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Isolation and characterization of collagen from the skin of Brama australis
Accepted Manuscript Title: Isolation and characterization of collagen from the skin of Brama australis Author: Alina Sionkowska Justyna Kozłowska Małgorzata Skorupska Marta Michalska PII: DOI: Reference:
S0141-8130(15)00503-6 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.07.032 BIOMAC 5236
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
2-1-2015 7-7-2015 10-7-2015
Please cite this article as: A. Sionkowska, J. Kozlowska, M. Skorupska, M. Michalska, Isolation and characterization of collagen from the skin of Brama australis, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.07.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Isolation and characterization of collagen from the skin of Brama australis
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Alina Sionkowska*, Justyna Kozłowska, Małgorzata Skorupska, Marta Michalska
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Nicolaus Copernicus University in Torun, Faculty of Chemistry, Department of Chemistry of
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Biomaterials and Cosmetics
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ul. Gagarina 7, 87-100 Toruń, Poland a
[email protected]
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Abstract Collagen was extracted from the skin of Brama australis, the fish from warm-water
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sea. The yield of collagen from skin of Brama australis was about 1.5% on a wet weight basis
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of raw material. The isolated protein was confirmed as collagen by different physico-chemical
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techniques such as: FTIR, SDS–PAGE, and amino acid analysis. The denaturation
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temperature (Td) of obtained collagen was found to be 24 °C, what is promising as an
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advantage for cosmetic application. According to the electrophoretic pattern, collagen
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consisted of two different α-chains (α1 and α2) and was classified as type I collagen.
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Although Td of obtained collagen is higher than 20°C it is still far from Td of mammalian
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collagen.
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Key words: collagen, fish skin, denaturation temperature.
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• Collagen type I from the skin of Brama australis was extracted and
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characterized by SDS-Page chromatography and amino acid analysis;
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• The denaturation temperature was found to be 24 °C;
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•
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Collagen from Brama australis can be promising as an advantage for cosmetic application.
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Introduction Collagen is the major fibrous protein in the extracellular matrix and in connective tissue
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[1,2]. This biopolymer is the most abundant protein found in animal body and it is widely
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used for biomedical, pharmaceutical and cosmetic applications [3,4]. However, due to high
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cost its applicability is severely limited and new source for collagen extraction are widely
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studied. In the literature one can find that collagen can be obtained from the skin of sailfish
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[5], from the skin of balloon fish [6], from the scales of marine fishes from Japan
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and Vietnam [7], and even from fish scales [8,9,10]. Collagen can be extracted not only from
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fish skin and scales. For example collagen can be extracted from marine sponge and such
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collagen was tested for potential cosmetic application [11]. For biomedical and cosmetic
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applications it is crucial to extract collagen with denaturation temperature closed to the value
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of denaturation temperature of mammalian collagen. This is why thermostable collagen from
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the marine sources have been studied [12]. Denaturation temperature of collagen depends on
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the amino acid composition of polypeptide chain. In each collagen chain there are 1000 amino
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acids, which form the sturdy structure by a repeated sequence of three amino acids. Every
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third amino acid is glycine, a small amino acid that fits perfectly inside the helix. In other
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positions in the chain are located: proline and a modified version of proline, hydroxyproline.
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It is believed, that hydroxyproline is responsible for collagen stability because of additional
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hydrogen bonds formed by OH group. Hydrogen bonds play the main role in the stabilization
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of the collagen triple helix [13]. Each collagen molecule undergoes a strong molecular
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connection with neighboring collagen molecules by hydrogen bonding and other crosslinks
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[14,15]. Thermal stability of collagens of fish from different areas can be different [16] as
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amino acid composition may differ. Several cross-linking methods can lead to the increase of
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the denaturation temperature [17]. There are two kinds of cross-linking methods, physical
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methods and chemical ones. Physical ones are cheap and easy to perform, nevertheless it is
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hard to control the process. For chemical cross-linking methods usually toxic chemicals are
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used. To avoid additional cross-linking of collagen several research groups are looking for
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collagen with high denaturation temperature. In our lab we have been working mainly with
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collagen extracted from rat tail tendon [18,19]. However, this type of collagen is not
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sufficiently good for preparation of materials for biomedical and cosmetic application, mainly
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due to limited source of such collagen.
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As fish processing waste can be promising and cost effective collagen source, we started to
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work with collagen extracted from fish scales [20,21,22]. Collagen type I was successfully
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isolated from the scale of Esox lucius (fish from fresh water). Thermal stability of isolated
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collagen was 28.5 °C and was higher than that of sea fishes [20]. The higher thermal stability
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of collagen from Esox lucius may be due to bigger contents of imino acids and may have
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correlation with the body temperature and living environment of this fish.
