C.ollagen Symp.osium， X (1973)

Flexibility of Tropocollagen

from Sedimentation and Viscocity

Hiroyasu Utiyama， Kuniaki Sakato， Kenji Ikehara，

Takashi Setsuiye and Michio Kurata

1n s ti tu te f.o r Chemical Research and Department

。f1ndusirial Chemistry， Ky.ot.o University

SYNOPSIS

Sedimentati.on c.onstant and int 1'insic visc.osity we1'e measu1'ed .on purified

tr.op.ocollagens ext1'ac ted fr.om ea1'thw.orm-cu ticle and la thy 1'itic ra tsl、in・

A cartesian diver visC.ometer was used t.o make visc.osity measu1'ements

at small shea1' st1'esses and t.o avoid th巴 effects.of surface f.o1'ces. By

c.ompa1'ing the expe1'imental data with the hyd1'.odynamic the.o1'ies .of w.o1'm-

like-c.oil .of Ullman， a value .of 1300 A has been assigned f.o1' the pe1'sist-

ence length .of these t1'.op.oc.ollagens. Othe1' fact.o1's which may affect the

estimate a1'e discussed.

INTRODUCTION

-29-

Like the a-helix .of p.olypeptide and the d.ouble-helix .of DNA， the t1'iple heli

cal st1'uctu1'e .of c.ollagen is .one .of the unique c.onf.o1'mati.ons .of bi.ol.ogical mac1'.om.ole-

cules. 1ts gene1'al featu1'es that a1'e n.ow widely accepted as c.o1'1'ect a1'e th1'ee

helical p.olypeptide chains a1'e w.ound a1'.ound each .othe1' t.o f.o1'm a th1'ee-st1'anded 1'.ope-

like st1'uctu1'e. Ramachand1'an and Ka1'tha 1) fi 1's t pr.op.osed this s truc tu1'e .on the

basis .of the fact that the1'e is a n.on-integ1'al sc1'ew axis which 1'elates equivalent

units by a t1'anslati.on .of 2.9λand a 1'.otati.on .of app1'.oximately 108" ， a吋 thateach

chain， has ev巴1'y thi1'd residue glycine. 1n additi.on， the1'e is n.ow little d.oubt in

that this t1'uctu1'e is unive1'sal t.o ve1'tebrate and inve1'teb1'ate c.ollagens. The ex-

pe1'imental evidences f.o1' this view a1'e the glycine 1'esidue c.ontents a1'e ve1'y cl.ose

t.o .one thi1'd， and the wide-angle X-1'ay patte1'ns .of samples .o1'iented by st1'etching a1'e

typical .of c.ollagens:;) The m.o1'e p1'ecise m.olecula1' c.onf.o1'mati.on has l.ong been c.on-

Aυ 。。

trovertial due to the limited detail of the X-ray pattern of collagen and the com-

plexity in the amino acid sequence 3-6). 1 t seems tha t the triple helical confor-

mation proposed by Yonath and Traub71， with one NH.. 0 interchain hydrogen bond per

。tripeptide of a hydrogen bond length of 2.86 A， is increasingly favored by the recent

8.9) mvestJgatJons 3)

This structure resembles collagen II type in its mode of hy-

drogen bonding， but the hydrogen-bond length is significantly longer than the values

previously reported.

The size and shape of tropocollagen in solution were established by Boedtker

and Doty in 1956 on collagen extracted from the carp swim bladder tunics (ichthyocol)

from measurements of light-scattering， osmotic pressure， intrinsic viscosity， sedi-

mentation constant， and flow birefringencelO). Comparison of experiment.al results

with a Iight-scattering th巴oryof thin rodsl1

) and hydrodynamic theories of prolate

ellipsoids of revolutionI2-15) lead them to an unequivocal and unified conclusion that tro-

pocollagen consists of rigid， rod-shaped molecules with a diameter and a length of 13.6

A and 3，000 A， respectively. This conclusion stood in remarkable agreement with

findings from X-ray diffraction j) and electron microscopic s tudiesI6). S imilar results

have been repeatedly obtained on numerous oth'er collagens17田:

1t may well be expected， however， that longer tropocollagen will show up some

flexibility， becaus巴 nomatter is completely rigid. The tropocollagen extracted from

the earthworm-cuticle is quite interesting in this respect; the molecular weight is 2

to 6 times as large as the usual vertebrate collagens， but otherwise similar24.251.

