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Structural and physical properties of collagen extracted from moon jellyfish under neutral pH conditions a
a
b
b
c
Ayako Miki , Satomi Inaba , Takayuki Baba , Koji Kihira , Harumi Fukada & Masayuki a
Oda a
Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan b
Jellyfish Research Laboratories, Inc., Kawasaki, Japan
c
Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Japan Published online: 26 May 2015.
Click for updates To cite this article: Ayako Miki, Satomi Inaba, Takayuki Baba, Koji Kihira, Harumi Fukada & Masayuki Oda (2015): Structural and physical properties of collagen extracted from moon jellyfish under neutral pH conditions, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2015.1046367 To link to this article: http://dx.doi.org/10.1080/09168451.2015.1046367
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Bioscience, Biotechnology, and Biochemistry, 2015
Structural and physical properties of collagen extracted from moon jellyfish under neutral pH conditions Ayako Miki1,a, Satomi Inaba1,a, Takayuki Baba2, Koji Kihira2, Harumi Fukada3 and Masayuki Oda1,* 1
Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan; 2Jellyfish Research Laboratories, Inc., Kawasaki, Japan; 3Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Japan
Received April 1, 2015; accepted April 21, 2015
Downloaded by [New York University] at 10:09 30 May 2015
http://dx.doi.org/10.1080/09168451.2015.1046367
We extracted collagen from moon jellyfish under neutral pH conditions and analyzed its amino acid composition, secondary structure, and thermal stability. The content of hydroxyproline was 4.3%, which is lower than that of other collagens. Secondary structure analysis using circular dichroism (CD) showed a typical collagen helix. The thermal stability of this collagen at pH 3.0 was lower than those from fish scale and pig skin, which also correlates closely with jellyfish collagen having lower hydroxyproline content. Because the solubility of jellyfish collagen used in this study at neutral pH was quite high, it was possible to analyze its structural and physical properties under physiological conditions. Thermodynamic analysis using CD and differential scanning calorimetry showed that the thermal stability at pH 7.5 was higher than at pH 3.0, possibly due to electrostatic interactions. During the process of unfolding, fibrillation would occur only at neutral pH. Key words:
jellyfish collagen; secondary structure; thermal stability; pH dependency
Collagens are the characteristic structural molecules of the extracellular matrix and have been used in various industries such as food, cosmetics, and biomaterials. They are organized in a triple helical conformation and contain the repeating sequence (Gly-X-Y)n, with Pro often located at positions X and Y, which contributes to the triple helical conformation by restricting the dihedral angle of the main chain. Furthermore, the Pro residues are often post-translationally modified to hydroxyproline (Hyp), which leads to an increase in the thermal stability of collagen.1,2) Possibly due to the native temperature of the organism, the thermal stability of collagen from mammals is higher than that from marine animals such as fish and jellyfish.3) Interestingly, the content of Hyp in the former collagen is higher than that in the latter.4) *Corresponding author. Email: [email protected] a These authors contributed equally to the work. © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
Collagen is the major components of jellyfish. In edible jellyfish, it was reported that more than 60% of the tissue is a collagen that is rich in hydroxylysinelinked carbohydrate.5) Most mesoglea collagens are extracted by limited enzymatic digestion under acidic conditions.6) The solubility of these collagens is high at acidic pH, but low at neutral pH. In contrast, we found that the collagen from moon jellyfish could be extracted under neutral pH conditions, as described below, and its solubility at neutral pH is quite high. The feature of high solubility especially at neutral pH is considered to be a desirable feature for applications using collagen. In addition, jellyfish collagen was recently found to be useful in the field of tissue engineering and has been analyzed for applications in cell culture.7,8) These results strongly indicate that the physical property of jellyfish collagen is different from those of other collagens. In this study, we analyzed the structural and physical properties of collagen extracted from moon jellyfish under neutral pH conditions. The thermodynamic properties analyzed using circular dichroism (CD) and differential scanning calorimetry (DSC) were compared between neutral and acidic pHs, and those measurements at acidic pH were also compared to those of other collagens.