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The aim of this work was extraction of collagen from the skin of fish Brama australis
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belonging to the family Bramidae. This family of fish lives in pelagic of subtropical water.
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Brama australis is widely present in the South Pacific Ocean. This species is valued as food in
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Chile, where is officially fished since 1994 [23]. For our best knowledge the skin of Brama
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australis was not previously used as a source of collagen. We would like to check whether the
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isolated fish collagen may serve as an attractive alternative to mammalian collagen for
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biomedical and cosmetic applications. Collagen from fish species for biomedical and
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cosmetic application should possess good thermal stability. Its denaturation temperature
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should be at least 20oC.
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1. Materials and methods
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2.1.
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Collagen was extracted from the skin of Brama australis, the fish from warm sea. The
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fishes were imported from Chile by Polish company of food production “Ikra” in Kurowo.
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The skin of Brama australis was a waste of food production in this company.
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At the beginning the skin was cleaned with distilled water and residual of soft tissue was
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removed. The skin was extracted with 0.1 M NaOH (at a solid to solution ratio 1:10 w/v) for 4
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days to remove non-collagenous proteins. Then washed with distilled water to obtain a neutral
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pH. From the skin we extracted fat with 10% butyl alcohol for 1 day, then washed with
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distilled water. The insoluble matter was extracted with 0.5 M acetic acid for 2 days, then the
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viscous solution was salted out by adding NaCl (to a concentration of 0.7 M) followed by
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precipitation of collagen by addition of NaCl and the extract was centrifuged by Eppendorf
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Centrifuge at 5804 R for 20 min. All the preparative procedures were performed a 4oC. The
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resultant precipitate was dissolved in 0.5 M acetic acid, dialyzed against 0.1 M acetic acid for
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3 days, changing the solution every 24h, and then lyophilized. For dialysis we used tubes
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with Molecular Weight Cut Off (MWCO) = 12-14 kDa, Serva, Heidelberg, Germany.
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The completely frozen samples were lyophilized at -20 ºC and 100 Pa until constant weight of
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sample was reached (ALPHA 1–2 LD plus, CHRIST, Germany).
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Preparation of collagen from fish skin
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2.2.
SDS-Page chromatography
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SDS-PAGE was carried out using the Hoefer SE600 system. The resolving gels (7.5%
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acrylamide of about 0.75 mm thickness) were run at constant voltage (200V) and prepared
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according to the method described by Laemmli [24]. Protein were visualized by silver
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staining described by Merril [25]. Spectra Multicolor High Range Protein Ladder was used as
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marker with apparent molecular weights of 40 to 300 kDa.
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2.3.
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Amino acids analysis
The amino acid composition of the Brama australis skin collagen was analyzed at
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BioCentrum Ltd. (Kraków, Poland). The protein samples were hydrolyzed in the gaseous
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phase with 6 M HCl at 115 °C for 24 h. The unhydrolyzed residue was also analyzed for the
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presence of free amino acids. The liberated amino acids were converted into
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phenylthiocarbamyl (PTC) derivatives and analyzed by high-pressure liquid chromatography
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(HPLC) on a PicoTag 3.9×150 mm column [26].
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2.4.
UV–Vis measurements
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The ultraviolet absorption spectra of the Brama australis skin collagen were recorded by a
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Shimadzu spectrophotometer (Model UV-1601PC). Data were collected and plotted using the
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UVPC program and computer data station supplied by the manufacturer. UV–Vis spectra
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were recorded using solution of samples.
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2.5.
Determination of denaturation temperature
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The denaturation temperature was measured by viscometric method [27]. The thermal
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denaturation curve was obtained by measuring the viscosity of 1.5 mg/mL collagen solution
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in 0.1 M acetic acid at several temperatures. The denaturation temperature, Td, was
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determined as the temperature that the change in viscosity was half completed. The
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denaturation temperature was measured for three samples from the same part of material.
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2.6.
Differential scanning calorimetry
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Differential scanning calorimetry analysis was carried out with DSC 204 F1 Phoenix,
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NETZSCH apparatus. The samples were heated heated from 20 to 250°C at a heating rate of
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10oC/min. The transition temperature on DSC curves was measured for three samples from
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the same part of material with precision of DSC apparatus and the average value was
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calculated.
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2.7.
FTIR Spectroscopy
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Infra red spectra of collagen from Brama australis were registered using a Genesis II
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FTIR spectrophotometer (Mattson, USA) equipped with an ATR device (MIRacleTM PIKE
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Technologies) with zinc selenide (ZnSe) crystal. All spectra were recorded in absorption
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mode at 4cm-1 intervals and 64 scans.
