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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Isolation and structural characterisation of acid- and pepsinsoluble collagen from the skin of squid Sepioteuthis lessoniana (Lesson, 1830) a
a
Pasiyappazham Ramasamy , Namasivayam Subhapradha , a
a
Vairamani Shanmugam & Annaian Shanmugam a
Centre of Advanced Study in Marine Biology, , Faculty of Marine Sciences, Annamalai University, Parangipettai 608 502, Tamil Nadu, India Published online: 31 Jan 2014.
To cite this article: Pasiyappazham Ramasamy, Namasivayam Subhapradha, Vairamani Shanmugam & Annaian Shanmugam (2014) Isolation and structural characterisation of acid- and pepsin-soluble collagen from the skin of squid Sepioteuthis lessoniana (Lesson, 1830), Natural Product Research: Formerly Natural Product Letters, 28:11, 838-842, DOI: 10.1080/14786419.2014.880914 To link to this article: http://dx.doi.org/10.1080/14786419.2014.880914
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [Florida International University] at 02:45 04 September 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 11, 838–842, http://dx.doi.org/10.1080/14786419.2014.880914
SHORT COMMUNICATION Isolation and structural characterisation of acid- and pepsin-soluble collagen from the skin of squid Sepioteuthis lessoniana (Lesson, 1830)
Downloaded by [Florida International University] at 02:45 04 September 2014
Pasiyappazham Ramasamy*, Namasivayam Subhapradha, Vairamani Shanmugam and Annaian Shanmugam Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai 608 502, Tamil Nadu, India (Received 7 October 2013; final version received 26 December 2013) Acid-solubilised collagen (ASC) and pepsin-solubilised collagen (PSC) were effectively isolated from squid skin with good yield and total protein content. ASC and PSC consist of two a-chains with an imino acid content of 182.6 and 184 imino acid residues/1000 residues. The molecular weight was determined to be between 73 and 107 kDa by using SDS-PAGE. For peptide mapping, collagens were digested with achromo endopeptidase, and all components, including a, b-chains, were markedly hydrolysed. Degradation peptides with molecular weights between 106.9 and 15.47 kDa were obtained. UV – vis absorption spectrum revealed distinct absorption at 220– 240 nm. FT-IR spectra of collagens were almost similar when compared with standard. In differential scanning calorimetry profile, ASC and PSC exhibited a To of 59.10, 62.188C and TP of 104.91, 98.10 8C, respectively. This investigation indicates that the collagen isolated from the squid skin, which is thrown as waste in the seafoodprocessing plant, might supplement the vertebrate collagen in industrial applications. Keywords: characterisation; collagen; Sepioteuthis lessoniana; squid skin
1. Introduction Collagen is a major protein present abundantly in animal tissues and constitutes approximately 30% of total animal protein (Muyonga et al. 2004). It is extensively distributed in skin, bone, cartilage, tendons, ligaments, blood vessels, teeth, cornea and other organs of vertebrates (Senaratne et al. 2006), and they contribute as a structural framework to the tissues of most of the organs. The highest utilisation of collagen is in pharmaceutical applications including production of wound dressings, vitreous implants and as carriers for drug delivery (Kittiphattanabawon et al. 2005). The collagen extracted from porcine sources cannot be used as a component of some foods, due to religious barriers. Therefore, finding alternative sources of collagen becomes a mandate. In this line, scientists reported that skin, bone, scale, fin and cartilage of freshwater and marine fish, scallop mantle (Shen et al. 2007), adductor of pearl oyster (Mizuta et al. 2002) and the skin of the cuttlefish (Nagai et al. 2001) can be used as new sources of collagen. The squid, Sepioteuthis lessoniana, is a commercially available cephalopod in the Palk Strait of the Indian Ocean. In most of the seafood industries, skin is thrown as waste during processing of cephalopods. If substantial amount of collagen could be obtained from these wastes, they would provide alternatives to mammalian collagen in food, cosmetics and biomedical materials. Hence,
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
Natural Product Research
839
in this study, an attempt has been made to use the S. lessoniana skin as a raw material for the isolation and characterisation of acid- and pepsin-soluble collagen.
