title: An investigation of the reaction kinetics of luciferase and the effect of ionizing radiation on the reaction rate.
abstract: 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.
authors.affiliation: School of Physics and Astronomy, The University of Birmingham, Birmingham, B15 2TT, UK. n.berovic@bham.ac.uk
authors.forename: Nikolas
authors.initials: N
authors.lastname: Berovic
authors.forename: David J
authors.initials: DJ
authors.lastname: Parker
authors.forename: Michael D
authors.initials: MD
authors.lastname: Smith
mesh_terms: D000818:Animals; D004307:Dose-Response Relationship, Radiation; D048888:Fireflies; D007700:Kinetics; D008027:Light; D008156:Luciferases / Q000378:metabolism* / Q000528:radiation effects*; D008163:Luminescent Measurements; D010100:Oxygen / Q000737:chemistry; D017785:Photons; D011839:Radiation, Ionizing; D013997:Time Factors
chemical_list: D008156:Luciferases; D010100:Oxygen