M 3. Results and discussion
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3.1.
SDS-PAGE chromatography
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The yield of collagen from the skin of Brama australis was 1.5% (9.7g of lyophilized
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collagen from 638g of wet raw fish skin). The obtained collagen was examined by SDS-
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PAGE using 4% and 7.5% gel. It was found that collagen comprised at least two different
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α chains; α1 and α2 and β dimmers (Fig. 1). In electrophoretic mobility the positions of α1
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and α2 chains of collagen were different. The molecular weight of β dimmers can be assessed
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as about 212 kDa, whereas α1 and α2 chains as 120 kDa and 112 kDa, respectively. If other α
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chains, such as α3 and α4, were present in these collagens, they were not separated from the
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corresponding α1 chain under the electrophoretic conditions employed. SDS -PAGE
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chromatography proved that the obtained protein is collagen, however it worth to mention that
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amino analysis has to be done to confirm whether hydroxyproline is present in protein just to
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be sure that the extracted protein is collagen. Hydroxyproline is amino acid representative
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only for collagen.
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3.2.
Amino acids analysis
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The amino acid composition of Brama australis collagen, expressed as nanomoles of
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amino acid per 1 mg of dry mass of collagen is shown in Table 1. As one can see in the Table
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1 glycine was the most abundant amino acid in Brama australis skin collagen. There were
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relatively high contents of alanine, proline and glutamic acid. Glycine accounted for about
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36% of all amino acids in this collagen may suggest that collagen type I was extracted.
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Glycine which is regularly spaced at every third residue throughout the central region of the
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α-chain in collagen, is the most abundant amino acid in collagen. This amino acid as the
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smallest amino acid having only a hydrogen atom as α side chain, allows the three helical α-
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chains to pack tightly together, to form the final collagen superhelix [28].
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In collagen extracted from Brama australis the contents of imino acids, proline and
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hydroxyproline, were relatively high. The presence of these amino acids is important factor in
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the formation of the triple-stranded helix in collagen. It may suggest that the collagen
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extracted from the skin of Brama australis can be relatively stable in comparison with the
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collagen of other species. Taking into account that the amount of imino acids, especially Hyp,
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depends on the temperature in which the fish lives and it affects the thermal stability of the
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collagens one can expect that collagen extracted from fish that lives in warm sea should
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exhibit higher thermal stability than those from fish living in cold environments [29].
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High temperature induces the structural melting or unfolding of the collagen molecule. In the
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case of collagen type I, thermal denaturation means unfolding of the triple helix and leads to
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loss of collagen’s unique characteristics [30]. For this reason the denaturation temperature
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(Td) is an important measure of the thermal stability of proteins and it delivers important
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information for future application of collagen. Denaturation temperature can be studied by
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viscometry techniques and by differential scanning calorimeter. In this study denaturation
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temperature was found by viscometric method. The decreasing viscosity with increasing
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temperature is shown in Fig. 2.
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3.3.
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The denaturation temperature of collagen from Brama australis was determined as the
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temperature that the change in viscosity was half completed and it was 24 ºC. Denaturation
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temperature of collagen from Brama australis is bigger than Td of collagen extracted from
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fish species from cold sea water [31]. However, several collagens with higher denaturation
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temperature than that one found for Brama australis were reported in scientific literature. For
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example Tiger puffer collagen had a Td of 28.4 °C similar to that of ocellate puffer fish (T.
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rubripes) or marine collagen from Japanese sea bass fin had 29.1 °C [29]. It is worth to
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mention, that fish species with Td of collagens higher than 30 °C are very limited. Most fish
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collagens undergo denaturation at temperatures below 30 °C. This fact clearly indicates that
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fish collagen is generally less stable than mammalian collagen. However, the collagen of
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eagle ray, red stingray and yantai stingray had Td values of 34.1 °C, 33.2 °C and 32.2 °C,
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respectively which values are very closed to Td of mammalian collagen [32].
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Determination of denaturation temperature
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3.4.
Differential scanning calorimetry
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Differential scanning calorimetry (DSC) is particularly suited for the study of the thermal
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denaturation of protein. It measures heat flow between sample and reference zone and 9 Page 9 of 21
provides information about the thermal transitions of protein. The DSC curve is shown in
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Figure 3. DSC analysis allowed to examine the thermal stability of collagen in solid state
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(lyophilizated collagen). The DSC curve was obtained during the heating of collagen in a
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nitrogen, at a heating rate 10 °C/min. The values of transition temperature of collagen
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extracted from Brama australis species, which were calculated from the maximum transition
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point (the endothermic peak) of the thermal transition curves, are shown in Table 2.
