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Contents lists available at ScienceDirect

Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Isolation and characterization of thermostable collagen from the

marine eel-fish (Evenchelys macrura)
Anguchamy Veeruraj ∗ , Muthuvel Arumugam, Thangavel Balasubramanian
Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai 608 502, India

a r t i c l e i n f o a b s t r a c t

Article history: Aim and methods: Collagen is the most abundant protein found in animal body, which is widely used for
Received 28 February 2013 biomedical and pharmaceutical applications. In the present study, acid soluble collagen (ASC) and pepsin
Received in revised form 15 July 2013 soluble collagen (PSC) from the skin wastes of marine eel fish (Evenchelys macrura) were isolated and
Accepted 16 July 2013
characterized.
Results: ASC and PSC extracted from eel fish skin showed the yields of 80 and 7.10 percent (based on dry
Keywords:
weight), respectively. ASC and PSC comprising different ␣-chains (␣1, ␣2 and ␣3) were characterized
Collagen
as type I and exhibited high solubility in acidic pH (1–4) and were soluble in the presence of NaCl at
Eel fish
Acid soluble collagen
concentration up to 3.0 and 4.0 percent (w/v) for ASC and PSC, respectively. Amino acids analysis of both
Pepsin soluble collagen ASC and PSC contained imino acid of 190 and 200 residues per 1000 residues, respectively. The present
UV absorption spectrum results of ASC and PSC from eel fish skin exhibited higher thermal stability of 39 ◦ C and 35 ◦ C, respectively.
Denaturation temperature Similar, Fourier transform infrared (FTIR) spectra of ASC and PSC were observed and suggesting that
pepsin hydrolysis did not affect the secondary structure of collagen, especially triple-helical structure.
Conclusion: These results suggest that the marine eel fish skin collagen close to the Td (denaturation
temperature) of mammalian collagen which could be used in the biomedical materials, food and phar-
maceutical industries as an alternative source.
© 2013 Elsevier Ltd. All rights reserved.

1. Introduction Collagen is a unique in its ability to form insoluble fibers that

have a high tensile strength and right-handed triple superhelical
A greater amount of food has been dumped as commercial and rod consisting of three polypeptide chains and is found in connec-
domestic waste. Although there is an attempt to decrease the waste tive tissues, including tendons, bones and skins (e.g. type I collagen)
in the world, the quantity of the waste produced is increasing [3]. Collagen has been, traditionally, isolated from the skins of
annually [1]. Recently, there has been much interest in investigat- land-based animals, such as cow and pig. Non-denatured collagens
ing possible means of making more effective use of under-utilized from these sources find applications in food, cosmetics, biomedi-
resources and industrial wastes [1]. Marine captured fisheries con- cal, and pharmaceutical industries. Denatured collagen, known as
tribute 50 percent of total world fish production and more than 70 gelatin, finds applications in the food and biomedical industries.
percent were utilized by processing industries. The annual discard- Biomedical and pharmaceutical applications of collagen include the
ing rate of world fisheries were estimated at 25 percent of the total treatment of hypertension, urinary incontinence and pain associ-
marine captured fisheries [2]. ated with osteoarthritis, use in tissue engineering for implants in
humans, inhibition of angiogenic diseases, such as diabetes com-
plications, obesity, and arthritis. In recent years, the outbreak of
bovine spongiform encephalopathy (BSE) transmissible spongi-
form encephalopathy (TSE), and the foot-and-mouth disease (FMD)
Abbreviations: ASC, acid-soluble collagen; PSC, pepsin-solubilized collagen; crisis, the uses of collagen and collagen derived products of land
NaCl, sodium chloride; NaOH, sodium hydroxide; Tris–HCl, tris hydrochloride;
animal origin have become of more concern. In addition, the col-
BSA, bovine serum albumin; ␤ME, ␤-mercaptoethanol; SDS-PAGE, sodium-dodecyl-
sulfate-polyacrylamide gel electrophoresis; DEAE, diethylaminoethyl cellulose-52; lagens extracted from bovine sources are prohibited for Sikhs and
HPLC, high performance liquid chromatography (or high pressure liquid chromatog- Hindus, whilst porcine collagen cannot be consumed by Muslims
raphy); KBr, potassium bromide; kDa, kiloDalton; Td , denaturation temperatures; and Jews, both of whom require bovine to be religiously prepared
FTIR, Fourier transform infrared. [4,5].
∗ Corresponding author at: Centre of Advanced Study in Marine Biology, Faculty
As a consequence, the alternative sources of collagen, especially
of Marine Sciences, Annamalai University, Parangipettai 608502, Tamil Nadu, India.
Tel.: +91 04144 243223; fax: +91 04144 243641. from aquatic animals including freshwater and marine fish and
E-mail address: [email protected] (A. Veeruraj). mollusks have received increasing attention. Furthermore the use

1359-5113/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.procbio.2013.07.011

