Firefly luciferase generates two low-molecular-weight light-emitting species

A bioluminescent D-luciferin-luciferase mixture is separated by gel filtrat ion during the time course of the reaction. A simultaneous analysis with an UV-visible diode array detector and an on-line luminometer gives nonsuperi mposable chromatograms. Luminescence recordings display three peaks, one as sociated with the enzyme (light-emitting species 1: LES1), and two other sp ecies free from the luciferase: LES2, with a luciferyl-adenylate-like spect rum and LES3. Production of these two species is nucleotide (ATP or 2'-dATP )- and pH-dependent. The chromatographic data presented here could lead to reconsideration of the generally assumed emission mechanism, which involves one emitter only. It could also suggest that each free emitting species is related to a colour of emission corresponding to the two defined wavelengt hs previously described (similar to 575 and similar to 620 nm). (C) 2000 Ac ademic Press.