Lee J, Wang Y Y, Gibson B G
Department of Biochemistry, University of Georgia, Athens 30602.
Biochemistry. 1991 Jul 16;30(28):6825-35. doi: 10.1021/bi00242a004.
Fluorescence dynamics measurements have been made on the bioluminescence reaction intermediates using Photobacterium leiognathi, Vibrio fischeri, and Vibrio harveyi luciferases, both alone and in mixtures with Photobacterium phosphoreum lumazine protein. Each luciferase produces a "fluorescent transient" intermediate on reaction with the bioluminescence substrates, FMNH2, tetradecanal, and O2, and all have a fluorescence quantum yield about 0.3, with a predominant lifetime around 10 ns. The P. leiognathi luciferase fluorescent transient has a rotational correlation time of 79 ns at 2 degrees C, as expected for the rotational diffusion of a 77-kDa macromolecule. In the presence of lumazine protein however a faster correlation time of about 3 ns predominates. This rapid channel of anisotropy loss is attributed to energy transfer from the flavin intermediate bound on the luciferase to the lumazine ligand, reflects the presence of protein-protein complexation, and is greatest in the case of P. leiognathi, but not at all for V. fischeri. This fact is consistent with the strong influence of lumazine protein on the bioluminescence reaction of P. leiognathi, and not at all with V. fischeri. The rate of energy transfer is of order 10(9) s-1, much greater than the 10(8) s-1 fluorescence rate of the donor. Thus the bioluminescence excitation of lumazine protein could occur by a similar photophysical mechanism of interprotein energy transfer from a chemically excited fluorescent transient donor to the lumazine acceptor.
利用鱼发光杆菌、费氏弧菌和哈氏弧菌荧光素酶，对生物发光反应中间体进行了荧光动力学测量，这些荧光素酶既单独使用，也与磷光杆菌的蝶啶单核苷酸蛋白混合使用。每种荧光素酶在与生物发光底物FMNH2、十四醛和O2反应时都会产生一种“荧光瞬态”中间体，且它们的荧光量子产率均约为0.3，主要寿命约为10纳秒。在2摄氏度下，鱼发光杆菌荧光素酶的荧光瞬态的旋转相关时间为79纳秒，这对于一个77千道尔顿大分子的旋转扩散来说是预期的。然而，在存在蝶啶单核苷酸蛋白的情况下，约3纳秒的更快相关时间占主导。这种各向异性损失的快速通道归因于从结合在荧光素酶上的黄素中间体到蝶啶单核苷酸配体的能量转移，反映了蛋白质 - 蛋白质络合的存在，并且在鱼发光杆菌的情况下最为显著，但费氏弧菌则完全没有。这一事实与蝶啶单核苷酸蛋白对鱼发光杆菌生物发光反应的强烈影响一致，而与费氏弧菌完全无关。能量转移速率约为10⁹ s⁻¹，远大于供体的10⁸ s⁻¹荧光速率。因此，蝶啶单核苷酸蛋白的生物发光激发可能通过一种类似的光物理机制发生，即从化学激发的荧光瞬态供体到蝶啶单核苷酸受体的蛋白质间能量转移。