Downloaded by [Florida International University] at 02:45 04 September 2014
2. Results and discussion 2.1. Yield of collagen The yield of acid-solubilised collagen (ASC) and pepsin-solubilised collagen (PSC) was 3.83% and 11.25% on the basis of dry weight of the skin of S. lessoniana. Nagai et al. (2001) isolated 2% of ASC and 35% of PSC (on dry weight basis) from the skin of Sepia lycidas, which was lower than that of the ASC and higher than that of the PSC reported in this study. In this study, ASC was higher when compared with the ASC (1.30%) of Thysanoteuthis rhombus, whereas the PSC of S. lessoniana was low when compared with the PSC (35.6%) of T. rhombus on dry weight basis (Nagai 2004). Shanmugam et al. (2011) also reported 1.70% of ASC and 3.61% of PSC from the skin of Sepia pharaonis (on dry weight basis). In this study, the yield of ASC and PSC was considerably good with some variations when compared with that of the previous findings. Difference in the yield of collagen may be obtained by employing different concentrations of acetic acid, which was probably due to different solubility of collagen in the acidic extracting medium. 2.2. Total protein content The protein content in ASC and PSC was 79.7% and 87.75% (on dry weight basis), respectively. It was far higher than the total protein content present in the arm (9.1%) and mantle (14%) of Octopus vulgaris (Mizuta et al. 2003) and total protein contents of ASC (16.4%) and PSC (31.65%) from skin of S. pharaonis (Shanmugam et al. 2011). The higher amount of total protein content in S. lessoniana may be attributed to the fact that the whole-body skin contains more protein than the arm and mantle of O. vulgaris. 2.3. SDS-PAGE The electrophoretic pattern of ASC comprising a1 was similar to PSC comprising a1 and a2 from S. lycidas as well as to porcine collagen (Nagai et al. 2001). In this study, SDS-PAGE recorded the presence of one and two a-chains in ASC and PSC, respectively. ASC revealed two bands with molecular weights of 107 and 75 kDa and PSC revealed three bands with molecular weights of 105, 98 and 73 kDa (Figure S1). The ASC from S. pharaonis exhibited single band with a molecular weight of 107 kDa and the PSC exhibited three distinct bands with molecular weights of 117, 84 and 73 kDa (Shanmugam et al. 2011). Muyonga et al. (2004) and Yan et al. (2008) reported ASC from the skin of Nile perch and Walley pollock by using SDS-PAGE and reported that collagen consists of a1 and a2, which demonstrated two distinct species varying in their mobility, their dimer (b chain). They concluded that the existence of at least two different subunits demonstrated that the major collagen from Walley pollock skin might be the type I collagen. This is in accordance with this study and the observed molecular weight between 73 and 107 kDa which is similar to that of type I collagen. 2.4. Peptide mapping The collagens digested by achromo endopeptidase were examined by using SDS-PAGE to easily compare the pattern of peptide fragment with standard collagen. As a result, the electrophoretic pattern of PSC was more or less similar to that of ASC and standard collagen (Figure S2). For peptide maps of collagens digested by achromo endopeptidase, all components, including
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a1, b-chains, were markedly hydrolysed and the degradation peptides with the molecular weights of 106.9, 63.5, 62.07, 41.91, 40.34, 31.26, 29.64 and 15.47 kDa were obtained in both ASC and PSC. Jekel et al. (1983) reported that achromo endopeptidase is a serine endoprotease which hydrolyses peptide bonds at the carboxyl side of lysyl residues. The result suggested that squid skin collagen might contain higher lysine content. 2.5. FT-IR spectral analysis Woo et al. (2008) studied the collagen from dorsal fin of yellow fin tuna Thunnus albacores by using FT-IR and observed amide-region bands of A, I, II and III at wavelengths of 3427, 1651, 1547 and 1544 cm21, respectively. Shanmugam et al. (2011) observed that the amide-region bands of amides A, B, I, II and III in both ASC and PSC have wavelengths of 3443, 2923, 1647, 1541 and 1241 cm21 and 3420, 2922, 1649, 1542 and 1238 cm21, respectively. In this study, the amide-region bands of amides A, B, I, II and III were recorded at 3429, 2922, 1644, 1545 and 1238 cm21 for ASC and 3443, 2923, 1649, 1542 and 1243 cm21 for PSC (Figure S3). Plepis et al. (1996) observed that the peak present between 1240 cm21 (Amide III) and 1454 cm21 band confirmed the triple helical structure of the collagen isolated from the skin, scale and bone of Sebastes mentella. The amide I band position was observed at 1644 cm21 in ASC and 1649 cm21 in PSC, which is the absorption band of CvO stretching and is responsible for the secondary structure of the peptide. Similarly, transmission peaks present between 1238 cm21 (amide III) and 1458 cm21 in ASC and PSC, respectively, confirm the triple helical structure of collagen from the skin of S. lessoniana. 