If we calculate the particle length， assuming a prolate ellipsoid of revolution， from

the intrinsic viscosity data obtained by Josse and Harrington25J， it will amount to only

about one half of the values expected for rigid rods. This clearly indicates that sol-

ution properties of earthworm-cuticle collagen need to be analyzed in terms of the

wormlike-coil modeI26-V;)

1n treating with this problem， there are two important aspects to be considered

with care. One is experimental in nature and the other is theoretical. First，

viscosity of solutions of very rigid and long particles such as earthworm-cuticle col-

lagen should be considerably subject to the influence of the sh巴ar stress. 1n addi-

tion， viscosity measurements will be badly influenced by the solid surface film formed

at the liquid-air interface. Second， the existing theories of intrinsic viscosity and

sedimentation constant on the wormlike-coil model include hydrodynamic diameter and

friction constant per unit length as the param巴terswhich are no way uniquely deter-

min巴d. Therefore， the persistence length is estimated with greater credence frbm

comparison of hydrodynamic properties of more than one samples of the same species

but of different molecular weight. With respect to the former， we may obtain accu-29)

rate viscosity data with use of a viscometer of cartesian-diver type For the

latter， we may regard earthworm-cuticlp collagen and other vertebrate collagens such

唱

EA

qd

as ratskin collagen as a homologous series， because in these collagens the content

of the glysine residue is very close to one third and the molecular w巴ight of dena-

tured gelatin is also approximately one third of the native tropocollagen.

Thus， in the present investigation we attempt to reinvestigate intrinsic viscosi-

ty and sedimentation constant of tropocollagens extracted from the earthworm-cuticle

and the ratskin in the hope of deducing a universal persistence length.

MATERIALS AND METHODS

Extrαc tionαηd purific日tionof co IIαgen

Earthworm-cuticle collagen and lathyritic ratskin collagen were prepared essen-

tially following the procedures described by Josse and Harr・ington251 and Nimni叫

respectively. The proc巴duresare briefly described here. Lathyritic collagen was

employed simply to use in a later work of renaturation kinetics on a-gelatin.

Earthworms， Phere tma communis s ima， were collec ted locally and s tored f rozen

in a refrigerator until use. After thawing， about 1， 000 earthworms were removed off

cuticles with forceps. The cuticles were minced with scissors， washed thoroughly

with water， and ground with a mortar and pestle until a thick slurry was obtained.

This material was suspended in 100 ml of 0.5 M NaCl and collagen was extracted for

48 hr with stirring. The resuItant viscous suspension was separated from undis-

sol ved material by centrifugation and subject巴d to precipitation twice with saturated

ammoniun sulfa te. The pellet collected by centrifugation was finally dissolved in

30ml of O. 2 M r、JaCl，centrifuged at 105， 000 x g for 1 hr， and th巴 supernatantliquid

collected. This solution was thoroughly dialyzed against 0.2 M NaCl or 0.06 M sodium

acetate buffer， pH4. 8， and stored at 40C.

Rats (Sprague-Dawley) were placed on a diet supplemented with 400 mg of D-peni-

cillamine per kg for 7 days. About 30 g of dorsal skin were cleaned of adhering

tissue and fat. After mincing with scissors into small pieces and washing with water，

they were homogenized for 20 min with 20 volume's of 0.15 M NaCl in Sakuma homoge-

nizer. The homogenized material was suspended in 30 ml of 0.15 M t、JaCl and collagen

was extracted for 48 hr under stirring. Undissolved material was then separated from

the solution by filtering through several layers of cheese cloth and by centrifugation

at 77，000 x g for 2 hr. The crude collagen solution was purifi巴dby precipita tions

with ac巴ticacid and saturated NaCl solution. The precipitate was collected by cen-

trifugation and dissolved in O. 5 M acetic acid with stirr・ing for 24hr. After cen-

trifugation， the supernatant was adjusted to pH 4.8 by addition of 1 M， potassium a-

cetate and thoroughly dialyz巴d against 0.06 M' potassium acetate buffer， pH4. 8， and

10'3 M sodium bisulfite. Sodium bisulfite was added to prevent the formation of

32

free aldehydes叫 Thecollagen solution was finally centrifuged at 105，000 x g for

4 hr and the supernatant collected as the sample solution.

until use.

Conceηtration determination

1t was stored at 40

C

Concentrations were determined as an average of at least thre巴 independentmi-

cro-Kieldahl determinations. A nitrogen content of 18.2% was used for both the

ra tskin and earthworm-cuticle collagens to convert nitrogen analysis to dry weight

of collagen 25.:121.

Intrins icγiscosity

Viscosities of sample liquids were measured in a rota ting cartesian diver vis-{~)

cometer which was described in detail elsewhere" Th巴 rotorused was like a test

tube in shape; 9mm in outer diameter and 50mm in length. A copper ring which

fitted tightly insid巴 the rotor was fixed at about 5 mm from the open end with epoxy

resin. The rotor was submerged upside-down in the test liquid in the stator with

an inner diameter of 10.66 mm. Driving torque was produced through the interaction

of the external rotating magnetic field with the magnetic moment induced by the eddy-

色urrents in the copper ring. The vertical position of the rotor was maintained

constant to 土 0.1 mm by manually regulating pressure inside the rotor while viewing

the rotor through a cathetometer. To achieve the stationary and concentric revo-

lution of the rotor， the copper ring should have b巴enappropria tely fi!ed in advance.

