Bioluminescence Technology, Development of its Multipul Function

生物发光系统，如萤火虫发光和鱿鱼发光，主要是需要荧光素酶-荧光素(或光蛋白)加第三因子(S)的典型案例。我们已经建立了利用固定化萤火虫荧光素酶的技术，该酶已由大肠杆菌通过克隆产生。发光可以用类似于高效液相色谱系统的设备进行修改：因此，通过自动进样器将含有ATP和镁的缓冲液泵入具有固定化荧光素酶的柱中。过去光是用光电倍增管探测的，现在已经改进为使用带有计算机化设备的单光子计数器。因此，数据可以传输到机器上，顺序检测可以在10摩尔水平上进行。在此条件下，线性关系为max&gt；2×10；-10&gt；，并将该体系的应用扩展到检测蛋白磷酸酶与其抑制剂冈田酸、交托霉素等的作用模式。冲绳鱿鱼产生一种特殊的光蛋白，提取、纯化和分析以确定(部分)氨基酸序列。流明探测系统被证明对这些结构研究是有用的。在东南亚也成功地搜索了其他生物发光生物。另一方面，在发光的分子机理方面也取得了很大的进展，成功地合成了富含荧光素类似物的常见中间体二氧杂环己酮-78ﾟC的合成(在光氧化条件下)和光谱检测。对荧光素酶和荧光素类似物分子-分子相互作用的分子力学计算的计算机化学进行了研究。

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.