Molecular Orientation Behavior of Silk Sericin Film ... - ACS Publications

Biomacromolecules 2005, 6, 2049-2057

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Molecular Orientation Behavior of Silk Sericin Film as Revealed by ATR Infrared Spectroscopy Hidetoshi Teramoto† and Mitsuhiro Miyazawa*,‡ New Silk Materials Laboratory, Insect Biotechnology and Sericology Department, National Institute of Agrobiological Sciences, 1-4-8 Gohda, Okaya, Nagano 394-0021, Japan, and Biomaterial Development Laboratory, Insect Biomaterial and Technology Department, National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan Received January 24, 2005; Revised Manuscript Received March 28, 2005

This paper reports the structure-dependent molecular orientation behavior of sericin, an adhesive silk protein secreted by silkworm, Bombyx mori. Although application of sericin as a biomaterial is anticipated because of its unique characteristics, sericin’s physicochemical properties remain unclear, mainly because of its vulnerability to heat or alkaline treatment during separation from fibroin threads. This study employed intact sericin obtained from fibroin-deficient mutant silkworm to investigate the relationship between molecular orientation and the secondary structure of sericin. Sericin films were artificially stretched after moistening with aqueous ethanol of various concentrations. The resulting molecular orientation was analyzed using polarized infrared spectroscopy. These analyses indicated that formation of aggregated strands among extended sericin chains induced by ethanol treatment is the key to generating molecular orientation. Strong intermolecular hydrogen bonds are inferred to allow aggregated strands’ stretching-force transmission, thereby causing molecular orientation. Introduction Sericin is a family of adhesive silk proteins that are synthesized exclusively in the middle silk glands of silkworm, Bombyx mori.1,2 Sericin is characterized by its unusually high serine content (ca. 32%),3,4 which gives it a high hydrophilicity5 and a sensitivity to chemical modification.6 Recent studies have revealed unique characteristics of sericin, such as induction of heterogeneous nucleation of apatite7 and enhanced attachment of primary cultured human skin fibroblasts.8 Sericin is therefore anticipated as a promising natural resource that offers specific properties for developing novel protein-based materials.9,10 Sericin’s physicochemical properties are not sufficiently elucidated because sericin is easily degraded by heat or alkaline treatment during processing for separation from fibroin threads. Yamamoto and colleagues recently developed a new strain of Bombyx mori named Sericin-hope,11,12 which is an improved strain of fibroin-deficient mutant silkworm.13,14 The Sericin-hope silkworm secretes sericin almost exclusively. Therefore, separation from fibroin threads is unnecessary, allowing easy obtainment of intact sericin. This intact sericin forms films or gels of better mechanical properties than those made from degraded sericin,15 and is quite useful not only as a biomaterial but also for investigating sericin’s physicochemical properties. * To whom correspondence should be addressed. E-mail: miyazawa@ nias.affrc.go.jp. † New Silk Materials Laboratory, National Institute of Agrobiological Sciences. ‡ Biomaterial Development Laboratory, National Institute of Agrobiological Sciences.

We previously investigated the structure and molecular orientation of sericin fiber derived from the Sericin-hope silkworm using infrared spectroscopy.16 The study demonstrated that sericin fiber has a disordered structure with little molecular orientation, indicating that it is difficult to orient sericin molecules by the natural spinning of silkworms. The characteristic of sericin not to form a fibrous structure by spinning is reasonable considering its putative function to facilitate the spinning of silk fiber.16 However, molecular orientation of polymeric materials is important because it is closely associated with their physicochemical properties. Sericin’s availability as a material will expand greatly if it becomes possible to control its molecular orientation. Orientation of sericin has been a matter of interest in association with silk spinning mechanisms and silk processing.17,18 Hirabayashi and colleagues analyzed the molecular orientation of stretched sericin films using infrared dichroism and X-ray diffraction.17 They suggested that stretching induced β-sheet formation with hydrogen bonds aligned perpendicular to the stretching direction. Komatsu reported that the degree of molecular orientation of sericin film increased with the stretching ratio.18 These studies suggest that artificial stretching of sericin film orients sericin chains parallel to the stretching direction. However, it seems unlikely that sericin transforms into oriented β-sheet structure by stretching similarly to fibroin fiber because sericin fiber obtained from the Sericin-hope silkworm has little molecular orientation.16 In addition, the secondary structure of sericin film varies according to its preparation condition, which might affect its orientation behavior. Therefore, the relationship between molecular orientation and the secondary

10.1021/bm0500547 CCC: $30.25 © 2005 American Chemical Society Published on Web 06/04/2005

