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

本项目计划用光谱方法阐明萤火虫生物发光在毫秒级时间分辨率下的初级反应过程。项目期限正式分为三年(2004~2006年)。在第一个任期内，存在一个明显的技术问题，涉及使用笼状ATP(CATP)，这是一种利用紫外光产生ATP的试剂。针对激光脉冲可以通过将过氧化氢酶光化学分解为三磷酸腺苷来诱导生物发光的预期，发现底物荧光素(Ln)的荧光强度太高，无法检测到生物发光的本征强度，而且由于光谱峰相似，无法将后者与前者分开。在第二阶段，推迟了对该装置的改进，以寻找试剂制备的实验条件，使生物发光的衰减曲线的谱带形状变得更窄。然而，它的信号很难被所制备的装置检测到。在上一学期，田中博士受邀担任毫秒量级的FAST现象光谱测量方面的专家。利用机械快门来防止Ln荧光遮挡生物发光的微弱光，并利用光计数技术来提高探测器的灵敏度。从而首次在毫秒级的时间尺度上观测到了激光触发的生物发光的衰减曲线。作为该项目的成果，在某些国际期刊上发表了两篇论文，在三本书中发表了六篇论文，其中一本是世界反兴奋剂机构主编的，在国内年会和国际生物发光和化学发光研讨会上发表了17个主题。

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.