Analytical and Bioanalytical Chemistry

, Volume 405, Issue 10, pp 3351–3358 | Cite as

Fluorescence properties of Ca2+-independent discharged obelin and its application prospects

  • Roza R. Alieva
  • Nadezhda V. Belogurova
  • Alena S. Petrova
  • Nadezhda S. Kudryasheva
Original Paper

Abstract

Discharged obelin, a complex of coelenteramide and polypeptide, is a fluorescent protein produced from the photoprotein obelin, which is responsible for bioluminescence of the marine hydroid Obelia longissima. Discharged obelin is stable and nontoxic and its spectra are variable, and this is why it can be used as a fluorescent biomarker of variable color in vivo and in vitro. Here we examined light-induced fluorescence of Ca2+-independent discharged obelin (obtained without addition of Ca2+). Its emission and excitation spectra were analyzed under variation of the excitation wavelength (260–390 nm) and the emission wavelength (400–700 nm), as well as the 40 °C exposure time. The emission spectra obtained with excitation at 260–300 nm (tryptophan absorption region) included three peaks with maxima at 355, 498, and 660 nm, corresponding to fluorescence of tryptophan, polypeptide-bound coelenteramide, and a hypothetical indole–coelenteramide exciplex, respectively. The emission spectra obtained with excitation at 310–380 nm (coelenteramide absorption region) did not include the 660-nm maximum. The peak in the red spectral region (λmax = 660 nm) has not been previously reported. Exposure to 40 °C under excitation at 310–380 nm shifted the obelin fluorescence spectra to the blue, whereas excitation at 260–300 nm shifted them to the red. Hence, red emission and variation of the excitation wavelength form a basis for development of new medical techniques involving obelin as a colored biomarker. The addition of red color to the battery of known (violet to yellow) colors increases the potential of application of obelin.

Keywords

Fluorescent protein Discharged photoprotein obelin Fluorescence color Thermoinactivation Excitation wavelength Proton transfer 

Abbreviations

EDTA

ethylenediaminetetraacetic acid

PIPES

piperazine-N,N′-bis(2-ethanesulfonic acid)