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structure of sericin must be examined to elucidate the orientation mechanism of sericin molecules. This study investigated the molecular orientation behavior of sericin films having different secondary structures. Sericin films are rigid when dry, but they become extensible when swollen with aqueous media. Alcohol generally changes the protein secondary structure. For those reasons, we stretched sericin films that were swollen with aqueous alcohol of varied concentrations to clarify the influence of the secondary structure on the sericin orientation. Infrared spectroscopy with polarized light was employed for analyzing the degree of molecular orientation after stretching. Correlation between molecular orientation and secondary structure was investigated; then we speculated upon the sericin molecule orientation mechanism. Experimental Section Materials. Cocoons of Sericin-hope silkworms were used in this study as the sericin source. Silkworms were reared on mulberry leaves. All chemicals used in this study were reagent grade and were used without further purification. Sample Preparation. The fresh cocoon shells of Sericinhope silkworms (600 mg) were dissolved into 8 M LiBr aqueous solution (24 mL) at 35 °C for 24 h. Centrifugation and filtration removed insoluble residues. The supernatant was adjusted to ca. pH 8 with 1 M Tris-HCl buffer of pH 9 (6 mL) to prevent sericin precipitation during subsequent dialysis. The solution was dialyzed thoroughly to deionized water using a membrane (Spectra/Por MWCO 6-8000; Spectrum Laboratories Inc., Rancho Dominguez, CA) to yield ca. 1 wt % sericin solution. Sericin films were obtained on a polystyrene dish (28.5 × 8.5 cm2) after water evaporation at 10 °C from 40 mL of the sericin solution. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The dialyzed sericin solution prepared as above (1 mL) was mixed with urea (960 mg) to denature sericin completely. It was then adjusted to 2 mL with water. The 8 M ureadenatured sericin solution was mixed with an equal amount of SDS-PAGE sample buffer (0.1 M Tris-HCl of pH 6.8, 4% SDS, 12% 2-mercaptoethanol, 20% glycerol) and boiled for 5 min. The sample solution was adjusted to pH 6.8 with dilute HCl solution immediately before electrophoresis. SDS-PAGE was performed according to the method of Laemmli19 using a ready-made 3-10% gradient gel (ATTO Bioscience, Tokyo, Japan). After electrophoresis, the gel was stained with Coomassie brilliant blue R-250. Molecular weights were estimated using a molecular weight marker (Precision Protein Standards; Bio-Rad Laboratories Inc., Hercules, CA). The relative abundance of protein components was estimated using a densitometer (AE-6920M; ATTO Bioscience, Tokyo, Japan). Stretching. Sericin films with 15 ( 3 µm thickness were cut to rectangular pieces (20 × 5 mm2) and attached to a tensile testing machine (RTA-100; Orientec Corp., Tokyo, Japan) with a gauge length of 10 mm. The sericin films were moistened thoroughly by spraying water or aqueous ethanol (10, 30, 50, or 70%). After equilibration for 2 min, the swollen films were stretched to a draw ratio of four at a

Teramoto and Miyazawa

crosshead speed of 20 mm/min. Tensile stress during stretching was recorded simultaneously. After stretching, films were dried for 1-2 h without removal from the instrument thereby preventing relaxation. Mechanical Testing. Mechanical tests were all performed at 20 °C and 65% relative humidity using a tensile testing machine (RTA-100; Orientec Corp., Tokyo, Japan). All samples were equilibrated at 20 °C and 65% relative humidity over 48 h before measurement. The sericin films used for mechanical tests were 20 × 2 mm2, with 20 ( 3 µm thickness. Mechanical tests of unstretched sericin films were carried out with a gauge length of 10 mm and a strain rate of 5 mm/min. Mechanical tests of stretched sericin films were performed as follows: films were attached to the instrument with a gauge length of 10 mm and were moistened thoroughly by spraying water or aqueous ethanol (30 or 70%). After equilibration for 2 min, the swollen films were stretched to a draw ratio of four at a crosshead speed of 20 mm/min. After equilibration over 48 h without removal from the instrument, mechanical testing was performed at a gauge length of 40 mm and a strain rate of 5 mm/min. The crosssection after stretching was approximated as one-fourth of that before stretching. Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) Analysis. Infrared spectra were collected using a Fourier transform infrared spectrometer (Herschel FT/IR-350; Jasco Inc., Tokyo, Japan) equipped with a liquidnitrogen-cooled mercury-cadmium-telluride (MCT) detector at a resolution of 4 cm-1. All samples and backgrounds were scanned 64 times. ATR measurements were performed using a single-reflection diamond ATR attachment (DuraSamplIR II; SensIR Technologies, Danbury, CT). ATRFTIR spectra of solvent-swollen sericin films were measured as follows: 100 µL of water or aqueous ethanol (10, 30, 50, or 70%) was dropped on a sericin film (10 × 5 mm2) that was fixed on the ATR substrate. Spectra were collected after equilibration for 2 min. Absorptions arising from solvents were subtracted to obtain a straight baseline from 2000 to 1750 cm-1. Perpendicularly polarized light to the surface normal of the ATR substrate was used to collect polarized spectra with a wire grid polarizer (Jasco Inc., Tokyo, Japan) placed after the sample. Parallel and perpendicular spectra of the stretched sericin films were obtained with the electric vector of the incident radiation vibrating parallel and perpendicular to the stretching direction by rotating samples 90°. ATR-FTIR Data Analysis. Prior to analyzing amide I bands of ATR-FTIR spectra, a straight baseline tangent to spectra at around 1710 and 1580 cm-1 was subtracted and seven-point Savitzky-Golay smoothing was performed. Second derivative spectra were calculated to identify the positions of overlapping component bands in the amide I region, which were used as initial parameters for curve fitting analysis. Lorentzian curves were fitted to amide I bands by leaving the parameters free to adjust iteratively except for the baseline, which was not corrected during the curvefitting procedure. The fitted band areas were used to estimate the proportions of secondary structures. All calculations