Please cite this article in press as: Veeruraj A, et al. Isolation and characterization of thermostable collagen from the marine eel-fish (Evenchelys
macrura). Process Biochem (2013), http://dx.doi.org/10.1016/j.procbio.2013.07.011
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of fish by-products as a source of collagen can beneficially impact 2.4.3. Extraction of pepsin soluble collagen (PSC)
waste management. Collagen molecules in solution denature close The residue was thoroughly rinsed with distilled water, suspended in 0.5 M
acetic acid and subjected to limited hydrolysis with 10 percent (weights per volume)
to the upper limit of the physiological temperature or the maximum
pepsin (HiMedia, Mumbai, India) at 4 ◦ C for 48 h. The viscous solution obtained was
body temperature of the animal species from which the collagen is centrifuged at 20,000 × g for 1 h at 4 ◦ C and the supernatant was dialyzed against
extracted [6]. Many researchers have focused on the practical uti- 0.02 M sodium phosphate buffer (pH 7.2) for 3 days. Additionally, the sample was
lization of marine animals to produce collagen [7]. Some concerned salted out by adding NaCl to a final concentration of 0.8 M followed by addition of
collagens from freshwater fish, such as carp [8,9] and grass carp NaCl to a final concentration of 2.3 M at neutral pH (0.05 M Tris–HCl, pH 7.5) for
precipitation of collagen. The resultant precipitate was collected and re-dissolved
[10]. However, relative lower denaturation temperatures, i.e., lower in 0.5 M acetic acid. Finally, the resultant solution was dialyzed against 0.1 M acetic
thermostability, have become one of the main limiting factors for acid and distilled water then freeze-dried and referred to PSC.
the application of fish collagens, especially for those from marine
fish. At present, the explanation for the reduced thermostability of 2.5. Quantification of ASC and PSC
collagens from fish is limited to the imino acid (hydroxyproline and
The protein content of isolated collagen was determined by the method of Lowry
proline) content of the samples. The denaturation temperatures of et al. [13] using bovine serum albumin as a standard.
collagens increase with their imino acid content. Hydroxyproline
may stabilize the triple helix by hydrogen-bonded water-bridges, 2.6. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
as originally proposed by Ramachandran et al. [11].
Sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was
The marine eel fish contains thick size of the skin, which is
performed by following the Laemmli [14] method using 12 percent acrylamide
treated as waste of the home, fish shops, fish processing and preser- gel in the absence of ␤-mercaptoethanol (␤ME). After electrophoresis, the gel was
vations industries. In view of utilizing this wastes, the present study stained with methanol: acetic acid: Coomassie brilliant blue R250 (80:20:0.3). The
aimed to extract acid soluble collagen (ASC) and pepsin soluble gels were destained by using de-staining solution containing the methanol, acetic
collagen (PSC) from the skin waste of marine eel fish (Evenchelys acid and water (40:10:50). After destaining, the gels were plotted for molecular
weight determination.
macrura). In addition, the isolated collagen was characterized for
its content and thermal stability. 2.7. Peptide mapping

