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 oxyl uciferin) bounded in the firefly luciferase emits the light, Also it is gen erally accepted that two forms (keto and enol) of the oxyluciferin emit red and yellow-green light, respectively. In order to demonstrate the existenc e of free emitting species using His-tagged luciferase (His-luc), N-termina l 6x His-tagged luciferase (Photinus pyralis) was prepared and expressed in E. coli. After immobilization of His-luc on the membrane by IDA-Ni2+-His c omplex, 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 o f 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. (C) 1999 Academic P ress.