Physical properties of type I collagen extracted from fish scales of Pagrus major and Oreochromis niloticas

International Journal of Biological Macromolecules 32 (2003) 199–204

Physical properties of type I collagen extracted from fish scales of Pagrus major and Oreochromis niloticas Toshiyuki Ikoma a,b,∗ , Hisatoshi Kobayashi a , Junzo Tanaka a,b , Dominic Walsh b,c , Stephen Mann b,c a

Biomaterials Center, National Institute for Materials Science, Namiki 1-1, Tsukuba, Ibaraki 305-0044, Japan CREST, Japan Science and Technology Corporation, Honcho 4-1-8, Kawaguchi, Saitama 332-0012, Japan c School of Chemistry, University of Bristol, Cantocks Close, Bristol BS8 1TS, UK

b

Received 22 October 2002; received in revised form 14 April 2003; accepted 28 April 2003

Abstract Type I collagens were extracted from fish scales of Pagrus major and Oreochromis niloticas as a possible underutilized resource for medical materials. The fish scales were demineralized with EDTA and digested by pepsin. The resultant type I collagens contained more than 33.6% of glycine as the most abundant amino acid. The denaturation temperatures of the collagens from P. major and O. niloticas were 303 and 308 K, respectively, both of which were relatively lower than that of porcine dermis collagen (314 K). CD spectra indicated that the denaturation temperatures were dependent on the amount of hydroxyproline, rather than proline residues. Raman spectra also indicated that the relative intensities of Raman lines at 879 and 855 cm−1 assigned to Hyp and Pro rings were changed due to the contents of the imino acids. Significantly, the content of sulphur-containing methionine was higher in the fish scales than in porcine dermis. The enthalpy and entropy estimated from thermal analyses could be correlated to amino acid sequences (Gly-Pro-Hyp) of type I collagens and the number of methionine amino acid residues. © 2003 Elsevier B.V. All rights reserved. Keywords: Fish scale; Type I collagen; Amino acid constituent; Raman spectrum

1. Introduction Type I collagen is the main component of extracellular matrix and has functions that include mechanical protection of tissues and organs or physiological regulation of the cell environment [1]. The use of type I collagen in industry for health foods, cosmetics, and biomaterials is expanding. The benefits of type I collagen for the use of biomaterials are its low antigenic and direct cell adhesion properties. At present, the main sources of type I collagen in many fields are limited to those of bovine or porcine dermis. Type I collagens have been extracted from the skin of aquatic species, e.g. jellyfish [2,3], starfish [4], octopus [5], paper nautilus [6], cuttlefish [7], purple sea urchin [8], and others [9–13]. On the other hand, extraction of type I collagen from fish scales, which are biocomposites of highly ordered type I collagen fibers and hydroxyapatite (Ca5 (PO4 )3 OH) [14–18], has been described only by ∗

Corresponding author. Tel.: +81-29-860-4387; fax: +81-29-851-8291. E-mail address: [email protected] (T. Ikoma).

0141-8130/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0141-8130(03)00054-0

Nomura et al. [19]. They extracted collagen from the fish scale of sardine and reported that the acid-solubilized collagen had a denaturation temperature of 300 K, which like the marine skin collagens was lower than the denaturation temperature for porcine dermis (314 K). In general, pepsin-solubilized collagen (PSC) degenerates at lower temperature than SC and has little antigenic property compared with SC because of the loss of N- and C-terminus domains in PSC. The times and temperatures used for pepsin digestion treatments are important for the production of lower molecular weight fragments than the intact alpha I (I) chains [20]. Raman spectroscopy is particularly useful for analyzing collagen and associated degradation products as shown by Fruchour and Koenig [20–22], who were able to assign specific functional groups associated with the amino acid backbone of collagen, and thereby identify natural collagens from other proteins. In this paper, we extract pure native type I collagens from fish scales of Pagrus major (seawater fish) and Oreochromis niloticas (freshwater fish) by a simple method and elucidate the denaturation temperatures with circular dichroism

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T. Ikoma et al. / International Journal of Biological Macromolecules 32 (2003) 199–204

(CD) spectroscopy. We use FT-Raman spectroscopy to correlate these temperatures with changes in the amino acid components.

