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Preparation of undegraded native molecular fibroin solution from silkworm cocoons
Materials Science and Engineering C 14 Ž2001. 41–46 www.elsevier.comrlocatermsec
Preparation of undegraded native molecular fibroin solution from silkworm cocoons Hiromi Yamada a , Hiroshi Nakao b, Yoko Takasu a , Kozo Tsubouchi a,) a
National Institute of Sericultural and Entomological Science, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan b Kowa Research Institute, 1-25-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan Received 16 January 2001; received in revised form 12 March 2001; accepted 3 April 2001
Abstract The molecular mass of solubilized fibroin prepared from silk was analyzed by SDS-PAGE. It was found that the fibroin molecule was degraded during reeling, degumming Žremoval of sericin., and dissolution of silk threads. A protocol for the preparation of solubilized fibroin conserving its native molecular size is offered, that is, Ž1. to use only fresh cocoons Žnot dry cocoons or reeled silk threads., Ž2. to degum by heating in 8 M aqueous urea, and Ž3. to dissolve by saturated Ž; 9 M. aqueous lithium thiocyanate at room temperature. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Silk fibroin; Fibroin dissolution; Molecular weight; SDS-PAGE
1. Introduction Silk is a well-known natural fiber produced by the silkworm, Bombyx mori, which has been used traditionally in the form of threads. It is composed of two kinds of protein: a fibrous one Žnamed fibroin. that forms the thread core, and a glue-like one Žnamed sericin. that surrounds the fibroin fibers to cement them together. Although the use of silk as threads is very popular, recently, interest has been increasing in the use of solubilized fibroin in biotechnological materials w1x, and biomedical applications w2x. In these studies, the source of fibroin was usually traditional Adegummed silk Žsericin-free silk fiber.B, which was prepared from cocoons by reeling and degumming. For decades, much biochemical research has been done on fibroin. These studies have shown that fibroin is a homogeneous protein with a molecular mass of 370 kDa w3x. In contrast, there have been few reports concerning the molecular mass of solubilized fibroin, even though the production of degummed silk from cocoons and dissolution of the silk fiber involve a number of drastic processes.
)
Corresponding author. Fax: q81-298-38-6028. E-mail address: [email protected] ŽK. Tsubouchi..
This report examines the factors affecting the molecular mass of fibroin in the solubilizing process, and subsequently describes a procedure for obtaining solubilized fibroin from cocoons without incurring molecular degradation.
2. Materials and methods 2.1. Cocoons Silkworms Ža hybrid strain. were reared with mulberry leaves by the usual method and cocoons were harvested. 2.2. Fresh cocoon shells Cocoons, harvested just after the completion of spinning, were cut and pupae were extracted before emerging as moths. 2.3. Raw silk (reeled silk threads) Raw silk was produced by standard method; that is, harvested cocoons Žcontaining pupae. were heated in an oven at 1608C for 1 h. They were soaked in hot water to soften the sericin, and then the thread was reeled by automatic reeling machine.
0928-4931r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 Ž 0 1 . 0 0 2 0 7 - 7
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H. Yamada et al.r Materials Science and Engineering C 14 (2001) 41–46
2.4. RemoÕal of sericin (degumming) of cocoon shells or raw silk In the standard method, cocoon shells or raw silk were kept in 50 times Žvrw. of boiling aqueous 0.05% Na 2 CO 3 for 60 min. Usually, this treatment was repeated twice. Degumming was also carried out with various concentrations of Na 2 CO 3 and lengths of heating time. Soap degumming was performed in 100 times ŽVrW. of 0.5% soap for 30 min at 1008C. Enzymatic degumming was done in 50 times ŽVrW. of 1% alkalase 2.5 l ŽKosindo Chemical. for 30 min at 608C. Degumming by urea was carried out by heating in 30 times ŽVrW. of 8 M aqueous urea containing 0.04 M Tris–SO4 ŽpH 7. and 0.5 M mercaptoethanol, under various conditions of time and temperature. Degumming was also done in 30 times ŽVrW. of hot water at 1008C for 5 to 60 min, or at 1208C Žusing an autoclave. for 5 to 30 min. After the above degumming treatments, the resulting silk materials were washed in water repeatedly and then air dried. 2.5. Dissolution of fibroin 2.5.1. Ajisawa’s method [4] Cocoon shells or raw silk fibers were degummed and then added to 15 times ŽVrW. of Ajisawa’s reagent ŽCaCl 2rethanolr water, 111r92r144 in weight.. The mixture was stirred at 758C to form a clear solution. The resulting fibroin solution was dialyzed in a cellulose tube ŽVisking. against running water until the dialyzate tested negative for chloride ion ŽAgNO 3 .. 2.5.2. Lithium thiocyanate (LiSCN) method: Raw silk fibers or cocoon shells were stirred in 30–100 times Žvrw. of saturated Žca. 9 M. aqueous LiSCN at room temperature until dissolved. The solution was dialyzed against 100 times ŽVrV. of water or 5 M aqueous urea for 2 h, while refreshing the outer solution every 30 min. 2.6. Silk gland fibroin Fibroin was extracted from the silk gland of mature five instar larvae according to the method of Tashiro and Otsuki w3x. 2.7. Determination of protein concentration Absorbance at 275 nm was measured ŽUV-1200 apparatus, Shimadzu., and protein content was calculated as 1 s 1 mgrml. If necessary, the baseline was corrected at 250 and 300 nm. Bradford protein assay ŽBIO-RAD. was also used.
