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The effect of UV radiation on the thermal parameters of collagen degradation
Polymer Degradafion and Stahiliry 0 1996Elsevier Printed
in Northern
Ireland.
0141-3910(95)00158-1
ELSEVIER
51 (1996) 15-1X Science Limited
All rights reserved 0141.3910/96/$15.00
The effect of UV radiation on the thermal parameters of collagen degradation A. Kamiriska &
A. Sionkowska
Department of Chemistry, N. Copernicus
(Received
University, 87-100 Toruri, Poland
16 June 1995: accepted 11 July 1995)
Thermal parameters of collagen before and after UV irradiation were determined in air and nitrogen atmospheres. After UV irradiation of collagen, mass decrement, activation energy and entropy changes were apparently changed and may give information about the phototransformations which occur in this protein. However, these processes do not affect distinctly the values of T” parameters. Comparison of the shape of the derivative curves in air and nitrogen atmospheres suggests that oxidation of collagen may occur during heating and that this may also occur under UV irradiation.
1 INTRODUCTION
the triple helix was transformed to the statistical coil form. This must be preceded by the breaking-up of inter- and intra-molecular hydrogen bonds and release of water, which controls H-O-H-collagen bonds, i.e. their number and the length and distance between protein chains.‘sm’x If these conclusions are true, thermal parameters of collagen obtained by a derivatographic method ought to show some changes in DTG, DTA and TG curves after UV-irradiation of this protein. The aim of our work was to determine the thermal parameters of collagen and answer the question if thermal analyses may give any information about the photochemical transformation in collagen.
Photochemical transformations in polymers occur only in thin layers of the samples.‘*2 This is one reason why the efficiency of these transformations is rather small. In spite of this, we observed clear changes of thermal parameters in synthetic polymers after their UV irradiation. These changes were in agreement with the results obtained by other methods.‘.” It is known that the course of photochemical reactions depends upon the structure of polymer chains, presence of chromophoric groups, impurities, temperature, pressure and wavelength.“” In synthetic polymers, photochemical reactions and their effect on the polymer properties have been accurately investigated. But the structure of synthetic polymers is simpler than that of protein. The variety of groups and bonds, and the complicated form of collagen molecules as a triple helix, with different amount of intermolecular hydrogen bonds, and water bound with this protein in different ways, mean that photochemical transformations are not easy to observe in collagen. In our previous investigationsi we found that radiation with a wavelength of 253.7 nm caused changes in the conformation of this protein and
2 MATERIALS
AND METHODS
Insoluble collagen type I (produced by Sigma Chemical Company, lot 128F-81451) was used. The samples, in the form of powder, were irradiated in air at room temperature using a mercury lamp Philips TUV-30 which emitted light of mainly 253.7 nm wavelength. The intensity of radiation was 0.263 J/cm’ min. The derivatographic analyses were carried out 15
16
A. Kamihka,
Paulik-Paulik-Erdey derivatograph type OD 102 in nitrogen atmosphere and at a heating rate of S”/min, heating programme 25-lOOo”C, sensitivity of TG-100 mg, DTG-l/5, DTA-l/5. From DTG curves it was possible to determine the temperatures of the starting point of decomposition T”, taking as initial reaction the point at which the DTG curve begins to deviate from its base line, and temperatures of the maximum speed process T”“” obtained from the intersection of tangents to the peak. From TG curves we determined mass decrement Am from the point T” up to the end of the reaction, that is, to the point at which the DTG curve returns to its base line. A three-stage process of thermal decomposition of collagen recorded in the form of three different peaks on the DTG and DTA curves and a three-stage mass decrement on the TG curve determination of the enables following parameters: of the starting point of T” (,,2,3,-temperature processes at the 1, 2 or 3 stage. of maximum speed T”“” (I,,,3,-temperature processes 1, 2 or 3. Am (,.2.3j-mass decrement in the processes 1, 2 or 3. energy in the processes 1, 2 E (,,,,,,--activation or 3. nc1.2,3j-Order of reaction in the stage 1, 2, or 3 were calculated by the Horovitz-Metzger method.‘”
A. Sionkowska
Changes of entropy the equation:
were then calculated
by
AS=Rln$ where: h-Planck’s constant, constant, R-gas constant
K-Boltzmann’s
EO ’
&--heating
=R(Tmax)2nc:-le”/RTm”x
rate, n-order
of reaction
w - Wf c, = ~ w0 - Wf
3 RESULTS AND DISCUSSION On the DTG and DTA curves, which characterize the thermal degradation of collagen in air atmosphere, three peaks arise (Fig. 1). They suggest a three-stage destruction of this protein. The first peak, which arises at 311 K, is accompanied by mass decrement (Am,) equal to 9% recorded on the TG curve (Fig. 2). The second peak, which appears at 463 K, is accompanied by significant mass decrement Am, equal to 58.5%. As Lim” suggested, at the first stage, evaporation of water which was absorbed into the collagen occurs. At the second stage water bound with collagen is released. Also
In this method the relation
log[
1- CzY’]
+@)
is a straight line, where: w,,starting mass; at the completion of the main wl-mass reaction stage; w-mass at the temperature T”““; n-order of reaction; T”““---temperature of maximum speed process. Activation relation:
energy
was calculated
E = bR(Tm”“)2
where b is the tangent angle slope.