2. Materials and methods 2.1. Extraction of type I collagen from fish scales 2.1.1. Demineralization process Fish scales were collected from P. major and O. niloticas. Initially, the fish scales were washed twice in 10 wt.% of NaCl solutions to remove unnecessary proteins on the surface for 48 h. Demineralization was achieved with 0.5 mol/l of EDTA, followed by extraction of collagen with Tris–HCl buffered at pH 7.5 for 24–48 h. After the suspension was centrifuged at 10,000×g, the residue was washed three times with distilled water. The demineralized fish scale was analyzed by thermogravimetry (TG-DTA) to confirm complete dissolution of calcium phosphate. 2.1.2. Isolation of acid- and pepsin-solubilized collagens Extraction of type I collagen from the fish scales was conducted at a relatively low temperature of 277 K to reduce chain fragmentation [20]. Acid-solubilized collagen was extracted from the thoroughly demineralized scales with 0.01 mol/l HCl solutions for 48 h. After insoluble collagen (IC) was separated with a centrifuge at 10,000 × g, acid-solubilized collagen fibers were obtained from the solution by adjusting to pH 7.0 for 24 h. The fibers were purified three times by washing with distilled water. The residue was washed again with distilled water for extracting PSC. Atelocollagen was obtained from the final residue by chemical treatment with pepsin (digestive power of 1/100: Wako Chemical Co.) for 72 h. The pepsin was added in 0.01 mol/l of HCl solution with a weight ratio of pepsin/IC = 1/6. The PSC was purified by a similar method to the soluble collagen. All the isolation treatments were conducted at 277 K. 2.2. Amino acid analysis The amino acid compositions of the collagens were analyzed with an amino acid analyzer (HITACHI L8800). Five milligrams of the lyophilized samples of type I collagen from porcine dermis (Nitta Gelatin Co.) and type I collagen extracted from the fish scales were dissolved in 6 mol/l HCl solutions and hydrolyzed at 383 K for 22 h. The solvents were removed by evaporation. Ten milliliters of 0.02 mol/l of the HCl solutions was then added to the residues followed by filtering with 0.45 ␮m membrane filters. 2.3. Circular dichroism measurement CD spectra were measured using a JASCO model 725 spectrometer. Lyophilized fish scale PSC (0.04 g) was di-

luted with 100 ml of HCl solution (pH 3; 0.001 mol/l) and the solution placed into a quartz cell with a path length of 1 mm. CD spectra measurements were performed between 278 and 353 K with temperature steps of 10 K and for wavelengths 250–190 nm at a scan speed of 50 nm/min with an interval of 0.5 nm. The data were accumulated three times. Similar experiments were undertaken with collagen from porcine dermis. In order to determine the collagen denaturation temperatures, a rotatory angle at a fixed wavelength of 221 nm, [θ]221 , was measured as a function of temperature. Measuring temperatures were raised at a rate of 1 K/min by controlling with a Peltier holder with experimental error of ±0.1 K, and the data collected every 0.2 K. The mean molar ellipticity (θ) was calculated using mean residue molecular weight and expressed in deg cm2 /dmol. The enthalpy and entropy values were calculated based on van’t Hoff plots using the software of TDA-360W. 2.4. Raman spectroscopy FT-Raman spectra were measured using a Perkin-Elmer spectrum GX with excitation at 1064 nm using an Nd:YAG laser. The backscattered light was collected at 180◦ . To avoid thermal damage to the species, the power of laser was adjusted to 100 mW and 128 scans at 4 cm−1 resolutions were accumulated. The collagens were washed with distilled water of pH 7.2 and freeze dried.

3. Results and discussion Demineralization of fish scales was completed after 24 h as shown by TG-DTA analysis. Under these conditions, the total yields of soluble and PSCs were about 2 wt.%. Table 1 shows the amino acid composition of the collagens per 1000 total residues. As expected, due to the characteristic (Gly-Pro-Hyp)n , triple helical repeat, glycine (Gly) was the most abundant amino acid being 34.6 and 33.6% of the total amino acid present for P. major and O. niloticas, respectively, compared to 34.1% for the type I collagen of porcine dermis. The number of imino acids per 1000 residues, proline (Pro) and hydroxyproline (Hyp), decreased in order from porcine dermis (220), O. niloticas (193) to P. major (180), which is likely to affect the stability of the collagen fibers and denaturation temperatures. The degrees of hydroxylation of proline for type I collagens from porcine dermis, O. niloticas and P. major were calculated to be 44.1, 43.0, 40.6% and those of lysine for were about 21%. The number of sulphur-containing methionine (Met) residues was significantly higher in the fish scale collagens (O. niloticas (12/1000); P. major (15/1000)) compared with porcine dermis (6/1000). Fig. 1 shows the CD spectra of the three species of type I collagens over the temperature range of 278–353 K. The collagens showed a rotatory maximum at 221 nm and

T. Ikoma et al. / International Journal of Biological Macromolecules 32 (2003) 199–204

Total

1000

1000

1000

Denaturation temperature (K)