Materials and methods Sample preparation. Moon jellyfish, Aurelia sp., was caught in Tokyo bay, Japan. The water-containing crushed jellyfish tissues were centrifuged at 10,000 × g at 4 °C for 10 min to remove insoluble materials. And then, the supernatant was mixed with isopropyl alcohol to 50% (w/w). The mixture was centrifuged at 10,000 × g at 4 °C for 10 min, and the pellet was remixed with isopropyl alcohol to 40% (w/w). The resultant mixture was centrifuged at 10,000 × g at 4 °C for 10 min, and the pellet was dissolved in water. After centrifugation at 10,000 × g at 4 °C for 10 min, the supernatant was purified using the 10-kDa cutoff
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membrane. The purified fractions were lyophilized. The collagen samples from fish scale and pig skin were purchased from Taki Chemical Co., Ltd. and Nitta Gelatin Inc., respectively. CD measurements. Far-UV (200–250 nm) CD spectra were recorded on a Jasco J-820 spectropolarimeter at 20 °C equipped with Peltier-type temperature control system. The spectra were obtained for the protein concentration, 0.1 mg/mL in 20 mM potassium phosphate buffer (pH 7.5) or 10 mM Gly-HCl buffer (pH 3.0), using quartz cell with 1.0 cm path-length. CD spectra were obtained using scanning speed of 20 nm/ min, a time response of 1 s, a bandwidth of 1 nm, and an average over 4 scans. The melting curves were recorded in temperature mode at 220 nm, from 10 to 60 °C with a heating rate of 1.0 °C/min. The analysis of the transition curves obtained by temperature-scanning CD measurements was performed on the basis of two-state transition model, as described previously.9) DSC measurements. DSC experiments were carried out on a Nano DSC calorimeter (TA instruments). The data were collected by heating the solution at a rate of 1 °C/min. The sample was reheated without exchanging the solution in the cells to check the reversibility. The outer buffer solution recovered from final dialysis experiment was used in the reference cell for each case. The protein concentrations were 0.5 mg/mL in 20 mM potassium phosphate buffer (pH 7.5) or 10 mM Gly-HCl buffer (pH 3.0). The data were analyzed using CpCalc software.10)
Results and discussion One of the unique features of jellyfish collagen (collagen-J) used in this study was its high water solubility at neutral pH (>10 mg/mL). This would be closely correlated with the extraction that the collagen could be liberated from Aurelia sp. jellyfish under neutral pH conditions. SDS-PAGE analysis for purified collagen-J under reducing conditions showed the bands around 130, 80, and 50 kDa molecular mass (Fig. 1(A)). At around 130 kDa, at least, two bands were observed. Reverse phase HPLC analysis of purified collagen-J showed three main peaks eluting around 17 min (Fig. 1(B)), indicating that collagen-J contains α1, α2, and α3 chains.6) The bands around 80 and 50 kDa are likely the hydrolyzed products of these α chains. The amino acid composition of collage-J summarized in Table 1 is comparable to that of edible jellyfish exumbrella as reported previously.3) The low content of Hyp theoretically results in a low thermal stability as described below. To analyze the secondary structure and thermal stability of collagen-J at neutral and acidic pHs, we first performed CD measurements. The CD spectrum of collagen-J at pH 7.5 is similar to that at pH 3.0 (Fig. 2(A) and (B)), which is similar to that of other collagens as previously reported.11,12) The CD spectra after heating until 60 °C showed random structures,
Fig. 1. SDS-PAGE and HPLC analysis. Notes: (A) Coomassie-stained polyacrylamide gel (8%) after SDSPAGE of collagen-J (10 μg). Protein molecular-mass markers are also shown. (B) Reverse-phased HPLC pattern of collagen-J. 20 μg of collagen-J was loaded onto Inertsil W300 C8 column (4.6 mm × 15 cm, GL Sciences). The proteins were eluted with a linear gradient of acetonitrile (1–80%) in 0.1% trifluoroacetic acid from 0 to 30 min. The eluate was monitored at 215 nm.
Table 1.
Amino acid composition of collagen-J.