Orientation Behavior of Sericin

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2800 cm-1 region to normalize band areas in this study. The normalized dichroic ratio, R, can be calculated as A| A|,CH R) A⊥ A⊥,CH

(1)

where A|,CH and A⊥,CH are the respective band areas of C-H stretching absorption in parallel and perpendicular spectra. Results and Discussion

Figure 1. SDS-PAGE pattern of sericin solution prepared from cocoons of Sericin-hope silkworms. Lane 1, molecular weight marker; lane 2, sericin solution.

Figure 2. ATR-FTIR spectrum of a sericin film. The C-H stretching absorption used to normalize band areas is marked with an asterisk.

including derivation and curve fitting were performed using analysis software (Spectra Manager; Jasco Inc., Tokyo, Japan). Calculation of Dichroic Ratio. The degree of molecular orientation in a stretched polymer can be evaluated using a dichroic ratio. The dichroic ratio is defined as A|/A⊥, where A| and A⊥ are the respective areas of the selected absorption band measured with radiation polarized parallel and perpendicular to the stretching direction. Hence, the dichroic ratio is calculable directly from infrared spectra. However, to obtain both parallel and perpendicular spectra, samples must be removed once from the ATR substrate, rotated, and then re-clamped. Consequently, it is difficult to obtain identical contact areas before and after rotation, thereby perturbing the measured band areas. Everall and Bibby proposed a practical method to minimize this perturbation by normalizing band areas of interest relative to a nondichroic band in the same spectrum.20 We inferred that the C-H stretching absorption that appears in the 3000-2800 cm-1 region (Figure 2) is useful as a nondichroic band because the relative intensity of this band seemed to be almost constant among samples having different molecular orientation. In fact, normalization using this band provided good reproducibility for calculation of the dichroic ratio. For that reason, we used the C-H stretching absorption that appeared in the 3000-

Characterization of Protein Components. SDS-PAGE patterns of sericin solution prepared from cocoons of Sericinhope silkworms are shown in Figure 1. Two major components were observed clearly at >250 and 180 kDa. A densitometric analysis showed that the larger sericin was more abundant than the smaller one. Takasu and colleagues previously reported the isolation of three sericin components whose molecular weights were 400, 250, and 150 kDa, and respectively named them sericin M, A, and P.4 We attributed the two sericin components of >250 and 180 kDa detected by SDS-PAGE (Figure 1) to sericin M and A from the comparison of molecular weight and abundance, respectively. We assume that the faint band observed at 100 kDa in Figure 1 corresponds to sericin P. The difference of the estimated molecular weights is probably attributable to the difference of analytical conditions including the buffer, gel, and marker. Takasu and colleagues4 analyzed amino acid compositions of each isolated sericin component and suggested that sericin M (the larger sericin; >250 kDa) corresponds to Ser1C protein deduced from Ser1 gene21,22 and that sericin A (the smaller sericin; 180 kDa) corresponds to S-2 protein synthesized from Src-2 (Ser2) gene.23,24 Ser1C protein encoded by Ser1 gene contains about 70 repeats of serinerich 38-amino acid motif, which was predicted to exhibit mostly β-sheets.22 S-2 protein is abundant in charged amino acids.23 Ser2 gene contains about 30 repeats of 45-bp encoding mostly hydrophilic amino acids.24 These characteristics are consistent with our knowledge that the smaller sericin exhibits higher solubility in water than the larger one. Assignment of Infrared Bands. The ATR-FTIR spectra of a sericin film prepared from cocoons of Sericin-hope silkworms exhibited characteristic amide absorption bands of protein, amide I, II, and III, at 1639, 1514, and 1236 cm-1, respectively (Figure 2). Amide I absorption primarily represents the CdO stretching vibration of the amide group. Amide II absorption contains contributions from N-H bending and C-N stretching vibrations; amide III arises mainly from the C-N stretching vibration coupled to the N-H in-plane bending vibration. The N-H stretching absorption of amide groups around 3300 cm-1 was overlapped with the strong O-H absorption of hydroxyl amino acid residues such as serine and threonine. Bands at 1394 and 1065 cm-1 are assignable to C-H and O-H bending vibrations and C-OH stretching vibration, which are also attributable to the abundance of hydroxyl amino acid side chains.

