Temperature Induced Denaturation of Collagen in Acidic SolutionChangdao Mu,1 Defu Li,1 Wei Lin,1 Yanwei Ding,2 Guangzhao Zhang21 Department of Pharmaceutics and Bioengineering, Key Lab of Leather Chemistry, and Engineering of Ministry of Education,

Sichuan University, Chengdu, China

2 Hefei National Laboratory for Physical Sciences at Microscale, Department of Chemical Physics,

University of Science and Technology of China, Hefei, Anhui, China

Received 3 March 2007; revised 19 March 2007; accepted 20 March 2007

Published online 12 April 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20742

This article was originally published online as an accepted

preprint. The ‘‘Published Online’’ date corresponds to the

preprint version. You can request a copy of the preprint by

emailing the Biopolymers editorial office at biopolymers@wiley.

com

INTRODUCTION

It is well known that the fibril-forming collagens including

type I, II, and III form the matrix of bone, skin, and tis-

sues of vertebrate animals. The most abundant collagen

in tissue is the type I which consists of two a1(I) polypep-tide chains and one a2(I) chain.1,2 The three chains are

coiled in a left-handed helices and thrown into a right-

handed triple superhelix dominantly stabilized by periodic

hydrogen bonds.3,4 Such triple-helices associate laterally and

longitudinally to form fibrils through covalent cross-links. As

the cross-links increase, the thermal stability of the collagen

increases.5 A thermal denaturation usually leads the hydrogen

bonds to break and induces the unfolding of the triple helix.6

So far, the denaturational transition process has been exten-

sively examined by circular dichroism (CD),7–10 fluorescence

depolarization,11 a combination of size exclusion chromatog-

raphy and light scattering (LS),12,13 NMR,14 and differential

scanning calorimetry (DSC).15–24 However, there still remain

some unsolved problems about the fibrillation and the stabi-

lization of the triple helices in the denaturation.

ABSTRACT:

The denaturation of collagen solution in acetic acid has

been investigated by using ultra-sensitive differential

scanning calorimetry (US-DSC), circular dichroism

(CD), and laser light scattering (LLS). US-DSC

measurements reveal that the collagen exhibits a bimodal

transition, i.e., there exists a shoulder transition before

the major transition. Such a shoulder transition can

recover from a cooling when the collagen is heated to a

temperature below 358C. However, when the heating

temperature is above 378C, both the shoulder and major

transitions are irreversible. CD measurements

demonstrate the content of triple helix slowly decreases

with temperature at a temperature below 358C, but it

drastically decreases at a higher temperature. Our

experiments suggest that the shoulder transition and

major transition arise from the defibrillation and

denaturation of collagen, respectively. LLS measurements

show the average hydrodynamic radius hRhi, radius ofgyration hRgiof the collagen gradually decrease before a

sharp decrease at a higher temperature. Meanwhile, the

ratio hRgi/hRhi gradually increases at a temperature

below � 348C and drastically increases in the range 34–

408C, further indicating the defibrillation of collagen

before the denaturation. # 2007 Wiley Periodicals, Inc.

Biopolymers 86: 282–287, 2007.

Keywords: collagen; denaturation, light scattering

Temperature Induced Denaturation of Collagen in Acidic Solution

Correspondence to: Guangzhao Zhang; e-mail: gzzhang@ustc.edu.cn

Contract grant sponsor: National Natural Science Foundation (NNSF) of China

Contract grant number: 20474060

Contract grant sponsors: The Chinese Academy of Sciences

Contract grant number: KJCX2-SW-H14

VVC 2007 Wiley Periodicals, Inc.

282 Biopolymers Volume 86 / Number 4

It has been reported that both acidic soluble collagen and

neutral fibrillar collagen exhibit multiple denaturational

transitions.15–18 Privalov and Tiktopulo suggested some pre-

denaturational conformation of collagen before the denatu-

ration,15 but there was no further interpretation. Wallace

et al.16 proposed that such multiple transitions are because of

sequential melting of distinct classes of molecules and fibril-

lar species formed by the reconstituted collagen. However,

such fibrillar species have not been directly observed. Note

that the origin of the multiple denaturational transitions is

important for our understanding the denaturation mecha-

nism and the stabilization of collagen.