Amino acid
Content (%)
Hyp Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe hydroxyLys Lys His Arg
4.3 8.3 2.7 3.8 9.3 7.8 32.2 7.6 2.8 0.2 2.3 3.5 0.8 1.0 3.5 2.8 1.0 6.1
indicating that this thermal transition is irreversible. At a CD value of 220 nm, the process of thermal denaturation could be monitored (Fig. 2(C) and (D)). As reported previously,13) collagen denaturation is a nonequilibrium process, and the transition temperature (Tm) of the collagen analyzed in this study was dependent on a heating rate. The Tm values of collagen-J at either pH 7.5 or 3.0 determined at the heating rate of 0.25 °C/min were 1.3 °C lower than those determined at a heating rate of 1.0 °C/min. At a heating rate of 1.0 °C/min, the thermodynamic parameters of collagenJ were determined and compared to different pHs and
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Structural and Physical Properties of Jellyfish Collagen
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Fig. 2. CD analysis. Notes: The far-UV spectra of collagen-J at 20 °C (solid line) and 20 °C after heating (broken line) at pH 7.5 (A) and pH 3.0 (B). The thermal denaturation curves of collagen-J at pH 7.5 (C) and pH 3.0 (D) with fitting curves. In the fitting to data at pH 7.5, the CD values between 35 and 43 °C were not used, because the additional transition would be observed. The molar ellipticity (θ) was determined using the mean residue weight, 96, calculated from the amino acid composition of collagen-J (Table 1).
to other collagens. While the transition at pH 3.0 seems to fit well with the linear functions for the folded and unfolded states, the transition at pH 7.5 seems to be complex, at least, an additional transition appeared to be occurring around 40 °C. The change in CD value at 220 nm was well reproducible, and the additional transition including an irreversible transition would be accelerated at temperatures over 35 °C at neutral pH. After repetitive heating until 35 °C at pH 7.5, the CD spectra of collagen-J at 20 °C were reproducible and recovered by approximately 50% of the original spectrum (data not shown). These suggest that 50% of secondary structure could be reversible after incubation at 35 °C. It can be assumed that the triple helical conformation of collagen dissociates into individual polypeptides, which simultaneously transition into the unfolded state with increasing temperature. During this process, additional events such as intermolecular interaction and fibrillation would be expected to occur only at a neutral pH.14) These thermodynamic parameters are summarized in Table 2, together with the results of collagens from fish scale and pig skin. As reported previously,15) the contents of Hyp in collagens from fish scale and pig skin are 8.3 and 9.7%, respectively, and are higher than that in collagen-J, 4.3% (Table 1). This is in good correlation with the difference in thermal stability (Table 2). The previous structural and thermodynamic analyses using collagen model peptides have shown that the increased stability
Table 2. Thermodynamic parameters for denaturation of collagen-J analyzed using CD.
pH 7.5 Collagen-J pH 3.0 Collagen-J Collagen from fish scale Collagen from fish skinb Collagen from pig skin
Tm (°C)
ΔHvH (kJ/mol)
32.92 ± 0.02
642 ± 6
29.11 ± 0.01 36.03 ± 0.01 35.52a 32.52b 40.69 ± 0.02 40.68a
731 ± 8 1007 ± 21 452a 344 ± 8 669a
Note: The error values indicated are derived from the fitting of the transition curves. a Data were taken from Ikoma et al.15) b Data were taken from Wang et al.16) The collagen from the skin of Amur sturgeon was dissolved in 0.01 M HCl.
of collagen with a higher Hyp content is mainly due to the hydration effect.1,2) When comparing the apparent Tm values of collagen-J at the different pHs, the thermal stability at pH 7.5 is higher than that at pH 3.0, indicating an important contribution from electrostatic interactions.11) As reported previously,1) the Asp (pK ≈ 3.9) and Glu (pK ≈ 4.3) residues can form ion pairs with the basic residues at neutral pH, resulting in increased stability, which might partially compensate for the decreased stability resulting from a lower Hyp content (Table 1).
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Fig. 3. DSC analysis. Note: The heat capacity curves of collagen-J at pH 7.5 (A) and pH 3.0 (B). The baseline to calculate ΔHcal using CpCalc software is also indicated.
