Study of the primary reaction process of firefly bioluminescence by measurements of its time-resolved absorption and emission spectra

本项目拟利用光谱学方法阐明萤火虫生物发光在微秒时间分辨率下的主要反应过程。项目期限正式划分为三年（2004 ~ 2006年）。在第一个学期，有一个明显的技术问题，关于笼型ATP （CATP）的使用，一个ATP生成试剂的紫外线。与预期激光脉冲可以通过CATP光化学分解成ATP来诱导生物发光相反，我们发现底物荧光素（Ln）的荧光强度太高，无法检测生物发光的内在强度，而后者由于光谱峰相似而无法与前者分离。在第二阶段，为了寻找试剂制备的实验条件，推迟了对器件的改进，使得生物发光衰减曲线的能带形状窄了很多。但其信号难以被所制备的器件检测到。上学期，田中博士被邀请为msec级快现象的光谱测量专家。采用机械快门防止Ln荧光遮挡生物发光弱光，采用光计数技术提高检测器灵敏度，构建了足以分辨CATP的设备。首次在微秒时间尺度上观察到激光脉冲触发生物发光的衰减曲线。项目成果在部分国际期刊上发表论文2篇，在Wada主编的3本专著中发表论文6篇，在国内生物发光、化学发光年会和国际学术研讨会上发表课题17篇。

This project has been planed to clarify the primary reaction process of firefly bioluminescence in msec time resolution by spectroscopic methods. Term of project was formally divided into three years (2004〜2006). In the first term, there was a conspicuous technical problem concerning the use of caged-ATP (CATP), an ATP producing reagent by UV light. Against expectation that a laser pulse can induce the bioluminescence by the photochemical decomposition of CATP to ATP, it was found that the fluorescent intensity of substrate luciferin (Ln) is too high to detect the intrinsic intensity of bioluminescence and the latter could not be separated from the former due to the similar spectral peaks each other. In the second term, the improvement of the device was postponed to search for experimental conditions of reagents' preparation so that band shape of the decay curve of bio-luminescence was much more narrow. However, its signal was hard to be detected by the prepared device. In the last term, Dr. Tanaka was invited as an expert on the spectroscopic measurement of fast phenomena in time of msec order. The equipment adequate to resolve CATP was constructed using the mechanical shutter to prevent Ln fluorescence from blocking the weak light of bioluminescence and photocounting technique to improve the sensitivity of the detector. Thus the decay curve of bioluminescence triggered by laser pulses was observed for the first time in the msec time scale. As the results of the project, there are two papers published in certain international journals, six papers appeared in three books one of which was edited by Wada and the publication of 17 subjects at the domestic annual meetings and the international symposiums on bioluminescence and chemiluminescence.