2.8. Polyacrylamide gel electrophoresis (PAGE) SDS-PAGE was performed on a 2–15 % gradient gel Ž10 = 10 cm, Daiichi Kagaku. according to the procedure of Weber and Osborn w5x. The sample usually contained a high concentration of salt. It was desalted by the following procedures. Ž1. A NAP-5 column ŽAmersham Pharmacia. was equilibrated with 8 M aqueous urea and centrifuged Ž2000 rpm for 3 min. using a swinging rotor to remove the liquid occupying the void volume. A sample Ž0.2 ml. was added onto the top of the column, which was again centrifuged as above. The recovered solution was used as the desalted sample. Ž2. Salt in the sample solutions was filtered off with Centricon ŽAmicon. by adding water at intervals. Ž3. Samples were dialyzed against water or 5 M aqueous urea. The samples were mixed with an equivalent amount of electrophoresis buffer Žcontaining 10% SDS, 1% mercaptoethanol, and 8 M urea., and kept at 508C for 10 min. Crosslinked phosphorylase b ŽSigma. and Bench Mark Ladder ŽGibco BRL. were used as protein standards. The gel was stained with CBB R-250 or silver-stain kit ŽAmersham Pharmacia. and scanned to record. 2.9. Chemicals All reagents were of analytical grade. Deionized water made by Elix ŽMilipore. was used throughout the experiment. 3. Results 3.1. Molecular mass of solubilized fibroin Silkworm cocoons were heat-dried and reeled by soaking in hot water. The resulting threads Žraw silk. were boiled in aqueous Na 2 CO 3 by the standard procedure to remove sericin. The fiber thus obtained Žnamed degummed silk. was dissolved in aqueous CaCl 2-ethanol ŽAjisawa’s reagent. to prepare a fibroin solution. SDS-PAGE analysis of the solution showed a broad smeared band at a position around 70 kDa ŽFig. 1, lane 2.. As shown in the figure, SDS-PAGE analysis of native fibroin from silk glands revealed clear protein bands having molecular masses of about 350 and 25 kDa Žlane 1., which correspond to the heavy and light chains of the protein. These results indicated that the native fibroin molecule was degraded to a mixture of polypeptides of various sizes during the preparation of the fibroin solution. As the procedure for preparing solubilized fibroin from cocoons comprises of many steps, we examined which stepŽs. cause degradation of the fibroin molecule. At first, fresh, untreated cocoons were dissolved by Ajisawa’s method. SDS-PAGE analysis of the resulting solution showed a broad smear at a position around 200 kDa and a clear band at 25 kDa ŽFig. 1, lane 3.. When fresh cocoons
H. Yamada et al.r Materials Science and Engineering C 14 (2001) 41–46
43
Fig. 1. SDS-PAGE analysis of protein components in various silk materials dissolved by Ajisawa’s method and by the lithium thiocyanate ŽLiSCN. method.
were dissolved by the LiSCN method, however, clear bands were observed at 350 and 25 kDa Žlane 4., the same pattern as for silk gland fibroin. These results indicated that Ajisawa’s method caused degradation of the heavy chain of fibroin, whereas LiSCN caused no damage to the protein. When raw Žbefore degumming. silk was solubilized by LiSCN, a faint 350-kDa band accompanied by a smear and a clear 25 kDa one were observed ŽFig. 1, lane 5.. In the case of degummed silk, only a smear at a position around 100 kDa was found Žlane 6.. These results revealed that a partial decomposition of the heavy chain of fibroin was taking place in the heat-drying andror reeling steps, and that degumming damaged both the heavy and light chains of fibroin. Heat-drying of the cocoons Žbefore reeling. was also revealed to degrade fibroin Ždata not shown.. The above experimental results indicated that the following conditions were indispensable for preparing solubilized fibroin having native molecular mass: Ž1. the use of fresh cocoons as a starting material but not dried cocoons or raw silk, Ž2. the removal of sericin Ždegum. without breaking the fibroin molecule, and Ž3. dissolution by the LiSCN method. 3.2. Conditions for remoÕal of sericin without degradation of fibroin Since the standard Na 2 CO 3 degumming Ž0.05% Na 2 CO 3 , 60 min, 1008C. caused degradation of fibroin molecules, the effects of heating time on sericin removal
and fibroin conservation were investigated. As shown in Table 1, the amount of weight lost by Na 2 CO 3 extraction Žwhich corresponded to the amount of removed sericin. was nearly equal for treatment times ranging from 5 to 60 min. SDS-PAGE analysis of the resulting threads indicated that sericin Ža smear near the top of lane 1 in Fig. 2. was removed by the treatment for 5 min at 1008C without damage to fibroin, whereas prolonged heating degraded the fibroin heavy chain Žlanes 2 to 6.. Soap and proteinase degumming procedures, which have been routinely adopted in the silk industry, were also evaluated from the viewpoint of fibroin degradation. It was demonstrated that these treatments caused decomposition of fibroin molecules to some extent ŽFig. 3.. Extraction of sericin from cocoon shells by aqueous urea has long been reported w6,7x; hence, urea degumming under various conditions was investigated. Fresh cocoon Table 1 Time-related removal of sericin by Na 2 CO 3 Extracteda for Žmin.