The thermal stabilities of collagen-J at pH 7.5 and 3.0 were also analyzed using DSC (Fig. 3). No peak was observed in the second scan during reheating, supporting the notion of irreversible unfolding. The peak temperatures of DSC curves at pH 7.5 and 3.0 were determined to be 32.8 and 28.2 °C, respectively, which are comparable to the Tm values determined using CD (Table 2). Using different concentrations of collagen-J, namely 0.2, 0.4, and 2 mg/mL, similar results were obtained (data not shown). It should be noted that the peak area corresponding to enthalpy change at pH 7.5 was smaller than that at pH 3.0 (Fig. 3), which is also supported by calculations of ΔHvH determined using CD (Table 2). The results of smaller enthalpy change at pH 7.5 indicate that the increased thermal stability of collagen-J at neutral pH is due to an entropic contribution. Under neutral pH conditions, an additional DSC peak was observed at not only around 40 °C but also around 50 °C. The former would correspond to the additional transition observed in the CD experiments (Fig. 2(C)), and this might be due to additional events like fibrillation as discussed above. The latter was only observed in the DSC experiments, and this might be due to the transition from fibrillation to unfolded monomers, as reported previously.14) In contrast, at pH 3.0, only one DSC peak was observed, indicating that each polypeptide of collagen exists as monomer without forming fibrils after unfolding. In conclusion, we extracted jellyfish collagen, collagen-J, under neutral pH conditions, and found that the low content of Hyp in this collagen results in a low thermal stability. The high solubility of collagen-J at neutral pH makes it possible to analyze its structural and physical properties under physiological conditions. Its thermal stability at pH 7.5 is higher than that at pH 3.0, the latter being lower than for collagens from fish scale and pig skin. These results highlight the potential of collagen-J in future applications such as tissue engineering.
Author contributions KK and MO designed the research, AM and TB prepared samples, AM and SI carried out CD, SI and HF carried out DSC, TB and MO wrote the paper.
Acknowledgements We thank Mr Shigenori Nishimura of Osaka Prefecture University for CD measurements. We also thank Dr Kazuki Kawahara of Osaka University for helpful discussion.
Disclosure statement No potential conflict of interest was reported by the authors.
References [1] Venugopal MG, Ramshaw JA, Braswell E, Zhu D, Brodsky B. Electrostatic interactions in collagen-like triple-helical peptides. Biochemistry. 1994;33:7948–7956. [2] Kawahara K, Nishi Y, Nakamura S, Uchiyama S, Nishiuchi Y, Nakazawa T, Ohkubo T, Kobayashi Y. Effect of hydration on the stability of the collagen-like triple-helical structure of [4(R)-hydroxyprolyl-4(R)-hydroxyprolylglycine]10. Biochemistry. 2005;44:15812–15822. [3] Nagai T, Ogawa T, Nakamura T, Ito T, Nakagawa H, Fujiki K, Nakao M, Yano T. Collagen of edible jellyfish exumbrella. J. Sci. Food Agric. 1999;79:855–858. [4] Burjanadze TV. Hydroxyproline content and location in relation to collagen thermal stability. Biopolymers. 1979;18:931–938. [5] Kimura S, Miura S, Park Y-H. Collagen as the major edible component of jellyfish (Stomolophus nomural). J. Food. Sci. 1983;48:1758–1760. [6] Miura S, Kimura S. Jellyfish mesogloea collagen. Characterization of molecules as α1α2α3 heterotrimers. J. Biol. Chem. 1985;260:15352–15356. [7] Jeong SI, Kim SY, Cho SK, Chong MS, Kim KS, Kim H, Lee SB, Lee YM. Tissue-engineered vascular grafts composed of marine collagen and PLGA fibers using pulsatile perfusion bioreactors. Biomaterials. 2007;28:1115–1122. [8] Sewing J, Klinger M, Notbohm H. Jellyfish collagen matrices conserve the chondrogenic phenotype in two- and three-dimensional collagen matrices. Tissue Eng. Regen. Med. Forthcoming. [9] Morii H, Uedaira H, Ogata K, Ishii S, Sarai A. Shape and energetics of a cavity in c-Myb probed by natural and non-natural amino acid mutations. J. Mol. Biol. 1999;292:909–920. [10] Inaba S, Fukada H, Ikegami T, Oda M. Thermodynamic effects of multiple protein conformations on stability and DNA binding. Arch. Biochem. Biophys. 2013;537:225–232. [11] Mohs A, Silva T, Yoshida T, Amin R, Lukomski S, Inouye M, Brodsky B. Mechanism of stabilization of a bacterial collagen triple helix in the absence of hydroxyproline. J. Biol. Chem. 2007;282:29757–29765.