In the present work, we have investigated the temperature

induced denaturation of type I collagen in acidic solutions

using ultrasensitive microcalorimetry (US-DSC) and CD. In

addition, using a combination of static and dynamic laser

light scattering (LLS), we have carefully characterized the

denaturation of the collagen as a function of temperature.

Our aim is to understand the origin of the multiple dena-

turational transitions.

EXPERIMENTAL SECTION

Materials

Collagen used in this study was isolated from fresh adult bo-

vine Archilles tendon using acetic acid.25 After successive

rinsing with acetone to remove the fattiness, the collagen was

extracted with 0.5M acetic acid at � 48C for 2 days under

stirring and salted out with sodium chloride. Then, the colla-

gen was collected by refrigerated centrifugation. It was fur-

ther dialyzed against deionized water at � 48C for 2 days

until a constant conductivity. The collagen was then freeze-

dried and kept in refrigerator. Collagen solutions in 0.5M

acetic acid were freshly prepared just before use.

US–DSC Measurements

Collagen solutions were measured on a VP-DSC microca-

lorimeter from Microcal Inc. with acetic acid as the reference.

The solution was degassed at 258C for half an hour and equi-

librated at 108C for 2 h before the heating process. The en-

thalpy change (DH) during the transition was calculated

from the area under each peak. The phase transition temper-

ature (Tp) was taken as that centered at the transitions. The

concentration of the collagen solution was 5.0 3 10�4g/mL.

In the case of bulk collagen, the freeze-dried sample was

placed in the chamber at the end of a stainless steel tube

closed with a nylon plug, which was inserted in the measure-

ment cell. Such a tube without sample was used as the refer-

ence. The water content (� 14 wt %) of the bulk collagen

was determined by a Shimadzu DTG-60H thermogravimetric

analyzer. The sample was heated from 25 to 8008C with a

heating rate of 108C/min in the protection of nitrogen.

Circular Dichroism Measurements

The collagen solution in acetic acid with a concentration of

2.0 3 10�4g/mL was equilibrated at 48C for 12 h before mea-

surement. CD spectra from 190 to 240 nm were measured at

25, 40, and 508C. The molar ellipticity (Em) at � 220 nm was

obtained at a heating rate of 18C/min in the range 25–508C.

Laser Light Scattering

LLS measurements were carried out on ALV/SP-125 LLS

spectrometer (ALV/DLS/SLS-5022F) equipped with a multi-sdigital time correlation (ALV5000) and a cylindrical 22 mW

UNIPHASE He-Ne laser (k0 ¼ 632 nm) as the light source.

In static LLS,26,27 the weight-average molar mass (Mw), the

root-mean-square radius of gyration hRg2iz1/2 (or written as

hRgi), and the second virial coefficient A2 were obtained

from the angular dependence of the absolute excess time-av-

erage scattering intensity, known as the Rayleigh ratio Rvv(q)

by using

KC

RvvðqÞ �1

Mw1þ 1

3R2g

D Ezq2

� �þ 2A2C ð1Þ

where K ¼ 4p2n2(dn/dC)2/(NAk04) and q ¼ (4pn/k0)sin(y/2)

with C, dn/dC, NA, and k0 being the concentration of the col-

lagen, the specific refractive index increment, the Avogadro’s

number, and the wavelength of light in vacuum, respectively.

