, 8/2, 401 26 Bologna, Italy; and 4Kobe Women's University, Surna, Kobe 654, Japan SYNOPSIS Structural changes of tussah (Antheraea pernyi) silk fibroin films treated with different water-methanol solutions at 2OoC were studied as a function of methanol concentration and immersion time. X-ray diffraction measurements showed that the a-helix structure, typical of untreated tussah films, did not change for short immersion times (2 min), regardless of methanol concentration. However, crystallization to &sheet structure was observed fol- lowing immersion of tussah films for 30 min in methanol solutions ranging from 20 to 60% (v/v). IR spectra of tussah films untreated and methanol treated for 2 min exhibited strong absorption bands at 1265, 892, and 622 cm-', typical of the a-helix conformation. The intensity of the bands assigned to the &sheet conformation (1245, 965, and 698 cm-') increased for the sample treated with 40% methanol for 30 min. Raman spectra of tussah films with a-helix molecular conformation exhibited strong bands at 1657 (amide I), 1263 (amide HI), 1106, 908, 530, and 376 cm-'. Following a + (? conformational transition, amide I and I11 bands shifted to 1668, and to 1241, 1230 cm-I, respectively. The band at 1106 cm-l disappeared and new bands appeared at 1095 and 1073 cm-', whereas the intensity of the bands at 530 and 376 cm-' decreased significantly. 0 1995 John Wiley & Sons, Inc. Keywords: tussah silk fibroin methanol treatment molecular conformation crystal- lization - Raman spectroscopy analysis INTRODl CTION Silk fibroin is a fibrous biopolymer produced by dif- ferent silkworm species. Its typical amino acid com- position, as well as primary and higher-order struc- tures, offers an attractive opportunity for studying the relationships between chemical structure, mo- lecular conformation, physical properties, and func- tionality.'s2 Besides its use as a textile fiber, it has been recently investigated as a starting material for nontextile application^,^ especially in the biomedical and biotechnological field. High purity silk fibroin can be easily obtained from cocoons, in fiber form, * To whom all correspondence should be addressed. Journal of Polymer Science: Part B: Polymer Physics. Vol. 33,1995-2001 (1995) 0 1995 John Wiley & Sons. Inc. CCC 0887-62%/95/141995-07 or from silk glands, as aqueous solution. The latter represents an ideal starting material for the prep- aration of various kinds of fibroin-based materials,' such as gel, powder, porous, and homogeneous membranes. By casting a silk fibroin solution prepared either from domestic (Bornbyx rnori) or wild (Antheraea pernyi, tussah) silk, a film is obtained, whose struc- ture is characterized by a high packing density of the fibroin molecules. The physicochemical prop- erties of these films strongly depend on their mo- lecular conformation and crystallinity. Crystalliza- tion can be easily induced by simple physicochemical treatments, such as immersion in selected organic solvents, thermal treatments, application of me- chanical forces (stretching), etc. Magoshi et aL5ex- tensively studied the structural changes of amor- 1996

Structure and molecular conformation of tussah silk fibroin films: Effect of methanol

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Structure and Molecular Conformation of Tussah Silk Fibroin Films: Effect of Methanol

MASUHIRO TSUKADA,'. GlULlANO FREDDI,' PATRlZlA MONTI,' ALESSANDRO BERTOLUZZA,' and NOBUTAMI KASAI'

'National Institute of Sericultural and Entomological Science, Tsukuba City, lbaraki 305, Japan; 'Stazione sperimentale per la Seta, via G. Colombo, 81, 201 33 Milano, Italy; 'Dipartimento di Biochimica, Centro di Studi sulla Spettroscopia Raman, Universiti d i Bologna, via Belrneloro, 8/2, 401 26 Bologna, Italy; and 4Kobe Women's University, Surna, Kobe 654, Japan