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[12] Addad S, Exposito JY, Faye C, Ricard-Blum S, Lethias C. Isolation, characterization and biological evaluation of jellyfish collagen for use in biomedical applications. Mar. Drugs. 2011;9:967–983. [13] Leikina E, Mertts MV, Kuznetsova N, Leikin S. Type I collagen is thermally unstable at body temperature. Proc. Natl. Acad. Sci. USA. 2002;99:1314–1318. [14] Kar K, Amin P, Bryan MA, Persikov AV, Mohs A, Wang YH, Brodsky B. Self-association of collagen triple helic peptides into higher order structures. J. Biol. Chem. 2006;281:33283–33290.
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[15] Ikoma T, Kobayashi H, Tanaka J, Walsh D, Mann S. Physical properties of type I collagen extracted from fish scales of Pagrus major and Oreochromis niloticas. Int. J. Biol. Macromol. 2003;32:199–204. [16] Wang L, Liang Q, Wang Z, Xu J, Liu Y, Ma H. Preparation and characterisation of type I and V collagens from the skin of Amur sturgeon (Acipenser schrenckii). Food Chem. 2014; 148:410–414.
Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Structural and physical properties of collagen extracted from moon jellyfish under neutral pH conditions a
a
b
b
c
Ayako Miki , Satomi Inaba , Takayuki Baba , Koji Kihira , Harumi Fukada & Masayuki a
Oda a
Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan b
Jellyfish Research Laboratories, Inc., Kawasaki, Japan
c
Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Japan Published online: 26 May 2015.
Click for updates To cite this article: Ayako Miki, Satomi Inaba, Takayuki Baba, Koji Kihira, Harumi Fukada & Masayuki Oda (2015): Structural and physical properties of collagen extracted from moon jellyfish under neutral pH conditions, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2015.1046367 To link to this article: http://dx.doi.org/10.1080/09168451.2015.1046367
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Bioscience, Biotechnology, and Biochemistry, 2015
Structural and physical properties of collagen extracted from moon jellyfish under neutral pH conditions Ayako Miki1,a, Satomi Inaba1,a, Takayuki Baba2, Koji Kihira2, Harumi Fukada3 and Masayuki Oda1,* 1
Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan; 2Jellyfish Research Laboratories, Inc., Kawasaki, Japan; 3Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Japan
Received April 1, 2015; accepted April 21, 2015
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http://dx.doi.org/10.1080/09168451.2015.1046367
We extracted collagen from moon jellyfish under neutral pH conditions and analyzed its amino acid composition, secondary structure, and thermal stability. The content of hydroxyproline was 4.3%, which is lower than that of other collagens. Secondary structure analysis using circular dichroism (CD) showed a typical collagen helix. The thermal stability of this collagen at pH 3.0 was lower than those from fish scale and pig skin, which also correlates closely with jellyfish collagen having lower hydroxyproline content. Because the solubility of jellyfish collagen used in this study at neutral pH was quite high, it was possible to analyze its structural and physical properties under physiological conditions. Thermodynamic analysis using CD and differential scanning calorimetry showed that the thermal stability at pH 7.5 was higher than at pH 3.0, possibly due to electrostatic interactions. During the process of unfolding, fibrillation would occur only at neutral pH. Key words:
jellyfish collagen; secondary structure; thermal stability; pH dependency
Collagens are the characteristic structural molecules of the extracellular matrix and have been used in various industries such as food, cosmetics, and biomaterials. They are organized in a triple helical conformation and contain the repeating sequence (Gly-X-Y)n, with Pro often located at positions X and Y, which contributes to the triple helical conformation by restricting the dihedral angle of the main chain. Furthermore, the Pro residues are often post-translationally modified to hydroxyproline (Hyp), which leads to an increase in the thermal stability of collagen.1,2) Possibly due to the native temperature of the organism, the thermal stability of collagen from mammals is higher than that from marine animals such as fish and jellyfish.3) Interestingly, the content of Hyp in the former collagen is higher than that in the latter.4) *Corresponding author. Email: [email protected] a These authors contributed equally to the work. © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
