26

フイブロインのメッセンジャーRNA

手 塚統夫ヘ大木

伊東広雄，

勲，山根昭子，

三浦義彰

(千葉大学医学部生化学教室〉

フイプロインは絹糸の主成分であり， 後部絹糸腺中で特に 5令後半に至って急激に合成され

る.その構造はグリシン p アラニンに富み結品部分が存在するなど， コラーゲンと似た点が多

く，ペプチド転移反応のような合成機構を考えても不自然ではない. しかし蚕の種によりその

アミノ酸配列が一定していることは， ここにもまた Jacob-Monodの図式があてはまることを

強く示唆する.それならフイブロインの情報をもったメッセンジャーRNAがある筈である.若

々が後部絹糸腺から 5%SDSおよびフエノールを用いて抽出した核酸ば，無細抱タンパク合成

系に加えるとグリシンのとりこみを促進することが見出された.この核酸は，大部分RNAであ

るにも拘らずDNA様の糸状沈践を形成し，吾々はこれを糸状核酸 Cf-NA)と呼んだ.f-NAは

薦糖密度勾配遠心でリボゾーム RNAを含むことが分ったがp これは塩祈によって除かれた.

後部絹糸腺をこわして分画しその各から核酸を抽出すると， f-NAは核および大顎粒(フイブロ

イン合成の場と考えられる)のみから得られた. 精製糸状核竣ば， 薦糖密度勾配遠心では 5~

6Sの軽い部にあり，超遠心分析ではいわゆる Dipを生じるが， S。は 8.7Sと計算された.

しかし SephadexG200のカラムでは， f-NAは voidvolumeの位置に溶出される.メチル

化アルブミンカラムでは， f-NAは DNAの部分iこ一致して溶出された.このような事は， f-

NA中の RNA部分が DNAと何らかの complexを作っているとし、う事を示凌ずる. ιNA

の希釈塩溶液について深色効果を観察し，精製 f-NAの helix含量は 54%と算出された.吸光

度一温度曲線は 3つの融点 C31---33"C，50....，53"C， 78......，81"C)を示し，後二者がそれぞれ A-U

CT) rich， G -C rich部分にもとずくと仮定すると，数値上よく一致する.この仮定のもとに，

無処理， 55"Cおよび 100"C加熱 f-NAのホルムアルデヒドとの反応性から算出した helix含

量は55%であった.f-NA中の DNA量から考えれば， この数値は RNAの一部にも二重鎖構

造あるいは塩基の stackingがあると推定させる.無細胞系のアミノ酸とりこみを f-NAが促

進させる点について，前報では 105，000X g， 60分の上清にのみ明瞭な添加効果が認められた

この上清をさらに 180分遠心して得た沈盗を化学的， 電顕的に分析して遊離リボゾームである

ことを確認し，グリシンのとりこみがリボゾームの添加量に依存する事を確かめた.f-NAの添

加効果が t-RNAによるものでないことを証明するためには，in vivoおよび仇 vityoでグリシ

ルーt-RNAを合成させ，それが f-NAに入らないこと invivoで ιNA中に僅かに見出され

る放射能は安定で，ヒドロキシルアミン処理によってもはずれないこと， Sephadex G -200お

*現在:昭和大学医学部生化学教室

27

よびメチル化アルブミンカラムの溶出位置， 塩基組成において， プソイドウリジンが見出され

ないこと，などが確かめられた.すなわち f-NAは， リボゾーム RNAでも t-RNAでもない

タンパク合成促進能を持つということで， これはメッセンジャーである可能性が強い. メッセ

ンジャーというと一般には代謝回転が速いことが特徴と考えられるが， 吾々の実験でも他の例

でも，後部絹糸腺の核酸 CRNA)は 5令前半に爆発的に合成され， ブイブロイン合成がさかん

になる 5日目以後ではその合成は著しく低下してしまに しかし このように代謝的に安定な

メッセンジャーRNAは近年続々見つかっており，このように保存された形のメッセンジャーは

量的にもかなり多くなければならず， 吾々のデータは合理的であるといえよう. このメッセン

ジャー RNAが DNAと complexを作っていることは nascentRNAには当然考えられる

ことで， DNA帽 RNAの二重鎖の存在も考えられる.しかし両者の量的関係， helix含量および

塩基組成から， この complexは大部分が RNA-DNAの二重鎖ではなく， DNA二重鎖と構

造を持った RNAとが何らかの形で結合を作っていると考えられる. (このことは Addendum

において電顕的に証明された.) f-NAのかなりの部分が RNase1に対して抵抗性を示すが，

この酵素処理後も f-NAは糸状性を失なわず，水によく溶ける.ところがごの f-NAをさらに

DNase処理すると，難溶性の沈肢を生じ ここに存在する RNAは著しく G含量が高い.フ

イブロインのアミノ酸組成から推定されるそのメッセンジャー RNAの塩基組成は， G-richの

筈であり，これがこのままでは溶解度が低いであろう事も容易に想像出来る.G-richのメッセ

ンジャーは， DNAと何らかの形で結合して可溶となりタンパク合成の場まで運ばれ，そこでリ

ボゾームなどに直接渡されると考えれば， DNaseや加熱等の処理で ιNAがテンプレート活性

を失なうことも理解出来よう.ここに想定したような RNA-DNAcomplexが核から細胞質中

へ出て行くという現象は， 田代らによって近年見出された所見から考えて見ると， けっして奇

異なものではないと考えられる.