In dynamic LLS,28 we were able to measure the intensity-in-

tensity time correlation function G(2)(t, q) which is related to

the normalized first-order electric field-electric field time

correlation function g ð1Þðt ; qÞ�� �� � Eð0; qÞEðt ; qÞh i� �as

Gð2Þðt ; qÞ ¼ Ið0; qÞIðt ; qÞh i ¼ A 1þ b g ð1Þðt ; qÞ�� ��2h ið2Þ

where t is the delay time, E(0,q) and E(t,q) are the electric

field strength at t ¼ 0 and t, respectively; A is the measured

baseline; b is an instrument constant depending on the opti-

cal coherence of the detection. Generally, g ð1Þðt ; qÞ�� �� is relatedto a characteristic line-width distribution G(G) as

g ð1Þðt ; qÞ�� �� ¼Z

GðCÞ e�CtdC ð3Þ

For diffusive relaxation, G is related to the translational diffu-

sion coefficient (D) of the scattering object in dilute solution

Temperature Induced Denaturation of Collagen in Acidic Solution 283

Biopolymers DOI 10.1002/bip

or dispersion by

C=q2 ¼ D 1þ kq2hR2g i þ � � �

� �ð4Þ

where the coefficient (k) depends upon the structure and

hydrodynamic interactions of the scattering object. Equation

(4) yields (G/q2) q?0, k?0 ¼ D. Hydrodynamic radius (Rh) is

obtained by the Stokes-Einstein equation, Rh ¼ kBT/6pgD,where g, kB, and T are the solvent viscosity, the Boltzmann

constant and the absolute temperature, respectively. For a nar-

rowly distributed system, the cumlant analysis of g ð1Þðt ; qÞ�� �� issufficient to generate in a reliable average hGior hDior hRhi.Hydrodynamic radius distribution f(Rh) was calculated from

the Laplace inversion of a corresponding measured G(2)(t, q)

using the CONTIN program in the correlator on the basis of

Eqs.(2)–(4). All dynamic LLS experiments were carried out

at a scattering angle of 158. The collagen solution was clari-

fied using a 0.45 lm Millipore filter. The concentration of

the collagen solution was 5.0 3 10�4g/mL. Each data point

was obtained after the solution was equilibrated for twenty

minutes so that the measured values were stable.

RESULTS AND DISCUSSIONSFigure 1 shows the temperature dependence of specific heat

capacity (Cp) of the collagen in acetic acid at different heat-

ing rates. As reported before,15,16 at any heating rate, a bi-

modal transition with a major peak at Tm � 408C and a

shoulder at Ts � 328C are observed. As the heating rate

increases from 0.5 to 1.338C/min, both of them shift to

higher temperatures, indicating that they involve kinetic

effect. From the area of each endothermic peak, the enthalpy

change in the transition can be determined. Figure 2 shows

the enthalpy change corresponding to the major transition

(DHm) linearly decreases with the heating rate, indicating an

incomplete denaturation; namely, the major transition with a

heating rate is not in equilibrium. On the other hand, the en-

thalpy change concerning the shoulder (DHs) is almost inde-

pendent of the heating rate, suggesting that the transition is

so quick that equilibrium can attain in the time of scanning.

The reversibility of the bimodal transition was also exam-

ined. After the collagen solution was heated to a certain tem-

perature (Th), the solution was cooled at � 48C for a certain

time (tc). Then, the solution was rescanned. Figure 3 shows

FIGURE 1 The specific heat capacity (Cp) of collagen solution in

acetic acid at the heating rate of 0.58C/min (a), 1.008C/min (b) and

1.338C/min (c), respectively.

FIGURE 2 Scanning rate dependence of transition temperature

(Tp) and enthalpy change (DH).

FIGURE 3 The cooling time (tc) dependence of the specific heat

capacity (Cp) of collagen solution in acetic acid in the rescanning

process with the heating temperature in the first scanning being

358C. (a) tc ¼ 12 h; (b) tc ¼ 36 h; (c) The first scanning result (25–

608C); (d) tc ¼ 60 h. The scanning rate was 1.08C/min.

284 Mu et al.

Biopolymers DOI 10.1002/bip

the effect of the cooling time on specific heat capacity (Cp) of

collagen solution when Th ¼ 358C, where the shoulder tran-sition is complete but the major transition has not started.

