Conformation Transition of Silk Fibroin Inducedby Blending Chitosan

XIN CHEN, WENJUN LI, TONGYIN YU

Department of Macromolecular Science, Laboratory of Molecular Engineering of Polymers, Fudan University,220 Handan Road, Shanghai, 200433, People’s Republic of China

Received 31 October 1996; revised 3 March 1997; accepted 5 March 1997

ABSTRACT: The conformation of silk fibroin in silk fibroin/chitosan (SF/CS) blendmembrane was analyzed by infrared spectrum, X-ray diffractometry, and Raman spec-trum. The results demonstrated that the SF could show b-sheet conformation whenthe SF content in blend membranes was 10% (w/w) and 60–80% (w/w), while thepure SF membrane showed random coil conformation. A mechanism of the conformationtransition was suggested in that the SF chain could use the rigid CS chain as a moldplate to stretch itself to form a b-sheet structure according to the strong hydrogen bondbetween CS and SF. Therefore, a new concept, named ‘‘Polymer-Induced ConformationTransition,’’ was proposed. q 1997 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 35:2293–2296, 1997Keywords: conformation transition; silk fibroin; chitosan; blending; induction

INTRODUCTION behavior of SF induced by blending another natu-ral biopolymer—chitosan (CS) has been investi-gated. CS [Fig. 1(a)] is one of the few naturalSilk fibroin (SF) is one of the most extensively

studied materials among the natural biopolymers. cationic polyelectrolytes yielded from chitin [Fig.1(b)] , one of the most abundant polysaccharides,An important reason for the interest in this natu-

ral biopolymer lies in the fact that it can exist in and has a widespread distribution in inverte-brates and lower plants. Furthermore, a new con-several conformations, such as random coil and

b-sheet.1 The SF membrane cast from an aqueous cept, named ‘‘Polymer-Induced ConformationTransition’’, was proposed.solution shows mostly random coil conformation,2

although the physical properties of SF membranemainly depend on the intermolecular hydrogenbond, i.e., b-sheet conformation.3 The most com- EXPERIMENTALmon method to convert the random coil conforma-tion of SF into b-sheet conformation is to treat

Materialsthe membrane by an organic solvent, such asmethanol.4,5 Recently, some studies reported that Raw silk was degummed twice with 0.5% (w/w)changing the solvation temperature when casting NaHCO3 solution at 1007C for 30 min and thenSF membrane6 or mixing some other polymer in washed with distilled water. Degummed silk wasSF membrane, such as sodium alginate3 could in- dissolved in 9.5 mol/L LiBr solution. After dial-crease the b-sheet conformation in SF membrane. ysis against distilled water for 3 days, the solutionIn the present study, the conformation transition was filtered, then the SF solution was obtained.

CS was prepared from chitin according to themethod described in our previous article.7 Its vis-

Correspondence to: X. Chencosity-average mol weight was 3.0 1 105, and the

Journal of Polymer Science: Part B: Polymer Physics, Vol. 35, 2293–2296 (1997)q 1997 John Wiley & Sons, Inc. CCC 0887-6266/97/142293-04 N-deacelylation degree was 86% (mol/mol) , re-

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tent in blend membrane was 60–80% (w/w), theamide II of SF showed the peak not only at 1542cm01 , which was attributed to the random coilconformation, but also at 1535 cm01 which wasattributed to the b-sheet conformation.9 There-fore, it could be suggested that when SF contentin the blend membrane was 10% (w/w), almostall SF conformation was b-sheet structure. In theFigure 1. Structure of chitosan (a) and chitin (b).meantime, when SF content in the blend mem-brane was 60–80% (w/w), a part of the SF alsoshowed b-sheet conformation.spectively. Therefore, the CS used in this study

actually was the copolymer of CS and chitin. TheCS solution was prepared by dissolving CS in 2% X-Ray Diffraction Curves(w/w) acetic acid.

To confirm the conformation transition of SF inThe SF/CS blend membranes were obtained bySF/CS blend membranes, X-ray diffraction curvescasting the mixture of 2% (w/w) SF solution andof blend membranes were examined (Fig. 3). Pure2% (w/w) CS solution onto a PET plate andSF membrane showed no obvious 2u peak, whichallowing the solvent to evaporate in the air atdenoted that the SF conformation in the pureroom temperature.membrane was mainly random coil. The pure SFmembrane, which was treated by ethanol, exhib-

Measurements ited 2u peaks at 15.77 and 27.97, which were as-signed to the a crystal (silk I) as well as the peaksInfrared spectra of SF/CS blend membranes were

measured with a Magna-IR550 spectrometer atroom temperature.

X-ray diffraction measurements on the mem-branes were performed with a Rigaku d/MAX-IIAdiffractometer, using CuKa radiation. Irradiationconditions were 40 kV and 20 mA, and the scan-ning rate was 87 /min of the diffraction angle 2u.

Raman spectra were acquired with a Spex 1403Ramanor spectrometer, using the 514.5 nm lineof an argon laser operated, at 100 mV, as the excit-ing source.