from
the
I
200
I
I
400 600 Temperature (“C)
I
800
Fig. 1. DTG and DTA curves which characterize the thermal decomposition of collagen in air (curve 1) and in nitrogen (curve 2).
17
Effect of UV radiation on thermal parameters of collagen degradation Table 2. Thermal
0
200
4ou
Temperature
600 (“C)
800
Fig. 2. TG curves which characterize
the thermal decomposition of collagen in air (curve 1) and in nitrogen (curve 2.)
small molecular products of the thermal degradation of collagen are liberated. The third peak on the DTG and DTA curves at 773 K also appears together with mass decrement (Am,) equal to 35%. Such a peak does not arise on the DTA and DTG curves for thermal destruction of collagen in nitrogen atmosphere (Fig. 1). This is evidence that this peak results from the decomposition of oxidative products of collagen which were formed during heating in air. The order of the reactions (n), which occur at these three stages of the collagen decomposition, successively decreases and ~1,> n2 > n, (Table 1). The activation energy of these reactions increases and E, is almost twice the value of E,. Entropy changes of this system also increase during heating and S, > S, > S,. This means that, during heating of collagen, its initially ordered structure is gradually destroyed. This may be possible after the break-up of inter- and intra-molecular hydrogen bonds which are responsible for maintenance of collagen chains in the form of a triple helix. After that, breaking up of C-C, C-H, C-NH, C-C=O, and C-COOH Table 1. Thermal
Stage
1 2 3
and thermodynamical parameters lagen determined in air
T” WI
Am (“/J
T”“” (K)
n
311 463 773
9.0 58.5 35.0
363 583 883
2.0 1.8 1.2
(kJ:mol) 73.82 104.88 158.30
of col-
(kJ,% 0.0838 0.1187 0.1262
and thermodynamical parameters lagen determined in N, -__
of col-
Stage
T” (K)
Am (“h)
T”“” (K)
n
E (kJ/mol)
AS (kJ/mol K)
1 2
311 453
9.6 64
358 603
2.1 2.4
67.68 91.98
0.0972 0.1482
bonds increases the state of disorder in the collagen structure. All these mean that the entropy clearly increases during thermal degradation of collagen. Thermal parameters T,” and T,“‘“” which characterize the first stage of the destruction of collagen in nitrogen atmosphere (Table 2) have similar values to ones determined in air. However, larger values of Am, and Am, and smaller ones of E, and El may suggest that the destruction of collagen in these conditions occurs more quickly and easily than in air. It is caused by absence in N, of the oxidative products which were formed when this process took place in air. Changes of entropy caused by the thermal transformation in N, are greater than those in air. This is evidence that in nitrogen the structural changes of collagen occur more easily than in air. It is so because in air some of the energy must be used for the oxidation processes. After UV irradiation of collagen, its thermal decomposition in air occurs at the temperature (TO) similar to that obtained before irradiation (Table 3). Also the temperatures of maximum speed were changed only by approximately a few degrees. However, mass decrements were changed significantly: Am, almost doubled, whilst Am, and Am, decreased. Especially clear is the Am, change (Table 3). It is evidence that UV Table 3. Values of thermal parameters of collagen (determined in air and nitrogen) before and after UV irradiation Stage
Irradiation time (h)
Air
(K)
(‘%) (k.J/mol)
T” (K)
Am (‘4))
E (kJ/mol)
0 4 8
311 313 311
9.0 12.8 15.5
73.82 76.38 70.29
311 311 30x