討 議

永井 裕氏 1) RNA部分は RNas巴 Iでかなり小さな仕ag-

f-NAを RNase1で、処理した場合， GC content m巴ntになっているが， G-richのため溶解度が低く

の高い fragmentが上清に得られなかったが， 小さ 上清には来ない，この場合にも DNAと直接あるい

な fragmentになっても依然 DNAと結合している は間接に結合していることは上の事実から推論出来

と考えてよいのですか? る.

手 塚統夫氏 2) f-NA中の RNAは塩基が扇在しており， poly

f-NAを RNas巴 Iで処理しても不溶性の物質は Gのような部分がある場合.RNase 1ではこの部分

出て来ませんが， これをさらに DNase処理，加熱 はそのまま残る. この場合も溶解度を示すように

あるいば pH12以上とする等の処理を行なうと沈殿 DNAと直接，間援に結合していると考えられる.

が生じ， これは中性~微アルカリ性ではきわめて難 この結合については，庶糖密度勾配遠心の結果な

i容であります. その為大体の大きさを求める事も困 どから考えても lipopolysaccharideを介してのもの

難ですが，次のよりな可能性を考えることは出来ま ではないかと考えられます.

しよう.

Collagen Symposium， VIlI (1970) 29

Conformation of Silk Proteins in Solution and the Fiber-Forming

Property of Silk Fibroin U nder Shearing Stresses

Eisaku Iizuka

(Faculty of Textile Sci巴nc巴 andTechnology， Shinshu University)

Nearly 10 years ago several reviewsl-4) on the structure of silk proteins were

published. Since then the interest of most of the researchers in this field have

seemingly been oriented to the conformation of synthetic polypeptides. In this

paper some recent works on silk proteins carried out by the author are summa-

rized.

1) The disordered andβ conformations of silk fibroin5，6)

Degummed Bombyx mori L. silk was dissolved in 9. 3お1:LiBr at 370C for・

Fig. 1

+1.5

~ +1.0 O

E 、u 曲、い+0.5E U

~ 曲、

棒、

。、、同

。

小、~-O..5

-1.0 180

。q 司、

200 220 240‘

WAVELENGTH， mμ 260

R巴ducedmean residue rotations of silk fiibroiri in mixed solvents (vJv) Curves: 1， 93 % methanol; 2， 50 % methanol: 3， 50 % dioxane; 4， water only.

30

2 hr， dialyzed thoroughly against pure water until an AgNOa t巴stcould not

detect any trace of bromide ion (4-5 days) ， and clarified by centrifugation.

The stock solution of silk fibroin was mixed with appropriate solvents to get the

desired concentration and composition. The apparent pH of the solution was

adjusted to 7.3， unless otherwise stated. The conformation of silk fibroin was

determined by optical rotatory dispersion (ORD) ， circular dichroism (CD) and

infrared absorption (lR) methods.

Silk fibroin in water lacks any secondary structure as judged from the ORD

and CD， which resemble those typical of a coiled structure (Figs. 1 and 2;

Table 1). The molecule contracted in the presence of salts， and expanded moderately in 8 M urea solution. The intrinsic viscosity of silk fibroin in 0.2

M NaCl was only 0.33， while its molecular weight is about 290，000， suggesting that the molecule was much less expanded than that of such a polyion as ionized

polY-L-glutamic acid. The main feature of the chemical composition of Bombyx mori fibroin is its high content in the hydrophobic residues， which dislike the

hydrophilic atmosphere and cause contraction of the fibroin molecule. Silk

fibroin displayed Cotton effects， though weak， in the aromatic absorption bands，

+3

.tI O +2 .睦U 曲、

¥¥ .. E u

+1 也

'b

nv

v'qxへそ

k e旬、ハ

/¥ 11l

160

4 ..