RESULTS AND DISCUSSION

IR Spectra

The IR spectra of pure SF, pure CS, and SF/CSblend membranes were shown in Figure 2. Thepure SF membrane showed absorption bands at1654 cm01 (amide I), 1542 cm01 (amide II) , 1241cm01 (amide III) , and 659 cm01 (amide V), attrib-uted to the SF with random coil conformation.8,9

The pure CS showed around 898 cm01 and 1153cm01 peaks of assigned saccharide structure. Italso showed the characteristic peaks of d (N{H)resonance of CS at around 1561 cm01 .10 However,when SF content in the blend membrane was 10%(w/w), the amide I of SF showed the peak at 1630 Figure 2. FTIR spectra of SF/CS blend membranes:cm01 , which was attributed to the b-sheet confor- (a) pure SF; (b) SF/CS 8/2; (c) SF/CS 7/3; (d) SF/CS

6/4; (e) SF/CS 1/9; (f ) pure CS.mation. It could also be found that when SF con-

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TRANSITION OF SILK FIBROIN 2295

(w/w), a clear 2u peak at 20.67 could be found,which was attributed to b crystal. Nevertheless,because the 2u peak of CS at 23.07 was so strong,the another 2u peak of SF at 24.87 was covered.When the SF content in the blend membrane was60–80% (w/w), the 2u peaks at 20.67 and 23.07could both be found.

Raman Spectra

The another method to confirm the conformationtransition of SF in the SF/CS blend membranesis Raman analysis. In our previous study, it hasbeen found that the SF exhibited characteristicpeaks at 1106 cm01 , 1252 cm01 , 1270 cm01 (amideIII) , and 1660 cm01 (amide I) for random coil con-formation, and 1004 cm01 , 1085 cm01 , 1233 cm01

(amide III) , and 1678 cm01 (amide I) for b-sheetconformation.9,11 Figure 4 showed that the pureSF membrane had characteristic peaks at 1108cm01 , 1252 cm01 (amide III) , and 1662 cm01 (am-ide I), which assigned random coil conformation.The pure CS membrane showed characteristicpeaks around 1000 cm01 , 1084 cm01 , 1292 cm01 ,and 1692 cm01 , respectively.

When SF content in the blend membrane was

Figure 3. Wide-angle X-ray diffraction profiles of SF/CS blend membranes: (a) pure SF treated by ethanol;(b) SF/CS 8/2; (c) SF/CS 7/3; (d) SF/CS 6/4; (e) SF/CS 1/9; (f ) pure CS.

at 20.67 and 24.87, which belonged to b crystal(silk II) .6 Meanwhile, pure CS membrane showedthe 2u peaks at 16.57, 23.07 (crystal I) , and 18.07 Figure 4. Raman spectra of SF/CS blend mem-(crystal II) , respectively. branes. (a) pure SF; (b) SF/CS 8/2; (c) SF/CS 7/3; (d)

SF/CS 1/9; (e) pure CS.When SF content in blend membrane was 10%

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2296 CHEN, LI, AND YU

peak of d (N{H) for CS, which attributed to thehydrogen bond, shifts at 1630 cm01 and 1561cm01 . It also could be found that the amide I ofSF shifted from 1654 cm01 to 1649 cm01 when SFcontent in the blend membrane was 60–80% (w/w). The another important reason that we consid-ered that led to the conformation transition of SFwas the rigidity of the CS chain. From Figure 5we could find that the SF chain could use the rigid

Figure 5. b-Sheet structure formation of SF by CS chain as a mold plate to stretch itself and,strong hydrogen bonding between SF and CS. therefore, form a b-sheet structure by the strong

hydrogen bond between CS and SF. Further in-vestigation of the facts into the mechanism of10% (w/w), it could be found that the SF showedPolymer-Induced Conformation Transition, suchcharacteristic peaks at 1004 cm01 (amide III) andas the influence of the composition of SF/CS blend1680 cm01 (amide I), which was assigned to themembranes and blending other polymers, are be-

b-sheet conformation, although some other char-ing undertaken.acteristic peaks were covered by CS because the

SF content was rather low. For the blend mem-The authors are grateful to the National Natural Sci-

branes whose SF content was 70% (w/w) and 80% ence Foundation of China for the support of this study.(w/w), it could be clearly seen that the character- The authors also wish to thank Dr. Wei Zhong and Missistic amide III peak at 1234 cm01 and amide I Yuhua Lu for their valuable suggestion on this work.peak at 1668 cm01 and 1670 cm01 . According tothe amide I, peak in the blend membrane ap-peared in the middle of the characteristic peak of REFERENCES AND NOTESrandom coil conformation (1660 cm01) and b-sheet conformation (1678 cm01) , it could be con- 1. J. Magoshi, Y. Magishi, and S. Nakamura, ACS

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9. D. W. Jones, Introduction to the Spectroscopy ofTo explain this interesting phenomenon, we Biological Polymers, Academic Press, New York,considered the hydrogen bond forming between 1976.SF and CS in blend membrane. For instance, 10. K. D. Yao, T. Peng, M. F. A. Goosen, J. M. Min, andwhen SF content in the blend membrane was 10% Y. Y. He, J. Appl. Polym. Sci., 48, 343 (1993).(w/w), it could be found that the 1638 cm01 ab- 11. S. Zheng, G. Li, W. Yao, and T. Yu, Appl. Spectrosc.,

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