SYNOPSIS

Structural changes of tussah (Antheraea pernyi) silk fibroin films treated with different water-methanol solutions a t 2OoC were studied as a function of methanol concentration and immersion time. X-ray diffraction measurements showed that the a-helix structure, typical of untreated tussah films, did not change for short immersion times (2 min), regardless of methanol concentration. However, crystallization to &sheet structure was observed fol- lowing immersion of tussah films for 30 min in methanol solutions ranging from 20 to 60% (v/v). IR spectra of tussah films untreated and methanol treated for 2 min exhibited strong absorption bands at 1265, 892, and 622 cm-', typical of the a-helix conformation. The intensity of the bands assigned to the &sheet conformation (1245, 965, and 698 cm-') increased for the sample treated with 40% methanol for 30 min. Raman spectra of tussah films with a-helix molecular conformation exhibited strong bands at 1657 (amide I), 1263 (amide H I ) , 1106, 908, 530, and 376 cm-'. Following a + (? conformational transition, amide I and I11 bands shifted to 1668, and to 1241, 1230 cm-I, respectively. The band a t 1106 cm-l disappeared and new bands appeared a t 1095 and 1073 cm-', whereas the intensity of the bands at 530 and 376 cm-' decreased significantly. 0 1995 John Wiley & Sons, Inc. Keywords: tussah silk fibroin methanol treatment molecular conformation crystal- lization - Raman spectroscopy analysis

INTRODl CTION

Silk fibroin is a fibrous biopolymer produced by dif- ferent silkworm species. Its typical amino acid com- position, as well as primary and higher-order struc- tures, offers an attractive opportunity for studying the relationships between chemical structure, mo- lecular conformation, physical properties, and func- tionality.'s2 Besides its use as a textile fiber, it has been recently investigated as a starting material for nontextile application^,^ especially in the biomedical and biotechnological field. High purity silk fibroin can be easily obtained from cocoons, in fiber form,

* To whom all correspondence should be addressed. Journal of Polymer Science: Part B: Polymer Physics. Vol. 33,1995-2001 (1995) 0 1995 John Wiley & Sons. Inc. CCC 0887-62%/95/141995-07

or from silk glands, as aqueous solution. The latter represents an ideal starting material for the prep- aration of various kinds of fibroin-based materials,' such as gel, powder, porous, and homogeneous membranes.

By casting a silk fibroin solution prepared either from domestic (Bornbyx rnori) or wild (Antheraea pernyi, tussah) silk, a film is obtained, whose struc- ture is characterized by a high packing density of the fibroin molecules. The physicochemical prop- erties of these films strongly depend on their mo- lecular conformation and crystallinity. Crystalliza- tion can be easily induced by simple physicochemical treatments, such as immersion in selected organic solvents, thermal treatments, application of me- chanical forces (stretching), etc. Magoshi et aL5 ex- tensively studied the structural changes of amor-

1996

1996 TSUKADA ET AL.

phous B. mori silk fibroin films with random coil conformation induced by various kinds of treatment. Organic solvents, such as methanol, proved to be highly effective, the crystallization mainly proceed- ing through random coil + @-sheet transition. However, only limited information is available on the conformational transitions accompanying crys- tallization of tussah silk fibroin films. Tussah films cast from aqueous solution contain a variable pro- portion of a-helix, as well as random coil confor- m a t i ~ n , ~ ~ due to the presence of - (ala), - repeats in the primary structure.' One of the authors inves- tigated the changes in molecular conformation and crystalline structure induced by drying temperature and drying rateY7 thermal treatments: and immer- sion in a 50% water-methanol s o l ~ t i o n . ~

The aim of this article is to study the crystalli- zation of tussah silk fibroin films by applying a sys- tematic approach, which consists in treating the films with water-methanol solutions by varying methanol concentration and immersion time. This approach proved to be useful when applied to B. m r i films." Untreated and methanol-treated tussah films have been characterized by x-ray diffracto- metry, IR, and Raman spectroscopy. Although x- ray and IR measurements have been extensively ap- plied to the study of the crystalline structure and molecular conformation of silk fibroin materials,' very little is known about Raman spectra of silk fibroins. The Raman spectrum of Bombyx mori silk fibroin was reported by some author^,^*'^-'^ but no Raman investigation has been reported so far about tussah silk fibroin. This technique is a powerful tool for studying proteins at a molecular level, because it provides information on the peptide backbone structure, as well as on conformational transitions and changes in side-chain environment. Because proteins usually show very complicated Raman spectra, the results obtained on tussah silk fibroin films are discussed referring to literature data on model polypeptides. The amino acid composition of tussah silk fibroin is characterized by a large amount of alanine residues (> 40 mol %), and - (ala),- sequences are reported to form the primary structure of the crystalline regions.' For this reason, poly-L- alanine has been used as model polypeptide for tus- sah silk fibroin.