The peptide mapping was determined through SDS-PAGE analysis performed by

2. Materials and methods
the method of Laemmli [14] using 15 percent gel. The lyophilized collagen samples
(0.5 mg) were dissolved in 1 ml of 0.1 M sodium phosphate buffer (pH 7.2) containing
2.1. Materials and reagents
0.5 percent (weights per volume) SDS and heated at 100 ◦ C for 5 min. After cooling
the samples, the digestion was carried out at 37 ◦ C for 30 min by adding 5 ␮g of
Acetic acid, sodium chloride (NaCl), Bovine serum albumin (BSA), trichloroacetic
achromopeptidase from Achromobacter lyticus (EC 3.4 21.50, Sigma–Aldrich, Mum-
acid, pepsin, sodium dodecyl sulfate (SDS), acrylamide, ammonium persulfate,
bai, India) and the proteolysis was stopped by increasing temperature to 100 ◦ C for
N,N,N ,N -tetramethyl ethylene diamine (TEMED) and Coomassie Brilliant Blue R-
5 min. The peptide mapping was ascertained by comparing with standard type IV
250 were purchased from Himedia Chemical Co. (India). The standard type IV
collagen and protein marker.
collagen from human placenta (Sigma–Aldrich) and Achromopeptidase from Achro-
mobacter lyticus were purchased from Sigma–Aldrich (EC 3.4 21.50, Mumbai, India).
The standard molecular weight protein markers were purchased from GeNei (Ban- 2.8. Collagen solubility test
galore, India). All Other chemicals and reagents used were of analytical grade.
The optimum solubility test was carried out by dissolving in 0.5 M acetic acid
at various pH and NaCl concentrations. Collagen samples were dissolved in 0.5 M
2.2. Collection of sample acetic acid with gentle stirring at 4 ◦ C for 12 h to obtain the final concentration of 3
and 6 mg/ml.
The outer skin wastes of eel fish were freshly collected from Parangipettai fish
landing centre (Lat.11◦ 29 N long.79◦ 46 E), Tamil Nadu, south east coast of India 2.8.1. Effect of pH
and were brought to the laboratory (4 ◦ C) and immediately washed with distilled 6 ml of collagen solution was transferred to the centrifuge tube and pH was
water and stored at −20 ◦ C until further used. adjusted with 6 N HCl to obtain final pH range between 1 and 10. The sample solution
was making up to 10 ml with distilled water and adjusted the same pH. The solution
was stirred gently for 30 min at 4 ◦ C and centrifuged at 10,000 × g for 30 min at 4 ◦ C.
2.3. Proximate composition The relative solubility of collagen was calculated in comparison with pH rendering
the highest solubility.
The portions of the skin were removed from marine eel fish E. macrura, and after
blending, the proximate composition was determined. The amount of moisture, 2.8.2. Effect of NaCl
fat, ash and protein content of skin were determined according to the AOAC [12] 5 ml of collagen solution in 0.5 M acetic acid was mixed with 5 ml of cold NaCl
methods. in acetic acid of various concentrations (0–12 percent, w/v) to obtain final concen-
trations of 1–6 percent (weights per volume). The mixture was stirred gently at 4 ◦ C
for 30 min and centrifuged at 10,000 × g for 30 min at 4 ◦ C. The relative solubility
2.4. Isolation of collagen
was calculated in comparison with that the salt concentration exhibiting highest
2.4.1. Pretreatment of outer skin waste solubility.
The collagen was extracted from the outer skin waste according to the method
of Nagai and Suzuki [1] with suitable modification. All the experiments were per- 2.9. Subunit composition
formed at 4 ◦ C unless otherwise indicated. Briefly, the outer skin was removed,
cut into small pieces and defatted with 10 percent n-butyl alcohol (1:8) for 48 h, The separation of subunit composition was done by ion exchange column chro-
and washed with distilled water. Further, the skin was treated with 0.1 M NaOH matography using DEAE (Diethyl amino ethyl) cellulose 52 (Sigma, India). The
to remove non-collagenous proteins for 3 days, washed with distilled water and collagen sample was dissolved in 5 ml of 0.02 M sodium acetate buffer (pH 4.8)
lyophilized. containing 6 M urea at 4 ◦ C and denatured at 45 ◦ C for 30 min. The denatured colla-
gen was applied to DEAE cellulose 52 columns (1.5 cm × 8.0 cm) equilibrated with
same buffer. Each subunit was eluted with a linear gradient of NaCl (0–0.2 M) at the
2.4.2. Extraction of acid soluble collagen (ASC) flow rate of 0.4 ml/min. The collected fractions were examined for the presence of
The lyophilized skin was soaked into 0.5 M acetic acid (1:3 weights per vol- protein at 280 nm followed by Lowry et al. [13], pooled together and dialyzed.
ume) for 3 days and the extracts were centrifuged at 20,000 × g for 1 h at 4 ◦ C. The
supernatant was collected and the residue was re-extracted by same procedure. The 2.10. UV absorption spectrum
supernatant was mixed and desalted out by adding NaCl to a final concentration of
0.8 M and followed by precipitation of collagen by the addition of NaCl (final con- UV absorption spectrum of ASC and PSC was measured using a Shimadzu-UV-
centration of 2.3 M) at a neutral pH (0.05 M Tris–HCl, pH 7.5). The precipitates were Spectrophotometer. The ASC and PSC (1 mg) were dissolved in 100 ml 0.02 M sodium
collected and re-dissolved in 0.5 M acetic acid and dialyzed against 0.1 M acetic acid acetate buffer (pH 4.8) containing 2 M urea. The UV spectrum was measured at
and distilled water for 2 days until the neutral pH was obtained. The dialyzed sample wavelength between 190 and 400 nm at scan speed of 2 nm/s with an interval of
was lyophilized and referred to ASC. 1 nm.