Collagen is the major components of jellyfish. In edible jellyfish, it was reported that more than 60% of the tissue is a collagen that is rich in hydroxylysinelinked carbohydrate.5) Most mesoglea collagens are extracted by limited enzymatic digestion under acidic conditions.6) The solubility of these collagens is high at acidic pH, but low at neutral pH. In contrast, we found that the collagen from moon jellyfish could be extracted under neutral pH conditions, as described below, and its solubility at neutral pH is quite high. The feature of high solubility especially at neutral pH is considered to be a desirable feature for applications using collagen. In addition, jellyfish collagen was recently found to be useful in the field of tissue engineering and has been analyzed for applications in cell culture.7,8) These results strongly indicate that the physical property of jellyfish collagen is different from those of other collagens. In this study, we analyzed the structural and physical properties of collagen extracted from moon jellyfish under neutral pH conditions. The thermodynamic properties analyzed using circular dichroism (CD) and differential scanning calorimetry (DSC) were compared between neutral and acidic pHs, and those measurements at acidic pH were also compared to those of other collagens.
Materials and methods Sample preparation. Moon jellyfish, Aurelia sp., was caught in Tokyo bay, Japan. The water-containing crushed jellyfish tissues were centrifuged at 10,000 × g at 4 °C for 10 min to remove insoluble materials. And then, the supernatant was mixed with isopropyl alcohol to 50% (w/w). The mixture was centrifuged at 10,000 × g at 4 °C for 10 min, and the pellet was remixed with isopropyl alcohol to 40% (w/w). The resultant mixture was centrifuged at 10,000 × g at 4 °C for 10 min, and the pellet was dissolved in water. After centrifugation at 10,000 × g at 4 °C for 10 min, the supernatant was purified using the 10-kDa cutoff
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membrane. The purified fractions were lyophilized. The collagen samples from fish scale and pig skin were purchased from Taki Chemical Co., Ltd. and Nitta Gelatin Inc., respectively. CD measurements. Far-UV (200–250 nm) CD spectra were recorded on a Jasco J-820 spectropolarimeter at 20 °C equipped with Peltier-type temperature control system. The spectra were obtained for the protein concentration, 0.1 mg/mL in 20 mM potassium phosphate buffer (pH 7.5) or 10 mM Gly-HCl buffer (pH 3.0), using quartz cell with 1.0 cm path-length. CD spectra were obtained using scanning speed of 20 nm/ min, a time response of 1 s, a bandwidth of 1 nm, and an average over 4 scans. The melting curves were recorded in temperature mode at 220 nm, from 10 to 60 °C with a heating rate of 1.0 °C/min. The analysis of the transition curves obtained by temperature-scanning CD measurements was performed on the basis of two-state transition model, as described previously.9) DSC measurements. DSC experiments were carried out on a Nano DSC calorimeter (TA instruments). The data were collected by heating the solution at a rate of 1 °C/min. The sample was reheated without exchanging the solution in the cells to check the reversibility. The outer buffer solution recovered from final dialysis experiment was used in the reference cell for each case. The protein concentrations were 0.5 mg/mL in 20 mM potassium phosphate buffer (pH 7.5) or 10 mM Gly-HCl buffer (pH 3.0). The data were analyzed using CpCalc software.10)