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Teramoto and Miyazawa Table 1. Assignment of Individual Amide I Components

Figure 3. Second-derivative (the upper) and curve-fitted (the bottom) spectra of a sericin film. Nine Lorentzian curves (solid line) were fitted iteratively to the amide I band (broken line) using the peak positions obtained from the second derivative spectrum as the initial parameters. Prior to data analysis, baseline correction and seven-point Savitzky-Golay smoothing were performed for the amide I band.

Amide bonds are involved in a polypeptide backbone. Therefore, amide absorptions are sensitive to the secondary structure and molecular orientation of protein. Amide I absorption, found in the 1700-1600 cm-1 region, is the most useful for determining protein secondary structures because it arises predominantly from the CdO stretching vibration. We therefore used the amide I region to analyze the sericin structure in this study. Secondary Structure Analysis by ATR-FTIR. Natural protein contains more than one secondary structure. Therefore, absorptions overlap in the amide I region to yield a featureless band, as seen in Figure 3 (the broken line). Hence, a second derivative spectrum of the amide I band of a sericin film was calculated to distinguish among individual overlapping absorptions.25-27 Nine components were detected from the second derivative spectrum (Figure 3; upper spectrum). The lowest component around 1612 cm-1 was detected as a shoulder peak in the second derivative spectrum overlapped with an adjacent component. These components are attributable to particular types of a protein secondary structure. Curve-fitting analysis was performed as a linear combination of the component bands to obtain quantitative information about secondary structure. Nine Lorentzian curves were fitted to the amide I band using the peak positions obtained from the second derivative spectrum as initial parameters (Figure 3; bottom spectra). Excellent agreement was found for the band positions of the fitted Lorentzian components with those determined from the second-derivative spectrum (Figure 3). We assigned each amide I component based on previous studies16,28-30 (Table 1). Correlation between the position of a CdO stretching absorption and a particular type of secondary structure remains on a semiempirical basis.25,29-32 It is now generally accepted that β-sheet, R-helix, and random

frequency (cm-1)

assignment

1613, 1621 1630 1641, 1649 1659 1668, 1680 1695

aggregated strands β-sheet random coil R-helix turn antiparallel β-sheet

coil respectively absorb around 1630, 1655, and 1645 cm-1.29,30 We therefore respectively assigned the bands at 1630, 1659, and 1641 cm-1 to β-sheet, R-helix, and random coil. The assignment of a 1649 cm-1 band was ambiguous, but we assigned it to random coil because this component band was similarly observed for native sericin solution in silk gland,16 which forms mostly random coil according to the previous circular dichroism analyses.18,33 The appearance of bands between 1610 and 1625 cm-1 is often observed for solvent and thermally denatured proteins.34-37 The stronger the hydrogen bond involving the amide CdO, the lower the electron density in the CdO group and the lower the amide I absorption appears. Hence, the absorptions at lower wavenumber, 1613 and 1621 cm-1, are attributable to aggregated strands having strong intermolecular hydrogen bonds among extended chains.29,30 The absorptions in the region of 1660-1700 cm-1 are generally attributable to several types of turns and the high-frequency component of antiparallel β-sheet.28-30 Fibroin fiber of Bombyx mori is known to contain antiparallel β-sheet crystal.38 FTIR spectra of fibroin fiber exhibit a strong parallel amide I band at around 1696 cm-1,16,28,39 which is assigned to the highfrequency component of the antiparallel β-sheet. We thus assigned the highest band at 1695 cm-1 to the antiparallel β-sheet. Then, we attributed the remaining bands at 1668 and 1680 cm-1 to turns. The proportion of each secondary structure of sericin film was estimated from the peak areas of the component bands: aggregated strands 23%; β-sheet 15%; random coil 30%; R-helix 11%; turn 17%; antiparallel β-sheet 4%. This estimation suggested that random coil was the most abundant structural component in sericin film. Structural Changes by Solvent Treatment. It is generally acknowledged that alcohol changes the structure of proteins, including silk fibroin.34,40-42 Tsukada reported that thermal treatment of sericin in the presence of organic solvents or water induced structural changes from random coil to β-sheet.43 Methanol has been frequently used for β-sheet formation of silk proteins,40,41 but we have found that ethanol is more effective than methanol for gelation of sericin.15 It has an equivalent effect with methanol on structural changes of sericin film. In addition, ethanol is less toxic than methanol, which is advantageous for sericin’s application as a biomaterial. Therefore, we used aqueous ethanol of various concentrations to induce structural changes of sericin film in this study. We measured ATR-FTIR spectra of sericin films swollen with aqueous ethanol (0-70%); absorption attributable to solvents was subtracted, and the proportion of each secondary structure was estimated by curve-fitting analysis as above.