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2.11. Determination of denaturation temperature (Td ) In the present study, the protein content in eel fish skin was
comparatively higher than the Nile perch skin and lower than the
The denaturation temperature (Td ) was measured from changes in viscosity,
brown backed toadfish skin. Similarly, the moisture content was
using an Ostwald’s type viscometer [10] with suitable modifications. Briefly, 20 ml
of 0.03 percent collagen solution in 0.1 M acetic acid with 0.2 M sodium acetate greater than the skin of balloon fish (62.23 percent) [18], shark skin
buffer (pH 5.0) was used for viscosity measurements. Thermal determination curve (61.96 percent) [16], and scale of spotted golden goatfish (Paru-
was obtained by measuring the viscosity of the same solution at various temper- peneus heptacanthus) (24.79 percent) [19]. Likewise, fat content in
atures (20–55 ◦ C) at 15 min interval. The fractional viscosity at given temperature the E. macrura fish skin was comparatively lower than the Nile
was determined as the temperature that the change in viscosity was half completed.
perch skin (5.5 percent) [17] and brown backed toad fish skin (0.05
percent) [15], but higher than the shark fish skin (0.4 percent) [16].
2.12. Circular dichroism measurement
Conversely, the ash content of E. macrura fish skin was lower than
Extracted collagen (5 mg) was diluted with 5 ml 0.1 M glycolic acid and the the deep-sea redfish (39.4 percent) [20], and balloon fish (15.87
solution placed in a quartz cell with a path length of 0.1 or 1 cm. CD spectra mea- percent) [18], but little higher than that of brown backed toad-
surements were performed by various temperatures at 25 ◦ C, 26 ◦ C, 27 ◦ C, 28 ◦ C, fish (8.4 percent) [15], because of the eel fish skin covered without
29 ◦ C, 30 ◦ C, 32 ◦ C, 34 ◦ C, 36 ◦ C, 38 ◦ C, 40 ◦ C, 42 ◦ C, 44 ◦ C and 45 ◦ C for wavelengths of
190–280 nm at a scan speed of 2 cm/min. The mean molar ellipticity (Â) was calcu-
scales and small spine. After demineralization, ash content was
lated using the mean residue molecular weight and expressed in deg × cm2 /dmol. about 8.80 percent, in which about 92 percent of inorganic matters
The data were cumulated three times. In order to determine collagen denatur- were removed. Almost complete demineralization might cause the
ation temperatures, the rotatory angle at a fixed wavelength of 221 nm [Â]2 2 1 , looser matrix of skin, which could be easier for collagen extraction.
was measured as a function of temperature. The denaturation temperature, Tm ,
was determined as the temperature at which the change in ellipticity (Â) was half
complete. 3.2. Yield of ASC and PSC from the skin of E. macrura

2.13. Amino acid profiling The final yield of ASC and PSC from the skin of marine eel fish
was about 80 and 7.10 percent (dry weight basis) and 9 and 4.7
The collagen samples were hydrolyzed under reduced pressure in 6 M HCl at percent (wet weight basis), respectively. The eel fish skin was not
110 ◦ C for 24 h. Amino acid composition was analyzed by using amino acid ana-
completely solubilized by 0.5 M acetic acid extraction. In this result
lyzer (Merck Hitachi LaChrome D-7000 HPLC System, Darmstadt, Germany). The
hydrolysates were analyzed on a Hitachi LaChrome liquid chromatography system. was in agreed with Jongjareonrak et al. [5,21] who reported that the
The amino acid content is expressed as the number of residues/1000 residues. incomplete solubilization of bigeye snapper (Priacanthus marcra-
canthus) and brownstripe red snapper (Lutjanus vitta) skin in 0.5 M
2.14. Analysis of Fourier transform infrared spectroscopy (FTIR) acetic acid. The result of the present study suggested that the colla-
gen molecules in E. macrura fish skin were most likely cross-linked
The collagen samples were analyzed using Fourier transform infrared (FTIR)
by covalent bonds through the condensation of aldehyde groups at
spectrum. The lyophilized collagen samples (3 mg) were mixed with dried KBr
(100 mg), ground in a mortar and pestle and subjected to a pressure of about the telopeptide region as well as the inter-molecular cross-linking,
5 × 106 Pa in an evacuated die to produce as 13 × 1 mm clear transparent disk. The leading to a decrease in solubility of collagen [10,22]. With fur-
absorption intensity of the peaks was calculated by the base-line method. The resul- ther limited pepsin digestion, the cross-linked molecules at the
tant spectra were analyzed using ORIGIN 8.0 software (Thermo Nicolet, USA). telopeptide region were cleaved without damaging the integrity
of the triple helix. Therefore, the collagen with the predominant
2.15. Observation of scanning electron microscopy (SEM)
monomeric molecules could be solubilized with acid. In the present
The morphological characteristics of the isolated collagen (ASC and PSC) were study, the freeze dried ASC was found to be colorless fibril-like
observed by JEOL JSM-5610LV Field Emission Gun SEM (Tokyo, Japan). Collagen sam- matters, whereas PSC was found to be a pale-black or grayish col-
ples were mounted on a standard SEM sample holder and fixed. The sample holder ored soft fiber due to pigment like ommochrome. Among the total
was used to prepare 20-s glow discharged carbon support adhesive films (tape) of collagen extracted, ASC (80 percent) based on extractable colla-
30 nm thickness and then smeared and coated with gold ion using auto fine coater.
The samples were then introduced into specimen chamber of SEM and examined for
gen weight was comparatively higher the PSC (7.10 percent). The
surface morphology. The SEM observations were made at 20 kV accelerating voltage greater content of ASC fraction in eel fish skin was in accordance
with a high vacuum (HV) mode and secondary electron image (SEI) was employed with those reported in bigeye snapper (Priacanthus marcracanthus)
to scan the microscopic images of collagen matrixes. The average diameter of the skin (85 percent) [21], brownstripe red snapper (Lutjanus vitta) skin
collagens matrix was measured different arbitrary from SEM image. Samples were
was ASC (66 percent) [5]. However, the PSC fraction in eel skin was
short-pulse coated with graphite to avoid damage due to overheating and analyzed
on their fibrils side at increasing magnifications (×250–2000). 2-fold higher than those obtained from those two species [5,21]. It
is suggested that collagen with more inter-molecular cross-links is
2.16. Statistical analyses present to a greater extent in the skin of E. macrura, than in hake
and bigeye snapper skin.
All the methods of extraction of collagen and analysis were replicated three
times. The results were presented with mean ± standard deviations (SD).
3.3. Gel electrophoresis patterns of collagen from the skin of E.
macrura
3. Results and discussion
The electrophoretic patterns revealed that both the ASC and PSC
3.1. Proximate composition of eel fish skin were consisting of ␣1- and ␣2-chains at a ratio of approximately 2:1
(Fig. 1) and high-molecular-weight components including ␤ chain
The proximate composition studies revealed that eel fish skin (dimmers) and ␥ chain (trimers) components, as well as their cross-
contained 75.89 ± 0.25 percent moisture, 90.05 ± 0.23 percent pro- linked molecules. These results suggested that both ASC and PSC
tein, 1.23 ± 0.02 percent lipid and 8.82 ± 0.05 percent ash on a dry was most likely to be classified as type I collagen. Similarly, Jong-
weight basis which was slightly different that of the moisture, pro- jareonrak et al., also reported about the electrophoretic patterns of
tein, fat and ash contents of brown backed toadfish skin contains type I collagen from the skin of brownstripe red snapper [5]. The
73.4, 90.3, 1.3 and 8.4 percent, respectively [15]. Moreover, the high MW cross-linked molecules in collagen was increases with
shark skin contained 61.96 percent moisture, 24.75 percent pro- animal age [22] and starving fish has more cross-linked collagen
tein, 0.19 percent fat and 12.12 percent ash [16], and Nile perch skin than well fed fish [23].
contained 68.4 percent moisture, 21.6 percent protein, 6.8 percent After digestion by pepsin, some ␤- and ␥-components of ASC
fat and 6.0 percent ash [17]. were cleaved into ␣-components, as evidenced by the increasing