The temperature of the sample liquid could be kept constant to 土1.01 'C by circu-

lating water in the mantle surrounding the stator from a constant temperature bath.

The range of shear stress obtainable by changingthe magnet speed was 0.003 -0.4

dynes・ cm-"forwater at 20 oC. The relative viscosity was estimated according to

the equa tion:

ηr = (P - Pml / (P 0-Pml (1)

where P and P 0 denote the speeds of rotation of the rotor in the collagen solution

and the salt solution used as solvent ，respectively， and P ~ the speed of the magnet. 町1

The relative viscosity could be m巴asuredcorr巴ctly to 士 0.1 %. 1ntrinsic viscosity

was estimated as the coincident intercept of th巴 plotsofη可、/c versus η( the 'ISp' - . ----- '/ sp、

S chulz and B laschke plot ) and in ηrel/ c versus c (the Mead and Fuoss plotl.

Partiα1 specific volume

Densities of solvent and solutions were measured in a measuring-cylinder pycno-

meter of the Lipkin-Davison type. For lathyritic ratskin collagen in 0.06 M potas-

sium buffer， pH 4.8， the partial specific volume was determined as 0.698土 0.005ml/

g. This vaJue is compared to the data reported in the Jiterature: 0.705 for dog-2:{)

fish shark skin collagen in sodium formate， pH 3.8， 20 oC 0.70t and 0.686 in so-

dium phosphate buffer， pH 7.4， and sodium citrate buffer， pH 3.7， respectively， for

qa qa

calf skin collagenl9J

. Density measurements on earthworm-cuticle collagen could not

be made due to shortage of the sample. We therefore used a value of 0.69 ml / g

which was calculated from the amino acid composition25J.

Sedimeηtation constαnt

Sedimentation velocity measurements were made at 20'C in a Beckmaim-Spinco

Mod巴1E ultracentrifuge at a rotor speed of 59，780 rpm. Sedimentation of the

boundary was measured with use of the Schlieren optics， and th巴 Schlierenpatterns

obtain巴d were read on a Nikon universal contour projector， Model V-16， accurate to

土 0.005mm. Sedimentation constant at infinite dilution was estimated from a plot

。f1/ S versus concentration， and the result obtained was converted to that for the

standard condition of water at 20'C by multiplying by (7Jsolvent/ザH20)andby (1予pw)/

(1ー予ρ ) . solvent

S edimenta tion equi lib rium

The short column m巴thodof Van Holde and Baldwin341 was employed for sedim巴nta-

tion eqilibrium measurements on ratskin collagen. The experim巴ntalprocedures were

describ巴d in detail elsewhere35J. Five test solutions of different concentration

were simultaneous¥y centrifuged in a multicell rotor of An-G type with a solution

co¥umn height of approximate¥y 0.17 cm. An inert ¥iquid was not inserted at the cell 361

bottom. The overspeeding t巴chniquedescrib巴d by Hexner et al.~' was used to reduce

the time r巴qUlr巴d for the attainment of巴qui¥ibrium. The attainment of sedimenta-

t10n 巴qui¥ibriumwas assured by plotting against time the horizontal distance between

two Ray¥巴igh fringes s巴paratedas distant as possible on the photograph. The cen-

trifugation was not stopped b巴fore the distance was observed constant at ¥east for 2

hr. Since the ratskin' collagen preparations were fair¥y mon odisperse， the mo¥ecu¥ar

weight and the second viria¥ coefficient can be estimated unambiguous¥y from

M.~~ =M+ B ( c.-c、)/2十higher terms in c and c.. app a b a h (2)

Here M is the molecu¥ar w巴ightof the so¥ute， c a and cb the so¥ute concentrations

in equilibrium at the meniscus and at the bottom， respective¥y， and B is twice the

second viria¥ coefficient. The apparent mo¥ecu¥ar weight M appis defined by

M ム二三三一 2 RT (3)

一 -app c 0 ( 1ーマグ)ω2(ri-r;)

with P" the so¥vent density，ωthe angu¥ar speed of the rotor， r a and r b the radi-

al distances from the center of rotation to the meniscus and bottom of the so¥ution

co¥umn， r巴spectively，R the gas contant， T the absolut巴 temperature，and c 0 the

solute concentration prior to centrifugation. One may obtain， as we did in the pre--1

sent investigation， accurate mo¥ecular weight by simp¥y p¥otting Mapp versus c 0 as in

eq. (5) below， but the s¥ope in this p¥ot does not give the s巴condviria¥ coefficient.

