Water-Collagen Interactions: Calorimetric and Mechanical Experiments M. H. PINERI, M. ESCOUBES,* and G. ROCHE, Centre d'Etudes Nucle'aires de Grenoble, De'partement de Recherche Fondamentale, Section de Physique du Solide, 85 X , 38041 Grenoble Cedex, France Synopsis The influence of hydration of rat-tail tendons has been studied by measuring the heat involved in the water fixation depending on the degree of hydration. The modulus and damping dependences have been measured when changing the temperature for different water uptakes. In situ hydrations have been realized. Evidence of several states of water absorption has been derived from both calorimetric and dynamic mechanical experiments. A model has been proposed taking into account the energies corresponding to the different regimes of fixation.
The role of water in biological processes has been under investigation for over a century. Considerable controversy exists concerning the nature of the water associated with cells and subcellular components. Water has been found to be essential in the maintenance of tropocollagen in its native conformation. Upon complete dehydration, collagen becomes inso1uble.l model of The structure of the collagen has been intensively ~ t u d i e d . ~A- ~ collagen has been proposed involving the participation of two water molecules in interchain hydrogen bonds.2 The absorption isotherm for water uptake by collagen a t 25OC has been defined.5 It is a sigmoidal type I1 isotherm form. Typical values of hydration are 0.1 g water/g collagen for a relative humidity (rel. hum.) of 20%, 0.3 g water/g collagen for 80% rel. hum., 0.4 g water/g collagen for 90%rel. hum., and must approach infinity at 100% rel. hum. This important water absorption is due both to the physical fibrous structure and to the chemical composition (large concentration of hydrophilic groups: C=O, N-H, COOH, OH, etc.). Many experiments have been performed to study the localization of water molecules and the state of bonding when changing the hydration percentage. The techniques most commonly used involve broad-line nmr, calorimetric, ir, and dynamic mechanical experiments. Because of the very low value (1G) of the dipolar splitting corresponding to some H20 molecules, Berendsen6 considers that these water molecules are rotating or reorientating about a single axis, with a water chain structure along the fiber axis. Another orientation of water molecules for high humidities would give a central line. From a comparison between the spectra of H20 and D20 in collagen Dehl and Hoeve7 concluded that there is only * Laboratoire de Chimie Appliquhe e t de Genie Chimique, U.E.R. de Chimie et Biochimie, Universitb Claude Bernard, 69621 Villeurbanne, France. Biopolymers, Vol. 17,2799-2815 (1978) 01978 John Wiley & Sons, Inc.
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PINERI, ESCOUBES, AND ROCHE
one kind of orientation of water molecules. The splitting which is observed would be caused by a slightly anisotropic but rapid reorientation of the water molecules. A third view has been proposed by Fung and Trautmann.8 According to their model, a part of the water adsorbed is bound to the collagen fiber through hydrogen bonding and the rest is randomly reorienting. The two types of water are in chemical exchange. The observed dipolar splitting for H20 are time-averaged values. Frozen water has been seen to appear a t water concentrations of more than 0.54 g/g collagen.9 A similar result has been found from calorimetric measurernents.l0 The heat capacity of collagen in the presence of different amounts of water has been plotted against the temperature. An endothermic peak appears around 260°K for a concentration of 0.48 g HzOIg collagen. When the water content is increased, this peak is more and more important and its maximum is shifted towards OOC. Infrared experiments as a function of relative humidity have led to the conclusion that water molecules are gradually attached to peptide NH bonds over a range of humidity of 0-75%.11 This conclusion is very different from the results obtained with other synthetic polypeptide films in which the water molecules are adsorbed a t specific sites and orientations with respect to the substrate molecules.12 The discrepancy about the role of water in the mechanical relaxations In a recent publication, Nomura has been outlined in a general a r t i ~ 1 e . l ~ et al.'* have been able to define four different regimes in the hydration of a collagenous tissue (human dura mater). These conclusions have been drawn mainly from the evolution of the rigidity modulus vs the hydration at different temperatures. In this article the interaction of water and collagen has been studied by dynamic mechanical experiments complemented by calorimetric measurements. Mechanical spectroscopy is able to evidence molecular relaxations and to define the energy implied in the motion. We have previously investigated rotations of alkyl side groups in some p01yolefins.l~ Using the same experiment, it is possible to show some chemical exchange reaction which can take place in polymers.16 Gravimetric and calorimetric experiments permit one to define the sorption mechanism of water. It is possible to define the enthalpy of absorption of water depending on the hydration of the specimens. Information about the kind of bonding can therefore be obtained.17
SAMPLES The collagen used in this investigation was from rat-tail tendons. After extraction, the tendons were kept in a 0.5M NaCl solution and washed in distilled water before experiments.
WATER-COLLAGEN INTERACTIONS
2801
Calorimetric and Gravimetric Experiments The same experiment permits one to obtain both the sorption isotherm and the differential heat absorption ~ a 1 u e s . l ~A Setaram thermobalance B 60 and a Richard Eyraud isotherm differential microcalorimeter were cal/sec, respectively. used. The accuracies were better than 0.1 mg and The relative humidity was obtained by changing the temperature of a w a t e r h e bath (accuracy: O.l°C) which was connected with the sample. Two collagen specimens were kept a t a temperature of 20 f 0.1"C and a Hg mm vacuum for 24 hr. A hydration corresponding to a low relative humidity was then realized. During the water sorption, we recorded the weight change and the heat involved in this absorption. It was therefore possible to plot the isotherm sorption heat vs the relative humidity or weight increase. A change in the humidity level was realized after 24 hr.
Electrical Resistivity Measurements A very sensitive electrometer was used for this experiment. The collagen sample, made of many parallel fibers, was mounted in series with a 360-V battery. We checked the ohmic behavior of the specimen by recording the intensity values corresponding to the different voltages. Dynamic Mechanical Experiments Dynamic mechanical experiments were performed by using an inverted torsion pendulum which has been previously described.18 The values plotted against temperature are
AW/W = 1- exp - (2/n In O,/O,) and
where O0 and 8, are the amplitudes of the oscillations o and n. P ( T , ~is)the period of the measurement corresponding to the temperature, T (in OK), and to the relative humidity, x. Because of the kind of specimen formed by the juxtaposition of many fibers, it was not possible to calculate the absolute values for the modulus. In order to compare the modulus between the different degrees of humidity, special attention was paid to the process of hydration. This hydration was realized in situ without moving the collagen specimen. The sample was exposed to a defined-humidity air for 12 hr. A rapid quenching down to liquid-nitrogen temperature was then realized. This procedure has the advantage of specifying exactly the water content of the specimen. In the usual procedure, the hydration takes place in a cell that is then mounted in the torsion pendulum; a drastic change
2802
PINERI, ESCOUBES, AND ROCHE
in the humiditylevel can occur during this transfer and a small change in the counterweight or geometry can change the modulus values drastically. Other advantages of our procedure are the accuracy of our humidity measurement (*a%) and the range of the values which can be obtained (0-100% rel. hum.). In Fig. 1 we can see the hydration procedure. Air is flowed through water or ice, which is maintained at temperature TI (-30 to +2OoC). A good contact between air and water is realized by bubbling the air through a porous glass and raschig beads. We therefore obtain air which is saturated at temperature TI. This air is then warmed to room temperature, and the hydration is measured with a LiCl cell. By changing this T1 temperature, we can obtain any water concentration at room temperature.