Figure 4. Proportion of each secondary structure in a sericin film swollen with aqueous ethanol of varied concentrations. Average values and standard deviations from five specimens were employed for each experimental plot.

We plotted the results against ethanol content (Figure 4). The proportion of aggregated strands in sericin film before solvent treatment was ca. 23%. Compared with this value, an increase of aggregated strands was observed for all solvent treatments. The proportion of aggregated strands at the swollen state exhibited a remarkable increase with ethanol content and reached ca. 43% when treated with 70% aqueous ethanol (Figure 4; closed squares). In conjunction with the increase of aggregated strands, the proportion of random coil at the swollen state decreased greatly with increased ethanol content (Figure 4; closed circles). Other structural components exhibited relatively small changes of their proportions upon swelling with aqueous ethanol. The increase of aggregated strands was observed by swelling with pure water. Sericin is a highly hydrophilic protein.3,4 For that reason, it swells readily in contact with water. We assumed that swelling with water activated molecular motion and caused transformation to stable aggregated strands. The increase of ethanol content implies an increase of the solvent’s hydrophobicity. Therefore, when treated with the solvents of higher ethanol content, hydrogen bonds among sericin chains will become stronger because of the hydrophobicity of ethanol, engendering the formation of a larger amount of aggregated strands (Figure 4). Interestingly, after evaporation of solvents, sericin films exhibited a similar secondary structure regardless of the ethanol content in the swelling solvents, indicating that structural changes also occur during the drying process (data not shown). Stretching and Mechanical Properties. Sericin film is rigid when dry, but it becomes extensible when moistened with aqueous solvent. The structural analyses above revealed that the secondary structure of sericin film in a swollen state depends on the ethanol content of the swelling solvent (Figure 4). We expect that stretching with aqueous ethanol can elucidate the relationship between molecular orientation and secondary structure of sericin. Sericin films were moistened thoroughly with aqueous ethanol (0-70%) and stretched to a draw ratio of four. The tensile stress applied to the films was recorded during stretching. The maximum stresses, recorded at the end of stretching, were plotted against ethanol content (Figure 5). The maximum stress during stretching increased linearly with ethanol content, showing that stretching with higher ethanol


Figure 5. Maximum stress applied to sericin films during stretching (at a draw ratio of four) plotted against ethanol content in the swelling solvents.

Figure 6. Representative stress-strain curves of sericin films: (a) unstretched and (b-d) stretched to a draw ratio of four with 0%, 30%, and 70% aqueous ethanol, respectively. Table 2. Mechanical Properties of Sericin Films before and after Stretchinga

unstretched stretched withb water 30% ethanol 70% ethanol

yield stress (MPa)

stress at failure (MPa)

Young’s modulus (GPa)

40.35 ( 4.67

26.60 ( 5.08

3.34 ( 0.12

96.41 ( 20.63 110.13 ( 12.96 119.56 ( 10.69

98.58 ( 21.72 121.02 ( 26.07 127.58 ( 15.92

8.02 ( 0.70 8.64 ( 0.60 9.27 ( 0.63

a The average values and standard deviations from five specimens were employed for each test. b Stretched to a draw ratio of four.

content requires greater tensile stress. Stretching with ethanol content over 70% often caused failure, which probably results from exceeding the permissible load of sericin film. This result indicated that ethanol strengthened the interaction among sericin chains, which increased the stretching stress. Mechanical properties of sericin films were measured before and after stretching. Figure 6 shows representative stress-strain curves. Table 2 summarizes mechanical properties of the respective samples. All samples exhibited obvious yield points at almost the same strain rate around 1.7% (Figure 6). Yield stresses of the stretched films were much higher than that of the unstretched film and increased with ethanol content. The stresses at stretched films’ failure points were often higher than the yield stresses, whereas the stress at failure of the unstretched film was lower than the yield stress. This fact means that the stretched films exhibited a stress-hardening effect beyond the yield point. The stretched films’ stresses at failure also increased with ethanol content.

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Figure 7. Polarized ATR-FTIR spectrum of an unstretched sericin film. The full curve was obtained with the electric vector vibrating parallel to the longitudinal axis of the film; the broken curve was obtained after the sample was rotated 90°.

