Analytical and Bioanalytical Chemistry

, Volume 410, Issue 26, pp 6837–6844 | Cite as

Protein-based fluorescent bioassay for low-dose gamma radiation exposures

  • Alena S. Petrova
  • Anna A. Lukonina
  • Dmitry V. Dementyev
  • Alexander Ya. Bolsunovsky
  • Anatoliy V. Popov
  • Nadezhda S. KudryashevaEmail author
Research Paper


The study suggests an application of a coelenteramide-containing fluorescent protein (CLM-CFP) as a simplest bioassay for gamma radiation exposures. “Discharged obelin,” a product of the bioluminescence reaction of the marine coelenterate Obelia longissima, was used as a representative of the CLM-CFP group. The bioassay is based on a simple enzymatic reaction—photochemical proton transfer in the coelenteramide-apoprotein complex. Components of this reaction differ in fluorescence color, providing, by this, an evaluation of the proton transfer efficiency in the photochemical process. This efficiency depends on the microenvironment of the coelenteramide within the protein complex, and, hence, can evaluate a destructive ability of gamma radiation. The CLM-CFP samples were exposed to gamma radiation (137Cs, 2 mGy/h) for 7 and 16 days at 20 °C and 5 °C, respectively. As a result, two fluorescence characteristics (overall fluorescence intensity and contributions of color components to the fluorescence spectra) were identified as bioassay parameters. Both parameters demonstrated high sensitivity of the CLM-CFP-based bioassay to the low-dose gamma radiation exposure (up to 100 mGy). Higher temperature (20 °C) enhanced the response of CLM-CFP to gamma radiation. This new bioassay can provide fluorescent multicolor assessment of protein destruction in cells and physiological liquids under exposure to low doses of gamma radiation.

Graphical abstract


Bioassay Enzymes Fluorescence/luminescence Fluorescent protein Radiotoxicity Gamma radiation 



The authors would like to thank Alejandro D. Arroyo, University of Pennsylvania, for critical review of the manuscript.