RESULTS Gravimetry and Calorimetry Figure 2 shows the plots of the sorption-desorption isotherm at a temperature of 2OoC up to a relative water pressure of 0.85. We note a small hysteresis which is more important for p / p o values below 0.60. Similar absorption isotherms have been previously ~ b t a i n e d .Figure ~ 3 gives the plots of the enthalpy values vs the water pressure during sorption and desorption runs. Small differences appear between the sorption and desorption, these differences being more important when plotting the enthalpy vs the water uptake (Fig. 4). Three different regimes of sorptiondesorption are evident from these curves; they correspond to energies of 16.5,14, and 9.5 kcal/mol, respectively. The hydration ranges and energy values differ between the sorption and desorption processes. The energy value of 9.5 kcal/mol corresponds to an energy close to the value of the
Room T r a t u r e
ymm I L1
Torsion
-
Fig. 1. Hydration procedure for the mechanical experiments.
WATER-COLLAGEN INTERACTIONS a %A
2803
i r
+
LO*4
+ l,
300
+
+
+
p
20 -
7
Lp’ + + /o /o , / -%’
,:
to ++
’0-
4
I
00
025
05
PIP0
desorption
Fig. 3. Changes 0 in L enthalpy - during 7 a cycle 5 of sorption and desorption plotted against the relative humidity.
liquefaction of water. It has not been possible to increase the humidity range above plpo = 0.8 because the accuracy of our measurements is not good enough to define the sorption energy in this humidity range. In Fig. 5 are plotted the water uptakes a t different humidities vs time. For low humidity levels, we obtained equilibrium very quickly. Diffusion has to be taken into account for humidity above 60%. The Fick law seems t o be
PINERI, ESCOUBES, AND ROCHE
2804
I I
I
desorptionl
I Ji
II
III
IV
Regimes
0 ’
Ib i0 30 a YO Fig. 4. Changes in enthalpy during a cycle of sorption and desorption plotted against the water uptake. valid up to 70% of the water uptake for a relative humidity greater than 60%. We have an abnormal sigmoid curve with two different regimes for the relative humidity.
Dynamic Mechanical Experiments We will be concerned only with the curves between 77 and 300°K. No additional relaxation peak has been found between 4 and 77°K. Because of the changes which occur in the humidity level when warming the sample above room temperature, no attention has been paid to the peaks which appear above 300°K. In Fig. 6 are plotted the different damping curves obtained when changing the relative humidity. Four peaks can be seen. The low temperature peak, which will be called 6, is the only one in the dehydrated sample. We want to emphasize that the sample corresponding mm Hg] for to 0% rel. hum. has been obtained by vacuum pumping one night a t room temperature. Such a procedure is known not to result in irreversible changes in the collagen, some water being left, as will be shown below. When increasing the humidity, the 6 peak shifts down from 210°K (0% rel. hum.) to 140°K (75% rel. hum.). No further change in temperature is observed when increasing the humidity above 75%. The amplitude of this peak does not seem to change much with humidity. However, it is impossible to define exactly this amplitude because of the superposition of other peaks on this 6 peak. The peak /3 between 260 and 280°K appears at very low humidity (9.5% rel. hum.) and does not seem to change in amplitude and position with further increases in humidity. The a peak appears only in the curves
WATER-COLLAGEN INTERACTIONS
2805
1
0.5
0.43 - 0.54; Fig. 5. Water uptake during a sorption run vs time. (+) p/po: 0 - 0.33; (0)
(A)0.515 - 0.618; (0) 0.66 - 0.814.
corresponding to 95 and 98%rel. hum. It is a very sharp peak located at 262'K and is superimposed on the p peak. A fourth peak, which we will call y, appears when the humidity is higher than 60%. Its location is around 210'K, and it is more apparent in the 77.5% rel. hum. sample. In the 95 and 98%rel. hum. samples, this peak is masked because of the superposition of the 7-P and a peaks. However, the important increase in the damping after the relaxation is indicative of the existence of this peak.
Rigidity Figures 7 and 8 give the plots of the relative modulus corresponding to the different humidities vs the temperature. There is a progressive increase of the modulus a t each temperature below 270°K when increasing the degree of humidity, as is shown in Fig. 9. In Fig. 9 we have plotted the evolution of the relative modulus vs the water concentration at different temperatures. We do not observe the decrease in modulus which has been observed by Nomura et al.I4between 0.25 and 0.5 glg for all temperatures below 250°K with a human dura mater sample. In this range of absorption
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PINERI, ESCOUBES, AND ROCHE
P
I
?+ +
g Ha0 g collagen
0,61 0,51
0,39
RH
0.37
98
0.27
95 77.5
0235 039
75
0.15
60 52
0.l
41
0.09 0,08
27.5 15.5 12
0.01
9.5 0 100
2b0
300
T(OK)
Fig. 6. Effect of hydration on the relaxation spectrum of collagen. For convenience, each curve has been translated by a 0.1 AW/W factor.
we simply observe a small increase in the rigidity values when increasing the concentration of water. For concentrations larger than 0.40 g waterlg collagen, there is a drastic increase in the modulus for temperatures lower than 210°K. Water therefore has a stiffening effect at all concentrations below 270°K. Note the large decreases in the modulus at 270°K observed in Fig. 8 for the two most hydrated samples.
WATER-COLLAGEN INTERACTIONS
RH
qH>O/qcdh= 0 20 0 15 0 10 0 09
L1 % + 275% L 155% .:
.
0
2807
12 % 95%
0
oa
om
o vo
*
200 360 I60 T O K Fig. 7. Relative rigidity modulus plotted against temperature for different degrees of hydration. 0
160
A
G -
GG% *=i
6-
-
gHzO/gcdkqm 0 61 051 0 37 077.5% 039 0 6 0 Yo 0 27 RH
98 % +95 % .75 %
x
5*+ +*
1-
f
X
3-
2-
1-
1
160 260 300 T O K Fig. 8. Relative rigidity modulus plotted against temperature for different degrees of hydration.
