Bioluminescence Technology, Development of its Multipul Function

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Bioluminescent systems such as firefly luminescenece and squid luminescence were mainly utilized as typical cases requiring luciferase-luciferin (or photoprotein) plus the third factor(s). We have already estalished the technology to use immobilized luciferase of firefly, which has already been produced by E.coli through cloning. The luminescence can be monitored with an equipment similar to HPLC system with modification : thus, pumping a buffer containing ATP and Mg through an autosampler into a column having immobilized luciferase. The light used to be detected with a photomultiplier, and this has been improved to use "single photon counter" with computerized equipment. Thus the data can be transfered to Machintosh and the ordinal detection can be operated at 10^<-14> molar level. The linearity under the condition was max>2x10^<-10> molarlevel.An application of this system was extended to detect the mode of action of protein phosphatase with its inhibitors such as okadaic acid, tautomycin etc.using both types of phosphatases (type I and II). Okinawan squid produced a specific photoprotein, which was extracted, purified and analized to determine the (partial) amino acid sequence. The lumi-detection system was proved to be useful for these structural studies. Other bioluminescent organisms were also searched in South East Asia with success. On the other hand, great progress was made in the molecular mechanism of luminescence ; thus, we have succeeded in the synthesis (under photooxygenation condition) and spectroscopic detection by ^<13>CNMR and IR of the common intermediate "dioxetanone" at-78ﾟC with ^<13>C enriched luciferin analog. Computer chemistry of the molecular mechanic calculation of the luciferase and luciferin analogs were studied for molecular-molecular interaction.