Before extraction Žmg.
After extraction Žmg.
Sericin removed Žmg.
5 10 15 20 60
39.6 40.1 39.5 39.8 40.3
30.7 30.4 28.7 29.5 31.2
8.9 9.7 10.8 10.3 9.1
a
Cocoon shells were kept in 2 ml of aqueous 0.05% Na 2 CO 3 at 1008C.
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H. Yamada et al.r Materials Science and Engineering C 14 (2001) 41–46
Fig. 2. SDS-PAGE analysis of protein components in cocoons after degumming by Na 2 CO 3 .
shells were kept in aqueous 8 M urea at various temperatures for 2 h. As shown in Table 2, only 10 to 15 wt.% of silk protein was removed from cocoon shells at temperatures below 708C, and 25% was extracted at 808C or higher. As the latter value almost equals that reported as the sericin content of cocoon shells, it was considered that sericin removal was nearly complete in this condition. SDS-PAGE analysis of the urea-treated cocoon shells ŽFig. 4. indicated that sericin removal was incomplete below 708C and that fibroin degradation occurred at 908C. Thus, heating at 808C seemed to be the preferable condition for the degumming in 8 M urea. Examination of time dependency of degumming at 808C showed that the treatment time of 10 min was sufficient to remove sericin. When water was used at 1008C to degum fresh cocoon shells, 5 wt.% of silk protein Žca. 20% of sericin. was removed from cocoon shells for 5 min and 12% Žca. 50 % of sericin. for 60 min without degradation of fibroin. Water treatment at 1208C for 5 min removed nearly 20% of silk protein Žca. 80% of sericin. without damaging the fibroin, but prolonged heating caused decomposition of the protein molecule.
3.3. Preparation of solubilized fibroin haÕing natiÕe molecular mass Based on the above experiments, a solution of intact fibroin was prepared as follows: 1.5 g of fresh cocoon shells were soaked in 150 ml of 8 M aqueous urea previously heated to 808C and maintained for 10 min under vigorous stirring to break the shell layers. The cocoon shells were separated from the urea solution and washed three times with water. After brief drying, the cocoon shells were dissolved in 30 ml of ca. 9 M aqueous LiSCN by stirring at room temperature for 15 min. The resultant solution was dialyzed against 3 l of water while changing the outer water every 30 min for 2 h. The deionized solution was centrifuged at 3000 rpm for 10 min to remove a small amount of insoluble material. 3.4. Properties of the intact fibroin solution The fibroin solution made by the above protocol was a highly viscous liquid having a slightly milky appearance. The protein concentration estimated by absorbance at 275
H. Yamada et al.r Materials Science and Engineering C 14 (2001) 41–46
Fig. 3. SDS-PAGE analysis of protein components in cocoons after degumming by soap and enzyme.
nm was nearly 20 mgrml. An assay by the pigment adsorption method according to Bradford gave a value of only 8 mgrml. This may be due to the unique amino acid composition of fibroin. SDS-PAGE without mercaptoethanol treatment showed a single band at ca. 350 kDa corresponding to native fibroin; and with mercaptoethanol, two bands Žca. 350 and 25 kDa. appeared representing the heavy and light chains of the protein formed by cleavage of an SS bond. The solution of intact fibroin showed a rapid increase in viscosity, and subsequently converted to a gel. The length of time before gelling depended on the degree of dialysis, that is, the residual amount of LiSCN in the fibroin solution. In the standard dialysis protocol Ž100 times of water, 30 min.= 4., the deionized fibroin solution gelled Table 2 Temperature-related removal of sericin by aqueous urea Extracteda at Ž8C.
Before extraction Žmg.
After extraction Žmg.
Sericin removed Žmg.