Fig. 2 Mean molar ellipticity of silk fibroin in mixed solvents Symbols th巴 s8.meas in Fig. J

200 220 240 WAVELENGTH， m，μ

260

31

Effects of salts， urea， and changes in pH on the optic且lrotatory dispersion of silk fibroin in aqueous solution

Table 1

Moffitt Solvent and pH

-a， b， 一kx10-6 入c(mμ〕

日リ

nunununUAUnu

t-nU凸

U

凸

yrO今

ム

凸

フ

今'h

今

4

今ん

'I'I'I

nUAUnuAUAリ凸

unu

ー

9.4 8.9 8.9 8.3

7.4

5.5 4.0

212

211 211 215 214 215 220

250 十1010.9 210

Salt (pH 7.3)

Non巴

0.20 M NaCl 0.35MKF

0.99恥1.LiBr 1.80 M NaCl 3.83 M LiBr 7.44 M LiBr

Urea (pH 7.3)

4M

260 +10 11. 5 209 8M

200 220 220

-10

0

十10

8.8

9.7

10.0

217

211 204

pH (0.2 M NaCl)

4.3

11. 2

12.2

indicating that the side groups such as tyrosine， being contained in only 5 moI

弘， are not completely free to rotate and are held together in some way.

With increasing salt concentration， a decline in magnitude of the levorotation

appeared， indicating that the contracted molecules had less restricted freedom of

rotation than the extended molecules. With increasing protein concentration， the

〔αDJ also became less negative; probably the polypeptide molecules became

contracted， because the molecules themselves are (poly)electrolyt←like salts.

This was consistent with the fact that the silk fibroin became less easy to make

a disordered-to-βtransition under shearing stresses with increasing protein con-

centration， as will be mentioned later. The fibroin molecule would fold on

itself many times to form a rod-like molecule， in which the adjacent segments

are bonded with hydrogen bondings through water molecules. Their number

decreases with increasing protein concentration， causing the decrease in length of

the rod-like molecule.

Addition of more than 30 %のjv)dioxane or methanol to the solution of

silk fibroin in water ，induced a coil-to-βtransition. This was based on the

observation of the amide 1 and V bands of the IR spectra (Figs.3 and 4). In

D20， the amide 1 band of deuterated silk fibroin appeared at 1650 cm-1， and

32

「//〆

1.0

5

0

G

1

φυCOOLOの円以《

0.5

0~800 1700 1600 1500 Wavenumber (cm-1)

FIg. 3 Infrared absorptIon spωtra of sIlk fibroIns In mIxed solvents From the top curv巴sto the bottom ones: Bombyx例 ori，

AnaPhe例 oloneyiand A時theraeaper刊yi.Lines : solId， H，O; dotted， 50 % (v/v) methanol; broken， 50 % (v/v) dIoxane.

the amide 11 band at 1450 cm-1. In the dioxane-D20 or deuterated methanol-

020， however， there appeared a band at 1620 cm-1 and a shoulder at 1690

cm-1， due to the formation of the βform. The transition was time dependent

and its rate varied with the solvent used. The maximumβcontent for silk

fibroin in variously mixtured solvents was estimated to be about 50 '" by resolving

the infrared spectrum into three bands， one centered near 1650 cm司 1 and two

near 1620 and 1690 cm-1. Formation of the βform would be a resu1t of the

extension of polypeptide segments in less hydrophilic atmosphere caused by the

addition of dioxane. ExtentioIl' of the segments would occur also as a result of

the dehydration caused by the addition of methanol to produce the βform.

The IR dichroism measurement carried out on the oriented films of silk

fibroin cast from the mixed solvents with polyvinyl alcohol (added beforehand)

indicated a strong paral1el dichroism. of the. amide 1 band and a perpendicular

33

50

4

30

1JIll-「llJ

LHγ/

/

/

JAd

一、

=

-戸一

戸-

/

-

/

d

d

--戸、

/

J

『

¥-

¥

一

、

¥

、、、、、

¥

J t

dF /

〆/ 〆/

/

」¥・、

、、、、

、、、

。主 60

O

O

R〕

4・FO

R

J

CO一山叫一「とのCOLド

3

800

Amide-V bands of the infrar巴dspectra of silk fibroins

From th巴 topcurves to the bottom ones : Bo隅 byx桝 orifrom the

middle silkgland， A時aPhe何 ticulataeand Antheraea ternyi. Lines: solid， cast from water; broken， cast from SO % (vJv) methanol.