34-

For eathworm-cuticle collagen solutions we employed the Yphantis procedure制，

because the concentration of the collagen preparation was rath巴r low. Centrifuga-

tion was made with the Schlieren optics using an eight-channel centerpiece. The

apparent molecular weight was estimated from

R T 1 dc =一一一←一一一一(-)

app (1-示p")ωrc0 dr r

(4)

whe re r， the radial position whe re the concentra tion remains unchanged upon cen tri-

fugation， is assumed as the midpoint of the liquid column. 1n this method， eq. (2)

is replaced by

M-l= M l十 Bco+・・・ (5)

RESULTS

Experimental results of M app obtained by s巴dimentation equilibrium measure ments

are plotted in Fig. 1 versus the solute concentration prior to centrifugation. Linear

extrapolation of the plotted points to infinite dilution yields the molecular weights

of 3ρ1 土 0.3 x10 and 169 士10 X 10' for ratskin and earthworm-cuticl巴 collagens

resp巴ctively. The former r巴sultstands in very good agreement with the data

of 29.4 X 10' reported by Lewis and Piez231

• The molecular weight of earthworm 23)

4 cuticle collagen may be compared with value of 190 X 10'， which was obtained by

25) combining the sedimentation constant and intrinsic viscosity~J' with a βvalue of 3.65

X 10 ぺ

Fig. 2 shows the results of s巴dimentationvelocity measurem巴nts. The plott巴d

points of each collagen follow a straight line， and the intercept at zero solute con-

centration yields S"20， wof 2.82 土 0.05and 5.08士 0.05for ratskin and earthworm

collagens， respectively. The present experim巴ntalresults on earthworm collage口 ar巴

251

contrasted with those of Josse and Harrington ，in which data points in the similar

plot exhibited marked d巴viation from linearity below the concentration of about 0.5 mg

/ ml. They obtained a value of 7.0 for SZO， win 0.2 M NaCl at pH 6. The sedト

mentation constant for ratskin collagen is compared to the value of 3.15， which was

obta.in巴d in citrate buffer， pH 3.7動.

Experimental results of viscosity measurements are shown in Figs. 3 and 4 for

ratskin and earthworm collagens， respectively. Th巴 shear stree used in the meas-

urements was 0.17 dynes / cm -. The plotted points wer巴 extrapolatedto infinite di-

lution so as obtain a common int巴rceptand the same value of Huggins' constant in

the two types .of plot. The intrinsic viscosity of 12.4 dl / g obtained on ratskin

collagen seems to b巴 in satisfactory agreement with a literature value of 13.7 dl / g

4.0

(0

~ 3.5 x '0. a ro

芝

1.0

0.5 O

.

0.5 1.0 1.5 Co (mg/ml)

Fig. 1 Results of sedimentation equilibrium on collagens at

200

C as a plot of reciprocal apparent molecular weight

versus concentration prior to centrifugation. Data

for ratskin collagen (open circles) were obtained in

0.06 M potassium acetate buffer， pH 4.8， with the

standard sedimentation equilibrium method (eq. 3) ，

and those for earthworm collagen (closed circles) 口

0.06 M sodium acetate buffer， pH 4.8， with the short

column method of Yphantis (eq. 4).

-35-

po nべυ

0.3

0.5

0.4

Tの

0.2

o 0.5 1.0 1.5

CONCENTRATION (mg/mt)

Fig. 2 Concent ration dependence of reciprocal sedimentation

constant at 2{)" C for ratskin collagen in 0.06 M po

tassium acetate buffer， pH 4.8 (open circles)， and

for earthworm collagen in 0.06 M sodium acetate buf

fer， pH 4.8 (c1osed circles)

~{)J

in citrate buffer， pH 3.1 The intrinsic viscosity of earthworm-cuticle col-

lagen ([η，) = 109 dl/ g} obtained is markedly higher than the 62 dl/ g reported by

Josse and Harrington Z51. The difference should be due， at least partly， to the

e ffec ts 0 f the s hear gradien t in the capill ary viscome te r emp loyed by them. We，

too， attempted to measure the viscosity with a viscometer of Ubbelohde type， but

the reproducibility of the measurement was observed very poor.

巧

tq宅U

O 22

。.. 20 u

‘句、

ra ω h

1 8 .、• .. 1 6 U “、

qhu • $::- 14 c ー

1 2

10 O

0.2

"7sp，。0.4 0.6 0.8 1.0 1.2

• 2 3 4

c (g Id l) x 1 03) •

5 6

Fig. 3 Viscosity results on ratskin collagen at 20"C in 0.06 M

potassium acetate buffer， pH 4.8， as a Schulz and Blasch-

ke plot (open circles) and a Mead and Fuoss plot (c losed

circles). Ext rapolations were made with a Huggins con

stant of 0.58.