50 60 70 80 90
50.1 50.7 50.8 50.6 51.0
44.8 44.9 43.1 37.8 37.0
5.3 5.1 7.7 12.8 14.0
a
Cocoon shells were kept in 1.5 ml of aqueous 8 M urea with 0.5 M mercaptoethanol ŽpH 7. for 2 h.
45
Fig. 4. SDS-PAGE analysis of protein components in cocoons after degumming by aqueous urea.
within 24 h. This is much shorter than the gelling time for the fibroin solution made from degummed silk, which conserved the liquid state for 1 week or more. Dialysis against 5 M urea instead of water brought the delay of gelling to 2 to 3 days. When the fibroin solution was diluted to less than 0.1%, the liquid state was maintained for over 1 week.
4. Discussion In all the studies on solubilized fibroin so far reported, the starting material has been traditional degummed silk, which was prepared through four steps: namely, Ž1. heating of cocoons to kill pupae, Ž2. soaking them in hot water to soften glue protein around the fiber, Ž3. reeling of threads, and Ž4. heating in alkaline solution to completely remove the glue protein. Fibroin thus obtained was converted to aqueous solution by the aid of chaotropic reagents. The present work clearly indicated that mild degumming is indispensable for the preparation of solubilized intact molecular fibroin. The most preferable degumming procedure involved heating in aqueous 8 M urea, because the results were highly reproducible in repeated experiments. Besides this, enzymatic degumming is also satisfactory; however, care must be taken to avoid contamination
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H. Yamada et al.r Materials Science and Engineering C 14 (2001) 41–46
by the residual enzyme protein. Treatment with 0.05% Na 2 CO 3 for 5 min at 1008C was also permissible, but repeated experiments showed various degrees of conservation of the fibroin molecule and, hence, was difficult to use routinely. As dissolution reagents, chaotropic reagents such as LiSCN, LiBr, NaSCN, and CaCl 2 have been reported w4,8x. LiSCN was selected as being most suitable for the present study, because it can dissolve fibroin to a concentration of 3% or more at room temperature. LiBr also dissolves fibroin at room temperature, but solubility is limited. Dissolution by CaCl 2 ŽAjisawa’s reagent. and NaSCN required heating process to 758C, though degradation of fibroin was not appreciable. Cuprammonium solution, which had been first introduced for the dissolution of fibroin w9x, could not be used because it is known to be highly degradative w8x. It was impressive that the solution of intact fibroin showed a strong tendency to form a gel. It gelled so much more rapidly than did decomposed fibroin that it was difficult to handle. To overcome this difficulty, it is better to store the dissolved fibroin in the presence of LiSCN or urea to delay the gelling. It may be dialyzed against water just prior to use, if necessary.
Acknowledgements The authors thank Dr. Takao Nagoya, Director of Kowa Research Institute for his encouragement throughout this work.
This work was supported by Enhancement of Center of Excellence, Special Coordination Funds for Promoting Science and Technology, Science and Technology Agency, Japan.
References w1x N. Minoura, S. Aiba, Y. Gotoh, M. Tsukada, Y. Imai, Attachment and growth of cultured fibroblast cells on silk protein matrices, J. Biomed. Mater. Res. 29 Ž1995. 1215–1221. w2x E. Momotani, H. Yamada, Y. Takasu, K. Tsubouchi, Histopathogical evaluation of wound dressing by cocoon derived silk fibroin films ŽSFFs., Bull. Soc. Fr.-Jpn. Sci. Vet. 9 Ž1998. 45. w3x Y. Tashiro, E. Otsuki, Studies on the posterior silk gland of the silkworm Bombyx mori: IV. Ultracentrifugal analysis of native silk proteins, especially fibroin extracted from the middle silk gland of the mature silkworm, J. Cell Biol. 46 Ž1970. 1–10. w4x A. Ajisawa, Dissolution of silk fibroin with calciumchloriderethanol aqueous solution, J. Seric. Sci. Jpn. 67 Ž1998. 91–94. w5x K. Weber, M. Osborn, The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis, J. Biol. Chem. 244 Ž1969. 4406–4412. w6x M. Shimizu, Eine rontgenographische untersuchung des sericins, Bull. Imp. Seric. Exp. Stn. Jpn. 10 Ž1941. 441–476. w7x T. Gamo, Electrophoretic analyses of the protein extracted with disulfide cleavage from cocoons of the silkworm, Bombyx mori L, J. Seric. Sci. Jpn. 42 Ž1973. 17–23. w8x S. Sridhara, J.C. Prudhomme, J. Daillie, Studies on silk fibroin of the silkworm Bombyx mori, Arch. Biochem. Biophys. 156 Ž1973. 158– 175. w9x D. Coleman, F.O. Howitt, Studies on silk proteins: I. The conversion of fibroin into water-soluble form and its bearing on the phenomenon of enaturation, Proc. R. Soc. London, Ser. A 190 Ž1947. 145.