Stretching similarly increased the values of Young’s modulus, calculated tangentially to the first regions of the stressstrain curves. These results demonstrated that the mechanical properties of sericin film can be improved by stretching with aqueous ethanol and that the extent of that improvement is proportional to the ethanol content (Table 2). Polarized Infrared Spectra of Stretched Films. Polarized ATR-FTIR spectra of the stretched sericin films were obtained with the electric vector vibrating parallel and perpendicular to the stretching direction. Its parallel and perpendicular spectra should be identical if a sample is isotropic. In fact, sericin films before stretching exhibited no dichroism (Figure 7), demonstrating that no molecular orientation occurred during preparation. Polarized ATR-FTIR spectra of a sericin film that was stretched with water exhibited dichroism (Figure 8a). Amide I absorption of the parallel spectrum was weaker than that of the perpendicular one, whereas the amide II absorption of the parallel spectrum was stronger than that of the perpendicular one. This observed dichroism verified that sericin film became anisotropic by stretching because of the sericin molecules’ orientation. Dichroism became more significant when stretched with aqueous ethanol. The degree of dichroism observed in amide I and II bands was apparently proportional to the increase in ethanol content (Figures 8b-8e). The degree of dichroism became largest at ethanol content of 70%. This observation, combined with the result in Figure 5, strongly suggests that ethanol affects the strength of the interaction among sericin chains and that the strong interchain interaction induced by aqueous ethanol treatment caused molecular orientation during stretching. When stretched with 70% aqueous ethanol, the parallel amide I band was centered at 1641 cm-1 with a broad feature, whereas the perpendicular amide I band exhibited a large sharp peak at 1614 cm-1 (Figure 8e). As mentioned above, individual secondary structures exhibit specific amide absorptions. Therefore, the difference of the absorbing position between parallel and perpendicular spectra indicates that the orientation behavior differs among secondary structures. We then performed curve-fitting analysis for the amide I bands of polarized ATR-FTIR spectra of stretched sericin films. Representative results of curve fitting obtained for the sericin films stretched with water and 70% aqueous ethanol are shown in Figure 9.

Figure 8. Polarized ATR-FTIR spectra of sericin films stretched with (a) water, (b) 10%, (c) 30%, (d) 50%, and (e) 70% aqueous ethanol. The full curves were obtained with the electric vector vibrating parallel to the stretching direction and the broken curves with the electric vector vibrating perpendicular to this direction.

Dichroic ratios of respective secondary structures in stretched films were calculated using eq 1. The band areas (A| and A⊥) of secondary structures having two absorption components were calculated as their sum. We plotted calculated dichroic ratios, R, against ethanol content in the swelling solvents (Figure 10). The y axis was plotted as a logarithmic scale to represent the dichroic ratios greater and less than unity on the same scale. The values of dichroic ratios greater and less than unity respectively represent parallel and perpendicular dichroism of the corresponding secondary structure. Aggregated strands exhibited only remarkable perpendicular dichroism, whereas the other structural components exhibited relatively small dichroism. The transition moment of the amide I band, arising predominantly from the CdO stretching vibration, is nearly parallel to the direction of the CdO bond.44,45 Hence, the orientation manner observed for the stretched sericin films demonstrates that CdO bonds in aggregated strands are aligned perpendicularly to the stretching direction and that the axis of the polypeptide backbone is parallel to this direction. A similar orientation tendency was observed for silk fibers from insects and spiders.39,46 Orientation Mechanism. This series of experiments demonstrates that the sericin orientation depends strongly



Figure 9. Curve-fitted polarized spectra of sericin films stretched with (a) water or (b) 70% aqueous ethanol-to-draw ratio of four. Nine Lorentzian curves (solid line) were fitted iteratively to the amide I bands (broken line) with the peak positions obtained from second derivative spectra as the initial parameter. Parallel and perpendicular respectively denote the spectra obtained with the electric vector vibrating parallel and perpendicular to the stretching direction.

Figure 10. Dichroic ratio of each secondary structure component involved in the sericin films stretched to a draw ratio of four plotted against ethanol content in the solvents used for stretching. The dichroic ratios were plotted as a logarithmic scale. Correspondence of the symbols to secondary structures is the same as that shown in Figure 4. The average values and standard deviations from five specimens were employed for each experimental plot.

on its structure during stretching. Scheme 1 illustrates the presumed orientation mechanism of sericin molecules. Infrared analyses of solvent-treated sericin films (Figure 4) showed that sericin is transformed into an aggregated state by aqueous ethanol treatment (Scheme 1; native state to aggregated state). Aggregated strands contain strong hydrogen bonds among extended chains. Therefore, the stretching stress will propagate through hydrogen bonds, generating an

oriented structure (Scheme 1; aggregated state to oriented structure). Stress during stretching increased linearly with ethanol content in the swelling solvents (Figure 5), which reflects increasing interaction among sericin chains concomitant with the increase of ethanol content. Improvement of mechanical properties by stretching (Figure 6 and Table 2) was caused by molecular orientation and structural change. The presumed orientation mechanism of sericin molecules mentioned above can be contrasted with the fiber formation mechanism of silk fibroin, which was recently clarified by Asakura and colleagues.47,48 According to their studies by solid-state NMR technique, fibroin is transformed rapidly into a highly oriented β-sheet structure having strong intermolecular hydrogen bonds created by the silkworm’s spinning. Our previous study suggested that sericin is not intrinsically given such properties to form an oriented structure by the natural spinning of silkworm.16 However, the present study revealed that a fiberlike oriented structure can be generated by artificial stretching once strong intermolecular hydrogen bonds are formed, even in the case of sericin. Gene analysis revealed that Ser1C protein deduced from Ser1 gene, probably corresponding to the larger sericin of >250 kDa observed by SDS-PAGE (Figure 1), contains about 70 repeats of serine-rich 38-amino acid motif that was predicted to exhibit mostly β-sheets.22 Huang and colleagues