Funding information

This work was supported by the state budget allocated to the fundamental research at the Russian Academy of Sciences, project 01201351504; the Russian Foundation for Basic Research, Grant No. 16-34-00695; and the Krasnoyarsk Regional Fund of Science and Technology Support. AVP’s research reported in this publication was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under award number UL1TR000003.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Ilyin LA, Kutsenko SA, Savateev NV, Sofronov GA, Tiunov LA. Toxicological problems in mitigation strategies of chemical industries. 1990;35:440–7.Google Scholar
  2. 2.
    Bisswanger H. Enzyme assays. Report Enzymol Data – STRENDA Recomm Beyond. 2014;1:41–55. Scholar
  3. 3.
    Acker MG, Auld DS. Considerations for the design and reporting of enzyme assays in high-throughput screening applications. Report Enzymol Data – STRENDA Recomm Beyond. 2014;1:56–73. Scholar
  4. 4.
    Kratasyuk VA, Esimbekova EN. Applications of luminous bacteria enzymes in toxicology. Comb Chem High Throughput Screen. 2015;18:952–9. Scholar
  5. 5.
    Zhikrevetskaya S, Peregudova D, Danilov A, Plyusnina E, Krasnov G, Dmitriev A, et al. Effect of low doses (5-40 cGy) of gamma-irradiation on lifespan and stress-related genes expression profile in Drosophila melanogaster. PLoS One. 2015;10:e0133840. Scholar
  6. 6.
    Bolsunovsky A, Frolova T, Dementyev D, Sinitsyna O. Low doses of gamma-radiation induce SOS response and increase mutation frequency in Escherichia coli and Salmonella typhimurium cells. Ecotoxicol Environ Saf. 2016;134:233–8. Scholar
  7. 7.
    Tigini V, Giansanti P, Mangiavillano A, Pannocchia A, Varese GC. Evaluation of toxicity, genotoxicity and environmental risk of simulated textile and tannery wastewaters with a battery of biotests. Ecotoxicol Environ Saf. 2011;74:866–73. Scholar
  8. 8.
    Thakur MS, Ragavan KV. Biosensors in food processing. J Food Sci Technol. 2013;50:625–41. Scholar
  9. 9.
    Roda A, Guardigli M, Michelini E, Mirasoli M. Bioluminescence in analytical chemistry and in vivo imaging. TrAC Trends Anal Chem. 2009;28:307–22. Scholar
  10. 10.
    Thomas DJL, Tyrrel SF, Smith R, Farrow S. Bioassays for the evaluation of landfill leachate toxicity. J Toxicol Environ Health Part B. 2009;12:83–105. Scholar
  11. 11.
    Ivask A, Rõlova T, Kahru A. A suite of recombinant luminescent bacterial strains for the quantification of bioavailable heavy metals and toxicity testing. BMC Biotechnol. 2009;9:41. Scholar
  12. 12.
    Kudryasheva N, Kratasyuk V, Esimbekova E, Vetrova E, Nemtseva E, Kudinova I. Development of bioluminescent bioindicators for analysis of environmental pollution. Field Anal Chem Technol. 1998;2:277–80.<277::AID-FACT4>3.0.CO;2-P.CrossRefGoogle Scholar
  13. 13.
    Ranjan R, Rastogi NK, Thakur MS. Development of immobilized biophotonic beads consisting of Photobacterium leiognathi for the detection of heavy metals and pesticide. J Hazard Mater. 2012;225–226:114–23. Scholar
  14. 14.
    Girotti S, Ferri EN, Fumo MG, Maiolini E. Monitoring of environmental pollutants by bioluminescent bacteria. Anal Chim Acta. 2008;608:2–29. Scholar
  15. 15.
    Efremenko EN, Maslova OV, Kholstov AV, Senko OV, Ismailov AD. Biosensitive element in the form of immobilized luminescent photobacteria for detecting ecotoxicants in aqueous flow-through systems. Luminescence. 2016;31:1283–9. Scholar
  16. 16.
    Roda A, Guardigli M. Analytical chemiluminescence and bioluminescence: latest achievements and new horizons. Anal Bioanal Chem. 2012;402:69–76. Scholar
  17. 17.
    Fedorova GF, Menshov VA, Trofimov AV, Tsaplev YB, Vasil’ev RF, Yablonskaya OI. Chemiluminescence of cigarette smoke: salient features of the phenomenon. Photochem Photobiol. 2017;93:579–89. Scholar
  18. 18.
    Tarasova AS, Kislan SL, Fedorova ES, Kuznetsov AM, Mogilnaya OA, Stom DI, et al. Bioluminescence as a tool for studying detoxification processes in metal salt solutions involving humic substances. J Photochem Photobiol B. 2012;117:164–70. Scholar
  19. 19.
    Kudryasheva NS, Tarasova AS. Pollutant toxicity and detoxification by humic substances: mechanisms and quantitative assessment via luminescent biomonitoring. Environ Sci Pollut Res. 2015;22:155–67. Scholar
  20. 20.