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Fig. 1. SDS–polyacrylamide gel electrophoresis of Eel fish (E. macrura) and human
placenta type IV collagen. Lane 1 contains ASC, Lane 2 contains PSC, Lane 3 contains
Human placenta type IV collagen, Lane 4 contains Standard protein marker.

Fig. 2. Peptide maps of ASC and PSC from E. macrura skin waste collagens digested
band intensity of the ␣-chains. Similar electrophoretic pro- by Achromopeptidase. Lanes: 1, 2 and 3 contains standard collagen Type IV, ASC and
tein patterns were found in ASC and PSC from the grass carp PSC, Lane 4 contains protein markers.
(Ctenopharyngodon idella) [10] and the pepsin cleaves the cross-
link containing the telopeptide, and the ␤-chain is concomitantly that of the PSC with different pH. The solubility variation was due
converted to two ␣-chains [24]. to ASC which consist of higher molecular weight protein than that
The type I collagen isolated from the present study consists of of PSC. The dissimilarity in solubility of collagens with pH has been
two ␣1- and one ␣2-chain as the major component ([␣1]2 ␣ 2). reported from big eye snapper skin and bone over pH ranges of
Since the ␣3-chain has a molecular mass indistinguishable from 1–10 [25]. In the present study, we have exposed that the higher
␣1 chain and as it cannot be separated from ␣1 chain under the degree of molecular cross linking of ASC fraction which is due to
electrophoretic conditions employed, the co-presence of ␣3 with the predominance of stronger bonds than PSC.
␣1 might be possible. The band intensity of ␣1-chain was 2-fold A drastic decrease in PSC solubility was observed with 3 percent
higher than that of ␣2-chain are consist of both ASC and PSC. The NaCl or above. For PSC, solubility decreased up to 4 percent and
␣2 chain was found to be minor component of eel fish collagen it slightly increased at 6 percent NaCl. Whereas, the solubility of
with consisted of two ␣1 and single ␣2 chain. The results of the ASC was serially decreased up to 3 percent and rapidly reached the
present study about ␣1 and ␣2 chain patterns were dissimilar to minimum of 4 percent and slightly decreased with 6 percent NaCl
that of standard type IV collagen from human placenta (Lane 3) and (Fig. 3B). The decrease in solubility of collagens could be described
similar to Nile perch collagen [17], and bigeye snapper [25]. by the salting out phenomenon which occurred at relatively low
NaCl concentration. An increase in ionic strength causes a reduc-
3.4. Peptide mapping of collagen from the skin of E. macrura tion in protein solubility by an enhanced hydrophobic-hydrophobic
interaction between protein chains, and competing for water of
The electrophoretic pattern of denatured eel fish collagen sug- ionic salts, leading to the induced protein precipitation [27]. How-
gested that the ASC and PSC gave very similar peptide maps; ever, PSC exhibited a greater solubility than ASC at 2 percent NaCl
however, these patterns were different from standard type IV colla- concentrations which was due to the partial hydrolysis of high
gen (Fig. 2). After hydrolysis, the ␣-chain and high molecular weight molecular weight cross-linked molecules by pepsin.
cross linked molecules of ASC and PSC from the skin of eel fish were
degraded into small molecular weight peptides ranging from 384 3.6. Subunit composition of collagen from the skin of E. macrura
to 73 kDa and 380 to 63 kDa, respectively. When compared to the
other fish skin collagens of bigeye snapper [25] and Brownstripe The subunit composition of ASC and PSC was determined by
red snapper [5] digested by V8 protease, the peptide maps were applying denatured collagen to DEAE–cellulose chromatography.
different. Peptide maps of collagens were reported to differ among As a result, a large peak was resolved having a shoulder on the right
from the other sources and species [26]. As a result, the pattern of and it was supposed that the second and third fractions contain ␣-
the peptide fragment of eel fish skin collagen may be closely similar chains as major components (Figs. 4 and 5) and small amount of ␤-
to that of mammalian skin. chain (dimer) was observed in the PSC. Kimura et al. [28] suggested
that two different heterotrimers of (␣1)2, ␣ 2 and ␣1, ␣ 2, ␣ 3 were
3.5. Effect of pH and NaCl concentration on collagen solubility present in skin collagen of eel fish. The results of the present study
evidenced about the occurrence of ␣3 chain in eel fish skin collagen.
The effect of the pH and NaCl concentrations on the solubility
of ASC and PSC extracted from the eel fish (E. macrura) skin were 3.7. UV absorption spectrum
presented in Fig. 3. The results of the present study revealed that
maximum stability of ASC and PSC was found to be at pH 4 and 3, The UV absorption spectrums of ASC and PSC at the wavelength
respectively (Fig. 3A). Generally, these types of collagen were exhib- ranges 190–400 nm were showed in Fig. 6(a) and (b). The results
ited higher solubilization in acidic pH from 1 to 4 and the lesser in of the present study revealed that the amount of tyrosine in
pH 7. In the solubility analysis, the ASC had higher solubility than ASC and PSC from E. macrura were 3 residues per 1000 residues,