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Scheme 1. Schematic Diagram of the Presumed Orientation Mechanism of Sericin Moleculesa

a

Shaded rectangles represent aggregated strands formed among sericin chains.

synthesized recombinant proteins based on the repetitive motif and showed that the recombinant proteins self-assemble into fibrillar structures during dialysis as a formation of β-sheets.49 We assume that this characteristic repetitive motif might be mainly responsible for formation of aggregated strands by ethanol treatment. Our observation that stretching orients aggregated strands of sericin implies the remarkable strength of the interchain interaction in the aggregated segment of sericin. This strong interchain interaction might result from the abundance of polar side chains such as hydroxyl and carboxyl groups, which can form interchain hydrogen bonds with other polar side chains or amide groups in peptide backbones.49 Conclusions We investigated the relationship between molecular orientation and secondary structure of sericin to elucidate the orientation mechanism of sericin molecules using ATR-FTIR spectroscopy. We clarified that secondary structure changes upon the swelling with aqueous ethanol depending on ethanol content. Polarized spectra of sericin films that were stretched with aqueous ethanol of varied concentrations revealed that the degree of molecular orientation increases with ethanol content. Structural analyses indicated that the formation of aggregated strands that have strong intermolecular hydrogen bonds plays a crucial role in the sericin molecules’ orientation: the amount of aggregated strands in swollen sericin films was proportional to ethanol content, which was thought to be responsible for the ethanol concentration-dependent orientation. We then inferred the molecular orientation mechanism of sericin as follows: aqueous ethanol treatment induces formation of aggregated strands that transfer the stretching stress through their strong intermolecular hydrogen bonds, thereby generating an oriented structure. Therefore, this study shows clearly that sericin exhibits structuredependent molecular orientation behavior, indicating that the molecular orientation of sericin can be controlled by its secondary structure. Sericin does not form an oriented structure by the natural spinning of silkworm, whereas fibroin rapidly forms a fibrous structure during spinning. However, it is interesting that sericin can form a fiberlike oriented structure through artificial alteration of its structure: it can thereby exhibit improved mechanical properties. Results obtained in this study suggest the availability of silk sericin as a fibrous material that will be beneficial for producing novel proteinbased materials. Acknowledgment. We thank the Sericultural Science Laboratory, National Institute of Agrobiological Sciences,