EXPERIMENTAL

Materials

Solution-cast native silk fibroin films were obtained as follows. Liquid silk was collected from the pos-

terior division of the silk gland in full grown larvae of tussah silkworm (A. pernyi). The liquid silk was gently dispersed into deionized water, until a final concentration of about 0.3% (w/v) was reached. Tussah silk fibroin films were prepared by casting the aqueous solution on a polyethylene plate as sub- strate, at 20°C and 65% relative humidity. Tussah films about 50 pm thick were obtained. They were first removed from the polyethylene substrate and then immersed in different water-methanol solu- tions [methanol concentration: 20, 40, 60, 80, and 100% (v/v)], for different immersion times (2 and 30 min). The temperature of the treatment bath was 20°C. Untreated and methanol-treated tussah films were dried under vacuum for 2 days before mea- surements.

Measurements

Wide-angle x-ray diffraction curves were recorded with a Rigaku Denki Co, Ltd. diffractometer, using the CuKa radiation ( A = 1.54 A), a t a scanning rate of l"/min. Voltage and current of the x-ray source were 30 kV and 20 mA, respectively.

Infrared spectra were measured with a Perkin El- mer 1760X Fourier Transform Infrared Spectro- photometer, under dried air, in the spectral region of 1400-400 cm-'.

NIR-FT-Raman spectra were obtained by means of a Bruker FRA-106 Raman module coupled to an IFS-66 FT-spectrophotometer. An Nd/YAG Laser (1064 nm) was used as exciting source.

RESULTS

X-Ray Diffraction Curves

The crystalline structure of tussah silk fibroin films treated with different water-methanol solutions for 2 and 30 min was studied by x-ray diffractometry. Figure 1 shows the x-ray diffraction curves of the films subjected to methanol treatment for 2 min. The 26 pattern is characterized by the presence of two peaks at 11.5" and 22.0°, corresponding to the crystalline spacings of 7.69 and 4.03 A, respectively. This pattern is characteristic of tussah films with a-helix crystalline structure,' as obtained by casting from aqueous solution. It is interesting to note that the x-ray diffraction curves did not show significant changes, following the treatment of tussah films with different methanol solutions for 2 min.

Figure 2 shows the x-ray diffraction profiles of tussah films immersed in different water-methanol

EFFECT OF METHANOL ON TUSSAH SILK FIBROIN 1997

- t

Y - .- C 3 % L e e a - .-

Figure 1. films treatc

I I I I I I 10 15 20 25 30 35

29 (deg)

K-ray diffraction curves of tussah silk fibroin with water-methanol solutions for 2 min.

Methanol concentration (v/v): (a) 20%, (b) 40%, (c) 60%, (d) 80%, (e) 100%.

solutions for 30 min. The samples immersed in 80% and 100% methanol solutions (Fig. 2d,e) showed the crystalline pattern typical of the a-helix structure. On the other hand, the films immersed in 20%,40%, and 60% methanol solutions (Fig. 2a-c) exhibited rather different diffraction curves, with a major peak at about 20.5", and two minor peaks, in shoulder form, at 16.5" and 25.5". The crystalline spacings corresponding to these peaks (4.33, 5.37, and 3.78 A) are characteristic of tussah films with 0-sheet structure.*

IR Spectra

Changes in molecular conformation of tussah silk fibroin films, induced by immersion in different wa- ter-methanol solutions for 2 and 30 min, were in- vestigated by IR spectroscopy. The spectral region 1400-400 cm-' presents some absorption bands sensitive to the molecular conformation of silk fi- broin.6 The untreated tussah film (Fig. 3a) showed strong absorption bands at 1270 (amide 111), 892 (amide IV), and 623 cm-' (amide V), attributed to the a-helix structure, together with a random coil band at 662 cm-'. The absorption bands assigned to the 0-sheet conformation (1247, 966, and 698 cm-') remained rather weak. This IR pattern did not show significant changes following immersion in different water-methanol solutions for 2 min (Fig. 3b-e).

I 1 I I 1 I 10 15 20 25 30 35

23 (deg)

Figure 2. X-ray diffraction curves of tussah silk fibroin films treated with water-methanol solutions for 30 min. Methanol concentration (v/v): (a) 20%, (b) 40%, (c) 60%, (d) 80%, (e) 100%.

On extending the immersion time up to 30 min, the film treated with 40% methanol (Fig. 4c) exhib- ited a dramatic increase in intensity of the absorp- tion bands assigned to the &sheet conformation. Among the other conformation-sensitive bands, those attributed to a-helix decreased slightly or re- mained almost unchanged, whereas the band at 662

12GJ L12L7 662

I I I I I I0 1200 1000 800 600 400

Wavenumber (cm-l)

Figure 3. IR spectra of tussah silk fibroin films un- treated and treated with water-methanol solutions for 2 min. Methanol concentration (v/v): (a) untreated, (b) 40%, ( c ) 60%, (d) 80%, (e) 100%.