All the results obtained are summarized in Table 1.

Table I Experimental results of sedimentation equilibrium，

sedime口tationvelocity， and viscosity at 200

C on

retskin collagen in 0.06 M potassium acetate buffer，

pH 4，8， and on earthworm-cuticle collagen in 0.06 M

sodium acetate buffer， pH 4.8.

CoIlagen sample M XW' S~， 20. w 〔甲 J(dl / g)

Earthworm、cuticle

Ratskin

169:!: 10

30. 1士0.3

5. 19:!: 0.05

2. 82:!:0. 05

109

12.4

-38一

O 170

;150 a. ω

た"

• ど 130GI

p

c

110

O

"'Isp~o 0.2 0.4 0.6

• 234

c (gldl)x103a.

Fig. 4 Viscosity results on earthworm-cuticle collagen at 200 C

in 0.06 M sodium acetate buffer， pH 4.8， as a Schulz and

Blaschke plot (open circlesl and a Mead and Fuoss plot

(closed circles). The extrapolations were made with a

Huggins constant of 0.56.

DISCUSSION

0.8

5 6

The hydrodynamic experimental results obtained are now compared with theory

to estimate th巴 pers is tence length. o f the presen tly available hydrodynamic the-，)9-42) 41. 42)

ories of the wormlil日 coil we employ those of U¥!man ..， "'， because sedimen-

tation and viscosity were calculated on th巴 same model of the wormlike chain. The

polymer chain was treated as a continuous body and was not divid巴d into segments.

It was assumed that the source of hydrodynamic r巴sistance was on the axis of a

-39-

molecule of finite cross section， and that the hydrodynamic interaction was experi-

enced at the surface of the molecule. The excluded volume effect was not consi-

dered. Th巴 theoreticalresults are given as a function of several variables:λ 。。

(A )， the reciprocal Kuhn statisticallength; L(Al， the contour lengt.h;d(Al， the

hydrodynamic diameter;a， a dimensionless factor defined by /2c/ 2rc7}o・ The Kuhn •

statistical length is twice the persistence length， and c denot巴s the fric tion con-

stant per unit length and 7}0 the solvent viscosity. Since the parameter c cannot

be determtned uniquely by any method， some uncertainty may be introduced in the

estimate of the persistence length.

The recent work of Edwards and Oliver431

is quite instructive in this r巴spect.

They hav巴 shownin the theory of the sedimentation constant on a flexible coil mod-

el， a cylinder with.a random flight axis， that we can dispence with the segmental

friction constant by taking into consideration the non-slip boundary condition for the

liquid-cylinde r su rface. This suggests the importance of the similar theory be

developed for the wormlike coil. In addition， in view of the fact that the ca¥cu-

lation of Edwards and Oliver agrees with that of Kirkwood and Riseman441

in the

non-draining limit， it may be suggested that the U¥lman ca¥culation be applied in the

range where the draining parameter h proportional to c /(12庁 3)lnb770(b:the ef

fective bond lengthl is large. The draining parameter h is given forthe wormlike

coil by (6λL/π)lnQ.

The structural par，.ameters for the tropocol¥agen triple-helical structure are a 。

diameter of 15 A and three residues in 2.9A translation in the axial direction. The

average molecular weight per unit residue is ca¥culated from the amino acid com-

position24.251 as 90 for both ratskin and earthworm col¥agens. Thus we used 93 dal-

tons per angstrom， which gives as the contour lengths of respective collagens 3，200

A and 18，000 A. Fig. 5 shows the double logarithmic plot of [ηJ versus L. The

curves drawn in the figure represent the theoretical relations for various values

。f入withα= 5. O. This value ofαwas chosen to obtain for the both collagens 5

a co口sistentvalue ofλ， which is evaluated from Fig. 5 as 42 x 10 On the con-

trary， diffe ren t values 0 fλare assigned to the two col¥agens from the analysis of

sedimentation constant as shown in Fig. 6. This -discrepancy may partly originate

from the error in the determination of molec.ular weight， because the more consis-

tent Kuhn statistical length is obtained from the double logarithmic plot of S~~ 20，w

a gains t[η(Fig. 7). However， it is possible as wel¥ that the theoretical values

of sedimentation constant for shorter chains are overestimated in comparison to

those for longer chains. This is suggested from the fact that the comparison of

the same experimental results with the theory of Hearst and Stockmayer yields for -~)

λa value of 38 x 10 common to the two collagens (Fig. 8). The two theories

10∞ -40一

500

200 64

100

50

(四三刀)〔

?

