Biochemistry of dinoflagellate bioluminescence: purification and characterization of dinoflagellate luciferin from Pyrocystis lunula.
Biochemistry of dinoflagellate bioluminescence: purification and characterization of dinoflagellate luciferin from Pyrocystis lunula.
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DOI：
10.1021/bi00507a052
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发表时间：
1981-02
期刊：
影响因子：
2.9
通讯作者：
J. Dunlap;J. W. Hastings
中科院分区：
文献类型：
--
作者：
J. Dunlap;J. W. Hastings
迄今为止研究的所有甲藻物种的生物发光都是由荧光素酶催化氧化一种新发现的荧光素产生的，这种荧光素被假设具有取代的聚吡咯型结构。本文报道了从月形火孢子虫中分离得到的荧光素的纯化和性质，并证实其为多荧光素型分子。荧光素在低pH值，高盐浓度和O2下非常不稳定，因此，在可能的情况下，纯化步骤是在缓冲还原剂和氩气下进行的。纯化的荧光素可溶于水和极性有机溶剂。在中性或碱性水溶液中，它呈黄色（λ max为245 nm和390 nm，在290 nm处有一个肩部），并显示出强烈的蓝色荧光（λ max为390 nm激发，474 nm发射），与生物发光发射光谱密切匹配[Bode， V. C., & Hastings， J. W. (1963) Arch]。物化学。[j].生物工程学报，2016,33(4):488—499。自氧化导致390 nm的吸光度、474 nm的荧光和生物活性同时下降；K3Fe(CN)6的氧化也发生了类似的变化，因此可以用滴定法定量荧光素。荧光素的分子量在500 ~ 600之间，表现出正的埃利希和施莱辛格反应，酸铬酸盐氧化片段的产率明显类似于取代马来酰亚胺；这些数据支持了甲藻荧光素含有胆色素类型的取代聚吡咯的建议。
Bioluminescence in all dinoflagellate species studied to date is produced by the luciferase-catalyzed oxidation of a newly elucidated type of luciferin, hypothesized to have a substituted polypyrrole-type structure. This paper presents the purification and characterization of the luciferin from Pyrocystis lunula along with evidence that it is a polypyrrole-type molecule. Luciferin is extremely labile at low pH, at high salt concentration, and to O2, so, where possible, the purification steps were carried out in the presence of a buffered reducing agent and under argon. Purified luciferin is soluble in water and polar organic solvents. It is yellow (lambda max 245 and 390 nm with a shoulder at 290 nm in neutral or basic aqueous solution) and displays a strong blue fluorescence (lambda max for excitation at 390 nm, for emission at 474 nm) that closely matches the bioluminescence emission spectrum [Bode, V. C., & Hastings, J. W. (1963) Arch. Biochem. Biophys. 103, 488--499]. Autoxidation leads to concomitant decreases in the 390-nm absorbance, 474-nm fluorescence, and biological activity; similar changes occurred with oxidation by K3Fe(CN)6, thus allowing a quantitation of luciferin by titration. Luciferin has a molecular weight between 500 and 600, displays positive Ehrlich and Schlesinger reactions, and yields on acid chromate oxidation fragments apparently resembling substituted maleimides; these data support the proposal that dinoflagellate luciferin contains a substituted polypyrrole of the bile pigment type.