Fisheries Science

, 75:1461 | Cite as

A novel fluorescent protein purified from eel muscle

Original Article Chemistry and Biochemistry


We discovered that some isolated eel skeletal muscle cells exhibited green fluorescence under a fluorescence stereomicroscope, and we successfully isolated a novel fluorescent protein from the eel muscle homogenate. The protein was a monomer with a molecular mass of 16.5–17 kDa and showed minor and major peaks at 280 and 493 nm, respectively, in the absorption spectrum. The molar extinction coefficient at 493 nm was 41,300 M−1 cm−1 and A280/A493 was 0.083. Excitation and emission spectra of the protein showed maxima at 493 and 527 nm, respectively. Heat treatment at 95°C for 10 min or 5% trichloroacetic acid treatment of the protein caused aggregation of the protein but did not release any fluorescent components such as FAD into the supernatant after centrifugation. Fluorescence of the protein remained after native PAGE, but not after SDS-PAGE. These results indicate that the purified fluorescent protein is not a flavoprotein, and that its fluorescent chromophore is a covalently bound one, such as green fluorescent protein (GFP) from jellyfish Aequoria victoria, but that its fluorescence requires its native conformation within the protein. Based on these results, we can conclude that the fluorescent protein obtained from eel skeletal muscle is a novel GFP-like protein.


Eel Fluorescent protein GFP GFP-like protein Isolated muscle cells Skeletal muscle 


  1. 1.
    Babin PJ, Vernier J-M (1989) Plasma lipoproteins in fish. J Lipid Res 30:467–489PubMedGoogle Scholar
  2. 2.
    Hayashi S, Komatsu M (1999) Primary culture of eel hepatocytes—synthesis and secretion of lipoprotein. In: Doyle A, Griffiths JB, Newell DG (eds) Cell and tissue culture: laboratory procedures. Wiley, New York, pp 23A10–23A21Google Scholar
  3. 3.
    Kagawa A (1983) Shokuhin seibun-hyo. (Standard tables of food composition in Japan.) In: The Resource Investigation Committee, The Science and Technology Agency, Japan (ed) Shokuhin seibun-hyo. (Standard tables of food composition in Japan, 4th edn.) Kagawa Nutrition University Publishing Division, Tokyo, p 89 (in Japanese)Google Scholar
  4. 4.
    Job V, Marcone GL, Pilone MS, Pollegioni L (2002) Glycine oxidase from Bacillus subtilis. Characterization of a new flavoprotein. J Biol Chem 277:6985–6993CrossRefPubMedGoogle Scholar
  5. 5.
    Simonetta MP, Pollegioni L, Casalin P, Curti B, Ronchi S (1989) Properties of d-amino-acid oxidase from Rhodotorula gracilis. Eur J Biochem 180:199–204Google Scholar
  6. 6.
    Wagner MA, Khanna P, Jorns MS (1999) Structure of the flavocoenzyme of two homologous amine oxidases: monomeric sarcosine oxidase and N-methyltryptophan oxidase. Biochemistry 38:5588–5595CrossRefPubMedGoogle Scholar
  7. 7.
    Mihalik SJ, McGuinness M, Watkins PA (1991) Purification and characterization of peroxisomal l-pipecolic acid oxidase from monkey liver. J Biol Chem 266:4822–4830Google Scholar
  8. 8.
    Rosenblatt JD, Lunt AI, Parry DJ, Partridge TA (1995) Culturing satellite cells from living single muscle fiber explants. In Vitro Cell Dev Biol 31:773–779CrossRefGoogle Scholar
  9. 9.
    Alam N, Nakamura K, Hayashi S (2004) Lipoprotein metabolism in a coculture system with eel skeletal muscle cells and hepatocytes. Fish Sci 70:326–335CrossRefGoogle Scholar
  10. 10.
    Rosenfeld J, Capdevielle J, Guillemot JC, Ferrara P (1992) In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal Biochem 203:173–179CrossRefPubMedGoogle Scholar
  11. 11.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  12. 12.
    Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85CrossRefPubMedGoogle Scholar
  13. 13.
    Morise H, Shimomura O, Johnson FH, Winant J (1974) Intermolecular energy transfer in the bioluminescent system of Aequorea. Biochemistry 13:2656–2662CrossRefPubMedGoogle Scholar
  14. 14.
    Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544CrossRefPubMedGoogle Scholar
  15. 15.
    Miyawaki A, Nagai T, Mizuno H (2003) Mechanisms of protein fluorophore formation and engineering. Curr Opin Chem Biol 7:557–562CrossRefPubMedGoogle Scholar
  16. 16.
    Karasawa S, Araki T, Yamamoto-Hino M, Miyawaki A (2003) A green-emitting fluorescent protein from Galaxeidae coral and its monomeric version for use in fluorescent labeling. J Biol Chem 278:34167–34171CrossRefPubMedGoogle Scholar
  17. 17.
    Karasawa S, Araki T, Nagai T, Mizuno H, Miyawaki A (2004) Cyan-emitting and orange-emitting fluorescent proteins as a doner/acceptor pair for fluorescence resonance energy transfer. Biochem J 381:307–312CrossRefPubMedGoogle Scholar
  18. 18.
    Cubitt AB, Woollenweber LA, Heim R (1999) Understanding structure-function relationships in the Aequorea victoria green fluorescent protein. Meth Cell Biol 58:19–30CrossRefGoogle Scholar
  19. 19.
    Bokman SH, Ward WW (1981) Renaturation of Aequorea green-fluorescent protein. Biochem Biophys Res Commun 101:1372–1380CrossRefPubMedGoogle Scholar
  20. 20.
    Ward WW, Bokman SH (1982) Reversible denaturation of Aequorea green-fluorescent protein: physical separation and characterization of the renatured protein. Biochemistry 21:4535–4540CrossRefPubMedGoogle Scholar
  21. 21.
    Shimomura O (1979) Structure of the chromophore of Aequorea green fluorescent protein. FEBS Lett 104:220–222CrossRefGoogle Scholar
  22. 22.
    Cody CW, Prasher DC, Westler WM, Prendergast FG, Ward WW (1993) Chemical structure of hexapeptide chromophore of the Aequorea green-fluorescent protein. Biochemistry 32:1212–1218CrossRefPubMedGoogle Scholar
  23. 23.
    Miyawaki A (2002) Green fluorescent protein-like proteins in reef Anthozoa animals. Cell Struct Funct 27:343–347CrossRefPubMedGoogle Scholar
  24. 24.
    Lippincott-Schwartz J, Patterson GH (2003) Development and use of fluorescent protein markers in living cells. Science 300:87–91CrossRefPubMedGoogle Scholar

Copyright information

© The Japanese Society of Fisheries Science 2009

Authors and Affiliations

  1. 1.Laboratory of Food Chemistry, Faculty of FisheriesKagoshima UniversityKagoshimaJapan
  2. 2.KagoshimaJapan

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