〕 256

20

10

5

2

105

し(久)104 103

Double logarithmic plot of intrinsic viscosity versus

contour length for ratskin a口dearthworm-cuticle col-

lagens. The curves represent values calculated ac-

cording to the theory of Ullman1lJ for the indicated

10ユtimesthe reciprocal Kuhn statistical length.

Fig.5

30

20

ぇ10O

oか4

cf) 5

3

2

103

Fig.6.

30 20

主10oおの 5

3 2

Fig. 7

10

256

104

し(A)

128

100

〔η](dl/g)

256

128 64 32 16

105

1000

41-

-42-

20

1 0

Fig. 6 Double logarithmic' plot of sedimentation constant ver

sus contour length for ratskin and earthworm-cuticle

collagens. The curves represent values calculated ac-42)

cording to the theory of Ullman .~. for the indicated t

10" times the reciprocal Kuhn statistical length.

Fig. 7 Double logarithmic plot of sedimention constant and

intrinsic viscosity of ratskin and earthworm collagens.

For the curves see legends to Figs. 5 and 6.

1250

予三J三F 三J三2蜘5∞

3 I ~岳ョ=1200，.ααm M3

103

Fig.8

104 105

し(A)

Sedime口tatlo口 datareplotted as in Fig. 6. The curves

represent values calculated according to the theory of

Hearst and Stockmayer澗 forthe indicated persistence

length.

differ 0111y il1 whethe1' 01' not the hydrodynamic interaction is allowed withil1 the

bond len引 h b of the chain， and fo1' most values of interest， there is a constant

diffe 1'ence be tween the two calcu la tion s i1' 1'e spec tive 0 f入， L， and a. This sug-

gests， as understood from the discussion above in connection to the f1'iction con-

stant， that the1'e is no 1'eason to p1'efer Ullman calculation to that of Hearst and

Stocl、maye1'. Mo reove 1'， sedimen tion cons tan t is quite insens itive to the magni-

tude of the Kuhn statistical length. The1'efo1'e， we tentatively conclude that

qtυ a-

the hydrodynamic properties of ratskin and earthworm collagens are consistent with 。

the wormlike-coil model of a persistence length of 1300 A， which is twice as large

as that for double-helical DNA. From the value so obtained the drainillg parameter

h is calculated as 7.8 and 18.5 for ratskin and earthworm collagens， respectively.

The other factors which should be considered in di，scussing the flexibility of tropo-

collagen are (1) the telopeptide46l at the amino end may assume a structure dif-

ferent from the standard triple helix; (2) the unusuaIly large molecular weight of the

earthworm-cuticle collagen suggests subunits of smaller molecular weight held together

by some other unknown covalent bond. As long as the hydrodynamic proper-

ties are concerned， the experimental data shown above do not clearly indicate the

importance of these possibilities. Needless to say， however， we are unable to ex

clude for earthworm collag巴11 other model conformations such as brokell rods.

ACKNOWLEDGMENTS

lt is our pleasure to thank Professor H. Fujita for a good review of the recent

work of Edwards and Oliver. This investigation was supported in part by the Grant-

in-Aid for Operative'R巴search (8007 and 8(13)， the Ministry of Education of Japan

(to H. U.l.

REFERENCES

1) Ramachandran， G. N.， and Kartha， G. Nature， 176， 593 (1955)

2 ) Ramachandran， G. N.， : in Tγeatse on Collagen， Vol. 1， Academic Press， New York

(1967)， p. 170

3) Rich， A.， and Crick， F. H. C. : J. Mol. J3iol.， 3， 483 (1961)

4) Cowan， P. M.， McGavin， S.， and North， A. C. T. : Nature， 176， 1062 (1955)

5) Ramachandran， G. N.， and Sasisekharan， V. : Biochim. Biophys. Acta， 109， 314 (1965)

6) Ramachandran， G. N.， and Chandrasek!祖 国n，R. : Biopoかmers，6， 1649 (1968)

7) Yonath， A.， and Traub， W. : J. Mol. Biol.， 43， 461 (1969)

8) Traub， W.， Yonath， A.， and Segal， D. M. : Nature， 221， 914 (1969)

9) Chapman， Go E.， Campbell， 1. D.， and McLauchlan， K. A. : Ibid， 225， 639 (1970)

10) Boedtker， H.， and Doty， P. : J. Am. Che冊 . Soc.， 78， 4267 ('1956)

11) Neugebauer， T. : Ann. Physik， ( 5) 42， 509 (1943)

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13) Perrin， F. : J. Phys. Radium， ( 7) 5， 33 (1934)

14) Peterlin， A.， and Stuart， H. A. Z. Physik， 112， 1， 129 (1939)

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( 1951)

16) Hall， C. E.， and Doty， P. : J. Am. Che叫 . Soc.， 80， 1269 (1958)

44

17) Doty， P.， and Nishihara. T. : in Recent Advances in Gelatin and Glue Research

(ed. G. Stainsby)， Pergamon Press， London (1958)， p. 92

18) Nishihara， T.， and Doty， P.: Pγoc. Natl. Acad. Sci.， 44， 411 (1958)

19) Rice， R. V.， Casassa， E. F.， Kerwin， R. E.， and Maser， M. D. : Arch. Biochem.

