Structure of the Enzyme-resistant Fraction of Tussah Silk (Anthema pernyi) Fibroin

INTRODUCTION

It is known that the fibroin of the tussab moth (Antherma pernyi) contains in its chains a large number of polyalanine redues and that the crystalline structure of the fibroin in the solid state has a close resemblance to that of polyalanine in the a-helix conformation. We' have recently demonstrated that the interhelix interaction of POly-D-alanine, a model compound related to tussah fibroin, becomes attenuated above 210°C because of disruption of the weak bonding. This is revealed by the change in the x-ray diffraction pattern in the course of heating. On the other hand, the intrahelix interaction mainly associated with intramolecular hydrogen bonds remains unchanged regardless of the heat treatment. In earlier papers',' it was shown from DSC data that the random-coil-to-8 transformation in tussah fibroin occurs a t 220°C. In addition to this structural transformation, previous studies' of the structural changes induced by immer- sion in methanol showed that the intersheet packing of the 8 crystal of the original tussah silk fibroin was imperfect and remained so in the early stages of the immersion treatment, but the crystallization proceeded further when the immersion time exceeded 10 min.

We7 studied the effect of drying on the structure of tussah fibroin, and concluded that drying conditions, including the drying rate and drying temperature, significantly affect the molecular conformation in fibroin film cast from aqueous solution. Wes also demonstrated that the fraction of the 8-sheet structure in poly-L-alanine remains almost constant when the specimen is heated below 325"C, and that the a-helix fraction does not change, regardless of heat treatment.

Bergmann and coworkers3 have shown that chymotrypsin splits peptide bonds involving the carboxyl groups of tyrosine, phenylalanine, tryptophane, and methionine. It is known that the amorphous regions of silk fibroin can be removed by chymotrypsin in fibroin solutions.

This paper deals with the structural characteristics of the chymotrypsin-resistant and phos- phatase-resistant fractions obtained by the action of these enzymes on tussah fibroin in solution. The relationship between the amount of the enzyme-resistant fraction and its chemical stability is also discussed.

EXPERIMENTAL

Materials

Liquid silk was collected from the posterior division of the silk gland in full-grown larvae of Anthraeu pemyi (1 day before spinning). The aqueous fibroin was diluted to a concentration of about 0.35%. Then the film obtained by casting from solution onto a thin polyethylene film was dried at 25°C.

a-Chymotrypsin (lot No. 92F-8035, 59 units/mg solid) and alkaline phosphatase (lot No. 213-0270, 1.4 units/mg solid) were obtained from Sigma Chemical Company and were used without further purification. When an aqueous tussah fibroin solution (0.35%) is hydrolyzed at 38°C for 18 h by a-chymotrypsin at pH 7.9 or by phosphatase at pH 10.0, a precipitate is formed, and the enzymeresistant fraction can be obtained by drying the precipitate under reduced pressure at 25°C. These samples are referred to hereafter as CPS and PST fractions, respectively.

Measurements

Differential scanning calorimetry (DSC) measurements were performed on a Rigaku Denki instrument (DSC-1OA) at a heating rate of 10°C/min. The DSC range and sample weight were 2.5 mcal/s and 2 mg. The open aluminum cell was swept with nitrogen gas during the c o r n of the analysis.

Journal of Polymer Science: Part B: Polymer Physics, Vol. 26,949-952 (1988) 0 1988 John Wiley & Sons, Inc. CCC 0098-1273/88/040949-04$04.@3

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Wavonumbor (Cm-') Fig. 1. (a) Infrared spectra of tussah (Antheraeu pernyyi) silk fibroin film, (b) chymotrypsin-

resistant fraction (CPS fraction), and (c) phosphatase-resistant fraction (PST fraction). Absorp- tion band: A, attributed to a helix; B, attributed to B structure; R, attributed to random coil.

Infrared absorption spectrum of the solid film and the CPS and PST fractions were mea- sured with a Japan Spectroscopic Co., Ltd. spectrophotometer (IR-G) in the spectral region 4000-400 cm-'.

X-ray photographs were taken using Nickel-filtered radiation with a flat-plate camera. The conditions for x-ray measurements are described in detail elsewhere.2

RESULTS AND DISCUSSION

Infrared Spectra

Figure 1 shows infrared spectra of the tussah fibroin film and of the CPS and PST fractions. The absorption bands at 1650 cm-' (amide I), 1545 cm-' (amide 11), 1274 cm-' (amide 111), and 620 cm-' (amide V) assigned to the a helix and the 655 cm-' (amide V ) band for the random coil4 were observed for the fibroin film (Fig. la), showing almost the same absorption bands as were previously desc~ibed.~.~ This finding suggests that the silk fibroin film (Fig. la) consists mostly of a helices and random coils. On the other hand, the CPS and PST fractions (Fig. Ib and c) exhibit absorption bands at 1630, 1530, 1220, and 700 cm-', which are characteristic of the B ~ t r u c t u r e , ~ together with a minor absorption band attributed to the a helix: Judging from the increase in the intensity of the absorption band of the specimens (Fig. l b and c) a t 1630 and 700 cm-', crystallization appears to proceed in the enzyme-resistant fraction obtained by removing the amorphous regions. However, the intensity of the absorption band of the enzyme- resistant fractions (Fig. l b and c) a t 620 cm-' attributed to the a helix remained unchanged regardless of the enzymatic reaction. It was assumed that the presence of the a helix may contribute to the chemical stability of fibroin, as reported in a previous paper!