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On the DSC curve of freeze-dried collagen there are two noticeable peaks. The first one
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(Tmax = 78.2 oC) is related to the release of the water bound in the collagen molecule and the
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degradation of collagen triple-helical structure. The second peak (Tmax = 189.2 °C) refers to
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the melting temperature of the crosslinked collagen parts. The heat of the first transformation
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is 238.1 J/g and it is much bigger than the heat produced by the second conversion (6.4 J /g)
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(Figure 3, Table 2).
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3.5.
FTIR and UV-Vis spectroscopy
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The IR spectra recorded for thin films made of collagen from fish Brama australis show
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typical bands for collagen type I: Amide A, Amide B, Amide I, Amide II and Amide III
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(Figure 4). The absorption characteristics of Amide A, commonly associated with N-H
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stretching vibration, occurs in the wave number range 3300 ~ 3440 cm-1. The maximum of
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absorption peak of collagen from Brama australis was found at 3318 cm-1. When the N-H
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group of a peptide is involved in a hydrogen bond, the position starts to shift to lower
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frequencies. Amide B peak was found at 3079 cm-1. The wavenumber of characteristic
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absorption in Amide I bond is usually in the range of 1600 ~ 1700 cm-1, which is generated
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by stretching vibration of C=O in polypeptide backbone of protein. This is the sensitive area
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of changes of protein secondary structure, and often used for protein secondary structure
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analysis. The absorption peak of Amide I was found at 1655 cm-1. The Amide II band was
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found at 1548 cm-1. The Amide III peak is complex, with intermolecular interactions in
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collagen, consisting of components from C-N stretching and N-H in plane bending from
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amide linkages, as well as absorptions arising from wagging vibrations from CH2. IR spectra
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as well as UV-Vis spectra (not shown) obtained for protein extracted from the skin of Brama
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australis clearly confirm that collagen was extracted.
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4. Conclusions
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Collagen type 1 was successfully extracted from the skin of Brama australis, the fish
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living in warm-water sea. The denaturation temperature (Td) of obtained collagen was found
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to be 24 °C, what is promising as an advantage for cosmetic application. The yield of collagen
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from the skin of Brama australis is rather law and it is about 1.5% on a wet weight basis of
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raw material. Although Td of obtained collagen is higher than 20°C, it is still far from Td of
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mammalian collagen.
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ACKNOWLEDGMENTS
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The authors would like to thank the National Science Center Programme (NCN, Poland,
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Grant no: UMO-2013/11/B/ST8/04444) for providing financial support to this project.
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Figures captions
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Figure 1. SDS-PAGE polyacrylamide gel electrophoresis of collagen from Brama australis:
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M –marker, K- collagen.
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Figure 2. Viscosity of collagen solution in 0.1 M CH3COOH versus temperature.
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Figure 3. DSC thermogram of collagen extracted from the skin of Brama australis (samples
317
were heated from 20 to 250°C at a heating rate of 10oC/min).
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Figure 4. FTIR spectra of collagen from Brama australis.
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Table 1. Amino acid composition of Brama australis collagen, expressed as nanomoles of
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amino acid per 1 mg of dry mass of collagen.
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[nmol/1 mg of dry mass]
Aspartic acid+ Asparagine
377.60
Glutamic acid+ Glutamine
737.28
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Amino acid
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Hydroxyproline Serine Glycine Histidine Arginine Threonine Alanine Proline Tyrosine Valine Methionine Cysteine Isoleucine Leucine Phenylalanine
630.58 448.57 3719.43 96.12 547.95 265.14 1321.69 1122.59 43.72 220.90 152.61 0.04 91.92 225.65 125.01
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Lysine 323
246.67
324 325
Table 2. Thermal parameters obtained by DSC for liophylized collagen from the skin of
327
Brama australis (Tmax) and (ΔH).