9.6 12.X 14.2
67.68 56.26 55.35
0
463
5X.5
IO4.XX
453
64.0
VI.98
4 8
476 463
50.2 47.5
X7.72 96.75
463 468
61.7 61.3
X2.83 1OX.06
0
776
35.0
158~30
4 8
763 763
35.5 32.2
124.35 149.09
-
-
T”
1
2
K)
3
Nitrogen
Anr
E
18
A. Kamiriska, A. Sionkowska
radiation enables the release of water bound with collagen because of the photolysis of hydrogen bonds between HOH and collagen. It is proved by its smaller loss at the second stage (463 K). It suggests that some of this water is released at the first stage at the lower temperature, i.e. at 311 K after break-up of the hydrogen bonds, caused by UV radiation. Greater negative changes of Am, than positive changes of Am, suggest that photodegradation of collagen also takes place and some of the products of this reaction were released from this protein before heating. The direction of the changes of the thermal parameters caused by UV radiation determined in nitrogen and in air is similar (Table 3). But evidently the greater changes of entropy suggest that in nitrogen the thermal destruction of UV-irradiated collagen occurs with greater efficiency than in air (Table 4). This is also confirmed by the greater mass decrement observed at the second stage of destruction and smaller values of activation energy (Table 3). The insignificant increase of T: (15’) after UV irradiation of collagen may be caused by photooxidative processes which can occur before thermal treatment of this protein, especially as nonirradiated collagen degraded in air atmosphere shows the same value (T,” = 463”).
4 CONCLUSION The values of T” and T”“” are not sensitive to the photochemical changes in collagen. But values of mass decrement, activation energy and entropy changes may give some information about changes of water content and the manner of its bonding with collagen, and also about the structural transformation as well as weakening of bonds in this protein after UV irradiation.
REFERENCES 1. Ulidska, A., KoScielecka, Polimery, 10 (1965) 394.
A.
&
Madkowski,
Z.,
2. Uliriska,
A.
&
Malikowski,
Z.,
A.,
KoScielecka,
Polimery, 10 (1965) 442.
3. Kamifiska,
H., Angew.
A. & Kaczmarek,
Chem., 120 (1984) 149.
4. Kamiliska,
A., Sanyal, S. & Kaczmarek,
Anal., 35 (1989) 2135.
Macromol.
H., J. Thermal
H., J. Thermal Anal., 32
5. Kamiriska, A. & Kaczmarek, (1987) 1791.
6. Rabek, J.F., Polymer Photodegradation Mechanisms and Experimental Methods. Chapman and Hall, London, 195.5. 7. Lawrence, J.B. & Weir, N.A., J. Polym. Sci., AI, 11 (1973) 105. 8. Kamal, M.R., El-Kaissy, M.M. & Avedesian, M.M., J. Appl. Polym. Sci., 16 (1972) 83.
9. Price, T.R. & Fox, R.B., J. Pofym. Sci. B., 4 (1966) 771. 10. Fox, R.B. & Price, T.R., J. Polym. Sci., AI, 3 (1965) 2303.
Table 4. Changes of AS parameter (after UV irradiation of collagen) which characterizes collagen after UV irradiation Stage
Irradiation time (h)
AS (kJ/mol K) ~~~~~~~~ Air N,
1
4 8
0.0018 0.0184
0.0369 0.0419
2
4 8
0.0340 0.0170
0.1714
3
4 8
0.0403 0.0095
-
11. Ranby, B. & Rabek, J.F., .I. Polym. Sci., Al. 12 (1974) 273. 12. Gesner, B.D. & Kelleher, P.G., .I. Appl. Polym. Sci., 13 (1969) 2183. 13. Grassie, N. & Weir, N.A., J. Appl. Polym. Sci., 9 (1965) 987.
14. Kaminska,
A.,
Sionkowska,
A.
&
Rozploch,
F..
Polimery, 39 (1994) 458.
1.5. Ramachandran,
G.N.
&
Chanrasekhran,
R.,
Bio-
polymers, 6 (1968) 1649.
G.N., Treatise on Collagen, p. 146. 16. Ramachandran, Academic Press, London, 1967. 17. Berendsen, H.J.C., J. Chem. Phys., 36 (1967) 329. 18. Lim, J.J. & Shannos, M.H., Biopolymers, 13 (1974) 1791.