Fig. 4

一一丁一

よ

ーし一一」1600 1500 1800 1700

Wovenumber (cm-1)

r ， t t 1

1

1

、I

¥f ，1 If

)00

δ0

O

;;- 6'

s: 340ト凶 1

戸 20トo

ト OL-一一」18001700 1500

Infrared dichroisms of stretched films of Bombyx制 orifibroin

Left curv巴s，cast from wat巴r;right curves， cast from SO 5￥(vJv) m巴thanolwith 1 : 1 (w J w) polyvinylalcohol

1600

Fig. S

34

dichrojsm of the amideIl band (Fig. 5)， suggesting that the conformation was

of the intramolecular cross-βtype. In contrast， when the disordered form of

silk fibroin in aqueous solution was cast into the oriented film， the sense of

Table II c:otton 巴町ectsof the s form of silk fibroin in mixed solvents (vjv)

1: 1 Diωox悶aneι H凡即2ρo I 1: 1 地胤附t山h伽叩anol-H叩2ρo 1げ93:汀7M陥巴t伽h凶la四m叩ar叩仰no叶1旧O

5却0μ 戸 10∞0μβ1“μ 戸 10仰0%βI 5臼2μ s I 10∞0%戸

Cm')229 x 10-3

Cm')加 X10-3

Cm')"， x 10-3

R2J8 x 10"

R'97 X 10"

a，

b。

3.5

十9.0

-5.0

-260

+ 90

-6

+20

-10

-2.8

+9.9

-6.8

5.8

+8.9

Nonlinear

5 - 1.7

+24

-17

-13

+22

+15.0

- 6.7

- 4.9

十17.2

-40

十30

The first∞lumn under each mix巴dsolvent is the experimental values， and the second column refers to th己 extr<，.porated.v8JUωto 100 % s form

0.122%

40

22%

42%

同 01 ¥ 。

ー予く

メ?，，--..

E 、ー..J 0.042%

O O

o 200 400 600 T r ME. m in

Fig. 6 Time dependence of the reduced viscosity and optical rotation (at 229 mμ) of silk fibroin in dioxane-water mixture (1 : 1， vJv) at 270C

The numb巴rsn巴arthe curves represent the protein concentration.

- 3

+27

-16

- 9

+33

35

dichroism was just the opposite， implying that in this case the βform was of

an intermolecular type.

The 0 RD of the βform displayed a large peak at 205 mμand two troughs

at 229-230 and 190 mμ， and the corresponding CD showed a ・'small negative

band at 217-218 mμand a large positive one at 197 mμ(Figs.1 and 2). These

Cotton effects differ from those characteristic of theαhelix and represent the β

form. In Table H， the Cotton effects of the βform of silk fibroin in mixed

solvents and also the Moffitt parameters ao， bo for the visible ORD are shown.

The ORD and CD of polY-L-lysine in the βform are very similar to those of

silk fibroin. The bo， based on data between 600 and 300 mμ， remained close

to zero. With increasing protein concentration， where viscosity data (Fig.6)

suggested an extensive aggregation， the 恥10ffitt equation could b3 applied only

within a narrow range of wavelength (say between 600 and 460 mμ). This

resulted in a large positive bo up to + 400 and a correspondingly large negative

ao・

2) Sρecies specificity of the conformαtion of silk fibroin7l

Two species of silk fibroin from three subfamilies were tested (Table III).

Subfamily

Bo伽 bycinae

Thau制 etopoe初日E

Satu円1ii四ae

Table III Silk fibroins tested

Gehus

Bo間 byx

A珂aPhe

A時the叩 ea

Philosa例 ia

Species

問 ori，隅 andalina

間 oloneyi，reticulatae

pernyi cynthiaγici刊i

The silkglands were extracted from mature Bombycinae and Saturniinαe silk-worms， and their cellular membranes were stripped off with a forceps. The

contents of the silkgland were dispersed and dialyzed in cellophane tubing against

pure water overnight. When the middle silkgland was to be used， pure water

was replaced several times to remove silk 日ricine which covers silk fibroin.

Degummed silk of AnaJうhemoloneyi was dissolved with a 9. 3 M LiBr solution

and dialyzed againstpure water at 50C， while degummed silk of AnaPhe reticulαtae was first dissolved with trifluoroacetic acid， and cast and dried on

a glass plate to get an amorphous film prior to be dissolved with the LiBr solu-

tion. These solutions of silk fibroin were clarified prior to m巴asurem巴nts.

Bombycinαe and Thαumeto poeinae silk fibroins essentially exist in a

disorder・ed conformation (Figs.7 and 8). Two kinds of the former fibroin，

however， showed the presence of a trace of theβform， when they were taken

straight from the posterior silkglands wh巴resilk fibroin only was ejected. The

36

0.5

O

-0.5 ~ -0.5 O X

r-寸

ふ O

ー0.5180

t t

200 220 240 Wavelength (mjJ)

」

260 280

Fig. 7 Reduced mean residue rotation of Bo拠 byx叫 orifibroin taken straight from th巴

middle silkgland ( upp巴rcurves) and from th巴 posteriorsilkgland (¥ower curv巴s)Lin巴s: solid， in water; dotted， in 0.2 M NaCl; broken， in 8 M urea‘

ORD of the fresh silk fibrOin of Bombyx mori taken from the middle silkglands was less negative than that of the regenerated one by roughly 30 % in water;

the feature of the Cotton effects， however， was the same. As for Thαumeto-iうoeinaefibroins， Anαiうhereticulatae fibroin contained a trace of the αhelix，

as judged from the shift of the 205 mμtrough to 210 mμThe βform orα

helix which was contained in these fibroins disappeared， when 8 M urea was

added to solution.