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Peak 2
Tmax [oC]
78±2
189±2
ΔH [J/g]
238.1±0.5
6.4±0.5
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S0141-8130(15)00503-6 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.07.032 BIOMAC 5236
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
2-1-2015 7-7-2015 10-7-2015
Please cite this article as: A. Sionkowska, J. Kozlowska, M. Skorupska, M. Michalska, Isolation and characterization of collagen from the skin of Brama australis, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.07.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Isolation and characterization of collagen from the skin of Brama australis
2
Alina Sionkowska*, Justyna Kozłowska, Małgorzata Skorupska, Marta Michalska
3
Nicolaus Copernicus University in Torun, Faculty of Chemistry, Department of Chemistry of
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Biomaterials and Cosmetics
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ul. Gagarina 7, 87-100 Toruń, Poland a
[email protected]
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Abstract Collagen was extracted from the skin of Brama australis, the fish from warm-water
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sea. The yield of collagen from skin of Brama australis was about 1.5% on a wet weight basis
10
of raw material. The isolated protein was confirmed as collagen by different physico-chemical
11
techniques such as: FTIR, SDS–PAGE, and amino acid analysis. The denaturation
12
temperature (Td) of obtained collagen was found to be 24 °C, what is promising as an
13
advantage for cosmetic application. According to the electrophoretic pattern, collagen
14
consisted of two different α-chains (α1 and α2) and was classified as type I collagen.
15
Although Td of obtained collagen is higher than 20°C it is still far from Td of mammalian
16
collagen.
17
Key words: collagen, fish skin, denaturation temperature.
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• Collagen type I from the skin of Brama australis was extracted and
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characterized by SDS-Page chromatography and amino acid analysis;
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• The denaturation temperature was found to be 24 °C;
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•
22
Collagen from Brama australis can be promising as an advantage for cosmetic application.
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Introduction Collagen is the major fibrous protein in the extracellular matrix and in connective tissue
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[1,2]. This biopolymer is the most abundant protein found in animal body and it is widely
29
used for biomedical, pharmaceutical and cosmetic applications [3,4]. However, due to high
30
cost its applicability is severely limited and new source for collagen extraction are widely
31
studied. In the literature one can find that collagen can be obtained from the skin of sailfish
32
[5], from the skin of balloon fish [6], from the scales of marine fishes from Japan
33
and Vietnam [7], and even from fish scales [8,9,10]. Collagen can be extracted not only from
34
fish skin and scales. For example collagen can be extracted from marine sponge and such
35
collagen was tested for potential cosmetic application [11]. For biomedical and cosmetic
36
applications it is crucial to extract collagen with denaturation temperature closed to the value
37
of denaturation temperature of mammalian collagen. This is why thermostable collagen from
38
the marine sources have been studied [12]. Denaturation temperature of collagen depends on
39
the amino acid composition of polypeptide chain. In each collagen chain there are 1000 amino
40
acids, which form the sturdy structure by a repeated sequence of three amino acids. Every
41
third amino acid is glycine, a small amino acid that fits perfectly inside the helix. In other
42
positions in the chain are located: proline and a modified version of proline, hydroxyproline.
43
It is believed, that hydroxyproline is responsible for collagen stability because of additional
44
hydrogen bonds formed by OH group. Hydrogen bonds play the main role in the stabilization
45
of the collagen triple helix [13]. Each collagen molecule undergoes a strong molecular
46
connection with neighboring collagen molecules by hydrogen bonding and other crosslinks
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[14,15]. Thermal stability of collagens of fish from different areas can be different [16] as
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amino acid composition may differ. Several cross-linking methods can lead to the increase of
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the denaturation temperature [17]. There are two kinds of cross-linking methods, physical
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methods and chemical ones. Physical ones are cheap and easy to perform, nevertheless it is
51
hard to control the process. For chemical cross-linking methods usually toxic chemicals are
52
used. To avoid additional cross-linking of collagen several research groups are looking for
53
collagen with high denaturation temperature. In our lab we have been working mainly with
54
collagen extracted from rat tail tendon [18,19]. However, this type of collagen is not
55
sufficiently good for preparation of materials for biomedical and cosmetic application, mainly
56
due to limited source of such collagen.
57
As fish processing waste can be promising and cost effective collagen source, we started to
58
work with collagen extracted from fish scales [20,21,22]. Collagen type I was successfully
59
isolated from the scale of Esox lucius (fish from fresh water). Thermal stability of isolated
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collagen was 28.5 °C and was higher than that of sea fishes [20]. The higher thermal stability
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of collagen from Esox lucius may be due to bigger contents of imino acids and may have
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correlation with the body temperature and living environment of this fish.
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The aim of this work was extraction of collagen from the skin of fish Brama australis
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belonging to the family Bramidae. This family of fish lives in pelagic of subtropical water.
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Brama australis is widely present in the South Pacific Ocean. This species is valued as food in
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Chile, where is officially fished since 1994 [23]. For our best knowledge the skin of Brama
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australis was not previously used as a source of collagen. We would like to check whether the
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isolated fish collagen may serve as an attractive alternative to mammalian collagen for
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biomedical and cosmetic applications. Collagen from fish species for biomedical and
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cosmetic application should possess good thermal stability. Its denaturation temperature
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should be at least 20oC.