Preparation of undegraded native molecular fibroin solution from silkworm cocoons Hiromi Yamada a , Hiroshi Nakao b, Yoko Takasu a , Kozo Tsubouchi a,) a
National Institute of Sericultural and Entomological Science, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan b Kowa Research Institute, 1-25-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan Received 16 January 2001; received in revised form 12 March 2001; accepted 3 April 2001
Abstract The molecular mass of solubilized fibroin prepared from silk was analyzed by SDS-PAGE. It was found that the fibroin molecule was degraded during reeling, degumming Žremoval of sericin., and dissolution of silk threads. A protocol for the preparation of solubilized fibroin conserving its native molecular size is offered, that is, Ž1. to use only fresh cocoons Žnot dry cocoons or reeled silk threads., Ž2. to degum by heating in 8 M aqueous urea, and Ž3. to dissolve by saturated Ž; 9 M. aqueous lithium thiocyanate at room temperature. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Silk fibroin; Fibroin dissolution; Molecular weight; SDS-PAGE
1. Introduction Silk is a well-known natural fiber produced by the silkworm, Bombyx mori, which has been used traditionally in the form of threads. It is composed of two kinds of protein: a fibrous one Žnamed fibroin. that forms the thread core, and a glue-like one Žnamed sericin. that surrounds the fibroin fibers to cement them together. Although the use of silk as threads is very popular, recently, interest has been increasing in the use of solubilized fibroin in biotechnological materials w1x, and biomedical applications w2x. In these studies, the source of fibroin was usually traditional Adegummed silk Žsericin-free silk fiber.B, which was prepared from cocoons by reeling and degumming. For decades, much biochemical research has been done on fibroin. These studies have shown that fibroin is a homogeneous protein with a molecular mass of 370 kDa w3x. In contrast, there have been few reports concerning the molecular mass of solubilized fibroin, even though the production of degummed silk from cocoons and dissolution of the silk fiber involve a number of drastic processes.
)
Corresponding author. Fax: q81-298-38-6028. E-mail address: [email protected] ŽK. Tsubouchi..
This report examines the factors affecting the molecular mass of fibroin in the solubilizing process, and subsequently describes a procedure for obtaining solubilized fibroin from cocoons without incurring molecular degradation.
2. Materials and methods 2.1. Cocoons Silkworms Ža hybrid strain. were reared with mulberry leaves by the usual method and cocoons were harvested. 2.2. Fresh cocoon shells Cocoons, harvested just after the completion of spinning, were cut and pupae were extracted before emerging as moths. 2.3. Raw silk (reeled silk threads) Raw silk was produced by standard method; that is, harvested cocoons Žcontaining pupae. were heated in an oven at 1608C for 1 h. They were soaked in hot water to soften the sericin, and then the thread was reeled by automatic reeling machine.
0928-4931r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 Ž 0 1 . 0 0 2 0 7 - 7
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H. Yamada et al.r Materials Science and Engineering C 14 (2001) 41–46
2.4. RemoÕal of sericin (degumming) of cocoon shells or raw silk In the standard method, cocoon shells or raw silk were kept in 50 times Žvrw. of boiling aqueous 0.05% Na 2 CO 3 for 60 min. Usually, this treatment was repeated twice. Degumming was also carried out with various concentrations of Na 2 CO 3 and lengths of heating time. Soap degumming was performed in 100 times ŽVrW. of 0.5% soap for 30 min at 1008C. Enzymatic degumming was done in 50 times ŽVrW. of 1% alkalase 2.5 l ŽKosindo Chemical. for 30 min at 608C. Degumming by urea was carried out by heating in 30 times ŽVrW. of 8 M aqueous urea containing 0.04 M Tris–SO4 ŽpH 7. and 0.5 M mercaptoethanol, under various conditions of time and temperature. Degumming was also done in 30 times ŽVrW. of hot water at 1008C for 5 to 60 min, or at 1208C Žusing an autoclave. for 5 to 30 min. After the above degumming treatments, the resulting silk materials were washed in water repeatedly and then air dried. 2.5. Dissolution of fibroin 2.5.1. Ajisawa’s method [4] Cocoon shells or raw silk fibers were degummed and then added to 15 times ŽVrW. of Ajisawa’s reagent ŽCaCl 2rethanolr water, 111r92r144 in weight.. The mixture was stirred at 758C to form a clear solution. The resulting fibroin solution was dialyzed in a cellulose tube ŽVisking. against running water until the dialyzate tested negative for chloride ion ŽAgNO 3 .. 2.5.2. Lithium thiocyanate (LiSCN) method: Raw silk fibers or cocoon shells were stirred in 30–100 times Žvrw. of saturated Žca. 9 M. aqueous LiSCN at room temperature until dissolved. The solution was dialyzed against 100 times ŽVrV. of water or 5 M aqueous urea for 2 h, while refreshing the outer solution every 30 min. 2.6. Silk gland fibroin Fibroin was extracted from the silk gland of mature five instar larvae according to the method of Tashiro and Otsuki w3x. 2.7. Determination of protein concentration Absorbance at 275 nm was measured ŽUV-1200 apparatus, Shimadzu., and protein content was calculated as 1 s 1 mgrml. If necessary, the baseline was corrected at 250 and 300 nm. Bradford protein assay ŽBIO-RAD. was also used.