Results and discussion One of the unique features of jellyfish collagen (collagen-J) used in this study was its high water solubility at neutral pH (>10 mg/mL). This would be closely correlated with the extraction that the collagen could be liberated from Aurelia sp. jellyfish under neutral pH conditions. SDS-PAGE analysis for purified collagen-J under reducing conditions showed the bands around 130, 80, and 50 kDa molecular mass (Fig. 1(A)). At around 130 kDa, at least, two bands were observed. Reverse phase HPLC analysis of purified collagen-J showed three main peaks eluting around 17 min (Fig. 1(B)), indicating that collagen-J contains α1, α2, and α3 chains.6) The bands around 80 and 50 kDa are likely the hydrolyzed products of these α chains. The amino acid composition of collage-J summarized in Table 1 is comparable to that of edible jellyfish exumbrella as reported previously.3) The low content of Hyp theoretically results in a low thermal stability as described below. To analyze the secondary structure and thermal stability of collagen-J at neutral and acidic pHs, we first performed CD measurements. The CD spectrum of collagen-J at pH 7.5 is similar to that at pH 3.0 (Fig. 2(A) and (B)), which is similar to that of other collagens as previously reported.11,12) The CD spectra after heating until 60 °C showed random structures,
Fig. 1. SDS-PAGE and HPLC analysis. Notes: (A) Coomassie-stained polyacrylamide gel (8%) after SDSPAGE of collagen-J (10 μg). Protein molecular-mass markers are also shown. (B) Reverse-phased HPLC pattern of collagen-J. 20 μg of collagen-J was loaded onto Inertsil W300 C8 column (4.6 mm × 15 cm, GL Sciences). The proteins were eluted with a linear gradient of acetonitrile (1–80%) in 0.1% trifluoroacetic acid from 0 to 30 min. The eluate was monitored at 215 nm.
Table 1.
Amino acid composition of collagen-J.
Amino acid
Content (%)
Hyp Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe hydroxyLys Lys His Arg
4.3 8.3 2.7 3.8 9.3 7.8 32.2 7.6 2.8 0.2 2.3 3.5 0.8 1.0 3.5 2.8 1.0 6.1
indicating that this thermal transition is irreversible. At a CD value of 220 nm, the process of thermal denaturation could be monitored (Fig. 2(C) and (D)). As reported previously,13) collagen denaturation is a nonequilibrium process, and the transition temperature (Tm) of the collagen analyzed in this study was dependent on a heating rate. The Tm values of collagen-J at either pH 7.5 or 3.0 determined at the heating rate of 0.25 °C/min were 1.3 °C lower than those determined at a heating rate of 1.0 °C/min. At a heating rate of 1.0 °C/min, the thermodynamic parameters of collagenJ were determined and compared to different pHs and
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Structural and Physical Properties of Jellyfish Collagen
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Fig. 2. CD analysis. Notes: The far-UV spectra of collagen-J at 20 °C (solid line) and 20 °C after heating (broken line) at pH 7.5 (A) and pH 3.0 (B). The thermal denaturation curves of collagen-J at pH 7.5 (C) and pH 3.0 (D) with fitting curves. In the fitting to data at pH 7.5, the CD values between 35 and 43 °C were not used, because the additional transition would be observed. The molar ellipticity (θ) was determined using the mean residue weight, 96, calculated from the amino acid composition of collagen-J (Table 1).
to other collagens. While the transition at pH 3.0 seems to fit well with the linear functions for the folded and unfolded states, the transition at pH 7.5 seems to be complex, at least, an additional transition appeared to be occurring around 40 °C. The change in CD value at 220 nm was well reproducible, and the additional transition including an irreversible transition would be accelerated at temperatures over 35 °C at neutral pH. After repetitive heating until 35 °C at pH 7.5, the CD spectra of collagen-J at 20 °C were reproducible and recovered by approximately 50% of the original spectrum (data not shown). These suggest that 50% of secondary structure could be reversible after incubation at 35 °C. It can be assumed that the triple helical conformation of collagen dissociates into individual polypeptides, which simultaneously transition into the unfolded state with increasing temperature. During this process, additional events such as intermolecular interaction and fibrillation would be expected to occur only at a neutral pH.14) These thermodynamic parameters are summarized in Table 2, together with the results of collagens from fish scale and pig skin. As reported previously,15) the contents of Hyp in collagens from fish scale and pig skin are 8.3 and 9.7%, respectively, and are higher than that in collagen-J, 4.3% (Table 1). This is in good correlation with the difference in thermal stability (Table 2). The previous structural and thermodynamic analyses using collagen model peptides have shown that the increased stability
Table 2. Thermodynamic parameters for denaturation of collagen-J analyzed using CD.