2.6. SEM images The SEM analysis of ASC and PSC in low magnification revealed highly porous collagen, interconnected with scaffolds, and their surface was rough and uneven. At high magnification, there was a significant difference between the microstructure of ASC and PSC (Figure S4). However, under high magnification, PSC appeared regular and has a uniform network with porous, spongiform and honeycomb-like structures with pore size on the surface ranging from few to ten micrometres. 2.7. UV –vis spectra As can be observed from the UV –vis spectra (Figure S5 B&B1), the distinct absorbance of the ASC and PSC was obtained near 220 –240 nm. In the ASC and PSC of S. pharaonis, absorbance was observed at 233.6 and 236 nm, respectively (Shanmugam et al. 2011). In general, tyrosine and phenylalanine are sensitive chromophores, and they absorb UV light at 283 and 251 nm (Liu & Liu 2006), whereas ASC and PSC exhibit no evident absorbance. Therefore, ASC and PSC of the skin from the squid S. lessoniana substantially support the property of collagen that there is absorbance at 220 –240 nm, with little or no absorbance near 280 nm. Thus, it is revealed that the protein is a collagen (Yan et al. 2008). 2.8. Differential scanning calorimetry thermogram The thermogram of ASC has one resolved peak with a To value of 59.108C and TP value of 104.918C; PSC revealed a To value of 62.188C and TP value of 98.108C. DH of 180.2 and 203.9 J/ g was found for ASC and PSC, respectively (Figure S5 A&A1). Slightly higher denaturation enthalpy (DH) value was found for PSC, as compared with ASC. The removal of telopeptides might lead to more order by structure of PSC, in which higher energy was required. The presence of imino acids, particularly hydroxyproline in ASC and PSC, might contribute to the
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stabilisation of the triple helix structure through hydrogen bonding in coil-coiled a-chains (Bae et al. 2008). In general, collagens obtained from fish species living in cold environments have lower content of hydroxyproline and they exhibit lower thermal stability than those from fish living in warm environments (Muyonga et al. 2004).
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2.9. Amino acid composition In ASC and PSC, glycine (287 – 315 residues/1000 residues) is their major amino acid, followed by proline (127.6 – 108 residues/1000 residues), alanine and hydroxyproline. The imino acid content (proline þ hydroxyproline) of ASC and PSC was 182.6 and 184 resiudes/1000 residues, respectively (Table S1), which was lower than that of calf skin collagen (215 residues/1000 residues) and skin of brown-banded bamboo shark 204 and 207 resiudes/1000 residues, but much higher than that of cod skin collagen (154 residues/1000 residues) (Duan et al. 2009; Kittiphattanabawon et al. 2010). The difference in imino acid content amongst animals was associated with the difference in the living environments of their sources, particularly habitat temperature (Regenstein & Zhou 2007). 3. Conclusion Considerable quantity of collagen (ASC and PSC) could be extracted from the skin of S. lessoniana. The collagen consists of two a-chains (a1 and a2) and was identified as type I collagen. Then, a higher imino acid content and similar ultraviolet and FT-IR spectra of ASC and PSC were observed. Pepsin-aided extraction was used as a tool for obtaining the greater yield without a marked effect on the triple-helical structure. From the results of this study, it may be inferred that there is a possibility to use the skin waste of squid S. lessoniana produced as an alternative source of collagen for industrial purposes. Supplementary material Experimental details are available online, alongside Table S1 and Figures S1 – S5. Acknowledgements Authors are thankful to the Director and Dean, Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University for providing necessary facilities. The authors (PR & NS) are thankful to the Centre for Marine Living Resources and Ecology (CMLRE), Ministry of Earth Sciences, Cochin for the financial assistance.