Electrical Resistivity Figure 10 presents the plots of the different values of the current vs the water uptake at three different temperatures. A continuous increase of the current is observed over the entire concentration range. Figure 11 presents the plots for the intensity values in a logarithmic scale vs 1/T a t different humidity levels.
PINERI, ESCOUBES, AND ROCHE
2808
c G I 150
o
180
t
210 270
.
a
If,
n
I
O K
I
4
O K
OK O K O K
I
O K
m
.
I
V.
IV
I 'c- 1 .
Rc
/ '
Fig. 9. Relative rigidity modulus plotted against the water uptake at different temperatures (OK).
Discussion We have been unable to draw some conclusions from the electrical resistivity measurements. The purpose of these experiments was to show the different regimes of conduction when changing the temperature because of the different mobilities of the water molecules. We obtained a continuous change of the conduction over a wide range of temperatures and for all the water concentrations. At each hydration level an activation energy can be obtained from the plots of In I vs 1/T,but, since no physical meaning can be attached to this value, we do not try to define it. In this discussion we propose a model of water fixation from both our results and from those obtained from previous experiments. Our water concentrations have been defined by taking 0% water for a sample maintained at 100°C for 24 hr under a vacuum of at least 10-4 Torr. This ex-
WATER-COLLAGEN INTERACTIONS
2809
6
I amp
-
10-6
+ -0
,
-
10-8
't
+ / ' O
9 / ,/
lrn -
w'2
-
//'
f A
10-8 -
10-w.
1 0 1 2
-
10-11
13
+
35
L
is
1,~.10-30E
Fig. 11. Plots of the current against 1/T for different degrees of hydration (% rel. hum.): 95; 0,85;X, 70; A, 64; 0,49;0,39;=, 25.
+,
periment has been done after all the calorimetric experiments were performed in order to get rid of a possible irreversible change. The difference mm between dehydration a t room temperature and at 100°C under Hg is 0.010 g water/g collagen. These 0.01 g water were not extractable at room temperature, which means that we have water molecules which are firmly attached to the collagen molecule. The double-hydrogen-bonded water molecules will be seen to be extracted under vacuum at room temperature. It is very tempting to analyze this first kind of water molecule as a water molecule fixed by three hydrogen bonds, one of them involving the hydroxyl group. Such a possibility of water fixation has been pointed out by Ramachandran and Reddi.lg The fixation of one water molecule per three residues corresponds to 0.06 g waterlg collagen. The average hydroxyproline concentration corresponds to one group per 10 residues,
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PINERI, ESCOUBES, AND ROCHE
giving an opportunity of fixing about 0.018 g waterlg collagen by a threehydrogen-bond mechanism. The second kind of water molecule corresponds to the first molecules that are absorbed after the sample has been kept a t room temperature Torr. We think that these water molecules corunder a vacuum of respond to the interchain hydrogen bond inside the triple helix corresponding to the water-bridged structure of collagen proposed by Ramachandran and Reddi.19 The energy of fixation of these water molecules is around 17 kcallmol, which corresponds to a double-hydrogen-bonded water. We have 0.11 g water, which corresponds to an energy of 18kcallmol during the desorption process and only 0.07 g with an energy of 17 kcallmol during the sorption process. This difference can be explained by a hysteresis process. During the sorption process, the water molecules are fixed on different sites corresponding to different energies. In contrast, during the desorption, the water molecules corresponding to the highest energies are extracted after all the others have been removed. No change of the equatorial spacing has been observed in this range of water absorption.14J0 The helical parameters remain intact following dehydration to a moisture level of 0.1%. Therefore, we have very small changes of the tropocollagen structure upon removal of these water molecules. The water sites are still available and are occupied by the first molecules. The increase in modulus is not too important in this region of absorption because we have a system of rigid rods (tropocollagen molecules), and we increase only the rigidity of the tropocollagen molecules without crosslinking the rods between them. The /3 peak which appears for a water concentration of 0.08 g waterlg collagen must be connected with the presence of water. It can be explained by some exchange reaction involving the breaking and reforming of hydrogen bonds inside the tropocollagen molecule. On further hydration we do not observe any change in the amplitude and temperature position of this peak involving a saturation of the water molecules associated with this process. In this range of hydration, we observe a drastic change in the temperature position of the 6 peak. Stefanou et aLZ1interpret this peak as corresponding to oscillations of the proline group. If such an interpretation is correct, the energy barrier involved in this motion must decrease when increasing the humidity level. A small change in the environment of the proline group can produce drastic changes of the intermolecular barrier and can therefore explain the observed displacement of the peak with the hydration. It seems difficult to interpret such a 6 peak in the same way as it has been interpreted by Nomura et al.14 A low-temperature relaxation peak has been found in ice around 160°K a t 1 Hz. Motions of defects in an ice lattice involving a rotation and a jump of water molecules have been proposed to explain such a peak.22,23 There is no possible comparison between such an ice lattice with Bjerrum defects and a collagen structure with water molecules fixed in different sites. The results of nmr are consistent with our interpretation corresponding to this double hydrogen bonding. Up to a concentration of 0.35 g waterlg collagen, water has been found to be rigidly
WATER-COLLAGEN
INTERACTIONS
2811
bound to the collagen molecule.lO The third kind of water molecule corresponds to water concentrations between 0.11 and 0.235 glg a t the desorption, 0.07 and 0.235 glg a t the sorption. In this range of concentration there is a drastic increase of the equatorial spacing.14 We therefore interpret this regime as corresponding to the fixation of water molecules between the triple helices and between the microfibrils. The energy involved in this regime corresponds to 12 kcal at desorption and 14 kcal during sorption. This energy corresponds to a double hydrogen bonding and is a little lower than in the previous regime because some energy is necessary to swell the microfibrils and possibly to break some bonding between the triple helices. We have here the same differences in energy between the sorption and desorption results, the same explanation being valid. The large increase in the modulus values a t low temperatures (Fig. 9) is normal because of the kind of crosslinking (inter-triple-helices and intermicrofibrils) that is involved in this regime. No drastic change is observed in the damping curves during this regime. The 6 peak moved from 175°K down to 140°K because of a further decrease in the intermolecular potential barrier. The nmr results define the state of the water molecules fixed during this regime as nonrotational water molecules.10 The fourth kind of water molecule corresponds to absorption values between 0.235 and 0.50 g waterlg collagen. The equatorial spacing does not change too much in this absorption range.14 The rigidity modulus tends to level off and there appears a new peak located around 225OK (7). Different mechanisms can be proposed to explain this state. One explanation corresponds to the fixation of the water molecules between the microfibrils by just one hydrogen bond. We therefore have some kind of complexes with water molecules, the motion of which gives a relaxation peak (7). Mobile water molecules have been shown to appear at a concentration of 0.35 g waterlg collagen from nmr experiments.1° Another explanation would imply the fixation of the water molecules inside the holes -400 A long adjacent to the end of the tropocollagen molecule (A. Miller, personal communication). In both explanations a structural change of this water phase corresponding to a glass transition is possible. The last kind of water molecule appears above 0.50 water glg collagen. It corresponds to water molecules which form clusters large enough to behave as ice. A new peak ( a )appears at 270°K superimposed on the p peaks. This a peak is very important and corresponds to the melting of the ice. A drastic decrease in the rigidity modulus is associated with this peak (Fig. 7). We emphasize that a t low temperatures, water increases the modulus considerably, whereas it acts as a plastifier above 273°K. Such results are in agreement with previous calorimetric and nmr experiments,1° where free water has been found for water concentrations larger than 0.47 and 0.54 g waterlg collagen. This water seems to be fixed preferentially between the microfibrils.