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1. Materials and methods
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2.1.
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Collagen was extracted from the skin of Brama australis, the fish from warm sea. The
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fishes were imported from Chile by Polish company of food production “Ikra” in Kurowo.
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The skin of Brama australis was a waste of food production in this company.
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At the beginning the skin was cleaned with distilled water and residual of soft tissue was
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removed. The skin was extracted with 0.1 M NaOH (at a solid to solution ratio 1:10 w/v) for 4
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days to remove non-collagenous proteins. Then washed with distilled water to obtain a neutral
81
pH. From the skin we extracted fat with 10% butyl alcohol for 1 day, then washed with
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distilled water. The insoluble matter was extracted with 0.5 M acetic acid for 2 days, then the
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viscous solution was salted out by adding NaCl (to a concentration of 0.7 M) followed by
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precipitation of collagen by addition of NaCl and the extract was centrifuged by Eppendorf
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Centrifuge at 5804 R for 20 min. All the preparative procedures were performed a 4oC. The
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resultant precipitate was dissolved in 0.5 M acetic acid, dialyzed against 0.1 M acetic acid for
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3 days, changing the solution every 24h, and then lyophilized. For dialysis we used tubes
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with Molecular Weight Cut Off (MWCO) = 12-14 kDa, Serva, Heidelberg, Germany.
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The completely frozen samples were lyophilized at -20 ºC and 100 Pa until constant weight of
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sample was reached (ALPHA 1–2 LD plus, CHRIST, Germany).
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Preparation of collagen from fish skin
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2.2.
SDS-Page chromatography
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SDS-PAGE was carried out using the Hoefer SE600 system. The resolving gels (7.5%
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acrylamide of about 0.75 mm thickness) were run at constant voltage (200V) and prepared
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according to the method described by Laemmli [24]. Protein were visualized by silver
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staining described by Merril [25]. Spectra Multicolor High Range Protein Ladder was used as
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marker with apparent molecular weights of 40 to 300 kDa.
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2.3.
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Amino acids analysis
The amino acid composition of the Brama australis skin collagen was analyzed at
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BioCentrum Ltd. (Kraków, Poland). The protein samples were hydrolyzed in the gaseous
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phase with 6 M HCl at 115 °C for 24 h. The unhydrolyzed residue was also analyzed for the
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presence of free amino acids. The liberated amino acids were converted into
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phenylthiocarbamyl (PTC) derivatives and analyzed by high-pressure liquid chromatography
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(HPLC) on a PicoTag 3.9×150 mm column [26].
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2.4.
UV–Vis measurements
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The ultraviolet absorption spectra of the Brama australis skin collagen were recorded by a
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Shimadzu spectrophotometer (Model UV-1601PC). Data were collected and plotted using the
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UVPC program and computer data station supplied by the manufacturer. UV–Vis spectra
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were recorded using solution of samples.
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2.5.
Determination of denaturation temperature
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The denaturation temperature was measured by viscometric method [27]. The thermal
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denaturation curve was obtained by measuring the viscosity of 1.5 mg/mL collagen solution
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in 0.1 M acetic acid at several temperatures. The denaturation temperature, Td, was
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determined as the temperature that the change in viscosity was half completed. The
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denaturation temperature was measured for three samples from the same part of material.
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2.6.
Differential scanning calorimetry
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Differential scanning calorimetry analysis was carried out with DSC 204 F1 Phoenix,
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NETZSCH apparatus. The samples were heated heated from 20 to 250°C at a heating rate of
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10oC/min. The transition temperature on DSC curves was measured for three samples from
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the same part of material with precision of DSC apparatus and the average value was
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calculated.
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2.7.
FTIR Spectroscopy
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Infra red spectra of collagen from Brama australis were registered using a Genesis II
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FTIR spectrophotometer (Mattson, USA) equipped with an ATR device (MIRacleTM PIKE
130
Technologies) with zinc selenide (ZnSe) crystal. All spectra were recorded in absorption
131
mode at 4cm-1 intervals and 64 scans.
M 3. Results and discussion
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3.1.
SDS-PAGE chromatography
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The yield of collagen from the skin of Brama australis was 1.5% (9.7g of lyophilized
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collagen from 638g of wet raw fish skin). The obtained collagen was examined by SDS-
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PAGE using 4% and 7.5% gel. It was found that collagen comprised at least two different
138
α chains; α1 and α2 and β dimmers (Fig. 1). In electrophoretic mobility the positions of α1
139
and α2 chains of collagen were different. The molecular weight of β dimmers can be assessed
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as about 212 kDa, whereas α1 and α2 chains as 120 kDa and 112 kDa, respectively. If other α
141
chains, such as α3 and α4, were present in these collagens, they were not separated from the
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corresponding α1 chain under the electrophoretic conditions employed. SDS -PAGE
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chromatography proved that the obtained protein is collagen, however it worth to mention that
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amino analysis has to be done to confirm whether hydroxyproline is present in protein just to
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be sure that the extracted protein is collagen. Hydroxyproline is amino acid representative
146
only for collagen.