References Bae I, Osatomi K, Yoshida A, Osako K, Yamaguchi A, Hara K. 2008. Biochemical properties of acid-soluble collagens extracted from the skins of underutilised fishes. Food Chem. 108(1):49–54. Duan R, Zhang J, Du X, Yao X, Konno K. 2009. Properties of collagen from skin, scale and bone of carp (Cyprinus carpio). Food Chem. 112(3):702–706. Jekel PA, Weijer WJ, Beintema JJ. 1983. Use of endoproteinase Lys-C from Lysobacter enzymogenes in protein sequence analysis. Anal Biochem. 134:347–354. Kittiphattanabawon P, Benjakul S, Visessanguan W, Nagai T, Tanaka M. 2005. Characterization of acid-soluble collagen from skin and bone of bigeye snapper (Priacanthus tayenus). Food Chem. 89:363–372. Kittiphattanabawon P, Benjakul S, Visessanguan W, Shahidi F. 2010. Isolation and properties of acid- and pepsin-soluble collagen from the skin of blacktip shark (Carcharhinus limbatus). Eur Food Res Technol. 230:475–483. Liu YK, Liu DC. 2006. Comparison of physical– chemical properties of type I collagen from different species. Food Chem. 99:244–251. Mizuta S, Miyagi T, Nishimiya T, Yoshinaka R. 2002. Partial characterization of collagen in mantle and adductor of pearl oyster (Pinctada fucata). Food Chem. 79:319–325.
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Mizuta S, Tanaka T, Yoshinaka R. 2003. Comparison of collagen types of arm and mantle muscles of the common octopus Octopus vulgaris. Food Chem. 81:527–532. Muyonga JH, Cole CGB, Duodu KG. 2004. Characterization of acid soluble collagen from skins of young and adult Nile perch (Lates nilotics). Food Chem. 85:81– 89. Nagai T. 2004. Collagen from diamondback squid (Thysanoteuthis rhombus) outer skin. Z Naturforsch. 59c:271–275. Nagai T, Yamashita E, Taniguchi K, Kanamori N, Suzuki N. 2001. Isolation and characterization of collagen from the outer skin waste material of cuttlefish (Sepia lycidas). Food Chem. 72:425–429. Plepis AMDG, Goissis G, Das GDK. 1996. Dielectric and pyroelectric characterization of anionic and native collagen. Polym Eng Sci. 36(24):2932– 2938. Regenstein JM, Zhou P. 2007. Collagen and gelatin from marine by-products. In: Shahidi F, editor. Maximising the value of marine by-products. 1st ed. Cambridge: Woodhead Publishing Limited; pp.279– 303. Senaratne LS, Park P, Kim S. 2006. Isolation and characterization of collagen from brown backed toadfish (Lagocephalus gloveri) skin. Bioresour Technol. 97:191–197. Shanmugam A, Ramasamy P, Bharti MK, Saravanan R, Subhapradha N, Vairamani S, Jayalakshmi K. 2011. Isolation and characterization of collagen from the skin of Sepia pharaonis (Ehrenberg, 1831). Int J Curr Res. 33(6):107–111. Shen XR, Kurihara H, Takahashi K. 2007. Characterization of molecular species of collagen in scallop mantle. Food Chem. 102:1187–1191. Woo JW, Yu SJ, Cho SM, Lee YB, Kim SB. 2008. Extraction optimization and properties of collagen from yellow fin tuna Thunnus albacares dorsal skin. Food Hydrocolloids. 22:879–887. Yan M, Li B, Zhao X, Ren G, Zhuang Y, Hou H, Zhang X, Chen L, Fan Y. 2008. Characterization of acid-soluble collagen from the skin of walleye Pollock Theragra chalcogramma. Food Chem. 107:1581–1586.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Isolation and structural characterisation of acid- and pepsinsoluble collagen from the skin of squid Sepioteuthis lessoniana (Lesson, 1830) a
a
Pasiyappazham Ramasamy , Namasivayam Subhapradha , a
a
Vairamani Shanmugam & Annaian Shanmugam a
Centre of Advanced Study in Marine Biology, , Faculty of Marine Sciences, Annamalai University, Parangipettai 608 502, Tamil Nadu, India Published online: 31 Jan 2014.
