The emitting species dissociated from the enzyme can emit the light in Photinus pyralis luciferase system.
By fluorescent results, it has been proposed that the product (excited oxyluciferin) bounded in the firefly luciferase emits the light. Also it is generally accepted that two forms (keto and enol) of the oxyluciferin emit red and yellow-green light, respectively. In order to demonstrate the existence of free emitting species using His-tagged luciferase (His-luc), N-terminal 6x His-tagged luciferase (Photinus pyralis) was prepared and expressed in E. coli. After immobilization of His-luc on the membrane by IDA-Ni(2+)-His complex, His-luc clearly showed spectral changes of the emission toward red light. The luciferase-free product obtained from enzymatic reaction mixture in the presence of ATP and dATP emits the light with maximal wavelengths of 575 and 620 nm, respectively. Based on these results, we suppose that two different emitting species or two different energy levels of the emitting species are responsible for two different color lights.
Min
K L
KL
College of Natural Sciences and Research Center for Molecular Microbiology, Seoul National University, Seoul, 151-742, Republic of Korea. kyunglyummin@yahoo.co.kr
Steghens
J P
JP
D000818:Animals; D001517:Coleoptera / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D049454:Firefly Luciferin / Q000737:chemistry* / Q000378:metabolism; D066298:In Vitro Techniques; D007211:Indoles; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements; D008567:Membranes, Artificial; D008956:Models, Chemical; D009532:Nickel / Q000494:pharmacology; D010777:Photochemistry; D011719:Pyrazines; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry
D007211:Indoles; D008567:Membranes, Artificial; D011719:Pyrazines; D011994:Recombinant Proteins; C014730:oxyluciferin; D049454:Firefly Luciferin; D009532:Nickel; D008156:Luciferases