1998 TSUKADA ET AL.

a

L I I I I I 1400 1200 1000 800 600 400

Wavenumber (crn-l)

Figure 4. IR spectra of tussah silk fibroin films un- treated and treated with water-methanol solutions for 30 min. Methanol concentration (v/v): (a) untreated, (b) 20%, (c) 4076, (d) 80%, (e) 100%.

cm-', attributed to the random coil conformation, became very weak. Tussah films treated with solu- tions containing an amount of methanol higher or lower than 40% (Fig. 4b,d,e) exhibited an IR pattern similar to that of the untreated sample (Fig. 4a), with a-helix and random coil absorption bands stronger than @-sheet bands.

Raman Spectra

Raman spectra of untreated and methanol-treated tussah films were measured in the spectral region 2000-200 cm-' (Fig. 5). The immersion time was 30 min, and methanol concentration was 20%, 50%, and 80%. Differences among the spectra can be ob- served not only in the amide I and I11 range, but also below 1200 cm-', due to the presence of some conformationally sensitive bands. Taking the Ra- man spectrum of the untreated tussah film as a ref- erence (Fig. 5a), the films treated with 20% and 50% methanol (Fig. 5b,c) exhibited noticeable changes, whereas that immersed in 80% methanol (Fig. 5d) remained almost unchanged, as far as the position and intensity of the bands are concerned.

Following the treatment with 20% and 50% methanol, the tussah films showed a shift to higher wave number of the amide I band (from 1657 to 1668 cm-'). Moreover, remarkable changes in the shape of the amide I11 bands were observed. In par- ticular, the band at 1263 cm-' decreased and a new

and more intense band appeared at 1241 cm-', with a shoulder at about 1230 cm-'. In the spectral region below 1200 cm-l, the sharp and intense band at 1106 cm-' almost completely disappeared and new bands at 1095 and 1073 cm-' appeared. The band at 530 cm-' dramatically decreased and that at 376 cm-' almost completely disappeared.

DISCUSSION

When tussah silk fibroin films are treated with dif- ferent water-methanol solutions, changes in molec- ular conformation and crystalline structure may be induced. From our results it appears that these structural transitions depend on both methanol concentration and immersion time.

The x-ray diffraction profiles did not show any modification of the a-form crystalline structure of tussah films immersed in various methanol solutions for 2 min. The shorter the treatment time, the lower the annealing effect of the solvent treatment, in good agreement with previously reported result^.^ The formation of @-sheet crystals was observed when the immersion time was increased to 30 min. However, only a specific range of methanol concentration, from 20% to 60%, was effective in inducing @-sheet crystallization. A t higher methanol concentration, the treatment conditions were less favorable to in- duce the conformational changes leading to for- mation of @-sheet crystals, even after 30 min of im- mersion time.

The results of the IR spectroscopy analysis are consistent with the above x-ray data. In fact, judging from the intensity of the conformation-dependent IR absorption bands, the prevailing molecular con- formation of tussah films treated for 2 min is a- helix and random coil. The IR bands assigned to the @-sheet conformation are present as well, but remain rather weak. On the other hand, the tussah film treated with 40% methanol for 30 min exhibited strong @-sheet absorption bands, whereas the other samples showed only slight differences, compared to the untreated film. These findings confirm the hy- pothesis, previously suggested on the basis of x-ray data, that there is a range of methanol concentration values that is most effective in inducing crystalli- zation of tussah films to @-sheet form. Above and below this range, the conditions may become rather critical, resulting either in a partial crystallization or in the absence of any structural change. In fact, the sample treated with 20% methanol for 30 min, although exhibiting the presence of @-sheet crystals

EFFECT OF METHANOL ON TUSSAH SILK FIBROIN 1999

I 1657 1655 1337 1263 1lO6 Kx)5 908 853 759 530 376

I I I I I I I I 2000 1800 1600 1400 1200 1000 800 600 400 200

Wavenum bers ( cm-' )

Figure 5. FT-Raman spectra of tussah silk fibroin films untreated and treated with water-methanol solutions for 30 min. Methanol concentration (v/v): (a) untreated, (b) 2096, (c) 50%, (d) 80%.

by x-ray diffractometry, appeared only partially transformed on the basis of IR analysis. On the other hand, both techniques agreed in detecting the ab- sence of appreciable transitions in tussah films treated with 80-100% methanol solutions for 30 min.