    Kudryasheva NS, Rozhko TV. Effect of low-dose ionizing radiation on luminous marine bacteria: radiation hormesis and toxicity. J Environ Radioact. 2015;142:68–77. Scholar
  21. 21.
    Min J, Lee CW, Gu MB. Gamma-radiation dose-rate effects on DNA damage and toxicity in bacterial cells. Radiat Environ Biophys. 2003;42:189–92. Scholar
  22. 22.
    Ptitsyn LR, Horneck G, Komova O, Kozubek S, Krasavin EA, Bonev M, et al. A biosensor for environmental genotoxin screening based on an SOS lux assay in recombinant Escherichia coli cells. Appl Environ Microbiol. 1997;63:4377–84.PubMedPubMedCentralGoogle Scholar
  23. 23.
    Rozhko TV, Kudryasheva NS, Kuznetsov AM, Vydryakova GA, Bondareva LG, Bolsunovsky AY. Effect of low-level [small alpha]-radiation on bioluminescent assay systems of various complexity. Photochem Photobiol Sci. 2007;6:67–70. Scholar
  24. 24.
    Rozhko TV, Badun GA, Razzhivina IA, Guseynov OA, Guseynova VE, Kudryasheva NS. On the mechanism of biological activation by tritium. J Environ Radioact. 2016;157:131–5. Scholar
  25. 25.
    Selivanova MA, Mogilnaya OA, Badun GA, Vydryakova GA, Kuznetsov AM, Kudryasheva NS. Effect of tritium on luminous marine bacteria and enzyme reactions. J Environ Radioact. 2013;120:19–25. Scholar
  26. 26.
    Alexandrova M, Rozhko T, Vydryakova G, Kudryasheva N. Effect of americium-241 on luminous bacteria. Role of peroxides. J Environ Radioact. 2011;102:407–11. Scholar
  27. 27.
    Esimbekova EN, Kondik AM, Kratasyuk VA. Bioluminescent enzymatic rapid assay of water integral toxicity. Environ Monit Assess. 2013;185:5909–16. Scholar
  28. 28.
    Kudryasheva NS, Kovel ES, Sachkova AS, Vorobeva AA, Isakova VG, Churilov GN. Bioluminescent enzymatic assay as a tool for studying antioxidant activity and toxicity of bioactive compounds. Photochem Photobiol. 2017;
  29. 29.
    Kudryasheva NS. Bioluminescence and exogenous compounds: physico-chemical basis for bioluminescent assay. J Photochem Photobiol B. 2006;83:77–86. Scholar
  30. 30.
    Nemtseva EV, Kudryasheva NS. The mechanism of electronic excitation in the bacterial bioluminescent reaction. Russ Chem Rev. 2007;76:91.CrossRefGoogle Scholar
  31. 31.
    Tarasova AS, Stom DI, Kudryasheva NS. Effect of humic substances on toxicity of inorganic oxidizer bioluminescent monitoring. Environ Toxicol Chem. 2011;30:1013–7. Scholar
  32. 32.
    Vetrova EV, Kudryasheva NS, Kratasyuk VA. Redox compounds influence on the NAD(P)H:FMN-oxidoreductase-luciferase bioluminescent system. Photochem Photobiol Sci. 2007;6:35–40. Scholar
  33. 33.
    Kirillova TN, Gerasimova MA, Nemtseva EV, Kudryasheva NS. Effect of halogenated fluorescent compounds on bioluminescent reactions. Anal Bioanal Chem. 2011;400:343–51. Scholar
  34. 34.
    Sachkova AS, Kovel ES, Churilov GN, Guseynov OA, Bondar AA, Dubinina IA, et al. On mechanism of antioxidant effect of fullerenols. Biochem Biophys Rep. 2017;9:1–8. Scholar
  35. 35.
    Vetrova EV, Kudryasheva NS, Visser AJWG, van Hoek A. Characteristics of endogenous flavin fluorescence of Photobacterium leiognathi luciferase and Vibrio fischeri NAD(P)H:FMN-oxidoreductase. Luminescence. 2005;20:205–9. Scholar
  36. 36.
    Alieva RR, Kudryasheva NS. Variability of fluorescence spectra of coelenteramide-containing proteins as a basis for toxicity monitoring. Talanta. 2017;170:425–31. Scholar
  37. 37.
    Belogurova NV, Kudryasheva NS. Discharged photoprotein obelin: fluorescence peculiarities. J Photochem Photobiol B. 2010;101:103–8. Scholar
  38. 38.
    Belogurova NV, Kudryasheva NS, Alieva RR, Sizykh AG. Spectral components of bioluminescence of aequorin and obelin. J Photochem Photobiol B. 2008;92:117–22. Scholar
  39. 39.
    Alieva RR, Tomilin FN, Kuzubov AA, Ovchinnikov SG, Kudryasheva NS. Ultraviolet fluorescence of coelenteramide and coelenteramide-containing fluorescent proteins. Experimental and theoretical study. J Photochem Photobiol B. 2016;162:318–23. Scholar
  40. 40.
    Alieva RR, Belogurova NV, Petrova AS, Kudryasheva NS. Effects of alcohols on fluorescence intensity and color of a discharged-obelin-based biomarker. Anal Bioanal Chem. 2014;406:2965–74. Scholar
  41. 41.
    Petrova AS, Alieva RR, Belogurova NV, Tirranen LS, Kudryasheva NS. Variation of spectral characteristics of coelenteramide-containing fluorescent protein from Obelia longissima exposed to dimethyl sulfoxide. Russ Phys J. 2016;59:562–7. Scholar
  42. 42.