0.010-0.110
I
0.010-0.070
0-0.010
Regime
I1
Water Concentration (g water/g collagen) Absorption Desorption
TABLE I
Water extractable a t 100°C under Torr High energy of fixation (>18 kcal/mol) Distinct sites Nonrotationally hydrated water (nmr)a Heat of sorption: 17 kcal/mol Heat of desorption: 18 kcal/mol No change in the equatorial spacing Small changes in the rigidity modulus p peak corresponding to an exchange reaction involving the water molecules Decrease in temperature of the 6-peak position Nonrotationally hydrated water (nmr)a
Characteristics
The Different Regimes of Water Fixation
Double hydrogen bonding of the water molecules in the available sites inside the triple helix.
Triple hydrogen bonding inside the triple helix and involving hydroxyproline.
Suggested Mechanism
N
?-
s"B
Eto
h
a
0.235-0.50
>0.50
IV
V
Ref. 10. Ref. 11.
0.071-0.235
I11
>0.50
0.235-0.50
0.110-0.235
Heat of sorption: 14 kcal/mol Heat of desorption: 12 kcal/mol Large increase in the modulus Large changes of the equatorial spacing No change of the p peak Nonrotationally hydrated water (nmr)a Heat of sorption and desorption -9 kcal/mol No change in the modulus a t low temperatures No large change in the equatorial spacing New y peak a t 225'K Mobile water molecules (nmr)a New, very important LY peak at 273°K Large decreases in the modulus a t 273°K Appearance of a freezing bound state for water corresponding to 0.54 g water/g collagen (nmr) and 0.48 (calorimetric)a No further change in the ir spectrumh Free water between the microfibrils.
Water fixed by one hydrogen bond between the microfibrils or water fixed in the hole zones adjacent to the end of the tropocollagen molecule.
Double hydrogen bonding of the water molecules between the triple helix and between the microfibrils.
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PINERI, ESCOUBES, AND ROCHE
%
'1' r o po c o 1 1a ge n Triple helix
36 A
(Cross-section)
Tetrngonnl 1.a t t i c e
l n t e r t r i p l e h e l i x and i n t e r m i c r o f i h r i l l a r phase i n which w a t e r i s f i x e d d u r i n g t h e r e p i m e s 111 a n d I V .
Fig. 12. Different fixation sites of water in collagen.
WATER-COLLAGEN INTERACTIONS
2815
CONCLUSION The first aim of this paper was to give very precise experimental calorimetric and mechanical results. From these results and from many others, we have been able to propose a model which seems, in general, to be consistent with all the experiments. However, many points have to be defined more clearly. Table I summarizes the characteristics of the different regimes of water fixation. In Fig. 12 the different sites of water fixation are defined. The most important conclusion is the evidence of different states of water fixation corresponding to different energies. However, more experiments are necessary in order to refine the analysis of these regimes of water fixation. The authors are grateful to Dr. A. Miller for helpful discussions. We are indebted to Mr.
M. Brotte for technical assistance in both the experimental work and the drawings.
References 1. Yannas, I. V. & Tobolsky, A. V. (1967) Nature 215,509-510. 2. Ramachandran, G. N. & Chandrasekharan, R. (1968) Biopolymers 6,1649-1662. 3. Miller, A. & Wray, J. S. (1971) Nature 230,437-438. 4. Miller, A. & Parry, D. A. D. (1973) J . Mol. Biol. 75,441-450. 5. Kuntz, I. D., Kauzmann, J. R. & Kauzmann, W., in Hydration of Proteins and Polypeptides. 6. Berendsen, H. J. C. (1962) J . Chem. Phys. 36,3297-3307. 7. Dehl, R. E. & Hoeve, C. A. J. (1969) J. Chem. Phys. 50,3245-3251. 8. Fung, B. M. & Trautmann, P. (1971) Biopolymers 10,391-397. 9. Dehl, R. E. (1970) Science 170,738-739. 10. Mrevlishvili, G. M. & Privalov, P. L. (1969) Water i n Biological Systems, Kayushin, L. P., Ed., Consultants Bureau, New York, p. 63. 11. Susi, H., Ard, J. S. & Carroll, R. J. (1971) Biopolymers 10, 1597. 12. Malcolm, B. R. (1970) Nature. 13. Chien, J. C. W. (1975) J. Macromol. Sci., Rev. Mucromol. Chem. 12, 1-80. 14. Nomura, S., Hiltner, A,, Lando, J. B. & Baer, E. (1977) Biopolymers 16,231-246. 15. Pineri, M. (1975) Polymer 16,595-600. 16. Meyer, C. T. & Pineri, M. H. (1976) Polymer 17,382-386. 17. Soulier, J. P., Escoubez, M., Douillard, A. & Chabert, B. (1976) J . Chim.Phys. 4, 423-429. 18. Pineri, M. H., Bonjour, E., Gerard, P. & Martin d’Hermont, F. (1972) Plast. Mod. Elustomeres 10, 19-27. 19. Ramachandran, G. N. & Reddi, A. H. (1976) Biochemistry of Collagen, Plenum, New York, 1976, p. 68. 20. Rougvie, M. A. & Bear, R. S. (1953) J . A m . Leather Chem. Assoc. 48,735-751. 21. Stefanou, H., Woodward, A. E. & Morrow, D. (1973) Biophys. J . 13,772-779. 22. Bass, R. (1958) Z. Phys. 153. 23. Vassoille R., Tatibouet, J., Perez, J. & Gobin, P. F. (1974) C. R. Acad. Sci., Ser. B , 409-412.
