THE LUMINESCENT OXIDATION OF REDUCED RIBOFLAVIN OR REDUCED RIBOFLAVIN PHOSPHATE IN THE BACTERIAL LUCIFERIN-LUCIFERASE REACTION.
THE LUMINESCENT OXIDATION OF REDUCED RIBOFLAVIN OR REDUCED RIBOFLAVIN PHOSPHATE IN THE BACTERIAL LUCIFERIN-LUCIFERASE REACTION.
复制标题
细菌荧光素-荧光素酶反应中还原型核黄素或还原型核黄素磷酸盐的发光氧化。
DOI：
10.1073/pnas.40.1.10
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发表时间：
1954
影响因子：
11.1
通讯作者：
M. J. Cormier
中科院分区：
文献类型：
--
作者：
B. Strehler;E. N. Harvey;J. J. Chang;M. J. Cormier
1953年12月11日通信许多早期的研究表明发光和黄素化合物之间的关系，1-8，尽管迄今为止还没有证明发光细菌提取物对还原黄素的酶促氧化会导致发光。当黄素单胞苷（FMN=核黄素磷酸盐）或还原型二磷酸吡啶核苷酸（DPNH 2 =还原型辅酶I）是最后一个添加到发光细菌提取物中的因子时，发光的动力学相似，因此DPNH 2在发光中的唯一功能似乎是产生还原型黄素，而且，还原型黄素可能被认为是细菌黄素。由于处理还原黄素的技术困难，这些化合物没有用于早期对从细菌中提取的无细胞发光系统的研究。我们现在可以报告，还原核黄素或还原黄素单核苷酸肽将支持在不存在添加的DPNH 2的情况下无色杆菌提取物的发光。对于本文报道的实验，培养了费氏无色杆菌，并根据Strehler和Corp.的方法制备了5%的提取物。核黄素和黄素单甘肽分别从Merck and Company和Sigma Chemical Company获得，并且以约100 μ g/ml的浓度使用。ml.这些化合物在铂化石棉存在下在催化纯化的氢气流中还原。对于目视定性实验，使用了前面所述的双室”A管”（经修改以滤除镀铂石棉）。当过滤除去铂化石棉的还原的favin化合物与氢通过的酶厌氧混合时，会出现微弱的发光（可能是由于氧气的痕迹），但很快就消失了。然而，在给试管充气时，出现了持续约20秒的亮光。用简单快速混合停流法进行了13次定量光测定和反应时间过程的研究。在直流操作下用1 P21光电倍增管测量光输出，并从阴极射线衍射图中记录光输出。用于定量实验的黄素化合物在带塞的离心管中还原，并用1 ml离心管从离心管中除去。将催化剂离心后注射到皮下注射器中。图1包含了发光时间过程的示波器描记图
Communicated December 11, 1953 Numerous earlier studies on various light-producing species have suggested a relation between luminescence and fiavin compounds, 1-8 although it has not been demonstrated heretofore that the enzymatic oxidation of reduced flavins by luminous bacterial extracts will result in luminescence. From the similar kinetics of light emission when either flavin mononucleotide (FMN= riboflavin phosphate) or reduced diphosphopyridin nucleotide (DPNH2= reduced coenzyme I) was the last factor added to luminous bacterial extracts, 9 it seemed probable that the sole function of DPNH2 in luminescence was to produce reduced flavin, and, further, that reduced flavin might be considered as bacterial luciferin. Becauseof technical difficulties in handling reduced flavins these compounds were not used in earlier studies on the extracted cell-free luminescent system from bacteria. We can now report that either reduced riboflavin or reduced flavin mononucleotide will support the luminescence of Achromobacterfischeri extracts in the absence of added DPNH2. For the experiments reported here Achromobacter fischeri was grown and 5 per cent extracts prepared according to the method of Strehler and Cormier. 5" 0 Riboflavin and flavin mononucleotide were obtained from Merck and Company and Sigma Chemical Company, respectively, and were used in concentrations of about 100 Ag./ml. These compounds were reduced in a stream of catalytically purified hydrogen in the presence of platinized asbestos. For visual qualitative experi-ments the 2-chamber" A tubes" described earlier'1 were used (modified to filter off the platinized asbestos). When the reduced favin compounds, filtered free of platinized asbestos, were mixed anaerobically with the enzyme through which hydrogen had been passed, a dim flash of luminescence (probably due to traces of oxygen) occurred, which soon disappeared. However, on aerating the tube, a bright light of about 20 seconds duration appeared. 13 Quantitative light determinations were made and the time course ofthe reaction studied witha simple rapid mixing stop-flow method. Light output was measured with a 1P21 photomultiplier under DC operation, and recorded photographically from a cathode ray oscillograph. The flavin compounds employed for the quantita-tive experiments were reduced ina stoppered centrifuge tube and removed from the tube with a 1-ml. hypodermic syringe after centrifuging down the catalyst. Figure 1 contains photographic oscilloscope tracings of the time courseof luminescence