313.84 (2)

302.97 (3)

308.68 (3)

H (kJ/mol) S (kJ/mol K)

−669 (8) −2.13 (3)

−522 (9) −1.72 (3)

−452 (7) −1.46 (2)

-1 2

83 47 24 36 72 110 336 126 0 20 12 11 21 3 13 7 25 5 49

4

73 43 24 41 71 107 346 133 0 19 15 7 18 3 13 7 26 7 49

(θ ) (10 deg cm dmol )

97 44 16 33 72 123 341 115 0 22 6 10 22 1 12 7 27 5 48

212nm

333K

-1 313K -1.5 -2

293K 200

210

220

230

240

250

240

250

240

250

Wavelength (nm)

(a) 0.5

221nm

0 -0.5

353K 333K

-1

298K

212nm

-1.5 -2 -2.5 190

293K

200

210

220

230

Wavelength (nm)

(b) 0.5

221nm

0 353K -0.5

212nm

333K

2 4

minimum at 191 nm and a consistent cross over point (zero rotation) at about 212 nm, which was characteristic of the triple helical conformation of the protein [23,24]. Fig. 2 shows the corresponding mean molar ellipticities, [θ]221 , as a function of temperature. The [θ]221 values decreased with temperature due to decomposition of the collagen triple helical structure, and indicated denaturation temperatures of 313.8, 308.7, and 302.9 K for porcine dermis, O. niloticas and P. major, respectively. The starting and final points of the denaturation were 309 and 319 K for porcine dermis, 301 and 316 K for O. niloticas, and 297 and 309 K for P. major. A good linear correlation was observed when the denaturation temperatures were plotted against the corresponding numbers of Hyp residues, but this was not so pronounced with respect to the proline content (Fig. 3). Changes in enthalpy (H) and entropy (S) associated with collagen denaturation were estimated from the thermal changes shown in Fig. 2 by van’t Hoff plots. Residues of fitting curves (sigma) were 0.18, 0.17, and 0.20 for O. niloticas, P. major, and porcine dermis, respectively. Values of H and S were −452 (7) kJ/mol and −1.46 (2) kJ/mol K for O. niloticas, −522 (9) kJ/mol and −1.72 (2) kJ/mol K for P. major, and −669 (8) kJ/mol and −2.13 (3) kJ/mol K, respectively, for porcine dermis. The values were not in direct accordance with the denaturation temperatures because the enthalpy and entropy changes associated with collagen denaturation processes depend on the positional preferences of ionized residues in Gly-X-Y [25].

353K -0.5

-2.5 190

-1

Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Hydroxylysine Lysine Histidine Arginine

221nm

0

2

Fish scale of O. niloticas

4

Fish scale of P. major

(θ ) (10 deg cm dmol )

Porcine dermis

-1

Resource

0.5

(θ ) (10 deg cm dmol )

Table 1 Amino acid composition (residues/1000), denaturation temperatures and thermodynamic data of H and S of pepsin-solubilized type I collagen

201

-1 308K -1.5 -2 -2.5 190

(c)

293K 200

210

220

230

Wavelength (nm)

Fig. 1. CD spectra of the dilute aqueous solutions of type I collagen from (a) porcine dermis and fish scales of (b) P. major and (c) O. niloticas.

For example, the thermal stability, enthalpy and entropy of Ac(Gly-Pro-Hyp)3 -Gly-X-Y-(Gly-Pro-Hyp)4 -Gly-Gly-NH2 where X and Y are ionizable residues Arg, Lys, Glu, and Asp have been previously studied [26]. When X is a charged residue, the enthalpy was greater and the entropy smaller for Gly-Pro-Hyp and Gly-Ala-Hyp sequences. We, therefore, suggest that the differences in the enthalpy and entropy of type I collagen from fish scales can be attributed to the specific sequences of amino acids and their influence on the stability of the triple helix. This is dependent on the formation of hydrogen bonds in the inner coil-coiled ␣ chains, and the Gly-Pro-Hyp tripeptide sequence is known to be the most stable in collagen [27]. Although the lower denaturation temperatures of the fish scales are reflected

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T. Ikoma et al. / International Journal of Biological Macromolecules 32 (2003) 199–204

3000

130 Porcine dermis Pagrus major Oreochromis niloticas

hydroxyproline residue proline residue

120

Imino acid residue / number

2

(θ) [221nm] / deg cm dmol

-1

2500 2000 1500 1000 500 0

100

Oreochromis niloticas

Pagrus major

90 80

-500 -1000

Porcine dermis

110

280

290

300

310

320

330

340

70 300

350

305

Temperature / K

310

315

320

Denaturation temperature / K

Fig. 2. Temperature effect on the CD spectra at 221 nm of type I collagen from porcine dermis and fish scales of P. major and O. niloticas.