Received December 8,1977 Accepted February 21,1978
The role of water in biological processes has been under investigation for over a century. Considerable controversy exists concerning the nature of the water associated with cells and subcellular components. Water has been found to be essential in the maintenance of tropocollagen in its native conformation. Upon complete dehydration, collagen becomes inso1uble.l model of The structure of the collagen has been intensively ~ t u d i e d . ~A- ~ collagen has been proposed involving the participation of two water molecules in interchain hydrogen bonds.2 The absorption isotherm for water uptake by collagen a t 25OC has been defined.5 It is a sigmoidal type I1 isotherm form. Typical values of hydration are 0.1 g water/g collagen for a relative humidity (rel. hum.) of 20%, 0.3 g water/g collagen for 80% rel. hum., 0.4 g water/g collagen for 90%rel. hum., and must approach infinity at 100% rel. hum. This important water absorption is due both to the physical fibrous structure and to the chemical composition (large concentration of hydrophilic groups: C=O, N-H, COOH, OH, etc.). Many experiments have been performed to study the localization of water molecules and the state of bonding when changing the hydration percentage. The techniques most commonly used involve broad-line nmr, calorimetric, ir, and dynamic mechanical experiments. Because of the very low value (1G) of the dipolar splitting corresponding to some H20 molecules, Berendsen6 considers that these water molecules are rotating or reorientating about a single axis, with a water chain structure along the fiber axis. Another orientation of water molecules for high humidities would give a central line. From a comparison between the spectra of H20 and D20 in collagen Dehl and Hoeve7 concluded that there is only * Laboratoire de Chimie Appliquhe e t de Genie Chimique, U.E.R. de Chimie et Biochimie, Universitb Claude Bernard, 69621 Villeurbanne, France. Biopolymers, Vol. 17,2799-2815 (1978) 01978 John Wiley & Sons, Inc.
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PINERI, ESCOUBES, AND ROCHE
one kind of orientation of water molecules. The splitting which is observed would be caused by a slightly anisotropic but rapid reorientation of the water molecules. A third view has been proposed by Fung and Trautmann.8 According to their model, a part of the water adsorbed is bound to the collagen fiber through hydrogen bonding and the rest is randomly reorienting. The two types of water are in chemical exchange. The observed dipolar splitting for H20 are time-averaged values. Frozen water has been seen to appear a t water concentrations of more than 0.54 g/g collagen.9 A similar result has been found from calorimetric measurernents.l0 The heat capacity of collagen in the presence of different amounts of water has been plotted against the temperature. An endothermic peak appears around 260°K for a concentration of 0.48 g HzOIg collagen. When the water content is increased, this peak is more and more important and its maximum is shifted towards OOC. Infrared experiments as a function of relative humidity have led to the conclusion that water molecules are gradually attached to peptide NH bonds over a range of humidity of 0-75%.11 This conclusion is very different from the results obtained with other synthetic polypeptide films in which the water molecules are adsorbed a t specific sites and orientations with respect to the substrate molecules.12 The discrepancy about the role of water in the mechanical relaxations In a recent publication, Nomura has been outlined in a general a r t i ~ 1 e . l ~ et al.'* have been able to define four different regimes in the hydration of a collagenous tissue (human dura mater). These conclusions have been drawn mainly from the evolution of the rigidity modulus vs the hydration at different temperatures. In this article the interaction of water and collagen has been studied by dynamic mechanical experiments complemented by calorimetric measurements. Mechanical spectroscopy is able to evidence molecular relaxations and to define the energy implied in the motion. We have previously investigated rotations of alkyl side groups in some p01yolefins.l~ Using the same experiment, it is possible to show some chemical exchange reaction which can take place in polymers.16 Gravimetric and calorimetric experiments permit one to define the sorption mechanism of water. It is possible to define the enthalpy of absorption of water depending on the hydration of the specimens. Information about the kind of bonding can therefore be obtained.17
SAMPLES The collagen used in this investigation was from rat-tail tendons. After extraction, the tendons were kept in a 0.5M NaCl solution and washed in distilled water before experiments.
WATER-COLLAGEN INTERACTIONS
2801
Calorimetric and Gravimetric Experiments The same experiment permits one to obtain both the sorption isotherm and the differential heat absorption ~ a 1 u e s . l ~A Setaram thermobalance B 60 and a Richard Eyraud isotherm differential microcalorimeter were cal/sec, respectively. used. The accuracies were better than 0.1 mg and The relative humidity was obtained by changing the temperature of a w a t e r h e bath (accuracy: O.l°C) which was connected with the sample. Two collagen specimens were kept a t a temperature of 20 f 0.1"C and a Hg mm vacuum for 24 hr. A hydration corresponding to a low relative humidity was then realized. During the water sorption, we recorded the weight change and the heat involved in this absorption. It was therefore possible to plot the isotherm sorption heat vs the relative humidity or weight increase. A change in the humidity level was realized after 24 hr.
Electrical Resistivity Measurements A very sensitive electrometer was used for this experiment. The collagen sample, made of many parallel fibers, was mounted in series with a 360-V battery. We checked the ohmic behavior of the specimen by recording the intensity values corresponding to the different voltages. Dynamic Mechanical Experiments Dynamic mechanical experiments were performed by using an inverted torsion pendulum which has been previously described.18 The values plotted against temperature are
AW/W = 1- exp - (2/n In O,/O,) and
where O0 and 8, are the amplitudes of the oscillations o and n. P ( T , ~is)the period of the measurement corresponding to the temperature, T (in OK), and to the relative humidity, x. Because of the kind of specimen formed by the juxtaposition of many fibers, it was not possible to calculate the absolute values for the modulus. In order to compare the modulus between the different degrees of humidity, special attention was paid to the process of hydration. This hydration was realized in situ without moving the collagen specimen. The sample was exposed to a defined-humidity air for 12 hr. A rapid quenching down to liquid-nitrogen temperature was then realized. This procedure has the advantage of specifying exactly the water content of the specimen. In the usual procedure, the hydration takes place in a cell that is then mounted in the torsion pendulum; a drastic change
2802
PINERI, ESCOUBES, AND ROCHE
in the humiditylevel can occur during this transfer and a small change in the counterweight or geometry can change the modulus values drastically. Other advantages of our procedure are the accuracy of our humidity measurement (*a%) and the range of the values which can be obtained (0-100% rel. hum.). In Fig. 1 we can see the hydration procedure. Air is flowed through water or ice, which is maintained at temperature TI (-30 to +2OoC). A good contact between air and water is realized by bubbling the air through a porous glass and raschig beads. We therefore obtain air which is saturated at temperature TI. This air is then warmed to room temperature, and the hydration is measured with a LiCl cell. By changing this T1 temperature, we can obtain any water concentration at room temperature.