147
3.2.
Amino acids analysis
149
The amino acid composition of Brama australis collagen, expressed as nanomoles of
150
amino acid per 1 mg of dry mass of collagen is shown in Table 1. As one can see in the Table
151
1 glycine was the most abundant amino acid in Brama australis skin collagen. There were
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relatively high contents of alanine, proline and glutamic acid. Glycine accounted for about
153
36% of all amino acids in this collagen may suggest that collagen type I was extracted.
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Glycine which is regularly spaced at every third residue throughout the central region of the
155
α-chain in collagen, is the most abundant amino acid in collagen. This amino acid as the
156
smallest amino acid having only a hydrogen atom as α side chain, allows the three helical α-
157
chains to pack tightly together, to form the final collagen superhelix [28].
158
In collagen extracted from Brama australis the contents of imino acids, proline and
159
hydroxyproline, were relatively high. The presence of these amino acids is important factor in
160
the formation of the triple-stranded helix in collagen. It may suggest that the collagen
161
extracted from the skin of Brama australis can be relatively stable in comparison with the
162
collagen of other species. Taking into account that the amount of imino acids, especially Hyp,
163
depends on the temperature in which the fish lives and it affects the thermal stability of the
164
collagens one can expect that collagen extracted from fish that lives in warm sea should
165
exhibit higher thermal stability than those from fish living in cold environments [29].
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High temperature induces the structural melting or unfolding of the collagen molecule. In the
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case of collagen type I, thermal denaturation means unfolding of the triple helix and leads to
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loss of collagen’s unique characteristics [30]. For this reason the denaturation temperature
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(Td) is an important measure of the thermal stability of proteins and it delivers important
170
information for future application of collagen. Denaturation temperature can be studied by
171
viscometry techniques and by differential scanning calorimeter. In this study denaturation
172
temperature was found by viscometric method. The decreasing viscosity with increasing
173
temperature is shown in Fig. 2.
174 175
3.3.
176
The denaturation temperature of collagen from Brama australis was determined as the
177
temperature that the change in viscosity was half completed and it was 24 ºC. Denaturation
178
temperature of collagen from Brama australis is bigger than Td of collagen extracted from
179
fish species from cold sea water [31]. However, several collagens with higher denaturation
180
temperature than that one found for Brama australis were reported in scientific literature. For
181
example Tiger puffer collagen had a Td of 28.4 °C similar to that of ocellate puffer fish (T.
182
rubripes) or marine collagen from Japanese sea bass fin had 29.1 °C [29]. It is worth to
183
mention, that fish species with Td of collagens higher than 30 °C are very limited. Most fish
184
collagens undergo denaturation at temperatures below 30 °C. This fact clearly indicates that
185
fish collagen is generally less stable than mammalian collagen. However, the collagen of
186
eagle ray, red stingray and yantai stingray had Td values of 34.1 °C, 33.2 °C and 32.2 °C,
187
respectively which values are very closed to Td of mammalian collagen [32].
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Determination of denaturation temperature
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3.4.
Differential scanning calorimetry
190
Differential scanning calorimetry (DSC) is particularly suited for the study of the thermal
191
denaturation of protein. It measures heat flow between sample and reference zone and 9 Page 9 of 21
provides information about the thermal transitions of protein. The DSC curve is shown in
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Figure 3. DSC analysis allowed to examine the thermal stability of collagen in solid state
194
(lyophilizated collagen). The DSC curve was obtained during the heating of collagen in a
195
nitrogen, at a heating rate 10 °C/min. The values of transition temperature of collagen
196
extracted from Brama australis species, which were calculated from the maximum transition
197
point (the endothermic peak) of the thermal transition curves, are shown in Table 2.
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On the DSC curve of freeze-dried collagen there are two noticeable peaks. The first one
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(Tmax = 78.2 oC) is related to the release of the water bound in the collagen molecule and the
200
degradation of collagen triple-helical structure. The second peak (Tmax = 189.2 °C) refers to
201
the melting temperature of the crosslinked collagen parts. The heat of the first transformation
202
is 238.1 J/g and it is much bigger than the heat produced by the second conversion (6.4 J /g)
203
(Figure 3, Table 2).