in the less negative H values, the more negative H for P. major compared to O. niloticas may be related to the stronger localized sulphur bonding interactions associated with the higher Met content. Fig. 4 shows the Raman spectra of type I collagens in the range 1800–800 cm−1 . Corresponding peak assignments are given in Table 2 according to literature values [20,21]. Two peaks at 1670 and 1640 cm−1 were ascribed to amide I vibrations of collagen, whereas those near 1270 and 1246 cm−1 were assigned to amide III deformations. Bands correspond-

Fig. 3. Correlation curves of the denaturation temperatures against the amount of hydroxyproline or proline residues.

ing to carboxyl groups (Asp, Glu; 1421 and 1062 cm−1 ) and protonated amino residues (1162 cm−1 ) were also observed. Significant differences between the spectra were observed in the region of 1000–800 cm−1 , where amino acids such as phenylalanine (Phe), Pro and Hyp show strong Raman scattering due to aromatic or saturated side chain rings. Two bands corresponding to Phe appeared at 1033 and 1004 cm−1 , whereas two bands for Pro and one peak for Hyp residues were observed at 921 and 855 cm−1 and 880 cm−1 , respectively. The latter was clearly more prominent

1451

1641

Intensity

884 855 921

1269 1247

1670

1093

1421

1004 816

(a) 937

(b)

(c)

1800

Hyp Pro

1600

1400

1200

1000

800

-1

Wavenumber / cm

Fig. 4. Raman spectra of type I collagen in the region of 1800–800 cm−1 : (a) type I collagen from porcine dermis, (b) P. major, (c) O. niloticas. The obvious differences of the shape and intensity were appeared at the region of 1000–800 cm−1 due to differences in the content of imino acids (hydroxyproline and proline).

T. Ikoma et al. / International Journal of Biological Macromolecules 32 (2003) 199–204

203

Table 2 Raman lines and their assignments in type I collagen from porcine dermis and fish scales of P. major and O. niloticas Porcine dermis

Fish scale of P. major

Fish scale of O. niloticas

Assignment

1670 s 1640 sh 1605 m 1586 m 1451 s 1421 m, sh 1379 w 1340 w, sh 1319 m 1270 s 1247 s 1206 m, sh 1164 w 1123 w, sh 1093 m 1062 m 1034 s 1004 s 972 w, sh 958 w, sh 936 m, sh 921 s 884 s 858 s, sh 816 s

1670 s 1642 sh 1605 m 1585 m 1450 s 1421 m, sh 1387 w 1340 w, sh 1316 m 1268 s 1247 s 1208 w, sh 1163 w 1124 w, sh 1097 m 1063 m 1033 s 1003 s 971 w, sh 957 w, sh 936 m, sh 921 s 879 m 855 s 817 s

1671 s 1641 sh 1605 m 1584 m 1450 s 1418 m, sh 1393 w 1343 w, sh 1319 m 1270 s 1244 s 1209 w, sh 1162 w 1123 w, sh 1093 m 1065 m 1032 s 1003 s 975 w, sh 959 w, sh 937 m, sh 921 s 879 m 853 s 816 s

ν(C=O); amide ν(C=O); amide Phe, Tyr Pro, Hyp d(CH2 ) νs(COO− ) d(CH2 ) γ w (CH2 ) γ t (CH2 ) d(NH2 ); amide d(NH2 ); amide Hyp, Tyr NH3 +

I I

III III

ν(C–N) Bend of carboxyl OH Phe Phe ν(C–C) of residue ν(C–C) of protein backbone ν(C–C) of Pro ring ν(C–C) of Hyp ring ν(C–C) of Pro ring ν(C–O–C); ν(C–C) of backbone

ν: stretching coordinate; d: deformation coordinate; γ w : wagging coordinate; γ t : twisting coordinate; s: strong; m: medium; w: weak; sh: shoulder; vw: very weak.

in P. dermis due the relatively high content of Hyp in this collagen.

4. Conclusions Type I collagens from fish scales of P. major and O. niloticas have been examined and correlations between the amino acid compositions and denaturation temperatures elucidated. CD spectra indicate that the denaturation temperatures are reduced as the number of imino residues decreases, and that a strong linear correlation with Hyp content is apparent. Corresponding changes in enthalpy and entropy have been determined. Raman spectra also highlight the compositional differences between the fish scale and porcine dermis type I collagens.

Acknowledgements We thank Kanda technology Co. Ltd for kind supply of fish scales.

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