To cite this article: Pasiyappazham Ramasamy, Namasivayam Subhapradha, Vairamani Shanmugam & Annaian Shanmugam (2014) Isolation and structural characterisation of acid- and pepsin-soluble collagen from the skin of squid Sepioteuthis lessoniana (Lesson, 1830), Natural Product Research: Formerly Natural Product Letters, 28:11, 838-842, DOI: 10.1080/14786419.2014.880914 To link to this article: http://dx.doi.org/10.1080/14786419.2014.880914
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [Florida International University] at 02:45 04 September 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 11, 838–842, http://dx.doi.org/10.1080/14786419.2014.880914
SHORT COMMUNICATION Isolation and structural characterisation of acid- and pepsin-soluble collagen from the skin of squid Sepioteuthis lessoniana (Lesson, 1830)
Downloaded by [Florida International University] at 02:45 04 September 2014
Pasiyappazham Ramasamy*, Namasivayam Subhapradha, Vairamani Shanmugam and Annaian Shanmugam Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai 608 502, Tamil Nadu, India (Received 7 October 2013; final version received 26 December 2013) Acid-solubilised collagen (ASC) and pepsin-solubilised collagen (PSC) were effectively isolated from squid skin with good yield and total protein content. ASC and PSC consist of two a-chains with an imino acid content of 182.6 and 184 imino acid residues/1000 residues. The molecular weight was determined to be between 73 and 107 kDa by using SDS-PAGE. For peptide mapping, collagens were digested with achromo endopeptidase, and all components, including a, b-chains, were markedly hydrolysed. Degradation peptides with molecular weights between 106.9 and 15.47 kDa were obtained. UV – vis absorption spectrum revealed distinct absorption at 220– 240 nm. FT-IR spectra of collagens were almost similar when compared with standard. In differential scanning calorimetry profile, ASC and PSC exhibited a To of 59.10, 62.188C and TP of 104.91, 98.10 8C, respectively. This investigation indicates that the collagen isolated from the squid skin, which is thrown as waste in the seafoodprocessing plant, might supplement the vertebrate collagen in industrial applications. Keywords: characterisation; collagen; Sepioteuthis lessoniana; squid skin
1. Introduction Collagen is a major protein present abundantly in animal tissues and constitutes approximately 30% of total animal protein (Muyonga et al. 2004). It is extensively distributed in skin, bone, cartilage, tendons, ligaments, blood vessels, teeth, cornea and other organs of vertebrates (Senaratne et al. 2006), and they contribute as a structural framework to the tissues of most of the organs. The highest utilisation of collagen is in pharmaceutical applications including production of wound dressings, vitreous implants and as carriers for drug delivery (Kittiphattanabawon et al. 2005). The collagen extracted from porcine sources cannot be used as a component of some foods, due to religious barriers. Therefore, finding alternative sources of collagen becomes a mandate. In this line, scientists reported that skin, bone, scale, fin and cartilage of freshwater and marine fish, scallop mantle (Shen et al. 2007), adductor of pearl oyster (Mizuta et al. 2002) and the skin of the cuttlefish (Nagai et al. 2001) can be used as new sources of collagen. The squid, Sepioteuthis lessoniana, is a commercially available cephalopod in the Palk Strait of the Indian Ocean. In most of the seafood industries, skin is thrown as waste during processing of cephalopods. If substantial amount of collagen could be obtained from these wastes, they would provide alternatives to mammalian collagen in food, cosmetics and biomedical materials. Hence,
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
Natural Product Research
839
in this study, an attempt has been made to use the S. lessoniana skin as a raw material for the isolation and characterisation of acid- and pepsin-soluble collagen.