Sαturniinae fibroins was ch.aracterized by a trough at 231-232 mμand a

peak at 198 mμwith a shoulder around 215 mμ， displa，ying the presence of the αhelix， and might be c1assified as the helical type (Fig. 8). The helical content

as calculated from the bo value， assuming that bo for 100 % helix is -630， was

15 % fo1' Antheraeαρernyi fibroin and 20 % for Philos仰 niacynthia ricini fibroin. It inc1'eased to some extent when 0.05-0.2 M 1、ifaClwas add巴dto

solution， p1'obably because the electrostatic 1'epulsions between the ionized sid号

chains were minimized by cations. Theαhelix disappeared in the presence of

8 M urea as a 1'esult of the destruction of hydrog号nbondings between the p巴ptide

groups.

In all silk fib1'oins tested， the cross-βform appeared when more than 30 00

(v jv) dioxane or methanol was added to the solution. Theαhelix which was

primarily present in Saturniinae fibroins remained unchanged， while in Thau-metoρoeinae fibroins the αhelix also appear吋 orincreased in the content. The

1.0

.5

O

ー.51.0

守 .5

0 - 0

× ー.5

E:1.0 '--'

.5

O

ー‘5

180 200 220 240 260 280 Wavelength (mド)

Fig. 8 Reduced mean residue rotation of Sat叫門ui叫aeand Tham叫etotoeinaefibroins Upper curves， Anthe叩 eapernyi taken straight from the post巴riorsilkgland; middle curves， A叫aPhe叩 oloneyiregen己rat己d;lower curv巴S，A叫aPheγetic廿

latae reg巴nerated.

37

cross-βform appeared aJso at acidic pH's below 3， in the presence of mOre

than 4 M NaCl， or when silk fibroins were gelatinized upon standing at roOm

temperature (1 week or more at 270C). These obser・vationswere based on th巴

ORD， amide 1， II and V bands of the IR spectrum (Figs.3 and 4). In the

amide V band， the disordered，α-helical and βconformations of silk fibroins

showed their characteristic absorption maxima around 650， 620 and 690 cm-1，

respectively.

It is known that in the skeletal frequencies region of the IR sp巴ctraof β-

polypeptides， the Gly-Ala-Gly sequence has characteristic bands at 998 and 975

mμ， and the Ala-Ala sequence at 965 mμThe IR observation on silk fibroins

(Fig.9) was consistent with the idea of Kirimura about the main primary struc-

ture of silk fibroins (Table IV). The conformation of silk fibroin is now known

38

80

ア0

60

0

0

0

0

0

5

4

3

7

6

c

。E2E同COLド

ぷ

50

、、、f

ノノ''

/ /

/ ，，

，，

J 、、

¥ 、、

‘、

-aF 〆

/ 〆

Ill

一、『

f ， ，r

ノ

1Ill14，

/ ノ

ノ/

〆〆

ぽ3¥0

、、戸

〆/ /

f

mw|ιv

『，F /

aF /

/ /

/ /

町ト、

∞ σ 。 |//f¥

(J) I /ーーーーーー-""|~/ / ................ ... . /........

ィ.で~・

1050 1000 950 900 850

Wavenumbe九 cm-I

Fig. 9 Infrared spectra of films of silk fibroins in the skel巳talfrequ巴nciesregion From the top curves to the bottom ones: A刊aPhereticulatae， Antheraea pe何日uand Bombyx隅 ori.

Lines : solid， cast from water; dotted， cast from water and dri巴dfollowed by stretching; broken， cast from 50 % (v / v) methanol.

to be prescr均色dby its main primary structure. The result that the sequence of

alternating glycine and alanine favors the disordered conformation might indicate

that the stability of the αhelix prescribed by polyalanine and that of the poly-

glycine 11 helix prescribed by polyglycine do not differ much.

3) Conformation of silk sericine8l

Middle silkglands were removed from mature sericin←silkworms， Bombyx mori L.， whose posterior silkglands degenerate and are suppos巴dly unable to

produce silk fibroin. With a forceps they were stripped off from cellular mem-

branes， dispersed and dialyzed against pure water in cellophane tubing overnight.

Posterior silkglands， removed on the first day of the fifth instar， were also used.