2.8. Polyacrylamide gel electrophoresis (PAGE) SDS-PAGE was performed on a 2–15 % gradient gel Ž10 = 10 cm, Daiichi Kagaku. according to the procedure of Weber and Osborn w5x. The sample usually contained a high concentration of salt. It was desalted by the following procedures. Ž1. A NAP-5 column ŽAmersham Pharmacia. was equilibrated with 8 M aqueous urea and centrifuged Ž2000 rpm for 3 min. using a swinging rotor to remove the liquid occupying the void volume. A sample Ž0.2 ml. was added onto the top of the column, which was again centrifuged as above. The recovered solution was used as the desalted sample. Ž2. Salt in the sample solutions was filtered off with Centricon ŽAmicon. by adding water at intervals. Ž3. Samples were dialyzed against water or 5 M aqueous urea. The samples were mixed with an equivalent amount of electrophoresis buffer Žcontaining 10% SDS, 1% mercaptoethanol, and 8 M urea., and kept at 508C for 10 min. Crosslinked phosphorylase b ŽSigma. and Bench Mark Ladder ŽGibco BRL. were used as protein standards. The gel was stained with CBB R-250 or silver-stain kit ŽAmersham Pharmacia. and scanned to record. 2.9. Chemicals All reagents were of analytical grade. Deionized water made by Elix ŽMilipore. was used throughout the experiment. 3. Results 3.1. Molecular mass of solubilized fibroin Silkworm cocoons were heat-dried and reeled by soaking in hot water. The resulting threads Žraw silk. were boiled in aqueous Na 2 CO 3 by the standard procedure to remove sericin. The fiber thus obtained Žnamed degummed silk. was dissolved in aqueous CaCl 2-ethanol ŽAjisawa’s reagent. to prepare a fibroin solution. SDS-PAGE analysis of the solution showed a broad smeared band at a position around 70 kDa ŽFig. 1, lane 2.. As shown in the figure, SDS-PAGE analysis of native fibroin from silk glands revealed clear protein bands having molecular masses of about 350 and 25 kDa Žlane 1., which correspond to the heavy and light chains of the protein. These results indicated that the native fibroin molecule was degraded to a mixture of polypeptides of various sizes during the preparation of the fibroin solution. As the procedure for preparing solubilized fibroin from cocoons comprises of many steps, we examined which stepŽs. cause degradation of the fibroin molecule. At first, fresh, untreated cocoons were dissolved by Ajisawa’s method. SDS-PAGE analysis of the resulting solution showed a broad smear at a position around 200 kDa and a clear band at 25 kDa ŽFig. 1, lane 3.. When fresh cocoons
H. Yamada et al.r Materials Science and Engineering C 14 (2001) 41–46
43
Fig. 1. SDS-PAGE analysis of protein components in various silk materials dissolved by Ajisawa’s method and by the lithium thiocyanate ŽLiSCN. method.
were dissolved by the LiSCN method, however, clear bands were observed at 350 and 25 kDa Žlane 4., the same pattern as for silk gland fibroin. These results indicated that Ajisawa’s method caused degradation of the heavy chain of fibroin, whereas LiSCN caused no damage to the protein. When raw Žbefore degumming. silk was solubilized by LiSCN, a faint 350-kDa band accompanied by a smear and a clear 25 kDa one were observed ŽFig. 1, lane 5.. In the case of degummed silk, only a smear at a position around 100 kDa was found Žlane 6.. These results revealed that a partial decomposition of the heavy chain of fibroin was taking place in the heat-drying andror reeling steps, and that degumming damaged both the heavy and light chains of fibroin. Heat-drying of the cocoons Žbefore reeling. was also revealed to degrade fibroin Ždata not shown.. The above experimental results indicated that the following conditions were indispensable for preparing solubilized fibroin having native molecular mass: Ž1. the use of fresh cocoons as a starting material but not dried cocoons or raw silk, Ž2. the removal of sericin Ždegum. without breaking the fibroin molecule, and Ž3. dissolution by the LiSCN method. 3.2. Conditions for remoÕal of sericin without degradation of fibroin Since the standard Na 2 CO 3 degumming Ž0.05% Na 2 CO 3 , 60 min, 1008C. caused degradation of fibroin molecules, the effects of heating time on sericin removal
and fibroin conservation were investigated. As shown in Table 1, the amount of weight lost by Na 2 CO 3 extraction Žwhich corresponded to the amount of removed sericin. was nearly equal for treatment times ranging from 5 to 60 min. SDS-PAGE analysis of the resulting threads indicated that sericin Ža smear near the top of lane 1 in Fig. 2. was removed by the treatment for 5 min at 1008C without damage to fibroin, whereas prolonged heating degraded the fibroin heavy chain Žlanes 2 to 6.. Soap and proteinase degumming procedures, which have been routinely adopted in the silk industry, were also evaluated from the viewpoint of fibroin degradation. It was demonstrated that these treatments caused decomposition of fibroin molecules to some extent ŽFig. 3.. Extraction of sericin from cocoon shells by aqueous urea has long been reported w6,7x; hence, urea degumming under various conditions was investigated. Fresh cocoon Table 1 Time-related removal of sericin by Na 2 CO 3 Extracteda for Žmin.