Downloaded by [Florida International University] at 02:45 04 September 2014
2. Results and discussion 2.1. Yield of collagen The yield of acid-solubilised collagen (ASC) and pepsin-solubilised collagen (PSC) was 3.83% and 11.25% on the basis of dry weight of the skin of S. lessoniana. Nagai et al. (2001) isolated 2% of ASC and 35% of PSC (on dry weight basis) from the skin of Sepia lycidas, which was lower than that of the ASC and higher than that of the PSC reported in this study. In this study, ASC was higher when compared with the ASC (1.30%) of Thysanoteuthis rhombus, whereas the PSC of S. lessoniana was low when compared with the PSC (35.6%) of T. rhombus on dry weight basis (Nagai 2004). Shanmugam et al. (2011) also reported 1.70% of ASC and 3.61% of PSC from the skin of Sepia pharaonis (on dry weight basis). In this study, the yield of ASC and PSC was considerably good with some variations when compared with that of the previous findings. Difference in the yield of collagen may be obtained by employing different concentrations of acetic acid, which was probably due to different solubility of collagen in the acidic extracting medium. 2.2. Total protein content The protein content in ASC and PSC was 79.7% and 87.75% (on dry weight basis), respectively. It was far higher than the total protein content present in the arm (9.1%) and mantle (14%) of Octopus vulgaris (Mizuta et al. 2003) and total protein contents of ASC (16.4%) and PSC (31.65%) from skin of S. pharaonis (Shanmugam et al. 2011). The higher amount of total protein content in S. lessoniana may be attributed to the fact that the whole-body skin contains more protein than the arm and mantle of O. vulgaris. 2.3. SDS-PAGE The electrophoretic pattern of ASC comprising a1 was similar to PSC comprising a1 and a2 from S. lycidas as well as to porcine collagen (Nagai et al. 2001). In this study, SDS-PAGE recorded the presence of one and two a-chains in ASC and PSC, respectively. ASC revealed two bands with molecular weights of 107 and 75 kDa and PSC revealed three bands with molecular weights of 105, 98 and 73 kDa (Figure S1). The ASC from S. pharaonis exhibited single band with a molecular weight of 107 kDa and the PSC exhibited three distinct bands with molecular weights of 117, 84 and 73 kDa (Shanmugam et al. 2011). Muyonga et al. (2004) and Yan et al. (2008) reported ASC from the skin of Nile perch and Walley pollock by using SDS-PAGE and reported that collagen consists of a1 and a2, which demonstrated two distinct species varying in their mobility, their dimer (b chain). They concluded that the existence of at least two different subunits demonstrated that the major collagen from Walley pollock skin might be the type I collagen. This is in accordance with this study and the observed molecular weight between 73 and 107 kDa which is similar to that of type I collagen. 2.4. Peptide mapping The collagens digested by achromo endopeptidase were examined by using SDS-PAGE to easily compare the pattern of peptide fragment with standard collagen. As a result, the electrophoretic pattern of PSC was more or less similar to that of ASC and standard collagen (Figure S2). For peptide maps of collagens digested by achromo endopeptidase, all components, including
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P. Ramasamy et al.
Downloaded by [Florida International University] at 02:45 04 September 2014
a1, b-chains, were markedly hydrolysed and the degradation peptides with the molecular weights of 106.9, 63.5, 62.07, 41.91, 40.34, 31.26, 29.64 and 15.47 kDa were obtained in both ASC and PSC. Jekel et al. (1983) reported that achromo endopeptidase is a serine endoprotease which hydrolyses peptide bonds at the carboxyl side of lysyl residues. The result suggested that squid skin collagen might contain higher lysine content. 2.5. FT-IR spectral analysis Woo et al. (2008) studied the collagen from dorsal fin of yellow fin tuna Thunnus albacores by using FT-IR and observed amide-region bands of A, I, II and III at wavelengths of 3427, 1651, 1547 and 1544 cm21, respectively. Shanmugam et al. (2011) observed that the amide-region bands of amides A, B, I, II and III in both ASC and PSC have wavelengths of 3443, 2923, 1647, 1541 and 1241 cm21 and 3420, 2922, 1649, 1542 and 1238 cm21, respectively. In this study, the amide-region bands of amides A, B, I, II and III were recorded at 3429, 2922, 1644, 1545 and 1238 cm21 for ASC and 3443, 2923, 1649, 1542 and 1243 cm21 for PSC (Figure S3). Plepis et al. (1996) observed that the peak present between 1240 cm21 (Amide III) and 1454 cm21 band confirmed the triple helical structure of the collagen isolated from the skin, scale and bone of Sebastes mentella. The amide I band position was observed at 1644 cm21 in ASC and 1649 cm21 in PSC, which is the absorption band of CvO stretching and is responsible for the secondary structure of the peptide. Similarly, transmission peaks present between 1238 cm21 (amide III) and 1458 cm21 in ASC and PSC, respectively, confirm the triple helical structure of collagen from the skin of S. lessoniana. 