X-ray Diffraction Studies

X-ray diffraction patterns of the tussah silk fibroin film, CPS, and PST fractions are shown in Figure 2. Compared with the tussah fibroin film described in previous paper^,^,^ a very similar x-ray diffraction pattern was observed for the tussah fibroin film in the present work (Fig. 2a), demonstrating the diffraction patterns corresponding to spacings of 3.69 and 7.40 A, which are attributed to the a helix."v6 The diffraction pattern of the CPS fraction (Fig. 2b) was similar to that observed fFr the PST fraction (Fig. 2c), with diffraction patterns corresponding to the 3.64, 4.34, and 5.30 A spacings, which are characteristic of the P-crystalline form with a comparably high degree of crystalline order in addition to the diffraction patterns due to the a helix (7.40 A),

NOTES 951

Fig. 2. (a) X-ray diffraction patterns of tussah fibroin film, (b) chymotrypsin-resistant frac- tion, and (c) phosphatase-resistant fraction.

although the diffraction intensity of the latter was low. I t seems that the major crystalline structures of the CPS and PST fractions correspond to the /3 structure and the a helix.

Thermal Behavior

Figure 3 shows DSC thermograms of the tussah fibroin film and of the CPS and PST fractions. The DSC thermograms of the tussah fibroin film (Fig. 3a) were quite similar to that of the tussah fibroin film reported in a previous paper.’ The DSC thermograms of the CPS and PST fractions (Figs. 3b and c) exhibited minor endothermic peaks at about 223°C in addition to the major endothermic peak a t 359°C. No difference in the thermal properties could be detected between the CPS (Fig. 3b) and PST (Fig. 3c) fractions. The exothermic peak of the fibroin film at 230°C (Fig. 3a) disappeared when the enzyme was added to solution. Thus the exothermic peak observed at 230’C on the DSC thermogram can be attributed to the transition from the random coil to the p structure, since the CPS fraction (PST fraction), which consists mainly of the a helix and p structures, did not display the exothermic peak. The endothermic peak a t 359°C is due to thermal decomposition. It appears that the decomposition temperature is shifted slightly to higher temperature (to 362’C) after the enzymatic reaction.

On the basis of the quantitative determination of the molecular conformation of the tussah fibroin by computer analysis of the CD s p e ~ t r u m , ~ the percentages of a helix, /3 structure, and random coil were 16, 27, and 57, respectively. These values for the conformational distribution would be a safe estimate in taking into account the a helix content (15-29’%), for example, of the tussah fibroin determined by Iizuka,” and Kondo et al.”

952 J. POLYM. SCI. POLYM. PHYS. ED, VOL. 26 (1988)

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(a) DSC thennograms of tussah fibroin film, (b) chymotrypsin-resistant Fig. 3. (c) phosphatase-resistant fraction.

fraction, and

When an aqueous tussah fibroin solution (0.35%) is hydrolyzed by chymotrypsin (or alkaline phosphatase), a precipitate is formed accounting for 78% (73%) of the fibroin content.

These results indicate that the enzyme-resistant fractions consist mainly of a helix and /3 structures arising directly from the corresponding conformation of original tussah fibroin and that the a helix is characterized by high chemical and thermal stability,',* regardless of the conditions of preparation and treatment, including enzyme treatment, thermal treatment,' and immersion in methanol.*

References

1. M. Nagura, S. Yamazaki, and M. Tsukada, Proceedings of the 7th International Wool

2. M. Tsukada, J . Polym. Sci. Polym. Phys. Ed., 24, 1227 (1986). 3. M. Bergmann and C. Niemann, J . Biol. Chem., 122, 576 (1938). 4. T. Miyazawa and E. R. Blout, J . Am. Ckem. SOC., 83, 712 (1961). 5. E. Elliot and B. R. Marcolm, Biochim. Biophys. Actu, 21, 466 (1956). 6. K. Hiyabayashi, Y. Kondo, and Y. Go, Sen-i Gakkaishi, 23, 199 (1967). 7. M. Tsukada, J . Polym. Sci. Polym. Phys. Ed., 24, 457 (1986). 8. M. Tsukada, M. Nagura, and H. Ishikawa, J . Polym. Sci. Polym. Phys. Ed., 25, 1325

9. M. Tsukada, J . Seric. Sci. Japan, 48, 347 (1979). 10. E. Iizuka, Biochim. Biophys. Acta, 16, 454 (1968). 11. Y. Kondo, K. Hirabayashi, E. Iizuka, and Y. Go, Sen-i Gakkaishi, 23,311 (1967).

Textile Research Conference, Tokyo, 1985, Vol. 1, p. 345.

( 1987).

M. TSUKADA

Sericultural Experiment Station, Ministry of Agriculture, Forestry and Fisheries, 1-2 Yatabe, Tsukuba, Ibaraki 305, Japan

Received July 9, 1987 Accepted September 14, 1987