An investigation of the reaction kinetics of luciferase and the effect of ionizing radiation on the reaction rate

  • Nikolas BerovicEmail author
  • David J. Parker
  • Michael D. Smith
Original Paper


The bioluminescence produced by luciferase, a firefly enzyme, requires three substrates: luciferin, ATP and oxygen. We find that ionizing radiation, in the form of a proton beam from a cyclotron, will eliminate dissolved oxygen prior to any damage to other substrates or to the protein. The dose constant for removal of oxygen is 70 ± 20 Gy, a much smaller dose than required to cause damage to protein. Removal of oxygen, which is initially in excess, leads to a sigmoidal response of bioluminescence to radiation dose, consistent with a Michaelis–Menten relationship to substrate concentration. When excess oxygen is exhausted, the response becomes exponential. Following the irradiation, bioluminescence recovers due to a slow leak of oxygen into the solution. This may also explain previous observations on the response of bioluminescent bacteria to radiation. We have studied the dependence of the reaction rate on enzyme and substrate concentration and propose a model for the reaction pathway consistent with this data. The light output from unirradiated samples decreases significantly with time due to product inhibition. We observe that this inhibition rate changes dramatically immediately after a sample is exposed to the beam. This sudden change of the inhibition rate is unexplained but shows that enzyme regulatory function responds to ionizing radiation at a dose level less than 0.6 Gy.


Protein Ionizing radiation Luciferase Radiation damage Oxygen depletion by radiation Cooperativity 



We wish to acknowledge the support of EPSRC.


  1. Bacq ZM, Alexander P (1967) Fundamentals of radiobiology, 2nd edn. Pergamon Press, OxfordGoogle Scholar
  2. Berovic N, Pratontep S, Bryant A, Montouris A, Green RG (2002) The kinetics of radiation damage to the protein luciferase and recovery of the enzyme activity after irradiation. Radiat Res 157:122–127. doi: 10.1667/0033-7587(2002)157[0122:TKORDT]2.0.CO;2 PubMedCrossRefGoogle Scholar
  3. Chittock RS, Lidzey DG, Berovic N, Wharton CW, Jackson JB, Beynon TD (1993) The quantum yield of luciferase is dependent on ATP and enzyme concentrations. Mol Cryst Liq Cryst (Phila Pa) 236:599–604. doi: 10.1080/10587259308055210 Google Scholar
  4. Conti E, Franks NP, Brick P (1996) Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes. Structure 4:287–298. doi: 10.1016/S0969-2126(96)00033-0 PubMedCrossRefGoogle Scholar
  5. Denburgh JL, McElroy WD (1970) Catalytic subunit of firefly luciferase. Biochemistry 9:4619–4624CrossRefGoogle Scholar
  6. Harada H, Kizaka-Kondoh S, Li G, Itasaka S, Shibuya K, Inoue M, Hiraoka M (2007) Significance of HIF-1_active cells in angiogenesis and radioresistance. Oncogene 26:7508–7516. doi: 10.1038/sj.onc.1210556 PubMedCrossRefGoogle Scholar
  7. Henriquez NV et al (2007) Advances in optical imaging and novel model systems for cancer metastasis imaging. Clin Exp Metastasis 24(8):699–705. doi: 10.1007/s10585-007-9115-5 PubMedCrossRefGoogle Scholar
  8. Hug O, Wolf I (1956) Progress in radiobiology. Oliver and Boyd, Edinburgh, p 23 (see also Bacq and Alexander 1967, chapter 14, p 375)Google Scholar
  9. Kasney M, Pamuk HO, Trindle C (1983) A MINDO/3 study of the properties of the ground state dioxetanone molecule and its dissociation potential energy surface into CH2O + CO2 Theochem-. J Mol Struct 13:459–470Google Scholar
  10. Kepner GR, Macey RI (1968) Membrane enzyme systems–molecular size determinations by radiation inactivation. Biochim Biophys Acta 163:188–203. doi: 10.1016/0005-2736(68)90097-7 PubMedCrossRefGoogle Scholar
  11. Lea DE (1962) Actions of radiations on living cells. Cambridge University Press, LondonGoogle Scholar
  12. Lemasters JJ, Hackenbrock CR (1977) Kinetics of product inhibition during firefly luciferase luminescence. Biochemistry 16:445–447. doi: 10.1021/bi00622a016 PubMedCrossRefGoogle Scholar
  13. Lidzey DG, Berovic N, Chittock RS, Beynon TD, Wharton CW, Jackson JB, Parkinson N (1995) A critical analysis of the use of radiation inactivation to measure the mass of protein. Radiat Res 143:181–186. doi: 10.2307/3579155 PubMedCrossRefGoogle Scholar
  14. McCapra F, Beheshti I (1985) Selected chemical reactions that produce light in bioluminescence and chemiluminescence, vol 1. CRC Press, Boca RatonGoogle Scholar
  15. Nakatsu T, Ichiyama S, Hiratake J, Saldanha A, Kobashi N, Sakata K, Kato H (2006) Structural basis for the spectral difference in luciferase bioluminescence. Nature 440:372–376. doi: 10.1038/nature04542 PubMedCrossRefGoogle Scholar
  16. Osborne JC, Miller JH, Kempner ES (2000) Molecular mass and volume in radiation target theory. Biophys J 78:1698–1702PubMedCrossRefGoogle Scholar
  17. Ugarova NN, Brovko LI, Beliaeva EI et al (1981) Dimers are catalytically active particles of gloworm luciferase. Dokl Akad Nauk SSSR 260(2):358–360Google Scholar
  18. Vlasova TN, Ugarova NN (2007) Quenching of the fluorescence of Tyr and Trp residues of firefly luciferase from Luciola mingrelica by the substrates. Biochemistry (Mosc) 72:962–967CrossRefGoogle Scholar
  19. Wood KV, Lam YA, McElroy WD (1989) Introduction to beetle luciferases and their applications. J Biolumin Chemilumin 4:289–301PubMedCrossRefGoogle Scholar
  20. Xu XD et al (2007) Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues. Proc Natl Acad Sci USA 104(24):10264–10269. doi: 10.1073/pnas.0701987104 PubMedCrossRefGoogle Scholar

Copyright information

© European Biophysical Societies' Association 2008

Authors and Affiliations

  • Nikolas Berovic
    • 1
    Email author
  • David J. Parker
    • 1
  • Michael D. Smith
    • 1
  1. 1.School of Physics and AstronomyThe University of BirminghamBirminghamUK

Personalised recommendations