When tc ¼ 12 h, the shoulder transition is absent, while the

major transition still exists. However, as tc is increased to 36 h,

the shoulder transition partially recovers with the major

transition. When tc is prolonged to a week, the shoulder tran-

sition fully recovers. The facts demonstrate that the shoulder

transition is reversible with a hysteresis in one heating-and-

cooling cycle when the heating temperature is below 358C.However, when the collagen solution is heated to a tempera-

ture above 378C, neither the shoulder nor the major transi-

tion can be recovered even the solution is cooled at � 48Cfor one week (Figure 4). Thus, both the shoulder and the

major transitions are irreversible when the heating tempera-

ture is above 378C. Namely, the collagen is completely dena-

tureated. Note that this does not mean that collagen in tissue

already unfolds at body temperature (378C). As we will dis-

cuss later, the denaturation is also determined by the fibrilla-

tion of collagen. As shown in the inset, the collagen in bulk

shows a single endothermic peak at as high as 1048C. This isbecause the bulk collagen with low water content (� 14 wt %)

has a high degree of fibrillation. Without enough free water

or solvent molecules, the fibrillation is difficult to dissociate.

The above results clearly indicate that the origins of the

shoulder and the major transitions are different. It has been

suggested that collagen forms several distinct fibrillar species,

and each of them has a characteristic melting temperature.16

Since all the species consist of the same collagen molecules, it

is impossible that some species have a reversible melting but

the others have an irreversible one. The reversibility of the

shoulder transition indicates that it is not due to the denatu-

ration of a certain collagen species. On the other hand, the

nonfibrillar collagen in acidic solution only exhibits the

major transition without the shoulder transition.16 The facts

suggest that the shoulder transition is probably associated

with the fibrillation of collagen. It is known that the triple

helix is stabilized by the intra-helix hydrogen bonds. The tri-

ple helices form fibrils via inter-triple helix hydrogen bonds

and covalent cross-links.3,4 The defibrillation with the break-

ing of hydrogen bonds between triple helices might be re-

sponsible for the shoulder transition.

Figure 5 shows the temperature dependence of the molar

ellipticity (Em) around 220 nm measured by CD. The inset

shows typical CD spectrum of collagen solutions in acetic

acid in the range 190–240 nm at 25, 40, and 508C. The bandat � 220 nm is attributed to the triple helix.9 Obviously, the

collagen holds the triple helix structure at 258C but has less

triple helices at a higher temperature. The disappearance of

the band at � 220 nm at 508C clearly indicates a complete

destruction of the triple helix.10 Figure 5 shows that Emslowly decreases with temperature in the range 25–348C,indicating a slight destruction of the triple helices. A faster

decrease of Em can be observed in the range 35–428C. Asharp transition occurs in the range 40–448C, which corre-

sponds to the major denaturation in US-DSC curves. At a

temperature above 458C, Em tends to be a constant, implying

the completion of the denaturation. It is interesting to note

that there is a turn at � 348C, which implies some small

change in collagen structure. Since the change occurs before

the denaturation, it could be attributed to the defibrillation,

which corresponds to the shoulder transition in US-DSC

FIGURE 4 The temperature dependence of specific heat capacity

(Cp) of collagen solution in acetic acid in the rescanning process

with the heating temperature in the first scanning being 378C (a)

and 608C (b), respectively. The solution had been cooled at � 48Cfor one week before the re-scanning. (c) The first scanning result

(25–608C). The inset shows the temperature dependence of specific

heat capacity (Cp) of bulk collagen. All the scanning rates were

1.08C/min.

FIGURE 5 Temperature dependence of molar ellipticity (Em) of

collagen solution in acetic acid. The inset shows the CD spectra at

25, 40, and 508C.

Temperature Induced Denaturation of Collagen in Acidic Solution 285

Biopolymers DOI 10.1002/bip

experiments. To further clarify the nature of the bimodal

transition, we also examined the collagen solution in acetic

acid at different temperatures by LLS.

