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Biochemical properties of collagen from skin of Gulf coast fish M. 1 OGAWA , M.W. 1 Moody , R.J. 3 Portier , J. 1 Bell , M. 2 Schexnayder , and J.N. 1 Losso 1. Department of Food Science and 2. Fisheries Agent, Louisiana State University Agricultural Center, Baton Rouge, LA 70803 3. Department of Environmental Studies, Louisiana State University, Baton Rouge, LA 70803 EXPERIMENTAL Acid-soluble collagen (ASC) was isolated from the skin of black drum (Pogonias cromis) according to a conventional method. The molecular properties of collagen were analyzed by SDS-PAGE and circular dichroism (CD) spectroscopy. Denaturation temperature (Tm) of ASC was determined based on the melting curve of collagen obtained by monitoring [θ] at the wavelength of a positive extreme at 220 nm which is characteristic of the CD spectrum for collagen triple helix. 20000 Molecular mass and Molecular type •CD spectrum of the native ASC sample showed that the ASC formed collagen triple helices (Fig. 2). • Black drum skin ASC has at least two different chains (1 and 2) and their cross-linked chains (Fig. 1). •The structure was lost at 45 ºC. •Molecular mass of each chain was 126 kDa for 1 and 116 kDa for 2. 200kDa •The molecular mass of ASC ([1] 22) was 360kDa. 116kDa 97kDa 67kDa 55kDa Amino acid composition • Black drum ASC was rich in Pro, Gly, and Ala, which are characteristic of all collagens (Table 1). • High levels of hydroxylysine (Hyl) and hydroxyproline (Hyp), similar to collagens from animal species, were measured in black drum ASC. 0 205.0 215.0 225.0 235.0 245.0 -10000 Denatured collagen (45 ºC, 30min) -20000 -30000 -40000 Native collagen (15 ºC) -50000 -60000 -70000 10000 Denaturation temperature 1 2 • Transition temperature Tm for black drum ASC was 34.8 ºC, which is similar to that of calf hide ASC (36.3 ºC) (Fig. 3). 8000 6000 4000 2000 0 -2000 -4000 10 20 30 40 50 Temperature (ºC) Fig. 3. Melting curve of black drum skin ASC CONCLUSIONS Table 1. Amino acid composition of skin collagens Black drum Coda Calfb Hyp 80.2±7.0 40.7 94 Asx 43.9±1.1 41.5 45 Thr 25.9±0.6 26.1 18 Ser 40.7±2.3 60.8 33 Glx 70.1±2.3 77.4 75 Pro 119.6±2.7 89.6 121 Gly 319.8±9.8 332.2 330 Ala 128.7±2.4 105.6 119 Cys 0.0±0.0 ND ND Val 19.6±0.5 25.2 21 Met 14.2±0.5 20.6 6 6.9±0.4 17.1 11 Leu 20.8±0.5 29.7 23 Tyr 4.0±0.3 5.0 3 Phe 13.8±0.8 13.8 13 Hyl 5.8±0.9 7.6 7 Lys 23.5±2.8 33.1 26 His 6.0±0.5 11.8 5 Arg 56.5±1.1 62.5 50 Ile • The distribution patterns of amino acid composition were closer, especially for Pro and Hyp, to calf ASC than to cod ASC. 10000 Fig. 2. CD spectra of black drum skin ASC Fig. 1. SDS-PAGE pattern of skin collagen using NuPAGE Tris-Acetate gel (3-8%) (Invitrogen) Black drum Collagen isolated from land-based animal skin is presently used for functional food, healthcare, and pharmaceutical applications. However, in light of BSE and food-and-mouth disease crisis, and the ban on European Union meat, the use of collagen and collagenderived products from land-based animal skin has been called into question. Collagens from some other sources are needed. Fish skin collagen is thought to be a good candidate. The objective in this study was to isolate fish skin collagen and investigate its biochemical properties in order to determine the suitability of fish skin collagen as an alternate of land-based animal collagen. Wavelength (nm) [θ]220 (deg cm2 dmol-1) Acid-soluble collagen (ASC) was isolated from the skin of black drum harvested in the Gulf of Mexico Coastal waters. Analyses of molecular weight profile, amino acid composition, and secondary structure showed that the black drum ASC was typical type-1 collagen. The molecular mass of 1 and 2 subunits, as determined by SDS-PAGE, was 127 kDa and 116 kDa, respectively. The amino acid composition of black drum ASC was closer to calf skin ASC than to cod skin ASC. Thermal denaturation temperature, measured by melting curve using circular dichroism, was 34.8 ºC. The literature value for the heat stability of calf skin collagen is 36.3 ºC. The potentials of collagen from black drum skin in the functional food, healthcare, and pharmaceutical industries are discussed. INTRODUCTION Secondary structure RESULTS and DISCUSSION [θ] (deg cm2 dmol-1) ABSTRACT Total 1000 1000 aYamaguchi et al.(1976); bHerbage et al. (1977). 1000 • Black drum skin ASC had molecular mass and secondary structure of a typical type I collagen. • Black drum skin ASC was similar to calf skin ASC rather than cod skin ASC in terms of thermal stability and amino acid composition. • High stability of black drum ASC may be due to high Hyl and Pro contents. • The ASC from Gulf coast fish skin may be useful for functional food, healthcare, and pharmaceutical applications. ACKNOWLEDGEMENTS This work was supported by a grant from the US Department of Commerce through Louisiana Sea Grant Project # 167-14-5114. We wish to thank Mr. Harlon Pearce of LA Fish (Kenner, LA) for supplying the fish skins used in this research. REFERENCES •Yamaguchi, K.; Lavéty, J.; Love, R.M. J. Fd. Technol. 1976, 11, 389399. •Herbage, D.; Bouillet, J.; Bernengo, J.-C. Biochem. J. 1977, 161, 303312.