Dinoflagellate luciferases: purification of luciferases from Gonyaulax polyedra, Pyrocystis lunula, and Pyrocystis fusiformis

The isolation and purification of three luciferases from Pyrocystis lunula, Pyrocystis fusiformis, and Gonyaulax polyedra are described in this paper. The three luciferases have low molecular weights, 30,000 for G. polyedra and 40,000 for P. lunala and P. fusiformis, and each is composed of a single polypeptidic chain. The molecular weight of these luciferases is independent of both the period of the circadian rhythm and the pH of the extraction medium between pH 5.4 and pH 8. These enzymes are probably metalloproteins. Indeed, chelating agents such as EDTA, EGTA, and chlorotetracycline and also sodium azide and potassium cyanide inhibit the light emission. Three cations (Mn²⁺, Mg²⁺, and Ca²⁺) increase the flash height and the total light emitted, whereas other cations (Fe²⁺, Fe³⁺, Cu²⁺, Ni²⁺, and Zn²⁺) inhibit the light emission. The three luciferases cannot be replaced by peridoxases or oxidases as in the Balanoglossus and the Pholas systems. The pH dependence of the luciferase activities is represented by a symmetrical function with optimum near pH 7. Thus, the flashing mechanism cannot be explained by means of a switch mechanism controlled by the pH. The presence of a specific luciferin-binding protein has not been observed in the three extracts of dinoflagellates. The difference between our observations and those described in the literature may be explained by the difference of the degree of purification of these enzymes.