Tu S C
Biochemistry. 1979 Dec 25;18(26):5940-5. doi: 10.1021/bi00593a028.
Nonsubstrate long-chain aliphatic alcohols, carboxylic acids, and their methyl esters were found to complex reversibly with and stabilize an oxygenated flavin-luciferase intermediate, with alcohols being more effective in stabilizing the intermediate. Dissociation constants for the binding of alcohols to luciferase intermediate are in the order of K8 greater than K10 greater than K12 congruent to K14 where the subscripts represent the numbers of carbon atoms of various alcohols. Thermodynamic activation parameters for the decay of oxygenated flavin-luciferase intermediate complexed with alcohols or aldehydes were determined, and similarities were noted between alcohol and aldehyde complexes. Luciferase intermediate complexes formed with 1-decanol and 1-tetradecanol were isolated at 0 degrees C in neutral phosphate buffer, and both showed absorption properties characteristic of 4a-substituted dihydroflavins. The 1-tetradecanol-intermediate species contained one favin per luciferase molecule. Initially this complex was weakly fluorescent, but upon exposure to 370-nm light it was transformed to a highly fluorescent species. The latter shows a fluorescence excitation peak at 370 nm, and its fluorescence emission (lambda max 505 nm) and quantum yield (0.17) closely correspond to that of bioluminescence in vitro. Both the weakly and the highly fluorescent species exhibit full bioluminescence activities when reacted with decanal.
非底物长链脂肪醇、羧酸及其甲酯被发现可与氧化型黄素 - 荧光素酶中间体可逆结合并使其稳定，其中醇类在稳定中间体方面更有效。醇类与荧光素酶中间体结合的解离常数顺序为K8大于K10大于K12约等于K14，下标表示各种醇的碳原子数。测定了与醇类或醛类复合的氧化型黄素 - 荧光素酶中间体衰变的热力学活化参数，并注意到醇类和醛类复合物之间的相似性。在0℃下于中性磷酸盐缓冲液中分离出由1 - 癸醇和1 - 十四醇形成的荧光素酶中间体复合物，两者均显示出4a - 取代二氢黄素的吸收特性。1 - 十四醇 - 中间体物种每个荧光素酶分子含有一个黄素。最初这种复合物荧光较弱，但在暴露于370nm光后转变为高荧光物种。后者在370nm处显示荧光激发峰，其荧光发射（λmax 505nm）和量子产率（0.17）与体外生物发光的非常接近。当与癸醛反应时，弱荧光和高荧光物种均表现出完全的生物发光活性。