Before extraction Žmg.
After extraction Žmg.
Sericin removed Žmg.
5 10 15 20 60
39.6 40.1 39.5 39.8 40.3
30.7 30.4 28.7 29.5 31.2
8.9 9.7 10.8 10.3 9.1
a
Cocoon shells were kept in 2 ml of aqueous 0.05% Na 2 CO 3 at 1008C.
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H. Yamada et al.r Materials Science and Engineering C 14 (2001) 41–46
Fig. 2. SDS-PAGE analysis of protein components in cocoons after degumming by Na 2 CO 3 .
shells were kept in aqueous 8 M urea at various temperatures for 2 h. As shown in Table 2, only 10 to 15 wt.% of silk protein was removed from cocoon shells at temperatures below 708C, and 25% was extracted at 808C or higher. As the latter value almost equals that reported as the sericin content of cocoon shells, it was considered that sericin removal was nearly complete in this condition. SDS-PAGE analysis of the urea-treated cocoon shells ŽFig. 4. indicated that sericin removal was incomplete below 708C and that fibroin degradation occurred at 908C. Thus, heating at 808C seemed to be the preferable condition for the degumming in 8 M urea. Examination of time dependency of degumming at 808C showed that the treatment time of 10 min was sufficient to remove sericin. When water was used at 1008C to degum fresh cocoon shells, 5 wt.% of silk protein Žca. 20% of sericin. was removed from cocoon shells for 5 min and 12% Žca. 50 % of sericin. for 60 min without degradation of fibroin. Water treatment at 1208C for 5 min removed nearly 20% of silk protein Žca. 80% of sericin. without damaging the fibroin, but prolonged heating caused decomposition of the protein molecule.
3.3. Preparation of solubilized fibroin haÕing natiÕe molecular mass Based on the above experiments, a solution of intact fibroin was prepared as follows: 1.5 g of fresh cocoon shells were soaked in 150 ml of 8 M aqueous urea previously heated to 808C and maintained for 10 min under vigorous stirring to break the shell layers. The cocoon shells were separated from the urea solution and washed three times with water. After brief drying, the cocoon shells were dissolved in 30 ml of ca. 9 M aqueous LiSCN by stirring at room temperature for 15 min. The resultant solution was dialyzed against 3 l of water while changing the outer water every 30 min for 2 h. The deionized solution was centrifuged at 3000 rpm for 10 min to remove a small amount of insoluble material. 3.4. Properties of the intact fibroin solution The fibroin solution made by the above protocol was a highly viscous liquid having a slightly milky appearance. The protein concentration estimated by absorbance at 275
H. Yamada et al.r Materials Science and Engineering C 14 (2001) 41–46
Fig. 3. SDS-PAGE analysis of protein components in cocoons after degumming by soap and enzyme.
nm was nearly 20 mgrml. An assay by the pigment adsorption method according to Bradford gave a value of only 8 mgrml. This may be due to the unique amino acid composition of fibroin. SDS-PAGE without mercaptoethanol treatment showed a single band at ca. 350 kDa corresponding to native fibroin; and with mercaptoethanol, two bands Žca. 350 and 25 kDa. appeared representing the heavy and light chains of the protein formed by cleavage of an SS bond. The solution of intact fibroin showed a rapid increase in viscosity, and subsequently converted to a gel. The length of time before gelling depended on the degree of dialysis, that is, the residual amount of LiSCN in the fibroin solution. In the standard dialysis protocol Ž100 times of water, 30 min.= 4., the deionized fibroin solution gelled Table 2 Temperature-related removal of sericin by aqueous urea Extracteda at Ž8C.
Before extraction Žmg.
After extraction Žmg.
Sericin removed Žmg.