pH 7.5 Collagen-J pH 3.0 Collagen-J Collagen from fish scale Collagen from fish skinb Collagen from pig skin
Tm (°C)
ΔHvH (kJ/mol)
32.92 ± 0.02
642 ± 6
29.11 ± 0.01 36.03 ± 0.01 35.52a 32.52b 40.69 ± 0.02 40.68a
731 ± 8 1007 ± 21 452a 344 ± 8 669a
Note: The error values indicated are derived from the fitting of the transition curves. a Data were taken from Ikoma et al.15) b Data were taken from Wang et al.16) The collagen from the skin of Amur sturgeon was dissolved in 0.01 M HCl.
of collagen with a higher Hyp content is mainly due to the hydration effect.1,2) When comparing the apparent Tm values of collagen-J at the different pHs, the thermal stability at pH 7.5 is higher than that at pH 3.0, indicating an important contribution from electrostatic interactions.11) As reported previously,1) the Asp (pK ≈ 3.9) and Glu (pK ≈ 4.3) residues can form ion pairs with the basic residues at neutral pH, resulting in increased stability, which might partially compensate for the decreased stability resulting from a lower Hyp content (Table 1).
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Fig. 3. DSC analysis. Note: The heat capacity curves of collagen-J at pH 7.5 (A) and pH 3.0 (B). The baseline to calculate ΔHcal using CpCalc software is also indicated.
The thermal stabilities of collagen-J at pH 7.5 and 3.0 were also analyzed using DSC (Fig. 3). No peak was observed in the second scan during reheating, supporting the notion of irreversible unfolding. The peak temperatures of DSC curves at pH 7.5 and 3.0 were determined to be 32.8 and 28.2 °C, respectively, which are comparable to the Tm values determined using CD (Table 2). Using different concentrations of collagen-J, namely 0.2, 0.4, and 2 mg/mL, similar results were obtained (data not shown). It should be noted that the peak area corresponding to enthalpy change at pH 7.5 was smaller than that at pH 3.0 (Fig. 3), which is also supported by calculations of ΔHvH determined using CD (Table 2). The results of smaller enthalpy change at pH 7.5 indicate that the increased thermal stability of collagen-J at neutral pH is due to an entropic contribution. Under neutral pH conditions, an additional DSC peak was observed at not only around 40 °C but also around 50 °C. The former would correspond to the additional transition observed in the CD experiments (Fig. 2(C)), and this might be due to additional events like fibrillation as discussed above. The latter was only observed in the DSC experiments, and this might be due to the transition from fibrillation to unfolded monomers, as reported previously.14) In contrast, at pH 3.0, only one DSC peak was observed, indicating that each polypeptide of collagen exists as monomer without forming fibrils after unfolding. In conclusion, we extracted jellyfish collagen, collagen-J, under neutral pH conditions, and found that the low content of Hyp in this collagen results in a low thermal stability. The high solubility of collagen-J at neutral pH makes it possible to analyze its structural and physical properties under physiological conditions. Its thermal stability at pH 7.5 is higher than that at pH 3.0, the latter being lower than for collagens from fish scale and pig skin. These results highlight the potential of collagen-J in future applications such as tissue engineering.
Author contributions KK and MO designed the research, AM and TB prepared samples, AM and SI carried out CD, SI and HF carried out DSC, TB and MO wrote the paper.
Acknowledgements We thank Mr Shigenori Nishimura of Osaka Prefecture University for CD measurements. We also thank Dr Kazuki Kawahara of Osaka University for helpful discussion.
Disclosure statement No potential conflict of interest was reported by the authors.
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