ABSTRACT
The bioluminescent reaction of luciferase isolated from Vibrio harveyi, strain M17, was initiated by mixing the intermediate luciferase-bound flavin-4a-hydroperoxide, purified in advance, with a long chain aldehyde (dodecanal or octanal) at -4°C. concurrent measurements of bioluminescence and absorbance changes from 300 to 600 nm during the course of the reaction revealed the existence of three sets of isosbestic points and three kinetic phases, the second of which corresponds to the step giving rise to the excited state. The first intermediate, demonstrated here for the first time, is postulated to correspond to the luciferase-bound flavin-4a-peroxyhemiacetal and the second to the enzyme-bound flavin-4a-hydroxide.