Figure 6 shows the hydrodynamic radius distributions

f(Rh) at 25, 33, 41, and 458C measured by dynamic LLS. The

bimodal distribution at 258C clearly indicates that collagen

molecules form fibrillar species before the denaturation. The

peaks at � 25 and � 230 nm could be attributed to fibrillar

aggregates at with different sizes. When the collagen is heated

to 338C, the former slightly changes, but the latter shifts to

� 220 nm, suggesting that the triple helix structure is slightly

destroyed. Further increasing the temperature to 418C or

higher leads the size of the aggregates to decrease due to the

denaturation. Obviously, the aggregates do not disappear at

an elevated temperature. As revealed by US-DSC and CD

above, the collagen is already denatured at such a tempera-

ture that is the hydrogen bonds between the helices have

been broken. Therefore, the aggregates should be formed by

the coils via other molecular interactions such as hydropho-

bic interactions.

Figure 7 shows the temperature dependence of the average

hydrodynamic radius hRhi and the average radius of gyration

hRgi. In the range 25–348C, both hRhi and hRgi decrease withtemperature. This is an indicative of the defibrillation. A

much sharper decrease can be observed for either of them in

the range 34–408C, clearly indicating the denaturation of col-

lagen. Note that such a denaturation might involve some

defibrillation of collagen. At a temperature above 418C, sincethe denaturation has completed, hRhi and hRgi no longer

change. The inset shows that the Rvv(q)/KC gradually

decreases with temperature in the range 25–408C. Since

Rvv(q) is proportional to the weight-average molar mass

(Mw) on basis of Eq. (1) in a dilute solution, it implies that

the Mw of the fibrillar species decrease with the increasing

temperature. This is understandable because both the defib-

rillation and the denaturation lead Mw to decrease, and their

roles are comparable. Mw slightly changes at the tempera-

tures above 418C, further indicating the completion of dena-

turation. Note that the temperature ranges for the defibrilla-

tion and the denaturation measured by LLS are some differ-

ent from those from US-DSC and CD measurements. This is

because LLS measurements were conducted when the system

was in a relatively equilibrium state, but US-DSC and CD

measurements were performed with a certain heating rate.

Figure 8 shows the temperature dependence of hRgi/hRhi.The conformation of a macromolecule and the structure of

an aggregate can be described by the ratio of hRgi/hRhi. Forrandom coil, hyperbranched cluster or micelle, and uniform

sphere, hRgi/hRhi is � 1.5–1.8, � 1.0–1.2, and � 0.774,

respectively.29 In the present study, hRgi/hRhi� 0.57 at 258Cindicates the densely packed structure of the triple helix. As

FIGURE 7 Temperature dependence of average hydrodynamic ra-

dius hRhi and average radius of gyration hRgi of the collagen in ace-

tic acid. The inset shows the temperature dependence of Rvv(0)/KC.

FIGURE 6 Typical hydrodynamic radius distributions f(Rh) of

collagen solution in acetic acid at 25, 33, 41, and 458C.

FIGURE 8 Temperature dependence of ratio of average radius of

gyration to average hydrodynamic radius hRgi/hRhiof the collagen

in acetic acid.

286 Mu et al.

Biopolymers DOI 10.1002/bip

temperature increases from 25 to 338C, hRgi/hRhi gradually

increases to 0.64, suggesting the structure become looser

because of the defibrillation. In the range 34–408C, hRgi/hRhisharply increases to 0.88, indicating the disintegration of the

triple helices into coils which form spherical-like aggregates.

As discussed earlier, in such aggregates, the inter-triple helix

hydrogen bonds and intra-helix hydrogen bonds are broken,

and the triple helices can not recover upon cooling. That is

why either the shoulder or major transitions in the US-DSC

curves is irreversible after the collagen is heated at a tempera-

ture above 378C.

CONCLUSIONThe investigations on the denaturation of collagen solution

in acetic acid by using ultra-sensitive differential scanning

calorimetry (US-DSC), CD, and LLS lead to the following

conclusion. The collagen in acetic acid exhibits a bimodal

transition with one shoulder transition and one major transi-

tion. The former arises from the defibrillation of collagen,

whereas the latter is due to the denaturation. When the heat-

ing temperature is below a certain value, the defibrillated tri-

ple helices can reversibly form fibrillation after they are

cooled for sufficient time. However, both of them are no lon-

ger irreversible when the heating temperature is above the

value. Our experiments suggests the inter- triple helix hydro-

gen bonds for the fibrillation is easier to break than the intra-

triple helix hydrogen bonds for the denaturation.