The Raman spectra of tussah silk fibroin films may be discussed on the basis of the spectroscopic studies of poly-L-alanine in the a -he l i~a l '~ , ' ~ and an- tiparallel /3-sheet15 conformations, as well as refer- ring to the vibrational analysis of /3-poly-L-alanine." The bands at 1657 (amide I), 1263 (amide 111), 1106, 908, 530, and 376 cm-' present in the Raman spec- trum of untreated tussah film are due to vibration of parts of protein chains containing prevalently L- alanine residues in a form structure. This assign- ment is consistent with previously reported struc- tural as well as with x-ray and IR results obtained in this work.

The changes in the Raman spectra of tussah films treated with 20% and 50% methanol solutions are mainly due to modification of L-alanine conforma- tion. The behavior of amide I and I11 bands, which shift to 1668, and to 1241, 1230 cm-', respectively, suggests the occurrence of an LY + 8 transition, be- cause they appear in a spectral region typical of pro- teins in the @-sheet conformation. As concerns amide I11 band, the 1241 and 1230 cm-' components were attributed to the presence of random coil and 8-sheet structures, respectively, in the Raman spec- trum of mechanically deformed poly-L-alanine, which underwent a + 8 tran~i t ion. '~ However, on

the basis of a subsequent vibrational analysis,16 it has been suggested that these bands are due to split- ting of amide I11 in the antiparallel @-sheet confor- mation, without excluding the possibility that the disordered polymer also has a band at 1243 cm-'. We agree with the vibrational analysis assignment that, in the case of tussah film, is consistent with previous result^,^ which suggest an almost complete crystallization into 8 structure after immersion in 50% methanol solution for more than 5 min.

Frushour and Koenig" observed the disappear- ance of the 1106 cm-' band and the appearance of two bands between 1060 and 1000 cm-' in the spec- trum of mechanically deformed poly-L-alanine. They suggested that the presence of the latter bands may be of general use in identifying the 8-sheet confor- mation. A similar feature was exhibited by the tussah films treated with 20% and 50% methanol, the new /3 form bands being located at 1095 and 1073 cm-'. Moreover, the same authors hypothesized that the strong line at 530 cm-', attributed to the C = O in plane bending, is useful in measuring the a-helix content of polypeptides. Accordingly, we observed a dramatic decrease in intensity of this band following the treatment of tussah film with methanol.

The results of Raman spectroscopy are in good agreement with the above discussed x-ray and IR data, confirming that the conformational transition of tussah silk fibroin films is dependent on both methanol concentration and treatment time. Pro- vided that an immersion time as long as 30 min is

2000 TSUKADA ET AL.

needed, the three analytical techniques well agree in detecting the absence of any significant transition for the samples treated with 2 80% methanol so- lutions. The optimum methanol concentration should fall in the range 40-60%. Below 40%, the treatment may result in a partial annealing of the film. In fact, P-sheet structure was detected by x- ray and Raman measurements, whereas both a-helix and @-sheet conformation-dependent absorption bands appeared in the IR spectrum of the film im- mersed in 20% methanol solution.

On the basis of Raman data, crystallization to 0- sheet structure seems to proceed mainly through an a + p conformational transition. However, the ab- sence of strong marker bands, specifically assigned to the random coil conformation, made it difficult to evaluate the contribution of the amorphous do- mains of the films, However, IR measurements con- tributed to clarify this aspect. In fact, the intensity of the band at 662 cm-’ dramatically decreased in the sample treated with 40% methanol for 30 min, therefore suggesting that both a + p and random coil + 0 conformational transitions might occur, following methanol treatment of tussah silk fibroin films. These results are in good agreement with those previously reported by one of the authors,’ who sug- gested that the mechanism of crystallization by sol- vent treatment should imply the contribution of both random coil and a-helix fiber domains.