RESULTS Gravimetry and Calorimetry Figure 2 shows the plots of the sorption-desorption isotherm at a temperature of 2OoC up to a relative water pressure of 0.85. We note a small hysteresis which is more important for p / p o values below 0.60. Similar absorption isotherms have been previously ~ b t a i n e d .Figure ~ 3 gives the plots of the enthalpy values vs the water pressure during sorption and desorption runs. Small differences appear between the sorption and desorption, these differences being more important when plotting the enthalpy vs the water uptake (Fig. 4). Three different regimes of sorptiondesorption are evident from these curves; they correspond to energies of 16.5,14, and 9.5 kcal/mol, respectively. The hydration ranges and energy values differ between the sorption and desorption processes. The energy value of 9.5 kcal/mol corresponds to an energy close to the value of the
Room T r a t u r e
ymm I L1
Torsion
-
Fig. 1. Hydration procedure for the mechanical experiments.
WATER-COLLAGEN INTERACTIONS a %A
2803
i r
+
LO*4
+ l,
300
+
+
+
p
20 -
7
Lp’ + + /o /o , / -%’
,:
to ++
’0-
4
I
00
025
05
PIP0
desorption
Fig. 3. Changes 0 in L enthalpy - during 7 a cycle 5 of sorption and desorption plotted against the relative humidity.
liquefaction of water. It has not been possible to increase the humidity range above plpo = 0.8 because the accuracy of our measurements is not good enough to define the sorption energy in this humidity range. In Fig. 5 are plotted the water uptakes a t different humidities vs time. For low humidity levels, we obtained equilibrium very quickly. Diffusion has to be taken into account for humidity above 60%. The Fick law seems t o be
PINERI, ESCOUBES, AND ROCHE
2804
I I
I
desorptionl
I Ji
II
III
IV
Regimes
0 ’
Ib i0 30 a YO Fig. 4. Changes in enthalpy during a cycle of sorption and desorption plotted against the water uptake. valid up to 70% of the water uptake for a relative humidity greater than 60%. We have an abnormal sigmoid curve with two different regimes for the relative humidity.
Dynamic Mechanical Experiments We will be concerned only with the curves between 77 and 300°K. No additional relaxation peak has been found between 4 and 77°K. Because of the changes which occur in the humidity level when warming the sample above room temperature, no attention has been paid to the peaks which appear above 300°K. In Fig. 6 are plotted the different damping curves obtained when changing the relative humidity. Four peaks can be seen. The low temperature peak, which will be called 6, is the only one in the dehydrated sample. We want to emphasize that the sample corresponding mm Hg] for to 0% rel. hum. has been obtained by vacuum pumping one night a t room temperature. Such a procedure is known not to result in irreversible changes in the collagen, some water being left, as will be shown below. When increasing the humidity, the 6 peak shifts down from 210°K (0% rel. hum.) to 140°K (75% rel. hum.). No further change in temperature is observed when increasing the humidity above 75%. The amplitude of this peak does not seem to change much with humidity. However, it is impossible to define exactly this amplitude because of the superposition of other peaks on this 6 peak. The peak /3 between 260 and 280°K appears at very low humidity (9.5% rel. hum.) and does not seem to change in amplitude and position with further increases in humidity. The a peak appears only in the curves
WATER-COLLAGEN INTERACTIONS
2805
1
0.5
0.43 - 0.54; Fig. 5. Water uptake during a sorption run vs time. (+) p/po: 0 - 0.33; (0)
(A)0.515 - 0.618; (0) 0.66 - 0.814.
corresponding to 95 and 98%rel. hum. It is a very sharp peak located at 262'K and is superimposed on the p peak. A fourth peak, which we will call y, appears when the humidity is higher than 60%. Its location is around 210'K, and it is more apparent in the 77.5% rel. hum. sample. In the 95 and 98%rel. hum. samples, this peak is masked because of the superposition of the 7-P and a peaks. However, the important increase in the damping after the relaxation is indicative of the existence of this peak.
Rigidity Figures 7 and 8 give the plots of the relative modulus corresponding to the different humidities vs the temperature. There is a progressive increase of the modulus a t each temperature below 270°K when increasing the degree of humidity, as is shown in Fig. 9. In Fig. 9 we have plotted the evolution of the relative modulus vs the water concentration at different temperatures. We do not observe the decrease in modulus which has been observed by Nomura et al.I4between 0.25 and 0.5 glg for all temperatures below 250°K with a human dura mater sample. In this range of absorption
2806
PINERI, ESCOUBES, AND ROCHE
P
I
?+ +
g Ha0 g collagen
0,61 0,51
0,39
RH
0.37
98
0.27
95 77.5
0235 039
75
0.15
60 52
0.l
41
0.09 0,08
27.5 15.5 12
0.01
9.5 0 100
2b0
300
T(OK)
Fig. 6. Effect of hydration on the relaxation spectrum of collagen. For convenience, each curve has been translated by a 0.1 AW/W factor.
we simply observe a small increase in the rigidity values when increasing the concentration of water. For concentrations larger than 0.40 g waterlg collagen, there is a drastic increase in the modulus for temperatures lower than 210°K. Water therefore has a stiffening effect at all concentrations below 270°K. Note the large decreases in the modulus at 270°K observed in Fig. 8 for the two most hydrated samples.
WATER-COLLAGEN INTERACTIONS
RH
qH>O/qcdh= 0 20 0 15 0 10 0 09
L1 % + 275% L 155% .:
.
0
2807
12 % 95%
0
oa
om
o vo
*
200 360 I60 T O K Fig. 7. Relative rigidity modulus plotted against temperature for different degrees of hydration. 0
160
A
G -
GG% *=i
6-
-
gHzO/gcdkqm 0 61 051 0 37 077.5% 039 0 6 0 Yo 0 27 RH
98 % +95 % .75 %
x
5*+ +*
1-
f
X
3-
2-
1-
1
160 260 300 T O K Fig. 8. Relative rigidity modulus plotted against temperature for different degrees of hydration.