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Fig. 3. Optimal solubility of both ASC and PSC from eel fish (E. macrura) skin waste in 0.5 M acetic acid at different pHs and NaCl concentration.

Fig. 4. Absorption capability of fractionated denatured ASC subunits at 230 nm in DEAE cellulose chromatography and the fractionation indicated by the numbers were
examined by SDS-PAGE.

respectively. In the present study, ASC and PSC isolated from skin of 3.8. Thermal stability of collagen from the skin of E. macrura
E. macrura showed maximum absorption at 225 and 228 nm which
is similar to the studies of Edwards et al. [29] which suggested that The thermal denaturation temperature (Td ) of eel fish skin colla-
the groups C O, COOH, CONH2 were accessible in polypeptides gen was calculated using thermal denaturation curve were shown
chains of collagen. in Fig. 7. ASC and PSC showed transition curves with maximum

Fig. 5. Absorption capability of fractionated denatured PSC subunits at 230 nm in DEAE cellulose chromatography and the fractionation indicated by the numbers were
examined by SDS-PAGE.

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Fig. 6. (a) UV absorption spectrum of ASC from E. macrura skin. (b) UV absorption spectrum of PSC from E. macrura skin.

denaturation temperatures of 38.5 ◦ C and 35.0 ◦ C, respectively The circular dichroism spectroscopy (CD) of ASC and PSC at the
which is higher than that of type IV collagen from human pla- wavelengths of 190–280 nm were showed in Fig. 8(a) and (b) and
centa (28.5 ◦ C). The present study suggested that the intramolecular the CD spectra of collagen over a temperature range of 25–45 ◦ C.
hydrogen bonds stabilizing the triple helix structure of collagen As a result, the CD curves showed a rotatory maximum at 230 nm,
might be disrupted to some levels in the presence of acetic acid, a minimum at 204 nm and a consistent crossover point (zero rota-
mainly due to the repulsion of collagen molecules in acidic solu- tion) at about 252 nm, which was characteristic of the triple helical
tion [30]. Furthermore, a higher cross-linkage of marine eel fish conformation of the protein [33,34]. Triple helical structure of col-
skin collagen more likely contributed to the higher Td of both ASC lagen molecule is more stable with higher imino acid content as
and PSC. Td of collagen from the skin of E. macrura was much these facilitate intra and inter molecular crosslinking. Interestingly,
higher than that of ocellate puffer fish, 28 ◦ C [31] and bigeye snap- the Td of skin collagen of pig and calf (terrestrial mammals) is 37 ◦ C
per (30.4 ◦ C) [21], but was close to that of pig skin collagen (37 ◦ C) and 40.8 ◦ C, respectively [32], both having high iminoacid contents,
[32]. whereas, cold-water fish collagens have low Td since their imino

