Biochemistry of Luminescence

技术摘要生物发光发生在多种生物体中，在发光的化学过程中涉及多种类型的化合物和反应机制。本研究的重点是对真菌生物发光的生物化学理解，这在过去的40年里受到了几个团体的挑战，但没有成功。真菌的生物发光是独特的，与目前已知的所有其他生物发光系统明显不同。它们的发光是连续的，几乎可以持续数天，发光反应是由表面活性剂催化的，而不是由传统的荧光素酶催化的。在生物发光的化学研究中，关键成分的鉴定和表征是最重要的。因此，本项目的中心主题是各种真菌荧光素及其前体的结构确定。夜光蘑菇的荧光素具有由3个前体分子（酮醛PS- a和PS- b）和2个甲胺分子组成的复杂结构；这种分子含有一个不寻常的9元环。为了确定P. stipticus萤光素的完整结构，将制备一个在分子关键位置含有C-13原子的萤光素样品，并将通过哈佛大学Kishi博士实验室新开发的核磁共振技术对样品进行分析。从发光真菌柠檬色迈锡纳（Mycena citricolor）中分离到10多种荧光素前体；它们的结构现在正在被阐明。这些前体在与癸胺反应时很容易形成荧光素；由此得到的柠檬色荧光素的结构将被确定。很明显，所有发光真菌的荧光素都来源于醛类前体，如PS-A和PS-B，以及伯胺，并且它们都含有相同的发光发色团。这种发色团将从两种以上不同类型的真菌荧光素的结构中鉴定出来。在真菌的发光反应中，荧光素在超氧阴离子和阳离子表面活性剂的存在下被分子氧氧化，其中发射的光子没有明显的作用，但通过这种反应可以消除组织中有害的超氧阴离子；这项提议的研究可能会揭示该反应的其他重要作用。一种模拟荧光素酶的表面活性剂的参与表明，这项研究可能有助于理解酶的作用。真菌荧光素对超氧阴离子的高敏感性表明它可能用作超氧探针。在生物发光的化学研究中，识别和表征参与发光反应的关键物质是最重要的。荧光素是一种存在于发光生物体内的物质，当它被氧化时就会发光。本研究的中心主题是找出各种真菌荧光素及其前体的结构。科学家发现，一种名为Panellus stipticus的发光蘑菇的荧光素是一种复杂的化合物，包括一个由9个碳原子组成的不寻常的环。PI计划通过制备荧光素样品来确定这种荧光素的完整结构，该荧光素样品在分子的关键位置含有比平均碳原子重的碳原子（13C），然后通过核磁共振（NMR）光谱分析该样品。哥斯达黎加发光烟草真菌（Mycena citricolor）含有10多种荧光素前体，这些前体是最近分离纯化的；我们现在正试图确定它们的化学结构。这些前体分子用一种叫做癸胺的物质处理后很容易转化为荧光素；我们计划研究这些荧光素的化学结构。现在看来很明显，所有发光真菌的荧光素都来自类似的材料，而且它们都含有一种相同的发光结构，称为发色团。我们计划根据两种以上不同类型真菌荧光素的化学结构来识别这样一种发色团。在发光真菌中，当荧光素被分子氧氧化时，在某些负离子（称为超氧阴离子）和洗涤剂的存在下发出光；在该反应中，发射的光子没有明显的作用，但该反应可以消除组织中有害的超氧阴离子。洗涤剂的参与，模仿一种酶表明，这里所描述的研究也可能有助于理解酶的作用。真菌荧光素对超氧阴离子的高敏感性也表明，使用这种材料作为检测生物系统中有害超氧阴离子存在的手段是可能的。

9630861 Shimomura Technical abstract Bioluminescence occurs in a wide variety of organisms, involving diverse types of compounds and reaction mechanisms in the chemical process of light emission. This research is focused on the biochemical understanding of the bioluminescence of fungi, which has been challenged by several groups in the past 40 years without success. The bioluminescence of fungi is unique and distinctly differs from all other bioluminescent systems presently known. Their glow is continuous and nearly constant for days, and the light-emitting reaction is catalyzed by surfactants, not by conventional luciferases. In the chemical study of bioluminescence, the identification and characterization of the key components involved are most important. Thus, the central theme of this project is the structure determination of various fungal luciferins and their precursors. The luciferin of the luminous mushroom Panellus stipticus was found to have a complicated structure consisting of three molecules of the precursor (ketoaldehydes PS- A and PS-B) and two molecules of methylamine; the molecule contains an unusual 9-membered ring. In order to determine the complete structure of the P. stipticus luciferin, a luciferin sample that contains C-13 atoms at the key positions of the molecule will be prepared, and the sample will be analyzed by a NMR technique newly developed at Dr. Kishi's lab at Harvard. From the luminous fungus Mycena citricolor, more than 10 kinds of luciferin precursor were recently isolated in pure states; their structures are now being elucidated. These precursors readily form luciferins when reacted with decylamine; the structures of the M. citricolor luciferins thus obtained will be determined. It seems apparent that the luciferin of all luminous fungi are derived from a precursor of aldehydic type, such as PS-A and PS-B, and a primary amine, and that all of them contain an identical light-emitting chromophore. Such a chromophore will be identified from t he structures of more than two different types of fungal luciferins. In the luminescence reaction of fungi, a luciferin is oxidized by molecular oxygen in the presence of superoxide anions and a cationic surfactant, in which the emitted photons play no apparent role, but by which the reaction can eliminate harmful superoxide anions in the tissue; the proposed study might uncover other important roles for the reaction. The involvement of a surfactant that mimics a luciferase suggests that the study may contribute to the understanding of enzymatic action. The high sensitivity of fungal luciferin to superoxide anions suggests its possible use as a superoxide probe. Non-technical abstract In the chemical study of bioluminescence, it is most important to identify and characterize the key substances involved in the light emitting reaction. Luciferin is a type of substance that exits in luminous organisms and emits light when it is oxidized. The central theme of this research is to find out the structures of various fungal luciferins and their precursors. The luciferin of one type of luminous mushroom, named Panellus stipticus, was found to be a complicated compound that includes and unusual ring of 9 carbon atoms. The PI plans to determine the complete structure of this luciferin by preparing a luciferin sample that contains a heavier-than-average carbon atom (13C) at the key positions of the molecule, and then analyzing this sample by a process known as nuclear magnetic resonance (NMR) spectroscopy. The luminous Costa Rican tobacco fungus (Mycena citricolor) contains more that 10 kinds of luciferin precursor which have been recently isolated and purified; we are now trying to determine their chemical structures. These precursor molecules are readily converted to luciferins when treated with a substance called decylamine; we plan to investigate the chemical structures of these luciferins. Now it seems apparent that the luciferins of all luminous fungi are derived from similar mat erials, and that all of them contain an identical light-emitting structure called a chromophore. We plan to identify such a chromophore based on the chemical structures of more than two different types of fungal luciferins In luminous fungi, light is emitted when a luciferin is oxidized by molecular oxygen, in the presence of certain negative ions (called superoxide anions) and a detergent; in this reaction, the emitted photons play no apparent role, but the reaction can eliminate the superoxide anions, which are harmful, from the tissue. The involvement of the detergent that mimics an enzyme suggests that the study described here may also contribute to the understanding of enzymatic action. The high sensitivity of fungal luciferin to superoxide anions also suggests the possibility of using this material as a means of detecting the presence of harmful superoxide anions in biological systems.