REFERENCES1. Vanderrest, M.; Garrone, R. FASEB J 1991, 5, 2814–2823.

2. Engel, J.; Bachinger, H. P. Top Curr Chem 2005, 247, 7–33.

3. Aberle, E. D.; Mill, E. W. Proc Recip Meat Conf 1983, 36, 125–

130.

4. Lim, V. I. FEBS Lett 1981, 132, 1–5.

5. Judge, M. D.; Aberle, E. D. J. Anim Sci 1982, 54, 68–71.

6. Veis, A. The Macromolecular Chemistry of Gelatin; Academic

Press: New York, 1964.

7. Hayashi, T.; Curran-Patel, S.; Prockop, D. J. Biochemistry 1979,

18, 4182–4187.

8. Watanabe, K.; Nakagawa, J.; Ebihara, T.; Okamoto, Y. Polymer

1996, 37, 1285–1288.

9. Gayatri, R.; Sharma, A. K.; Rajaram, R.; Ramasami, T. Biochem

Biophys Res Commun 2001, 283, 229–235.

10. Burjanadze, T. V.; Bezhitadze, M. O. Biopolymers 1992, 32,

951–956.

11. Watanabe, K.; Tezuka, Y.; Ishii, T. Macromolecules 1997, 30,

7910–7913.

12. Claire, K.; Pecora, R. J Phys Chem B 1997, 101, 746–753.

13. Meyer, M.; Morgenstern, B. Biomacromolecules 2003, 4, 1727–

1732.

14. Rochdi, A.; Foucat, L.; Renou, J. P. Biopolymers 1999, 50, 690–

696.

15. Privalov, P. L.; Tiktopulo, E. I. Biopolymers 1970, 9, 127–139.

16. Wallace, D. G.; Condell, R. A.; Donovan, J. W.; Paivinen, A.;

Rhee, W. M.; Wade, S. B. Biopolymers 1986, 25, 1875–1893.

17. Finch, A.; Ledward, D. A. Biochem Biophys Acta 1972, 278,

433–439.

18. Flandin, F.; Buffevant, C.; Herbage, D. Biochem Biophys Acta

1984, 791, 205–211.

19. Brown, E. M.; Farrell, H. M.; Wildermuth, R. J. J Protein Chem

2000, 19, 85–92.

20. KomsaPenkova, R.; Koynova, R.; Kostov, G.; Tenchov, B. G. Bio-

chem Biophys Acta 1996, 1297, 171–181.

21. Miles, C. A.; Burjanadze, T. V.; Bailey, A. J. J Mol Biol 1995, 245,

437–446.

22. Miles, C. A.; Knott, L.; Sumner, I. G.; Bailey, A. J. J Mol Biol

1998, 277, 135–144.

23. Miles, C. A.; Avery, N. C.; Rodin, V. V.; Bailey, A. J. J Mol Biol

2005, 346, 551–556.

24. Leikina, E.; Mertts, M. V.; Kuznetsova, N.; Leikin, S. Proc Natl

Acad Sci USA 2002, 99, 1314–1318.

25. Zimm, B. H. J Chem Phys 1948, 16, 1099.

26. Friess, W. Eur J Pharm Biopharm 1998, 45, 113–136.

27. Chu, B. Laser Light Scattering, 2nd ed.; Academic Press: New

York, 1991.

28. Berne, B.; Pecora, R. Dynamic Light Scattering; Plenum: New

York, 1976.

29. Burchard, W. In Light Scattering Principles and Development;

Brown, W., Ed.; Clarendon Press: Oxford, 1996; p 439.

Reviewing Editor: Laurence Nafie

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Biopolymers DOI 10.1002/bip