2.6. SEM images The SEM analysis of ASC and PSC in low magnification revealed highly porous collagen, interconnected with scaffolds, and their surface was rough and uneven. At high magnification, there was a significant difference between the microstructure of ASC and PSC (Figure S4). However, under high magnification, PSC appeared regular and has a uniform network with porous, spongiform and honeycomb-like structures with pore size on the surface ranging from few to ten micrometres. 2.7. UV –vis spectra As can be observed from the UV –vis spectra (Figure S5 B&B1), the distinct absorbance of the ASC and PSC was obtained near 220 –240 nm. In the ASC and PSC of S. pharaonis, absorbance was observed at 233.6 and 236 nm, respectively (Shanmugam et al. 2011). In general, tyrosine and phenylalanine are sensitive chromophores, and they absorb UV light at 283 and 251 nm (Liu & Liu 2006), whereas ASC and PSC exhibit no evident absorbance. Therefore, ASC and PSC of the skin from the squid S. lessoniana substantially support the property of collagen that there is absorbance at 220 –240 nm, with little or no absorbance near 280 nm. Thus, it is revealed that the protein is a collagen (Yan et al. 2008). 2.8. Differential scanning calorimetry thermogram The thermogram of ASC has one resolved peak with a To value of 59.108C and TP value of 104.918C; PSC revealed a To value of 62.188C and TP value of 98.108C. DH of 180.2 and 203.9 J/ g was found for ASC and PSC, respectively (Figure S5 A&A1). Slightly higher denaturation enthalpy (DH) value was found for PSC, as compared with ASC. The removal of telopeptides might lead to more order by structure of PSC, in which higher energy was required. The presence of imino acids, particularly hydroxyproline in ASC and PSC, might contribute to the
Natural Product Research
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stabilisation of the triple helix structure through hydrogen bonding in coil-coiled a-chains (Bae et al. 2008). In general, collagens obtained from fish species living in cold environments have lower content of hydroxyproline and they exhibit lower thermal stability than those from fish living in warm environments (Muyonga et al. 2004).
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2.9. Amino acid composition In ASC and PSC, glycine (287 – 315 residues/1000 residues) is their major amino acid, followed by proline (127.6 – 108 residues/1000 residues), alanine and hydroxyproline. The imino acid content (proline þ hydroxyproline) of ASC and PSC was 182.6 and 184 resiudes/1000 residues, respectively (Table S1), which was lower than that of calf skin collagen (215 residues/1000 residues) and skin of brown-banded bamboo shark 204 and 207 resiudes/1000 residues, but much higher than that of cod skin collagen (154 residues/1000 residues) (Duan et al. 2009; Kittiphattanabawon et al. 2010). The difference in imino acid content amongst animals was associated with the difference in the living environments of their sources, particularly habitat temperature (Regenstein & Zhou 2007). 3. Conclusion Considerable quantity of collagen (ASC and PSC) could be extracted from the skin of S. lessoniana. The collagen consists of two a-chains (a1 and a2) and was identified as type I collagen. Then, a higher imino acid content and similar ultraviolet and FT-IR spectra of ASC and PSC were observed. Pepsin-aided extraction was used as a tool for obtaining the greater yield without a marked effect on the triple-helical structure. From the results of this study, it may be inferred that there is a possibility to use the skin waste of squid S. lessoniana produced as an alternative source of collagen for industrial purposes. Supplementary material Experimental details are available online, alongside Table S1 and Figures S1 – S5. Acknowledgements Authors are thankful to the Director and Dean, Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University for providing necessary facilities. The authors (PR & NS) are thankful to the Centre for Marine Living Resources and Ecology (CMLRE), Ministry of Earth Sciences, Cochin for the financial assistance.
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