19. Horowitz, H.H. & Metzger, G., Anal. Chem., 35 (1963) 1464.
in Northern
Ireland.
0141-3910(95)00158-1
ELSEVIER
51 (1996) 15-1X Science Limited
All rights reserved 0141.3910/96/$15.00
The effect of UV radiation on the thermal parameters of collagen degradation A. Kamiriska &
A. Sionkowska
Department of Chemistry, N. Copernicus
(Received
University, 87-100 Toruri, Poland
16 June 1995: accepted 11 July 1995)
Thermal parameters of collagen before and after UV irradiation were determined in air and nitrogen atmospheres. After UV irradiation of collagen, mass decrement, activation energy and entropy changes were apparently changed and may give information about the phototransformations which occur in this protein. However, these processes do not affect distinctly the values of T” parameters. Comparison of the shape of the derivative curves in air and nitrogen atmospheres suggests that oxidation of collagen may occur during heating and that this may also occur under UV irradiation.
1 INTRODUCTION
the triple helix was transformed to the statistical coil form. This must be preceded by the breaking-up of inter- and intra-molecular hydrogen bonds and release of water, which controls H-O-H-collagen bonds, i.e. their number and the length and distance between protein chains.‘sm’x If these conclusions are true, thermal parameters of collagen obtained by a derivatographic method ought to show some changes in DTG, DTA and TG curves after UV-irradiation of this protein. The aim of our work was to determine the thermal parameters of collagen and answer the question if thermal analyses may give any information about the photochemical transformation in collagen.
Photochemical transformations in polymers occur only in thin layers of the samples.‘*2 This is one reason why the efficiency of these transformations is rather small. In spite of this, we observed clear changes of thermal parameters in synthetic polymers after their UV irradiation. These changes were in agreement with the results obtained by other methods.‘.” It is known that the course of photochemical reactions depends upon the structure of polymer chains, presence of chromophoric groups, impurities, temperature, pressure and wavelength.“” In synthetic polymers, photochemical reactions and their effect on the polymer properties have been accurately investigated. But the structure of synthetic polymers is simpler than that of protein. The variety of groups and bonds, and the complicated form of collagen molecules as a triple helix, with different amount of intermolecular hydrogen bonds, and water bound with this protein in different ways, mean that photochemical transformations are not easy to observe in collagen. In our previous investigationsi we found that radiation with a wavelength of 253.7 nm caused changes in the conformation of this protein and
2 MATERIALS
AND METHODS
Insoluble collagen type I (produced by Sigma Chemical Company, lot 128F-81451) was used. The samples, in the form of powder, were irradiated in air at room temperature using a mercury lamp Philips TUV-30 which emitted light of mainly 253.7 nm wavelength. The intensity of radiation was 0.263 J/cm’ min. The derivatographic analyses were carried out 15
16
A. Kamihka,
Paulik-Paulik-Erdey derivatograph type OD 102 in nitrogen atmosphere and at a heating rate of S”/min, heating programme 25-lOOo”C, sensitivity of TG-100 mg, DTG-l/5, DTA-l/5. From DTG curves it was possible to determine the temperatures of the starting point of decomposition T”, taking as initial reaction the point at which the DTG curve begins to deviate from its base line, and temperatures of the maximum speed process T”“” obtained from the intersection of tangents to the peak. From TG curves we determined mass decrement Am from the point T” up to the end of the reaction, that is, to the point at which the DTG curve returns to its base line. A three-stage process of thermal decomposition of collagen recorded in the form of three different peaks on the DTG and DTA curves and a three-stage mass decrement on the TG curve determination of the enables following parameters: of the starting point of T” (,,2,3,-temperature processes at the 1, 2 or 3 stage. of maximum speed T”“” (I,,,3,-temperature processes 1, 2 or 3. Am (,.2.3j-mass decrement in the processes 1, 2 or 3. energy in the processes 1, 2 E (,,,,,,--activation or 3. nc1.2,3j-Order of reaction in the stage 1, 2, or 3 were calculated by the Horovitz-Metzger method.‘”
A. Sionkowska
Changes of entropy the equation:
were then calculated
by
AS=Rln$ where: h-Planck’s constant, constant, R-gas constant
K-Boltzmann’s
EO ’
&--heating
=R(Tmax)2nc:-le”/RTm”x
rate, n-order
of reaction
w - Wf c, = ~ w0 - Wf
3 RESULTS AND DISCUSSION On the DTG and DTA curves, which characterize the thermal degradation of collagen in air atmosphere, three peaks arise (Fig. 1). They suggest a three-stage destruction of this protein. The first peak, which arises at 311 K, is accompanied by mass decrement (Am,) equal to 9% recorded on the TG curve (Fig. 2). The second peak, which appears at 463 K, is accompanied by significant mass decrement Am, equal to 58.5%. As Lim” suggested, at the first stage, evaporation of water which was absorbed into the collagen occurs. At the second stage water bound with collagen is released. Also
In this method the relation
log[
1- CzY’]
+@)
is a straight line, where: w,,starting mass; at the completion of the main wl-mass reaction stage; w-mass at the temperature T”““; n-order of reaction; T”““---temperature of maximum speed process. Activation relation:
energy
was calculated
E = bR(Tm”“)2
where b is the tangent angle slope.