Biophys.， 105， 409 (1964)

20) Burge， R. E.， and Hynes， R. D. : J. Mol. Biol.， 1， 155 (1959)

21) Young， E. G.， and Lorimer， J. W. : Arch. Biochem. Biophys.， 88， 373 (1960)

22) Engel， J.， and Beier， G. : Hoppe-Seyler's Z. Physiol. Chem.， 334， 201 (1963)

23) Lewis. M. S.， and Piez， K. A. : J. Biol. Chem.， 239， 3336 (1964)

24) Maser， M. D.， and Rice， R. V. Biochim. Biophys. Acta， 63， 255 (1962)

25) Josse， J.， and Harrington， W. F. : J. Mol. Biol.， 9， 269 (1964)

26) Kratky， 0.， and Porod， G. : Rec. Tγav. Chim.， 68， 1106 (1949)

27) Daniels， H. E. : Pγoc. Roy. Soc. Edinburgh， 63， 290 (1952)

28) Hermans， J. J.， and Ullman， R. : Physica， 18， 951 (1952)

29) Gill， S. J.， and Thompson， D. S. Pγoc. Natl. Acad. Sci.， 57， 562 (1967)

30) Nimni， M. E. : J. Biol. Chem.， 243， 145 (1968)

31) Deshmukh， K.， and Nimni， M. E. : Ibid.， 244， 1787 (1969)

32) Piez， K. A.， Weiss， E.， and Lewis， M. S. : Ibid.， 235， 1987 (1960)

33) Sugi， N.， Tsunashima， Y.， Ikehara， K.， and Utiyama， H. : Japan. J. Appl. Phys.， 11，

No. 10 (1972)

34) Van Holde， K. E.， and Baldwin， R. L. : J. Phys. Chem.， 62， 734 (1958)

35) Utiyama， H.， Tagata， N.， and Kurata， M. : Ibid.， 73， 1448 (1967) 1969

36) Hexner， P. E.， Radford， L. E.， and Beams， J. W. : Pγoc. Natl. Acad. Sci.， 47， 1848

( 1961)

37) Yphantis， D. A. : Aπ札 N.Y. Acad. SCi.， 88， 586 (1960)

38) o rekhovich， V. N.， and Shpikiter， V. O. : Biokhi miya， 20， 438 (1955)

39) Hearst. J.， and Stockmayer， W. : J. Chem. Phys.， 37， 1425 (1962)

40) Ptitsyn， O. B.， and Eizner， Yu. E. Vysokomol.， Soedin.， 3， 1863 (1961)

41) Ullman， R. : J. Chem. Phys.， 49， 5486 (1968)

42) Ullman， R. : Ibid.， 53， 1734 (1970)

43) Edwards， S. F.， and Oliver，加lA. : J. Phys. A: Gen. Phys.， 4， 1 (1971)

44) Kirkwood， J. G.， and R;'5eman， J. : J. Chys.， 16， 565 (1948)

45) Piez， K. A.， Eigner， E. A.， and Lewis， M. S. Biochemistγy， 2，・58(1963)

46) Rubin， A. L.， Drake， M. f'.， Davison， P. F.， Speakman， P. T.， and Schmitt， F. O.・

Ibid.， 4， 181 (1965) Kang， A. H.， Bornstein， P.， and Piez， K. A. : Ibid.， 6， 788

(1967)

phυ

daτ

沈降および粘度測定から評価した

トロポコラーゲンのかたさ

内山敬康¥坂戸邦昭，池原健二，節家孝志，倉田道夫

(京都大学化学研究所，京都大学工学部*)

