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Dehydrocoelenterazine is the Organic Substance Constituting the Prosthetic Group of Pholasin

The bivalve mollusc Pholas dactylus uses the luciferin–luciferase reaction for light emission. Its luciferin molecule is known as Pholasin, 2] a glycoprotein with a prosthetic group that is responsible for light emission. The luciferase molecule is a copper-containing peroxidase that catalyzes the oxidation of Pholasin to emit light in the presence of peroxides. The amount of light emission is proportional to the amount of the photoprotein Pholasin present. Henry and Michelson found that the superoxide radical produced by the reaction of luciferase and peroxides catalyzes the oxidation of luciferin. Because of their finding, it is now possible to initiate the light emission of Pholasin in a test tube in the presence of reactive oxygen species (ROS) without luciferase. Pholasin is now commercially available as an indicator of ROS. Despite the long research history of Pholasin (Dubois discovered a luciferin–luciferase reaction in the mollusc 122 years ago), the structure of its prosthetic group is unclear. Although Pholasin has been cloned, the expressed molecule lacks the prosthetic group and thus it cannot be activated in the same way as natural Pholasin. Elucidation of the prosthetic group structure would enable Pholasin to be widely used in many disciplines following simple activation by the addition of the organic substance. ROS are involved in many biological events, and thus selective monitoring of ROS is an important tool that would be more easily attainable by the commercial availability of Pholasin. We recently noticed a similarity between Pholasin and the oceanic squid Symplectoteuthis photoproteins: both emit strong light following the addition of a mixture of hydrogen peroxide and catalase or peroxidase. Symplectin, a photoprotein of S. oualaniensis, contains 501 amino acids, with eleven cysteines and a prosthetic group composed of dehydrocoelenterazine (DCL) bound to the eleventh cysteine (Cys390). Pholasin has half the number of amino acids as symplectin, and has five cysteines; the existence of one cysteine in free thiol form could constitute a prosthetic group by binding with DCL. We therefore hypothesized that the prosthetic group of Pholasin might be constituted from apoPholasin (Pholasin lacking a prosthetic group) and DCL (Scheme 1). Indeed, we previously reported that DCL could be used as an organic substance for Pholasin luminescence, although it is conceivable that it was mimicking the real organic substance. In the present study, we attempted to isolate DCL from Pholasin and show that it is the organic substance that forms the prosthetic group of Pholasin. DCL alone is not able to emit light even in the presence of ROS, however, after reacting with a thiol compound, the resulting DCL–thiol adduct then reacts with oxygen to emit light. The addition of DCL to a thiol compound forms an equilibrium, and the adduct might be stabilized in a photoprotein (Scheme 1). It was expected that extraction of DCL directly from Pholasin would be problematic as no liberated DCL