Electrical Resistivity Figure 10 presents the plots of the different values of the current vs the water uptake at three different temperatures. A continuous increase of the current is observed over the entire concentration range. Figure 11 presents the plots for the intensity values in a logarithmic scale vs 1/T a t different humidity levels.
PINERI, ESCOUBES, AND ROCHE
2808
c G I 150
o
180
t
210 270
.
a
If,
n
I
O K
I
4
O K
OK O K O K
I
O K
m
.
I
V.
IV
I 'c- 1 .
Rc
/ '
Fig. 9. Relative rigidity modulus plotted against the water uptake at different temperatures (OK).
Discussion We have been unable to draw some conclusions from the electrical resistivity measurements. The purpose of these experiments was to show the different regimes of conduction when changing the temperature because of the different mobilities of the water molecules. We obtained a continuous change of the conduction over a wide range of temperatures and for all the water concentrations. At each hydration level an activation energy can be obtained from the plots of In I vs 1/T,but, since no physical meaning can be attached to this value, we do not try to define it. In this discussion we propose a model of water fixation from both our results and from those obtained from previous experiments. Our water concentrations have been defined by taking 0% water for a sample maintained at 100°C for 24 hr under a vacuum of at least 10-4 Torr. This ex-
WATER-COLLAGEN INTERACTIONS
2809
6
I amp
-
10-6
+ -0
,
-
10-8
't
+ / ' O
9 / ,/
lrn -
w'2
-
//'
f A
10-8 -
10-w.
1 0 1 2
-
10-11
13
+
35
L
is
1,~.10-30E
Fig. 11. Plots of the current against 1/T for different degrees of hydration (% rel. hum.): 95; 0,85;X, 70; A, 64; 0,49;0,39;=, 25.
+,
periment has been done after all the calorimetric experiments were performed in order to get rid of a possible irreversible change. The difference mm between dehydration a t room temperature and at 100°C under Hg is 0.010 g water/g collagen. These 0.01 g water were not extractable at room temperature, which means that we have water molecules which are firmly attached to the collagen molecule. The double-hydrogen-bonded water molecules will be seen to be extracted under vacuum at room temperature. It is very tempting to analyze this first kind of water molecule as a water molecule fixed by three hydrogen bonds, one of them involving the hydroxyl group. Such a possibility of water fixation has been pointed out by Ramachandran and Reddi.lg The fixation of one water molecule per three residues corresponds to 0.06 g waterlg collagen. The average hydroxyproline concentration corresponds to one group per 10 residues,
2810
PINERI, ESCOUBES, AND ROCHE
giving an opportunity of fixing about 0.018 g waterlg collagen by a threehydrogen-bond mechanism. The second kind of water molecule corresponds to the first molecules that are absorbed after the sample has been kept a t room temperature Torr. We think that these water molecules corunder a vacuum of respond to the interchain hydrogen bond inside the triple helix corresponding to the water-bridged structure of collagen proposed by Ramachandran and Reddi.19 The energy of fixation of these water molecules is around 17 kcallmol, which corresponds to a double-hydrogen-bonded water. We have 0.11 g water, which corresponds to an energy of 18kcallmol during the desorption process and only 0.07 g with an energy of 17 kcallmol during the sorption process. This difference can be explained by a hysteresis process. During the sorption process, the water molecules are fixed on different sites corresponding to different energies. In contrast, during the desorption, the water molecules corresponding to the highest energies are extracted after all the others have been removed. No change of the equatorial spacing has been observed in this range of water absorption.14J0 The helical parameters remain intact following dehydration to a moisture level of 0.1%. Therefore, we have very small changes of the tropocollagen structure upon removal of these water molecules. The water sites are still available and are occupied by the first molecules. The increase in modulus is not too important in this region of absorption because we have a system of rigid rods (tropocollagen molecules), and we increase only the rigidity of the tropocollagen molecules without crosslinking the rods between them. The /3 peak which appears for a water concentration of 0.08 g waterlg collagen must be connected with the presence of water. It can be explained by some exchange reaction involving the breaking and reforming of hydrogen bonds inside the tropocollagen molecule. On further hydration we do not observe any change in the amplitude and temperature position of this peak involving a saturation of the water molecules associated with this process. In this range of hydration, we observe a drastic change in the temperature position of the 6 peak. Stefanou et aLZ1interpret this peak as corresponding to oscillations of the proline group. If such an interpretation is correct, the energy barrier involved in this motion must decrease when increasing the humidity level. A small change in the environment of the proline group can produce drastic changes of the intermolecular barrier and can therefore explain the observed displacement of the peak with the hydration. It seems difficult to interpret such a 6 peak in the same way as it has been interpreted by Nomura et al.14 A low-temperature relaxation peak has been found in ice around 160°K a t 1 Hz. Motions of defects in an ice lattice involving a rotation and a jump of water molecules have been proposed to explain such a peak.22,23 There is no possible comparison between such an ice lattice with Bjerrum defects and a collagen structure with water molecules fixed in different sites. The results of nmr are consistent with our interpretation corresponding to this double hydrogen bonding. Up to a concentration of 0.35 g waterlg collagen, water has been found to be rigidly
WATER-COLLAGEN
INTERACTIONS
2811
bound to the collagen molecule.lO The third kind of water molecule corresponds to water concentrations between 0.11 and 0.235 glg a t the desorption, 0.07 and 0.235 glg a t the sorption. In this range of concentration there is a drastic increase of the equatorial spacing.14 We therefore interpret this regime as corresponding to the fixation of water molecules between the triple helices and between the microfibrils. The energy involved in this regime corresponds to 12 kcal at desorption and 14 kcal during sorption. This energy corresponds to a double hydrogen bonding and is a little lower than in the previous regime because some energy is necessary to swell the microfibrils and possibly to break some bonding between the triple helices. We have here the same differences in energy between the sorption and desorption results, the same explanation being valid. The large increase in the modulus values a t low temperatures (Fig. 9) is normal because of the kind of crosslinking (inter-triple-helices and intermicrofibrils) that is involved in this regime. No drastic change is observed in the damping curves during this regime. The 6 peak moved from 175°K down to 140°K because of a further decrease in the intermolecular potential barrier. The nmr results define the state of the water molecules fixed during this regime as nonrotational water molecules.10 The fourth kind of water molecule corresponds to absorption values between 0.235 and 0.50 g waterlg collagen. The equatorial spacing does not change too much in this absorption range.14 The rigidity modulus tends to level off and there appears a new peak located around 225OK (7). Different mechanisms can be proposed to explain this state. One explanation corresponds to the fixation of the water molecules between the microfibrils by just one hydrogen bond. We therefore have some kind of complexes with water molecules, the motion of which gives a relaxation peak (7). Mobile water molecules have been shown to appear at a concentration of 0.35 g waterlg collagen from nmr experiments.1° Another explanation would imply the fixation of the water molecules inside the holes -400 A long adjacent to the end of the tropocollagen molecule (A. Miller, personal communication). In both explanations a structural change of this water phase corresponding to a glass transition is possible. The last kind of water molecule appears above 0.50 water glg collagen. It corresponds to water molecules which form clusters large enough to behave as ice. A new peak ( a )appears at 270°K superimposed on the p peaks. This a peak is very important and corresponds to the melting of the ice. A drastic decrease in the rigidity modulus is associated with this peak (Fig. 7). We emphasize that a t low temperatures, water increases the modulus considerably, whereas it acts as a plastifier above 273°K. Such results are in agreement with previous calorimetric and nmr experiments,1° where free water has been found for water concentrations larger than 0.47 and 0.54 g waterlg collagen. This water seems to be fixed preferentially between the microfibrils.