Tussah films obtained by casting can be consid- ered almost anhydrous, as they contain only a small amount of bound water. When a film is immersed in water or water-methanol solutions, diffusion from the solution into the film should take place, owing to the sharp concentration gradient existing in the contact area between the bath and the film surface. The rate of diffusion into the film, as well as the effectiveness in inducing swelling and rearrange- ment of the inter-/intramolecular hydrogen bonds, might depend on the composition of the water- methanol solution. In fact, both water and methanol molecules may give rise to structures stabilized by hydrogen bonds, the size of these structures being dependent on the steric hindrance of H and CH3 side groups involved in their formation. Moreover, methanol is a poor solvent for silk fibroin, and its diffusion into the film matrix might take place very slowly, in the absence of water, or in the presence of small amounts of water. Therefore, without the necessary swelling action of water, ineffective or in- complete annealing of tussah films may result. From our data it has been elucidated that a methanol con- centration not exceeding 50-60%, and an immersion

time of 30 min, can be considered optimum condi- tions for swelling the film matrix, loosening the in- tramolecular hydrogen bonds in the a-helix struc- ture, as well as inducing conformational transition leading to crystallization to P-sheet structure of tus- sah films on drying.

CON CLUS I0 NS

Structural changes of tussah silk fibroin films, ob- tained by casting from aqueous solution, can be in- duced by treatment with water-methanol solutions at room temperature (20°C). The optimum meth- anol concentration ranges from 40% to 60% whereas the immersion time should be long enough to permit diffusion of the solvent system into the film, followed by swelling and rearrangement of the hydrogen bonds.

Crystallization is likely to proceed through con- formational transition from both a-helix to &sheet (as confirmed by x-ray and Raman measurements), and random coil to &sheet structure (as shown by IR spectra). The Raman spectra of tussah silk fi- broin, first reported in this study, showed signifi- cative changes of the bands sensitive to the molec- ular conformation of both untreated and methanol- treated films. Shift of amide I band, change in intensity of amide I11 bands, as well as changes in the spectral pattern below 1200 cm-’, can be used as a probe for the conformational analysis of fibroin- based materials.

REFERENCES AND NOTES

1. R. D. B. Fraser and T. P. MacRae, in Conformation in Fibrous Proteins and Related Synthetic Polypeptides Academic Press, New York, 1973, p. 293.

2. D. Kaplan, W. Wade Adarns, B. Farmer, and C. Viney, in Silk Polymers. Materials Science and Biotechnology, ACS Symposium Series 544, American Chemical SO- ciety, Washington, DC, 1994, P. 2.

3. T. J. Bunning, H. Jiang, W. Wade Adams, R. L. Crane, B. Farmer, and D. Kaplan, in Silk Polymers. Materials Science and Biotechnology, ACS Symposium Series 544, American Chemical Society, Washington, DC, 1994, p. 353.

4. T. Asakura, M. Demura, Y. Watanabe, and K. Sato, J. Polym. Sci., Polym. Phys. Ed., 30, 693 (1992).

5. J. Magoshi, Y. Magoshi, and S. Nakamura, J. Appl. Polym. Sci., Appl. Polym. Symp., 41, 187 ( 1985).

6. J. Magoshi, Y. Magoshi, and S. Nakamura, J. Appl. Polym. Sci., 21, 2405 (1977) .

EFFECT OF METHANOL ON TUSSAH SILK FIBROIN 2001

7. M. Tsukada, J. Polym. Sci., Polym. Phys. Ed., 24, 457 ( 1986 ) .

8. M. Nagura, S. Yamazaki, and M . Tsukada, Proc. 7th Znt. Wool Text. Res. Conf., Tokyo 1985, Vol. 1, p. 345.

9. M. Tsukada, J. Polym. Sci., Polym. Phys. Ed., 24, 1227 (1986).

10. M. Tsukada, Y. Goto, M. Nagura, N. Minoura, N. Kasai, and G. Freddi, J. Polym. Sci., Polym. Phys. Ed., 3 2 , 9 6 1 (1994).

11. B. G. Frushour, and J. L. Koenig, in Advances in In- frared and Raman Spectroscopy, R. J . H. Clark and R. E. Hester (eds.), Heyden, London, 1975, p. 35.

12. K. Itoh, J. Magoshi, Y. Magoshi, and S. Nakamura, Proc. 6th Int. Conf. on Raman Spectroscopy, Bangalore, Heyden, London, Vol. 2, p. 290, 1978.

13. D. B. Gillespie, C. Viney, and P. Yager, in Silk Poly- mers. Materials Science and Bwtechnology, ACS Sym- posium Series 544, American Chemical Society, Washington, DC, 1994, p. 155.

14. M. C. Chen and R. C. Lord, J. Am. Chem. SOC., 96, 4750 (1974).

15. B. G. Frushour and J. L. Koenig, Biopolymers, 13, 455 ( 1974).

16. W. H. Moore and S. Krimm, Biopolymers, 15, 1227 (1986) .

Received October 10, 1994 Revised February 15, 1995 Accepted April 6, 1995