Besides these， silk sericine was regenerated using Okamoto's method; it was

extracted from shells of unheated cocoons with 0.04 M NaOH at 30-350C， and

Table IV Amiriu" a:c~d ∞加positiohåi1d今市aÌÎl-pfîm~ry stl'ucture of silk fibroin

Amino acid B.刑 ori B.Ma時・ jωyi I P.c. ri叫dali向。

Gly 42.8 42.4 24.7 28.6 37.1 27.8

Ala 30.7 30.0 50.1 49.3 53.6 56.7

Ser 10.0 10.0 8.1 5.0 0.7 3.9

Tyr 4.9 4.5 4.3 4.5 0.6 1.2

Asp， Glu 1.9 1.8 4.6 3.2 0.7 2.1

Lys， Arg 0.6 0.6 2.8 . 1.9 0.1 0.8

Others 4.8 5.0 3.6 3.2 1.5 3.3

Main primary GXI，y-AXl-a GoIr y-SX er Ala-Ala-Ala-Ala Ala-X-Ala-X structure X， other than Ala

This table was prepar巴drefering to the data of Shimizu， Fukuda and Kirimura'). Data are given in mole %. * : silk sericin巴.

39

11.6

5.0

28.1

2.7

19.3

6.2

12.5

separated into two components by adjusting the solution to pH 4.0 with acetic

acid. The soluble part， sericine A， was adjusted to a neutral pH and dialyzed

against pure water overnight， while the insoluble part， sericine'B， was redissolved

with 0.04 M NaOH and dial.Yzed. Ser匂 inesA and B were in the ratio of 5: 2.

The ORD of fresh silk sericine displayed shalIow troughs near 230 and 205

mμand a peak at 190 mμ(Fig. 10); its feature resembled the calculated disper-

sion of a hypotheticaI mixed conformation of the βform and of the coil of silk

fibroin. The CD of fresh silk sericine showed two shallow， negative bands

centered at 217 and 200 mμThese results indicate that the conformation of

silk sericine is disordered， containing a smaIl amount of the βform.

Moffitt parameters ao and bo of fresh silk sericine were -270 and 0 at neutral

pH's and remained unchanged in a pH range between 5 and 11. No difference

in the Cotton effects was detected aIso， showing no drastic change in the con-

formation. This is probably because acidic and basic residues balanced the

dissociation of the po1ypeptide chain. The p1'esence of s且1tup to 3.6 M caused

a contraction of the polypeptide chain， while the βform of silk sericine dis-

appea1'ed in 8 M urea. As was the case in silk fibroins， the cross-βform

amounted to about 50 %， when 50 '/'0 (v jv) dioxane 01' methanol was added to

aqueous solution.

The confo1'mation of 日 ricin巴sA and B in the regenerated silk sericine was

similar to that of fresh silk sericine (Fig. 11). The content of the βform‘

however， is higher in the Iatter. Although any quantitative estimation of the β

form by CD and ORD is hazardous at the moment， the βcontent is tentatively

40

6

21-ー

-2

c")

O

X r-寸

8 ー1.5

40L 180

O

4

円

'OFX

〔主

iニエイ7

O

220 240 入(mμ)

Reduc巴dmean residue rotation and mean molar ellipticity of fresh silk s巴ricin巴 inaqueous solutions Lines -ー 1:pH 7.3;ユ 8M urea (pH 7.3); 3: 3.6 M NaCl (pH 7.3); 4: pH 5.0; 5: pH 11.0.

Fig.10

calculated as between 5 % and 10 %， assuming the mean molar el1ipticity， Cθ)，

of the 100 %βis -16，000 at 218 mμThe Cotton effects of the fresh silk

sericine were expressed from those of two components of the regenerated silk

sericine， taking into account of their respective contents.

Fiber-formation of silk fibroin under shearing stresses9-1l1

With a Farol rheogoniometer (a cone and plate viscometer)， dynamic visco-

sity was measured in the solutions of regenerated and fresh silk fibroins， Bombyx mori L.， at a bout 25 predetermined shear rates for 1. 5 min respectively， i. e. ，

b巴tween6 X 10-3 and 1. 5 x 102 sec-1. (lt is supposed that the silk fibroin is

under the shear rates of this range in the spinning process.) An interval of 0.5

min was spent to change the gears to raise the shear rate by degrees. The

measurements were carried out at 14土 20C and at pH 6. 5， unless otherwise stat巴d.

4)

41 r-ーーー，

6

2

O

4

や

02hE]

-2

α F】

O '; -1.5 ~ 目、C:J

~3.0

180 220 240

入(mμ)

Reduced mean residue rotation and mean molar ellipticity of the two components of reg巴neratedsilk sericine in aqueous solution

Lines一一一 1: sericin巴 A;2: sericine B.

一一一:water (pH 7.3);一一一一:8 M urea (pH 7.3).