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DENATURATION TEMPERATURE (Td) both the collagens from the eel fish skin being found to have
1.2
26.3–27.8 percent glycine of total amino acid, and to be low in
ASC PSC STD cystine, valine, leucine isoleucine, methionine, tyrosine and histi-
1 dine, similar to other collagens [10,16]. Relatively high contents of
alanine, proline, hydroxyproline and glutamic acid were observed
FRACTIONAL VISCOSITY

0.8 and no tryptophan was detected, like other source of collagens

[5,10,17,25,38].
The imino acid (proline + hydroxyproline) content of ASC and
0.6
PSC was about 190 and 200 residues per 1000 residues, respec-
tively, which was slightly lower than that of balloon fish collagen
0.4 (197 and 203 residues per 1000 residues) [18], but much higher
than that of cod skin collagen (179 and 174 residues per 1000
0.2 residues) [35]. The difference in imino acid content among the
animals associated with different living environments of their
sources, particularly habitat temperature [39]. Additionally, the
0 imino acid content was reported to have a major influence on
0 10 20 30 40 50 60 thermal stability of collagen [17]. The stability of collagen was
TEMPERATURE (ºC) proportional to the total content of pyrrolidine imino acids.
Pro + Hyp rich zones of the molecules are most likely involved
Fig. 7. Thermal denaturation curves of ASC and PSC from E. macrura skin and type
IV collagen from human placenta as a standard. in the formation of junction zones stabilized by hydrogen bond-
ing. Hydroxylysine (4–6 residues per 1000 residues) was found in
both ASC and PSC from eel fish skin. Hydroxylysine contributes
acid contents are very low [35]. Thermal denaturation temperature
to the formation and stabilization of cross links in the collagen
of collagens from different sources has been direct correlation with
[40].
the imino acid (proline and hydroxyproline) content [36]. There are
also interesting consequences of variation in Td with variation in
3.10. FTIR spectra of collagen from the skin of E. macrura
temperature of their living environment. Therefore, this isolated
type I collagen may find wide application due to its close denatur-
FTIR spectra of ASC and PSC from the eel fish (E. macrura) skin
ation temperature with mammalian collagen.
are shown in Fig. 9 and Table 2. The amide A band of ASC and PSC
were found at 3421 and 3395 cm−1 , respectively. This band is gen-
3.9. Amino acid composition of collagen from the skin of E. erally associated with the N H stretching vibration and shows the
macrura existence of hydrogen bonds, probably with a carbonyl group of the
peptide chain. A free N H stretching vibration occurs in the range
The amino acid composition of ASC and PSC extracted from of 3400–3440 cm−1 . The major peaks in the spectra of both ASC and
the eel fish skin had similar amino acid profiles (Table 1) which PSC from the skin of eel fish (E. macrura) were similar to those of
was similar to the previously obtained from other aquatic ani- collagen from others fish species [17,20,37]. When the NH group of
mals [16,21,37]. In general, glycine is their major amino acid and a peptide is involved in a hydrogen bond, the position is shifted to
one-third of total amino acid residues of collagen, because glycine lower frequency, usually 3300 cm−1 [41]. Amide B band of ASC and
could be found as being every third residue throughout the cen- PSC was observed at 3079 and 3079 cm−1 respectively, represent
tral region of the ␣-chain other than the first 14 amino acid the asymmetrical stretch of CH2 [17].
residues from the N-terminus and the first 10 amino acid residues The sharp amide I band of ASC and PSC was observed at 1649
from the C-terminus of the collagens [22]. In the present study, and 1653 cm−1 , respectively. The amide I band, with characteris-
tic frequencies in the range from 1600 to 1700 cm−1 , was mainly
Table 1 associated with stretching vibrations of the carbonyl groups (C O
Amino acid composition of acid-soluble collagen and pepsin soluble collagen from bond) along the polypeptide backbone [42], which is a sensitive
E. macrura skin (residues per 1000 total amino acid residues).
marker of the peptide secondary structure [43]. The amide I peak
Amino acids Three letter ASC PSC underwent a decrease in absorbance, followed by a broadening
Alanine Ala 125 117 accompanied by the appearance of additional shoulders when col-
Arginine Arg 56 40 lagen was heated at higher temperature. Due to the similarity in the
Asparagine Asn 8 5 amplitude, both collagens were most likely not denatured during
Aspartic acid Asp 60 56 the extraction. This was reconfirmed by the ratio of approximately
Cystine Cys 12 15
1.17 between amide III and 1454 cm−1 band of both collagens. Ratio
Glutamic acid Glu 80 83
Glycine Gly 278 263 of approximately 1 revealed the triple-helical structure of collagen
Histidine His 3 4 [44]. The amide II of both collagens appeared at 1542–1541 cm−1 ,
Hydroxylysine OH-Lys 4 6 resulting from N H bending vibration coupled with CN stretching
Hydroxyproline OH-Pro 94 98
vibration [45]. Thus, both ASC and PSC showed a similar secondary
Isoleucine Ile 8 26
Leucine Leu 9 13 structure.
Lysine Lys 76 60
Methionine Met BDL 18 3.11. Morphological characterization of collagen from the skin of
Phenylalanine Phe 5 3 E. macrura
Proline Pro 96 102
Serine Ser 31 36
Threonine Thr 24 22 The morphological structures of the isolated collagens (ASC and
Tryptophan Trp UND 2 PSC) were observed under SEM micro-photography with higher
Tyrosine Tyr 3 3 magnification (Fig. 10). The fibrillar structure was noted in both
Valine Val 28 23
ASC and PSC and also mentioned that the porous three dimen-
BDL, below detectable level; UND, undetectable. sional collagen fibril sponges were observed by SEM (Fig. 10).