Tris

tris(hydroxymethyl)aminomethane

References

  1. 1.
    Vysotski ES, Lee J (2004) Ca2+-regulated photoproteins: structural insight into the bioluminescence mechanism. Acc Chem Res 37:405–415CrossRefGoogle Scholar
  2. 2.
    Vysotski ES, Markova SV, Frank LA (2006) Calcium-regulated photoproteins of marine coelenterates. Mol Biol 40:355–367CrossRefGoogle Scholar
  3. 3.
    van Oort B, Eremeeva EV et al (2009) Picosecond fluorescence relaxation spectroscopy of the calcium-discharged photoproteins aequorin and obelin. Biochemistry 4:10486–10491CrossRefGoogle Scholar
  4. 4.
    Belogurova NV, Kudryasheva NS (2010) Discharged photoprotein obelin: fluorescence peculiarities. Photochem Photobiol B 101:103–108CrossRefGoogle Scholar
  5. 5.
    Liu ZJ, Stepanyuk GA, Vysotski ES, Lee J, Markova SV, Malikova NP, Wang BC (2006) Crystal structure of obelin after Ca2+-triggered bioluminescence suggests neutral coelenteramide as the primary excited state. Proc Natl Acad Sci USA 103:2570–2575CrossRefGoogle Scholar
  6. 6.
    Frank LA, Petunin AI, Vysotski ES (2004) Conjugates of the Ca2 + -regulated photoprotein obelin with immunoglobulins: synthesis and use as labels in bioluminescent immunoassay. Bioorg Chem (Russia) 30:364–368Google Scholar
  7. 7.
    Frank LA, Borisova VV, Markova SV, Malikova NP, Stepanyuk GA, Vysotski ES (2008) Violet and greenish photoprotein obelin mutants for reporter application in dual-color assay. Anal Bional Chem 391:2891–2896CrossRefGoogle Scholar
  8. 8.
    Malikova NP, Stepanyuk GA, Frank LA, Markova SV, Vysotski ES, Lee J (2003) Spectral tuning of obelin bioluminescence by mutations of Trp92. FEBS Lett 554:184–188CrossRefGoogle Scholar
  9. 9.
    Belogurova NV, Kudryasheva NS, Alieva RR, Sizykh AG (2008) Spectral components of bioluminescence of aequorin and obelin. Photochem Photobiol B Biol 92:117–122CrossRefGoogle Scholar
  10. 10.
    Allen DG, Вlinks JR, Prendergast FG (1977) Аеquorin luminescence: relation of 1ight emission to calcium concentration—а calcium-independent соmponent. Science 195:996–998CrossRefGoogle Scholar
  11. 11.
    Shimomura O, Johnson FH, Saiga Y (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. Cell Comp Physiol 59:223–239CrossRefGoogle Scholar
  12. 12.
    Shimomura O (1995) Cause of spectral variation in the luminescence of semisynthetic aequorins. Biochemistry 306:537–543Google Scholar
  13. 13.
    Head JF, Inouye S, Teranishi K, Shimomura O (2000) The crystal structure of the photoprotein aequorin at 2.3 Å resolution. Nature 405:372–376CrossRefGoogle Scholar
  14. 14.
    Mithofer A, Mazars C (2002) Aequorin-based measurements of intracellular Ca2+-signatures in plant cells. Biol Proc 4:105–118CrossRefGoogle Scholar
  15. 15.
    Illarionov BA, Frank LA, Illarionova VA, Bondar VS, Vysotski ES, Blinks JR (2000) Recombinant obelin: cloning, and expression of cDNA, purification, and characterization as a calcium indicator. Methods Enzymol 277:223–249CrossRefGoogle Scholar
  16. 16.
    Markova SV, Vysotski ES, Blinks JR, Burakova LP, Wang BC, Lee J (2002) Obelin from the bioluminescent marine hydroid Obelia geniculata: cloning, expression, and comparison of some properties with those of other Ca2+-regulated photoproteins. Biochemistry 41:2227–2236CrossRefGoogle Scholar
  17. 17.
    Yacimirski KB, Malikova TV (1984) Spectroscopic methods in chemistry of complex. Khimiya, MoscowGoogle Scholar
  18. 18.
    Mikhailenko VI, Redkin YuR (1979) Resolution of overlapped asymmetric bands. Zh Prikl Spectrosk 31:919–921Google Scholar
  19. 19.
    Shimomura O, Teranishi K (2000) Light-emitters involved in the luminescence of coelenterazine. Luminescence 15:51–58CrossRefGoogle Scholar
  20. 20.
    Imai Y, Shibata T, Maki S, Niwa S, Ohashi M, Hirano T (2001) Fluorescence properties of phenolate anions of coelenteramide analogues: the light-emitter structure in aequorin bioluminescence. Photochem Photobiol A 146:95–107CrossRefGoogle Scholar
  21. 21.
    Mori K, Maki S, Niwa H, Ikedab H, Hirano T (2006) Real light emitter in the bioluminescence of the calcium-activated photoproteins aequorin and obelin: light emission from the singlet-excited state of coelenteramide phenolateanion in a contact ion pair. Tetrahedron Lett 62:6272–6288CrossRefGoogle Scholar
  22. 22.
    Hirano T, Ohmiya Y, Maki S, Niwa H, Ohashi M (1998) Bioluminescent properties of fluorinate semisynthetic aequorins. Tetrahedron Lett 39:5541–5544CrossRefGoogle Scholar
  23. 23.
    Deng L, Vysotski ES, Lui ZJ, Markova SV, Malikova NP, Lee J, Rose J, Wang BC (2001) Structural basis for the emission of violet bioluminescence from a W92F obelin mutant. FEBS Lett 506:281–285CrossRefGoogle Scholar
  24. 24.
    Vysotski ES, Liu ZJ, Markova SV, Blinks JR, Deng L, Frank LA, Herko M, Malikova NP, Rose JP, Wang BC, Lee J (2003) Violet bioluminescence and fast kinetics from W92F obelin: structure-based proposals for the bioluminescence triggering and the identification of the emitting species. Biochemistry 42:6013–6024CrossRefGoogle Scholar
  25. 25.
    Tomilin FN, Antipina LU, Vysotski ES, Ovchinnikov SG, Gitelzon II (2008) Fluorescence of calcium-discharged obelin: the structure and molecular mechanism of emitter formation. Biochem Biophys Mol Biol 422:279–284Google Scholar
  26. 26.
    Tomilin FN, Antipina LU, Eremeeva EV, Ovchinnikov SG, Vysotski ES (2010) Quantum chemical study of mechanism of active photoprotein generation. Luminescence 25:210–211Google Scholar
  27. 27.
    Vekshin NL (2002) Photonics of biopolymers. Springer, HeidelbergCrossRefGoogle Scholar
  28. 28.
    Titushin MS, Markova SV, Frank LA, Malikova NP, Stepanyuk GA, Lee J, Vysotski ES (2008) Coelenterazine-binding protein of Renilla muelleri: cDNA cloning, overexpression, and characterization as a substrate of luciferase. Photochem Photobiol Sci 7(2):189–196CrossRefGoogle Scholar
  29. 29.
    Zubova NN, Bulavina AY, Savitsky AP (2003) Spectral and physicochemical properties of green (GFP) and red (drFP583) fluorescent proteins. Usp Biol Khim 43:163–224Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Roza R. Alieva
    • 1
  • Nadezhda V. Belogurova
    • 1
    • 2
  • Alena S. Petrova
    • 1
  • Nadezhda S. Kudryasheva
    • 1
    • 2
  1. 1.Siberian Federal UniversityKrasnoyarskRussia
  2. 2.Institute of Biophysics SB RASKrasnoyarskRussia

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