from
the
I
200
I
I
400 600 Temperature (“C)
I
800
Fig. 1. DTG and DTA curves which characterize the thermal decomposition of collagen in air (curve 1) and in nitrogen (curve 2).
17
Effect of UV radiation on thermal parameters of collagen degradation Table 2. Thermal
0
200
4ou
Temperature
600 (“C)
800
Fig. 2. TG curves which characterize
the thermal decomposition of collagen in air (curve 1) and in nitrogen (curve 2.)
small molecular products of the thermal degradation of collagen are liberated. The third peak on the DTG and DTA curves at 773 K also appears together with mass decrement (Am,) equal to 35%. Such a peak does not arise on the DTA and DTG curves for thermal destruction of collagen in nitrogen atmosphere (Fig. 1). This is evidence that this peak results from the decomposition of oxidative products of collagen which were formed during heating in air. The order of the reactions (n), which occur at these three stages of the collagen decomposition, successively decreases and ~1,> n2 > n, (Table 1). The activation energy of these reactions increases and E, is almost twice the value of E,. Entropy changes of this system also increase during heating and S, > S, > S,. This means that, during heating of collagen, its initially ordered structure is gradually destroyed. This may be possible after the break-up of inter- and intra-molecular hydrogen bonds which are responsible for maintenance of collagen chains in the form of a triple helix. After that, breaking up of C-C, C-H, C-NH, C-C=O, and C-COOH Table 1. Thermal
Stage
1 2 3
and thermodynamical parameters lagen determined in air
T” WI
Am (“/J
T”“” (K)
n
311 463 773
9.0 58.5 35.0
363 583 883
2.0 1.8 1.2
(kJ:mol) 73.82 104.88 158.30
of col-
(kJ,% 0.0838 0.1187 0.1262
and thermodynamical parameters lagen determined in N, -__
of col-
Stage
T” (K)
Am (“h)
T”“” (K)
n
E (kJ/mol)
AS (kJ/mol K)
1 2
311 453
9.6 64
358 603
2.1 2.4
67.68 91.98
0.0972 0.1482
bonds increases the state of disorder in the collagen structure. All these mean that the entropy clearly increases during thermal degradation of collagen. Thermal parameters T,” and T,“‘“” which characterize the first stage of the destruction of collagen in nitrogen atmosphere (Table 2) have similar values to ones determined in air. However, larger values of Am, and Am, and smaller ones of E, and El may suggest that the destruction of collagen in these conditions occurs more quickly and easily than in air. It is caused by absence in N, of the oxidative products which were formed when this process took place in air. Changes of entropy caused by the thermal transformation in N, are greater than those in air. This is evidence that in nitrogen the structural changes of collagen occur more easily than in air. It is so because in air some of the energy must be used for the oxidation processes. After UV irradiation of collagen, its thermal decomposition in air occurs at the temperature (TO) similar to that obtained before irradiation (Table 3). Also the temperatures of maximum speed were changed only by approximately a few degrees. However, mass decrements were changed significantly: Am, almost doubled, whilst Am, and Am, decreased. Especially clear is the Am, change (Table 3). It is evidence that UV Table 3. Values of thermal parameters of collagen (determined in air and nitrogen) before and after UV irradiation Stage
Irradiation time (h)
Air
(K)
(‘%) (k.J/mol)
T” (K)
Am (‘4))
E (kJ/mol)
0 4 8
311 313 311
9.0 12.8 15.5
73.82 76.38 70.29
311 311 30x
9.6 12.X 14.2
67.68 56.26 55.35
0