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3.5.
FTIR and UV-Vis spectroscopy
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The IR spectra recorded for thin films made of collagen from fish Brama australis show
207
typical bands for collagen type I: Amide A, Amide B, Amide I, Amide II and Amide III
208
(Figure 4). The absorption characteristics of Amide A, commonly associated with N-H
209
stretching vibration, occurs in the wave number range 3300 ~ 3440 cm-1. The maximum of
210
absorption peak of collagen from Brama australis was found at 3318 cm-1. When the N-H
211
group of a peptide is involved in a hydrogen bond, the position starts to shift to lower
212
frequencies. Amide B peak was found at 3079 cm-1. The wavenumber of characteristic
213
absorption in Amide I bond is usually in the range of 1600 ~ 1700 cm-1, which is generated
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by stretching vibration of C=O in polypeptide backbone of protein. This is the sensitive area
215
of changes of protein secondary structure, and often used for protein secondary structure
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analysis. The absorption peak of Amide I was found at 1655 cm-1. The Amide II band was
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found at 1548 cm-1. The Amide III peak is complex, with intermolecular interactions in
218
collagen, consisting of components from C-N stretching and N-H in plane bending from
219
amide linkages, as well as absorptions arising from wagging vibrations from CH2. IR spectra
220
as well as UV-Vis spectra (not shown) obtained for protein extracted from the skin of Brama
221
australis clearly confirm that collagen was extracted.
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4. Conclusions
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Collagen type 1 was successfully extracted from the skin of Brama australis, the fish
225
living in warm-water sea. The denaturation temperature (Td) of obtained collagen was found
226
to be 24 °C, what is promising as an advantage for cosmetic application. The yield of collagen
227
from the skin of Brama australis is rather law and it is about 1.5% on a wet weight basis of
228
raw material. Although Td of obtained collagen is higher than 20°C, it is still far from Td of
229
mammalian collagen.
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ACKNOWLEDGMENTS
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The authors would like to thank the National Science Center Programme (NCN, Poland,
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Grant no: UMO-2013/11/B/ST8/04444) for providing financial support to this project.
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[32] J.H. Muyonga, C.G.B. Cole, K.G. Duodu, Characterization of acid soluble collagen
310
from skins of young and adult Nile perch (Lates niloticus), Food Chemistry 85 (2004) 81-88.
311
Ac ce pt e
d
M
an
us
cr
ip t
312
15 Page 15 of 21
Figures captions
313
Figure 1. SDS-PAGE polyacrylamide gel electrophoresis of collagen from Brama australis:
314
M –marker, K- collagen.
315
Figure 2. Viscosity of collagen solution in 0.1 M CH3COOH versus temperature.
316
Figure 3. DSC thermogram of collagen extracted from the skin of Brama australis (samples
317
were heated from 20 to 250°C at a heating rate of 10oC/min).
318
Figure 4. FTIR spectra of collagen from Brama australis.
319
Table 1. Amino acid composition of Brama australis collagen, expressed as nanomoles of
320
amino acid per 1 mg of dry mass of collagen.
us
cr
ip t
312
[nmol/1 mg of dry mass]
Aspartic acid+ Asparagine
377.60
Glutamic acid+ Glutamine
737.28
d
Amino acid
M
Ac ce pt e
322
an
321
Hydroxyproline Serine Glycine Histidine Arginine Threonine Alanine Proline Tyrosine Valine Methionine Cysteine Isoleucine Leucine Phenylalanine
630.58 448.57 3719.43 96.12 547.95 265.14 1321.69 1122.59 43.72 220.90 152.61 0.04 91.92 225.65 125.01
16 Page 16 of 21
Lysine 323
246.67
324 325
Table 2. Thermal parameters obtained by DSC for liophylized collagen from the skin of
327
Brama australis (Tmax) and (ΔH).
ip t
326
Peak 2
Tmax [oC]
78±2
189±2
ΔH [J/g]
238.1±0.5
6.4±0.5
us
Peak 1
an
329
cr
328
M
330 331
Ac ce pt e
d
332
17 Page 17 of 21
Ac ce p
te
d
M
an
us
cr
ip t
Figure 1
Page 18 of 21
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)2
Page 19 of 21
Ac
ce
pt
ed
M
an
us
cr
i
Figure 3
Page 20 of 21
ip t
Figure 4
cr
1
us
0.9
an
0.8
M
0.6
ed
0.5
0.2
0.1
0 4000
ce
0.3
pt
0.4
Ac
Absorbance
0.7
3500
3000
2500
2000
1500
1000
Wavenumber [cm-1]
Page 21 of 21