280

Fig.l1

刈 0-2 )(10'・ )(100 x 10+1 Shear rate (5ec.') 2.34 5，89 1.47 1.47 一一一

1.47 3.71 9.32 1.47

6.32a/IOOcc 2

re3enen止なd.fLbrOill

O

14・c2，363//00cc Ji...broifL γl..G.:Liv色

(曲一司山由

hMgzf320注目

』

55∞

2

20

TlME， min

Shear stress vs. shear rate for r巴generatedsilk fibroin (a) and for fresh silk fibroin (b)

30 10

Fig.12

O

42

Concentrated solutions of regenerated silk fibroin showed the New10nian

behavior (Fig.12a) at the beginning. A tremendous increase in the shear stress

appeared suddenly， when the shear rate reached a certain valueラ andafter that

the shear stress no longer vanish巴d promptly， when the plate of the viscometer

was stopped in rotation， indicating that some structure was formed in solution.

After this sudden increase in shear stress， a number of water-insoluble clots were

nuiformly observed in solution. X-ray diffraction patterns of thes巴 showedt出ha抗t

， a fiゐb巴ぽrstructure s計imi日la但rtωo t白ha飢t(i凶nt印er印cha幻111β fo印rm)of bave was fo印rm巴d. As

there had original1y been no fib巴rstructure in solution， this was undoubtedly

produced under the shearing stresses.

Di1ute solutions of the ragenerated fibroin showed an extremely high torque

at very low shear rates， due 10 a film formed on the surface of the solutions

around the edge of the cone and plate. The conformation of the silk fibroin

molecule now seemed 10 be different in dilute and concentrated solutIons.

With increasing shear rate， the extinction angle decreas巴d and the curve of

the double refractive index showed an upward increase， indicating that the fibroin

molecule was not rigid but flexible， or being able to be extended (Fig. 13). It

45

40

30

35

4

2

U-MW己吋ロOニUC門戸刊以

る{×ロバー

2000 4000

s)1ear rate， sec-1

Double refractive index and extinction angle vs. shear rate for regenerated silk fibroin The numbers near th巴 curvl巴srepresent th巴 proteinconcentration in 5百，Temperature， 270C.

6000 O

Fig.13

43

is now clear that the fibroin molecule is scarcely ori巴nted，when it begins to

form the fiber structure (the shear rate being below 100 sec・1)，and that the fiber

structure is not attained by simple orientation of the molecule but by its unfold-

ing. This is achieved when the stability of the folded structure has been overcome

by the force between neighboring fibroin molecules， f10wing at different velocities

and colliding with each other to combine in the sheared fiow.

The critical shear rate， at which the silk fibroin begins to

structure， was plotted against the protein concentration (Fig. 14).

form the fiber

The fibroin

103

140C

O

O

o After being hea十ed

ot 980C， for 1・5hr

at 500C for 1・5hr

〉唱

SOLV.，0・2MBorate buffer

pH 7・5

102

10-'

10'

.>ど10

-UU由

10-2

0.1

C， g/I旧O∞OmCritical shear rat巴世S. concentration for solution of regen巴ratedfibroin (dialyzed mainly with tap water)

Fig.14

used was regenerated with LiBr and dialyzed against tap water (3-4 days) ，

followed by an overnight dialysis with pure water. With increasing fibroin

concentration， the critical shear rate first decreased， because of the increase in

opportunity for the fibroin molecule to collide with each other， and th巴nincreased

remarkably， probably because the fibroin . molecule became less extended and

stabilized in the structure; it also increased with the increasing ionic strength as

the molecule became less extcnded.

44

When dialyzed against pure water over the whole period， the regen巴rated

silk fibroin showed higher critical shear rates. When the dialysis was undertaken

against a O. 01 M CaClz solution during the first half period， the critical shear

rate-concentration relationship was the same as that shown in Fig.14. Dialysis

against a 0.01 M KCl solution， however， could not lower the critical shear rat巴.

It is thus obvious that divalent metal ions such as Ca汁 andMg什 promotethe

formation of fiber structure under shearing stress号s. Their role would be to join

the fibroin molecules by combining COO-groups of their side chains. These

metal ions are originally contained abundantly in the aqueous silk of silkwonns

(Table V)， but are considered to be dialyzed out mostly when the LiBr solution

of the silk fibroin has been dialyzed.

Table V Composition of dried cocoon shell

g component/100 g of dried cocoon shell Component

white race yellow rac巴

Fibroin 73.59 70.08

Sericine 22.28 24.29

Ash (total) 1.06 1.92

Ash (contained in fibroin) 0.09 0.16

Wax 3.02 3.46

After attaining the full growth， the silkworm attaches itself to any object

such as straw or twigs; from its two large silkglands (Fig.15) on either side of

its body， it ejects through a small orifice on its head (the spinneret) two fine

strands of the viscous fluid， joining into a double thread which becomes hardend

while passing through the nozzle. The rate of spinning bave ranges from 0.4

to 1. 5 cm/sec (mean rate 1 cm/sec) along the length of the bave. The mean

shear rate in the cross section accompanying the extrusion of aqueous silk was

calculated， assuming a Newtonian liquid (Fig. 16).