50 60 70 80 90
50.1 50.7 50.8 50.6 51.0
44.8 44.9 43.1 37.8 37.0
5.3 5.1 7.7 12.8 14.0
a
Cocoon shells were kept in 1.5 ml of aqueous 8 M urea with 0.5 M mercaptoethanol ŽpH 7. for 2 h.
45
Fig. 4. SDS-PAGE analysis of protein components in cocoons after degumming by aqueous urea.
within 24 h. This is much shorter than the gelling time for the fibroin solution made from degummed silk, which conserved the liquid state for 1 week or more. Dialysis against 5 M urea instead of water brought the delay of gelling to 2 to 3 days. When the fibroin solution was diluted to less than 0.1%, the liquid state was maintained for over 1 week.
4. Discussion In all the studies on solubilized fibroin so far reported, the starting material has been traditional degummed silk, which was prepared through four steps: namely, Ž1. heating of cocoons to kill pupae, Ž2. soaking them in hot water to soften glue protein around the fiber, Ž3. reeling of threads, and Ž4. heating in alkaline solution to completely remove the glue protein. Fibroin thus obtained was converted to aqueous solution by the aid of chaotropic reagents. The present work clearly indicated that mild degumming is indispensable for the preparation of solubilized intact molecular fibroin. The most preferable degumming procedure involved heating in aqueous 8 M urea, because the results were highly reproducible in repeated experiments. Besides this, enzymatic degumming is also satisfactory; however, care must be taken to avoid contamination
46
H. Yamada et al.r Materials Science and Engineering C 14 (2001) 41–46
by the residual enzyme protein. Treatment with 0.05% Na 2 CO 3 for 5 min at 1008C was also permissible, but repeated experiments showed various degrees of conservation of the fibroin molecule and, hence, was difficult to use routinely. As dissolution reagents, chaotropic reagents such as LiSCN, LiBr, NaSCN, and CaCl 2 have been reported w4,8x. LiSCN was selected as being most suitable for the present study, because it can dissolve fibroin to a concentration of 3% or more at room temperature. LiBr also dissolves fibroin at room temperature, but solubility is limited. Dissolution by CaCl 2 ŽAjisawa’s reagent. and NaSCN required heating process to 758C, though degradation of fibroin was not appreciable. Cuprammonium solution, which had been first introduced for the dissolution of fibroin w9x, could not be used because it is known to be highly degradative w8x. It was impressive that the solution of intact fibroin showed a strong tendency to form a gel. It gelled so much more rapidly than did decomposed fibroin that it was difficult to handle. To overcome this difficulty, it is better to store the dissolved fibroin in the presence of LiSCN or urea to delay the gelling. It may be dialyzed against water just prior to use, if necessary.
Acknowledgements The authors thank Dr. Takao Nagoya, Director of Kowa Research Institute for his encouragement throughout this work.
This work was supported by Enhancement of Center of Excellence, Special Coordination Funds for Promoting Science and Technology, Science and Technology Agency, Japan.
References w1x N. Minoura, S. Aiba, Y. Gotoh, M. Tsukada, Y. Imai, Attachment and growth of cultured fibroblast cells on silk protein matrices, J. Biomed. Mater. Res. 29 Ž1995. 1215–1221. w2x E. Momotani, H. Yamada, Y. Takasu, K. Tsubouchi, Histopathogical evaluation of wound dressing by cocoon derived silk fibroin films ŽSFFs., Bull. Soc. Fr.-Jpn. Sci. Vet. 9 Ž1998. 45. w3x Y. Tashiro, E. Otsuki, Studies on the posterior silk gland of the silkworm Bombyx mori: IV. Ultracentrifugal analysis of native silk proteins, especially fibroin extracted from the middle silk gland of the mature silkworm, J. Cell Biol. 46 Ž1970. 1–10. w4x A. Ajisawa, Dissolution of silk fibroin with calciumchloriderethanol aqueous solution, J. Seric. Sci. Jpn. 67 Ž1998. 91–94. w5x K. Weber, M. Osborn, The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis, J. Biol. Chem. 244 Ž1969. 4406–4412. w6x M. Shimizu, Eine rontgenographische untersuchung des sericins, Bull. Imp. Seric. Exp. Stn. Jpn. 10 Ž1941. 441–476. w7x T. Gamo, Electrophoretic analyses of the protein extracted with disulfide cleavage from cocoons of the silkworm, Bombyx mori L, J. Seric. Sci. Jpn. 42 Ž1973. 17–23. w8x S. Sridhara, J.C. Prudhomme, J. Daillie, Studies on silk fibroin of the silkworm Bombyx mori, Arch. Biochem. Biophys. 156 Ž1973. 158– 175. w9x D. Coleman, F.O. Howitt, Studies on silk proteins: I. The conversion of fibroin into water-soluble form and its bearing on the phenomenon of enaturation, Proc. R. Soc. London, Ser. A 190 Ž1947. 145.