for providing Sericin-hope silkworm cocoons and for densitometric analysis. We thank Drs. Chiyuki Takabayashi and Tsunenori Kameda, National Institute of Agrobiological Sciences, for invaluable discussion and advice. This work was partially supported by the Insect Technology Project from the Ministry of Agriculture, Forestry, and Fisheries of Japan. References and Notes (1) Fedicˇ, R.; Zˇ urovec, M.; Sehnal, F. J. Insect Biotechnol. Sericol. 2002, 71, 1. (2) Craig, C. L.; Riekel, C. Comp. Biochem. Physiol. B 2002, 133, 493. (3) Gamo, T.; Inokuchi, T.; Laufer, H. Insect Biochem. 1977, 7, 285. (4) Takasu, Y.; Yamada, H.; Tsubouchi, K. Biosci. Biotechnol. Biochem. 2002, 66, 2715. (5) Voegeli, R.; Meier, J.; Blust, R. Cosmetics Toiletries 1993, 108, 101. (6) Teramoto, H.; Nakajima, K.; Takabayashi, C. Biomacromolecules 2004, 5, 1392. (7) Takeuchi, A.; Ohtsuki, C.; Miyazaki, T.; Tanaka, H.; Yamazaki, M.; Tanihara, M. J. Biomed. Mater. Res. 2003, 65A, 283. (8) Tsubouchi, K.; Igarashi, Y.; Takasu, Y.; Yamada, H. Biosci. Biotechnol. Biochem. 2005, 69, 403. (9) Zhang, Y. Q. Biotechnol. AdV. 2002, 20, 91. (10) Zhang, Y. Q.; Tao, M. L.; Shen, W. D.; Zhou, Y. Z.; Ding, Y.; Ma, Y.; Zhou, W. L. Biomaterials 2004, 25, 3751. (11) Yamamoto, T.; Mase, K.; Miyajima, T.; Hara, K. Japanese Patent No. 3374177. (12) Yamamoto, T.; Miyajima, T.; Mase, K.; Iizuka, T. Breeding of the silkworm race “Sericin-hope” secreting silk protein in which sericin is contained in high concentration. In Annual Report 2002; National Institute of Agrobiological Sciences: Tsukuba, Japan, 2002; pp 2425. (13) Gamo, T. Jpn. J. Genet. 1973, 48, 99. (14) Takei, F.; Oyama, F.; Kimura, K.; Hyodo, A.; Mizuno, S.; Shimura, K. J. Cell Biol. 1984, 99, 2005. (15) Teramoto, H.; Nakajima, K.; Takabayashi, C. Biosci. Biotechnol. Biochem. 2005, 69, 845. (16) Teramoto, H.; Miyazawa, M. J. Insect Biotechnol. Sericol. 2003, 72, 157. (17) Hirabayashi, K.; Tsukada, M.; Sugiura, S.; Ishikawa, H.; Yasumura, S. J. Seric. Sci. Jpn. 1972, 41, 349 (in Japanese). (18) Komatsu, K. Bull. Seric. Exp. Stn. 1975, 26, 135 (in Japanese). (19) Laemmli, U. K. Nature 1970, 227, 680. (20) Everall, N. J.; Bibby, A. Appl. Spectrosc. 1997, 51, 1083. (21) Okamoto, H.; Ishikawa, E.; Suzuki, Y. J. Biol. Chem. 1982, 257, 15192. (22) Garel, A.; Deleage, G.; Prudhomme, J. C. Insect Biochem. Mol. Biol. 1997, 27, 469. (23) Gamo, T. Biochem. Genet. 1982, 20, 165. (24) Michaille, J. J.; Garel, A.; Prudhomme, J. C. Gene 1990, 86, 177. (25) Byler, D. M.; Susi, H. Biopolymers 1986, 25, 469. (26) Surewicz, W. K.; Mantsch, H. H. Biochim. Biophys. Acta 1988, 952, 115. (27) Surewicz, W. K.; Mantsch, H. H.; Chapman, D. Biochemistry 1993, 32, 389. (28) Miyazawa, T.; Blout, E. R. J. Am. Chem. Soc. 1961, 83, 712. (29) Jackson, M.; Mantsch, H. H. Can. J. Chem. 1991, 69, 1639. (30) Jackson, M.; Mantsch, H. H. Crit. ReV. Biochem. Mol. Biol. 1995, 30, 95. (31) Susi, H.; Byler, D. M.; Purcell, J. M. J. Biochem. Biophys. Methods 1985, 11, 235. (32) Susi, H.; Byler, D. M. Methods Enzymol. 1986, 130, 290. (33) Iizuka, E. Biochim. Biophys. Acta 1969, 181, 477. (34) Purcell, J. M.; Susi, H. J. Biochem. Biophys. Methods 1984, 9, 193.

Orientation Behavior of Sericin (35) Liu, C.; Bo, A.; Cheng, G.; Lin, X.; Dong, S. Biochim. Biophys. Acta 1998, 1385, 53. (36) Dong, A.; Randolph, T. W.; Carpenter, J. F. J. Biol. Chem. 2000, 275, 27689. (37) Murayama, K.; Tomida, M. Biochemistry 2004, 43, 11526. (38) Takahashi, Y. Crystal structure of silk of Bombyx mori. In Silk Polymers: Materials Science and Biotechnology; Kaplan, D., Adams, W. W., Farmer, B., Viney, C., Eds.; American Chemical Society: Washington, DC, 1994; pp 168-175. (39) Suzuki, E. Spectrochim. Acta 1967, 23A, 2303. (40) Tsukada, M.; Gotoh, Y.; Nagura, M.; Minoura, N.; Kasai, N.; Freddi, G. J. Polym. Sci., Polym. Phys. Ed. 1994, 32, 961. (41) Sonoyama, M.; Miyazawa, M.; Katagiri, G.; Ishida, H. Appl. Spectrosc. 1997, 51, 545.

Biomacromolecules, Vol. 6, No. 4, 2005 2057 (42) Chen, X.; Shao, Z.; Marinkovic, N. S.; Miller, L. M.; Zhou, P.; Chance, M. R. Biophys. Chem. 2001, 89, 25. (43) Tsukada, M. J. Seric. Sci. Jpn. 1983, 52, 157 (in Japanese). (44) Fraser, R. D. B.; Price, W. C. Nature 1952, 170, 490. (45) Fraser, R. D. B. J. Chem. Phys. 1953, 21, 1511. (46) Rousseau, M.-E.; Lefe`vre, T.; Beaulieu, L.; Asakura, T.; Pe´zolet, M. Biomacromolecules 2004, 5, 2247. (47) Asakura, T.; Yao, J. Protein Sci. 2002, 11, 2706. (48) Yamane, T.; Umemura, K.; Nakazawa, Y.; Asakura, T. Macromolecules 2003, 36, 6766. (49) Huang, J.; Valluzzi, R.; Bini, E.; Vernaglia, B.; Kaplan, D. L. J. Biol. Chem. 2003, 278, 46117.

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