463
5X.5
IO4.XX
453
64.0
VI.98
4 8
476 463
50.2 47.5
X7.72 96.75
463 468
61.7 61.3
X2.83 1OX.06
0
776
35.0
158~30
4 8
763 763
35.5 32.2
124.35 149.09
-
-
T”
1
2
K)
3
Nitrogen
Anr
E
18
A. Kamiriska, A. Sionkowska
radiation enables the release of water bound with collagen because of the photolysis of hydrogen bonds between HOH and collagen. It is proved by its smaller loss at the second stage (463 K). It suggests that some of this water is released at the first stage at the lower temperature, i.e. at 311 K after break-up of the hydrogen bonds, caused by UV radiation. Greater negative changes of Am, than positive changes of Am, suggest that photodegradation of collagen also takes place and some of the products of this reaction were released from this protein before heating. The direction of the changes of the thermal parameters caused by UV radiation determined in nitrogen and in air is similar (Table 3). But evidently the greater changes of entropy suggest that in nitrogen the thermal destruction of UV-irradiated collagen occurs with greater efficiency than in air (Table 4). This is also confirmed by the greater mass decrement observed at the second stage of destruction and smaller values of activation energy (Table 3). The insignificant increase of T: (15’) after UV irradiation of collagen may be caused by photooxidative processes which can occur before thermal treatment of this protein, especially as nonirradiated collagen degraded in air atmosphere shows the same value (T,” = 463”).
4 CONCLUSION The values of T” and T”“” are not sensitive to the photochemical changes in collagen. But values of mass decrement, activation energy and entropy changes may give some information about changes of water content and the manner of its bonding with collagen, and also about the structural transformation as well as weakening of bonds in this protein after UV irradiation.
REFERENCES 1. Ulidska, A., KoScielecka, Polimery, 10 (1965) 394.
A.
&
Madkowski,
Z.,
2. Uliriska,
A.
&
Malikowski,
Z.,
A.,
KoScielecka,
Polimery, 10 (1965) 442.
3. Kamifiska,
H., Angew.
A. & Kaczmarek,
Chem., 120 (1984) 149.
4. Kamiliska,
A., Sanyal, S. & Kaczmarek,
Anal., 35 (1989) 2135.
Macromol.
H., J. Thermal
H., J. Thermal Anal., 32
5. Kamiriska, A. & Kaczmarek, (1987) 1791.
6. Rabek, J.F., Polymer Photodegradation Mechanisms and Experimental Methods. Chapman and Hall, London, 195.5. 7. Lawrence, J.B. & Weir, N.A., J. Polym. Sci., AI, 11 (1973) 105. 8. Kamal, M.R., El-Kaissy, M.M. & Avedesian, M.M., J. Appl. Polym. Sci., 16 (1972) 83.
9. Price, T.R. & Fox, R.B., J. Pofym. Sci. B., 4 (1966) 771. 10. Fox, R.B. & Price, T.R., J. Polym. Sci., AI, 3 (1965) 2303.
Table 4. Changes of AS parameter (after UV irradiation of collagen) which characterizes collagen after UV irradiation Stage
Irradiation time (h)
AS (kJ/mol K) ~~~~~~~~ Air N,
1
4 8
0.0018 0.0184
0.0369 0.0419
2
4 8
0.0340 0.0170
0.1714
3
4 8
0.0403 0.0095
-
11. Ranby, B. & Rabek, J.F., .I. Polym. Sci., Al. 12 (1974) 273. 12. Gesner, B.D. & Kelleher, P.G., .I. Appl. Polym. Sci., 13 (1969) 2183. 13. Grassie, N. & Weir, N.A., J. Appl. Polym. Sci., 9 (1965) 987.
14. Kaminska,
A.,
Sionkowska,
A.
&
Rozploch,
F..
Polimery, 39 (1994) 458.
1.5. Ramachandran,
G.N.
&
Chanrasekhran,
R.,
Bio-
polymers, 6 (1968) 1649.
G.N., Treatise on Collagen, p. 146. 16. Ramachandran, Academic Press, London, 1967. 17. Berendsen, H.J.C., J. Chem. Phys., 36 (1967) 329. 18. Lim, J.J. & Shannos, M.H., Biopolymers, 13 (1974) 1791.
19. Horowitz, H.H. & Metzger, G., Anal. Chem., 35 (1963) 1464.