Fresh silk fibroin showed a complicated behavior in shear stress VS. shear

rate relation， because of the presence of high divalent metal ions which cause

the association of fibroin molecules. The critical shear rate could not unques-

tionably be determined. After the fourth incr・easein the shear stress appeared

when the shear rate was raised dy degress (after 40 min in Fig. 12b)， the

insoluble clots were observed in solution; the fourth increase could not b巴

distinguished from the third， sometimes. The shear rate at which the fourth

increase in shear stress appeared ranged from 50 to 100 see-1 for the concentra司

tion between 0.8 and 4.0%. The critical shear rate for the aqueous silk could

not be determined， but it might be considered to be below 100 sec-1. N uc lei of

Fig.15 Silk glands (parts named in Fig. 16)

0・05-0'3mmや 1・2mmや岳5mmや2・Ommや 0'8-0Ammや

一一 二二二二二コ

-35mm ご二 60mm----T二 200mm一一-4x102-2xl sec-I 3xI0-2sec-1 3xlσ3 sec-' 6x10-3 sec' 1 xlσJ... 8 X 10-1 sec-I

Anlerior division Middle division Poslerior divisi町、

MTe 川町 2竺三笠?μ2x2

ょ←J 二一品、こ 10Anlerior div 1085μ ¥Outlel 01 Ihe glond 01 Filippi

SPINNERET

Fig.16 Profile of th巴 dimensionand the distribution of shear rate for th巴 silkglandand the spinneret

4S

the extended βform would appear first in the ，anterior silkgland (anterior divi-sion of the silkgland) to grow into microfibrils under the constantly increasing

shear rate.

The role of silk sericine whi.ch covers the area adjacent to the wall of silk-

gland would be not only to let the aqueous silk coagulate more uniformly， but

also to render it pass easily through the spinneret after hardening.

Silk brin (fibroin monofilament) forms its fiber structure under shearing

46

5

V

0

5

o

a--4・ス

Mnv

%と

z一--u-帥

h』

U.2

c Q)

~

¥150 巴n

.、凶

100

Fig.17

、.

。¥¥叫

寄れ〉お¥.一@子丸、

~~\ ， 70 cps

200

C.60%RH

1.0 1.5 Size. denier

Crystallinity and dynamic Young's modulus vs. size for brin Taken from 1， outer layer; 2， middle layer; 3， inner layer

0.5 2.0

Table VI Young's modulus of silk sericine

Frequency (cjs) Layer Young's modulus (gjdenier)

0.028 static

170

Outer 30 ::l:: 13

Middle 35土 18

Inner 37士 21

Outer 61土 12

Middle 61土 18

lnner 65土 18

5000 75.2土 0.4Mixed

stress from aqueous solution， and has a higher crystallinity with the decreasing

area of cross section (Fig. 17). This would be due 10 the fact that the shear

rate is higher in the thinner silkgland (which produces the thinner brin) ， if the

spinning rate is same. . Actually the spinning rate is higher when the brin is

47

thinner. Y oung' s modulus is higher again for the thinner brin， reflecting the relationship of crystallinity VS. area of its cross section. Young's modulus of

silk sericine， as determined by comparing the brin and the bave in the modulus，

showed no dependence upon the thickness of the bave (Table VI)".

REFERENCES

1) Bamford，C.H.， Elliott，A. and Hanby，W.E. :Synthetic Polypeptides， Academic Press， N目Y.，p.369 (1956).

2) Shimizu， M.， Fukuda， T. and Kirimura， J. Protein Chemistry， Vo1.5， Kyoritsu Pub. Co.. Tokyo， p.317 (1957).

3) 1to， T. (ed.): Structure 01 the Silk， Chikuma-kai Press， Ueda (1957). 4) Lucas，F.， Shaw，J.T.B. and Smith，S.G. : Adv. Protein Chemistry， 13， 107 (1958)，

5) lizuka， E. and Yang， J. T. : Pγ'oc. Natl. Acad. Sci. U. S.， 55， 1175 (1966). 6) Iizuka， E. and Yang， J. T. : BiochemistγY， 7， 2218 (1968) 7) lizuka， E. : Biochi刑. Biophys. Acta， 160， 454 (1968). 8) lizuka，E.: Ibid.， 181， 477 (1969) 9) Iizuka， E. : Biorheology， 3， 141 (1966). 10) lizuka，E.: Ibid.， 3， 1 (1965). 11) 1izuka，E. : Seikagaku， 39， 197 (1967).