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Fig. 8. (a) CD spectra of the ASC from outer skin of the Eel fish measured at temperatures 25–45 ◦ C. (b) CD spectra of the PSC from outer skin of the Eel fish measured at
temperatures 25–45 ◦ C.

The morphological structures of ASC and PSC were fibrillar in that can be used for wound healing purposes because, for wound
nature. However, the fibril width range 1 ␮m of the ASC was lower management, freeze-dried collagen sponges are frequently placed
than that of pepsin soluble collagen. The width of collagen fibril- onto wounds without cells [47].
lar tubes was uniform in size (∼1 ␮m) and PSC had nodular-like The epineurium consists of thick bundles of collagen fibrils mea-
structures with tubular in nature. The results of the present study suring about 10–20 ␮m width; they were wavy and ran slightly,
revealed that SEM images has been confirmed that both ASC and obliquely to the nerve axis. Between these collagen bundles, very
PSC showed the cross-section in contact with the mold that exposes coarse meshwork of randomly oriented collagen fibrils was present.
an inter-connected network pore configuration with a pore size of The outer one consisted of longitudinally oriented bundles of about
90–250 ␮m. The regular porous structure was clearly visible in ASC 1–3 ␮m in width. The collagen fibril arrangement described above
when compared to PSC. The pore size of collagen was increased at may protect the nerve fibers against external forces. Although these
higher water content during preparation and the size was moder- collagen fibrils formed bundles, they were not parallel but were
ate for in vivo studies and comparatively similar to the previous entangled in individual bundles. These collagen bundles varied
reports [46]. In addition, the collagen extracted from the bone of in width and thickness, often gave off branches, and intertwined
horse mackerel and croaker could be used as biofilm or scaffold with each other. Pore size, porosity and surface areas are widely

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Fig. 9. Fourier transforms infrared spectra of standard type IV collagen from human placenta, acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the skin of
E. macrura.

recognized as important parameters for a biomaterial to under- formation. Generally, uniform and regular network structure
stand their biomedical importance [48]. In addition, other of collagen as drug carrier is propitious, for well-proportioned
architectural features such as pore shape, pore wall morphology distribution for other drugs [10]. Based on the forgoing account,
and interconnectivity have also suggested for cell seeding, migra- the isolated marine eel fish skin collagen could be used as an
tion, growth, mass transport, gene expression, and new tissue appropriate drug carrier system.

Table 2
General peak assignments of the FTIR spectra consist of control standard (STD) type IV collagen, ASC and PSC from eel fish E. macrura skin.

Peak wave numbers cm−1

Control (standard type Acid soluble Pepsin soluble Region peak assignments
IV collagen) collagen collagen

3545 3529 – N H asymmetric stretching of primary amide (Monomer)

3421 3421 3395 Amid A: mainly N H stretching of proteins
2959 3079 3079 Amid B: CH3 -asymmetric stretch
2922 2923 2924 Amid B: CH2 -asymmetric stretch
2852 2853 2854 CH3 -symmetric stretch: mainly proteins
1743 1743 1742 Carbonyl C O stretch: lipids
1644 1649 1653 Amide I: C O stretching of proteins
1578 1542 1541 Amide II: N H bending/C N stretching of proteins
1541 – – Amide II: N H bending/C N stretching of proteins
1461 1458 1458 CH3 -asymmetric bending: mainly lipids
– 1339 1338 COO-symmetric stretch: mainly phospholipids
1249 1241 1243 Amide III (␤-sheet, protein)
1156 – 1165 CO O C asymmetric stretch: glycogen and nucleic acids
– 1079 – PO2 -symmetric stretching: mainly nucleic acids
1020 1022 1022 C O stretching/C O banding of the C O H carbohydrates
833 – – Out of plane breathing Tyr; PO2 – asymmetric stretch DNA (B-form)
723 – 723 Adenine
670 669 670 CH2 banding, carbohydrates, proteins, and lipids (sterols of fatty acids)
– 562 559 N H out of plane banding
467 467 467 Out of plane ring banding

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Fig. 10. Scanning electron microscopic plates exhibiting the fibrillar structure of ASC and PSC isolated from E. macrura (1 ␮m resolution).

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