    Alieva RR, Belogurova NV, Petrova AS, Kudryasheva NS. Fluorescence properties of Ca2+−independent discharged obelin and its application prospects. Anal Bioanal Chem. 2013;405:3351–8. Scholar
  43. 43.
    Petrova AS, Lukonina AA, Badun GA, Kudryasheva NS. Fluorescent coelenteramide-containing protein as a color bioindicator for low-dose radiation effects. Anal Bioanal Chem. 2017;409:4377–81. Scholar
  44. 44.
    Kudryasheva NS, Petrova AS, Dementyev DV, Bondar AA. Exposure of luminous marine bacteria to low-dose gamma radiation. J Environ Radioact. 2017;169-170:64–9. Scholar
  45. 45.
    Rozhko TV, Guseynov OA, Bondar АА, Guseynova VE, Devyatlovskaya AN, Kudryasheva NS. Is bacterial luminescence response to low-dose radiation associated with mutagenicity. J Environ Radioact. 2017;177:261–5. Scholar
  46. 46.
    Illarionov BA, Frank LA, Illarionova VA, Bondar VS, Vysotski ES, Blinks JR. Recombinant obelin: cloning and expression of cDNA, purification, and characterization as a calcium indicator. Methods Enzymol Academic Press. 2000;305:223–49.CrossRefGoogle Scholar
  47. 47.
    Bolsunovsky AY, Tcherkezian VO. Hot particles of the Yenisei River flood plain, Russia. J Environ Radioact. 2001;57:167–74.CrossRefPubMedGoogle Scholar
  48. 48.
    Bolsunovsky A, Melgunov M, Chuguevskii A, Lind OC, Salbu B. Unique diversity of radioactive particles found in the Yenisei River floodplain. Sci Rep. 2017;7:1–10. Scholar
  49. 49.
    Shimomura O, Teranishi K. Light-emitters involved in the luminescence of coelenterazine. Luminescence. 2000;15:51–8.<51::AID-BIO555>3.0.CO;2-J.CrossRefPubMedGoogle Scholar
  50. 50.
    Mori K, Maki S, Niwa H, Ikedab H, Hirano T. Real light emitter in the bioluminescence of the calcium-activated photoproteins aequorin and obelin: light emission from the singlet-excited state of coelenteramide phenolate anion in a contact ion pair. Tetrahedron. 2006;62:6272–88.CrossRefGoogle Scholar
  51. 51.
    Imai Y, Shibata T, Maki S, Niwa S, Ohashi M, Hirano T. Fluorescenceproperties of phenolate anion of coelenteramide analogues: the light-emitter structure in aequorin bioluminescence. J Photochem Photobiol A. 2001;146:95–107.CrossRefGoogle Scholar
  52. 52.
    Hirano T, Ohmiya Y, Maki S, Niwa H, Ohashi M. Bioluminescent properties offluorinate semisynthetic aequorins. Tetrahedron. 1998;39:5541–4.CrossRefGoogle Scholar
  53. 53.
    Vysotski ES, Liu ZJ, Markova SV, Blinks JR, Deng L, Frank LA, et al. Violet bioluminescence and fastkinetics from W92F obelin: structure-based proposal for the bioluminescence triggering and the identification of the emitting species. Biochemistry. 2003;42:6013–24.CrossRefPubMedGoogle Scholar
  54. 54.
    Li Z-S, Zou L-Y, Min C-G, Ren A-M. The effect of micro-environment on luminescence of aequorin: the role of amino acids and explicit water molecules on spectroscopic properties of coelenteramide. J Photochem Photobiol B. 2013;127:94–9. Scholar
  55. 55.
    Min C, Li Z, Ren A, Zou L, Guo J, Goddard JD. The fluorescent properties of coelenteramide, a substrate of aequorin and obelin. J Photochem Photobiol Chem. 2013;251:182–8. Scholar
  56. 56.
    Tomilin FN, Antipina LY, Vysotski ES, Ovchinnikov SG, Gitelzon II. Fluorescence of calcium-discharged obelin: the structure and molecular mechanism of emitter formation. Dokl Biochem Biophys. 2008;422:279–84. Scholar
  57. 57.
    Vysotskiĭ ES, Markova SV, Frank LA. Calcium-regulated photoproteins of marine coelenterates. Mol Biol (Mosk). 2006;40:404–17.CrossRefGoogle Scholar
  58. 58.
    Frank LA. Ca2+-regulated photoproteins: effective immunoassay reporters. Sensors. 2010;10:11287–300. Scholar
  59. 59.
    Vysotski ES, Lee J. Ca2+-regulated photoproteins: structural insight into the bioluminescence mechanism. Acc Chem Res. 2004;37:405–15. Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Alena S. Petrova
    • 1
  • Anna A. Lukonina
    • 2
  • Dmitry V. Dementyev
    • 3
  • Alexander Ya. Bolsunovsky
    • 3
  • Anatoliy V. Popov
    • 4
  • Nadezhda S. Kudryasheva
    • 2
    • 3
    Email author
  1. 1.Krasnoyarsk State Agrarian UniversityKrasnoyarskRussia
  2. 2.Siberian Federal UniversityKrasnoyarskRussia
  3. 3.Institute of Biophysics SB RAS, FRC KSC SB RASKrasnoyarskRussia
  4. 4.Department of RadiologyUniversity of PennsylvaniaPhiladelphiaUSA

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