0.010-0.110
I
0.010-0.070
0-0.010
Regime
I1
Water Concentration (g water/g collagen) Absorption Desorption
TABLE I
Water extractable a t 100°C under Torr High energy of fixation (>18 kcal/mol) Distinct sites Nonrotationally hydrated water (nmr)a Heat of sorption: 17 kcal/mol Heat of desorption: 18 kcal/mol No change in the equatorial spacing Small changes in the rigidity modulus p peak corresponding to an exchange reaction involving the water molecules Decrease in temperature of the 6-peak position Nonrotationally hydrated water (nmr)a
Characteristics
The Different Regimes of Water Fixation
Double hydrogen bonding of the water molecules in the available sites inside the triple helix.
Triple hydrogen bonding inside the triple helix and involving hydroxyproline.
Suggested Mechanism
N
?-
s"B
Eto
h
a
0.235-0.50
>0.50
IV
V
Ref. 10. Ref. 11.
0.071-0.235
I11
>0.50
0.235-0.50
0.110-0.235
Heat of sorption: 14 kcal/mol Heat of desorption: 12 kcal/mol Large increase in the modulus Large changes of the equatorial spacing No change of the p peak Nonrotationally hydrated water (nmr)a Heat of sorption and desorption -9 kcal/mol No change in the modulus a t low temperatures No large change in the equatorial spacing New y peak a t 225'K Mobile water molecules (nmr)a New, very important LY peak at 273°K Large decreases in the modulus a t 273°K Appearance of a freezing bound state for water corresponding to 0.54 g water/g collagen (nmr) and 0.48 (calorimetric)a No further change in the ir spectrumh Free water between the microfibrils.
Water fixed by one hydrogen bond between the microfibrils or water fixed in the hole zones adjacent to the end of the tropocollagen molecule.
Double hydrogen bonding of the water molecules between the triple helix and between the microfibrils.
2814
PINERI, ESCOUBES, AND ROCHE
%
'1' r o po c o 1 1a ge n Triple helix
36 A
(Cross-section)
Tetrngonnl 1.a t t i c e
l n t e r t r i p l e h e l i x and i n t e r m i c r o f i h r i l l a r phase i n which w a t e r i s f i x e d d u r i n g t h e r e p i m e s 111 a n d I V .
Fig. 12. Different fixation sites of water in collagen.
WATER-COLLAGEN INTERACTIONS
2815
CONCLUSION The first aim of this paper was to give very precise experimental calorimetric and mechanical results. From these results and from many others, we have been able to propose a model which seems, in general, to be consistent with all the experiments. However, many points have to be defined more clearly. Table I summarizes the characteristics of the different regimes of water fixation. In Fig. 12 the different sites of water fixation are defined. The most important conclusion is the evidence of different states of water fixation corresponding to different energies. However, more experiments are necessary in order to refine the analysis of these regimes of water fixation. The authors are grateful to Dr. A. Miller for helpful discussions. We are indebted to Mr.
M. Brotte for technical assistance in both the experimental work and the drawings.
References 1. Yannas, I. V. & Tobolsky, A. V. (1967) Nature 215,509-510. 2. Ramachandran, G. N. & Chandrasekharan, R. (1968) Biopolymers 6,1649-1662. 3. Miller, A. & Wray, J. S. (1971) Nature 230,437-438. 4. Miller, A. & Parry, D. A. D. (1973) J . Mol. Biol. 75,441-450. 5. Kuntz, I. D., Kauzmann, J. R. & Kauzmann, W., in Hydration of Proteins and Polypeptides. 6. Berendsen, H. J. C. (1962) J . Chem. Phys. 36,3297-3307. 7. Dehl, R. E. & Hoeve, C. A. J. (1969) J. Chem. Phys. 50,3245-3251. 8. Fung, B. M. & Trautmann, P. (1971) Biopolymers 10,391-397. 9. Dehl, R. E. (1970) Science 170,738-739. 10. Mrevlishvili, G. M. & Privalov, P. L. (1969) Water i n Biological Systems, Kayushin, L. P., Ed., Consultants Bureau, New York, p. 63. 11. Susi, H., Ard, J. S. & Carroll, R. J. (1971) Biopolymers 10, 1597. 12. Malcolm, B. R. (1970) Nature. 13. Chien, J. C. W. (1975) J. Macromol. Sci., Rev. Mucromol. Chem. 12, 1-80. 14. Nomura, S., Hiltner, A,, Lando, J. B. & Baer, E. (1977) Biopolymers 16,231-246. 15. Pineri, M. (1975) Polymer 16,595-600. 16. Meyer, C. T. & Pineri, M. H. (1976) Polymer 17,382-386. 17. Soulier, J. P., Escoubez, M., Douillard, A. & Chabert, B. (1976) J . Chim.Phys. 4, 423-429. 18. Pineri, M. H., Bonjour, E., Gerard, P. & Martin d’Hermont, F. (1972) Plast. Mod. Elustomeres 10, 19-27. 19. Ramachandran, G. N. & Reddi, A. H. (1976) Biochemistry of Collagen, Plenum, New York, 1976, p. 68. 20. Rougvie, M. A. & Bear, R. S. (1953) J . A m . Leather Chem. Assoc. 48,735-751. 21. Stefanou, H., Woodward, A. E. & Morrow, D. (1973) Biophys. J . 13,772-779. 22. Bass, R. (1958) Z. Phys. 153. 23. Vassoille R., Tatibouet, J., Perez, J. & Gobin, P. F. (1974) C. R. Acad. Sci., Ser. B , 409-412.
Received December 8,1977 Accepted February 21,1978