トロポコラーゲン構造の形や大きさについては， Boedtkerと Dotyが ichthyocolコラーゲ

ンを使用して光散乱や流動複屈折などの測定から，長さ 3000A，直径 13.6Aの棒状であるこ

とを見出して以来，他の多くのコラーゲンについて研究が行なわれ，同校の結果が繰り返し得

られてきた O しかし二本鎖 DNAについて見られる様に，分子が長くなると分子全体としては

可携性が表われてくる筈である. ミミズの表皮から抽出されるコラーゲンは，分子量が1.9X

106 にも達し，ゴラーゲン三本鎖構造のかたさについて，より詳細な情報が得られる可能性が

ある.事実 Josseと Harr・Ingtonの得た粘度の結果を回転惰円体の理論と比較して得られる粒

子の長さは，実際の長さの約去にしかならない.この様な粒子は，いわゆる Kratkyと Porod

の wormlike-coil 模型を用いて解析する必要があり，分子のかたさは persistenelengthに

より定量化される.但し，この模型による沈降系数や極限粘度の現在の理論には，流体力学的

半径および単位長あたりの摩擦係数が変数として含まれており，かっこれらは一義的に決めら

れないので，構造が同じで長さの異なる試料の結果を統」的に記述できる様に，二れらの変数

むよび persistencelength が決'JE・される/

試仰としてミミズの表皮およびラット外皮から抽出したコラーゲンを使用し，沈降係数や粘

度の測定には0.06M K acetate buffer， pH 4.8を月Jい200

Cで行った.

分子量の決定は， Spinco E型超遠心機による沈降平衡法によった.但しミミズのコラーゲン

については， Schlieren光学系，および Yphantisのふチャンネルセルを使用し， ラット・コ

ラーゲンについては， Rayleigh干渉光学系を使用する通常の沈降平衡法によった. 得られた

分子量は，夫々 169X104， 30.1 X104

である Ratskinの分子量は，文献値の29.4X104

(Lewis & Piez， J. Biol. Chem.， 239， 3336 (1964)) とよく一致していと3が， ミミズの結果

は，粘度と沈降{系数から JosseとHarrington (J. Mol. Biol.， 9，269(1964)) が得た190X

104 より 10%程小さい.但し彼らの粘度測定には問題がある.

沈降係数 S020， w は，夫々 2.82士0.05(ratskin )， 5.19土0.05(ミミズ)であった. Josse

と Harr・ingtonがミミズ表皮コラーゲンについて報告している沈降係数の濃度依存性の異常は，

見られなかった.

粘度測定には，気~液界面に形成される膜の影響を除き，かっ低速度勾配で実験を行なうた

め，特別に設計したcartesian-divertYIieの回転粘度計を使用した.得られた結果は， 12.4dl

/ g (ratskin) および109dl/g (ミミズ)で， 後者は通常の毛管粘度計による測定値62dl/g

46-

に比し著しく大きい.

分子軸に沿って lAあたりの分子量を93として ratskinおよびミミズ表皮コラーゲンの

contour lengthは，夫々 3200λ，18，oooAと求められる.粘度の結果をUllmanの理論と比較

すると，分子量に無関係に persistencelength は1250Aとなる.一方沈降係数の結果からも，

大約一致した値1300Aが得られる.但し沈降係数の persistencelength依存性はごくわずか

で，粘度から求められた perslstenc巴 length の方が信頼度は高い.これらはDNAの persis

tence lengthの約 21音である.

以上は，流体力学的にミミズ表皮のコラーゲンと ratskin コラーゲンが完全に同じ構造をも

ち，その相異は長さだけであるという仮定のもとに導かれた結論であることは，注意する必要

があるが，グリシンの含量が共にすに近いこと，変性後の分子量が約すになる事実などは，上

記の仮定を支持するものと考えられる.

イ寸 記

第22回コラーゲン・シンポジウムにおける話題 I細胞内でのトロポコラーゲン構造形成の機構に

に関する物理化学的イメージ、」については，別に発表する予定である.

但し， HCHOにより架橋したコラーゲンを使用したコラーゲン構造再生の動力学的実験に関

する質問への回答は，ここに記載させて項きます.

討 議

御 橋広真氏 の系では回復しません.

Recoveryしなかった fraction の ORDへの (2) 使用したトロポコラーゲンは，全ての分

contributionをどう考えられますか? 子が架橋される様な条件で反応を行ないました.

内山敬康氏 結合した HCHO の平均数は20~100で，また実際

変性ゼラチンのORD と同一で、あると仮定して の架橋数には分布がある筈であります.原理的

います. には，鎖のregistrationさえできればよいので

林 利彦氏 すが，完全な構造再生を行なうには，温度・濃

(1) 37'Cでの回復は如何ですか? 度の領域を適当に定めねばならない筈でありま

(2) HCHOによる cross-linksがどの{立少なく す.

てもよく戻るものですか? (3) 架橋の数，位置が適当であれば， トロポ

(31 Spontaneous renaturationの%の大きさ コラーゲン分子聞の非特異性結合による構造形

と生体内での 3本鎖構造形成の調節との関係は 成のおくれは，問題にならないと考えられ，コ

如何ですか? ラーゲン合成の前駆体 procollagenでは，構造

内山敬康氏 形成の速度は非常に速いものと考えています.

(1) 使用した系でのTmは38.6"cであり，こ