Determination of rotational correlation times from deconvoluted fluorescence anisotropy decay curves. Demonstration with 6,7-dimethyl-8-ribityllumazine and lumazine protein from Photobacterium leiognathi as fluorescent indicators.
Determination of rotational correlation times from deconvoluted fluorescence anisotropy decay curves. Demonstration with 6,7-dimethyl-8-ribityllumazine and lumazine protein from Photobacterium leiognathi as fluorescent indicators.
复制标题
从解卷积荧光各向异性衰减曲线确定旋转相关时间。
作者：
Visser,AJ;Ykema,T;vanHoek,A;O'Kane,DJ;Lee,J
1984年8月10日收到的修订稿摘要：描述了从荧光各向异性衰减测量获得生物大分子旋转相关时间所需的实验和分析方案。作为一个实例，使用了来自发光杆菌（Photobacterium leiognathi）的lumazine蛋白。这种稳定的蛋白质（Mr 21 200）含有非共价结合的天然荧光标记物6，7-二甲基-8-ribityllumazine，其在结合状态下具有长的荧光寿命（r= 14 ns）。通过加入碰撞荧光猝灭剂碘化钾，实现了在室温下的荧光寿命缩短至2.6 ns。的缩短几乎没有影响的旋转相关时间的鲁马嗪蛋白（0= 9.4 ns，19 C）。测量双指数各向异性衰减的能力通过添加发光杆菌萤光素酶（Mr 80000）来测试，所述发光杆菌萤光素酶与鲁马嗪蛋白形成平衡复合物。在所用的实验条件下（2 ℃），双指数各向异性衰减可以用20和60 ns的相关时间最好地描述，分别代表未复合的和酶相关的lumazine蛋白。未结合的6，7-二甲基-8-核糖基鲁马嗪本身（= 9 ns）被用作用于确定皮秒时间范围内的相关时间的模型化合物。在后一种情况下，需要从激发曲线中进行严格的解卷积，以恢复相关时间，其比测量的激光激发脉冲宽度（500 ps）短（100-200 ps）。近20年来，人们对大分子转动扩散的研究进展进行了综述（Tao，1969; Yguerabide，1972; Rigler & Escherberg，1973; Wahl，1983）。通过偏振光的短脉冲产生偏振荧光的方法是弛豫方法。它测量先前创建的荧光分子的各向异性分布的随机化。该过程的时间常数（旋转相关时间）与荧光颗粒的尺寸成比例。荧光各向异性的时间依赖性与平行偏振的荧光强度有关。||（r）]和垂直于偏振激发光的[_L（i）]：
Revised Manuscript Received August 10, 1984 abstract: The experimental and analytical protocols required for obtaining rotational correlation times of biological macromolecules from fluorescence anisotropy decay measurements are described. As an example, the lumazine protein from Photobacterium leiognathi was used. This stable protein (Mr 21 200) contains the noncovalently bound, natural fluorescent marker 6, 7-dimethyl-8-ribityllumazine, which has in the bound state a long fluoresence lifetime (r= 14 ns). Shortening of the fluorescence lifetime to 2.6 ns at room temperature was achieved by addition of the collisional fluorescence quencher potassium iodide. The shortening of had virtually no effect on the rotational correlation time of the lumazine protein (0= 9.4 ns, 19 C). The ability to measure biexponential anisotropy decay was tested by the addition of Photobacterium luciferase (Mr 80000), which forms an equilibrium complex with lumazine protein. Under the experimental conditions used (2 C) the biexponential anisotropy decay can best be described with correlation times of 20 and 60 ns, representingthe uncomplexed and luciferase-associated lumazine proteins, respectively. The unbound 6, 7-dimethyl-8-ribityllumazine itself (= 9 ns) was used as a model compound for determining correlation times in the picosecond time range. In the latter case rigorous deconvolution from the excitation profile was required to recover the correlation time, which was shorter (100-200 ps) than the measured laser excitation pulse width (500 ps).Brownian rotation of fluorescent biopolymers can be directly followed via the time course of the polarized emission com-ponents after excitation with polarized light pulses. Infor-mation concerning rotational diffusion of macromolecules has been reviewedin the last two decades (Tao, 1969; Yguerabide, 1972; Rigler & Ehrenberg, 1973; Wahl, 1983). The method in which polarized fluorescence is created by a short pulse of polarized light is a relaxation method. It measures the return to randomization of a previously created, anisotropic distributionof fluorescent molecules. The time constant of this process (the rotational correlation time) is proportional to the size of the fluorescent particle. Thetime dependence of the fluorescence anisotropy is related to the fluorescence intensity polarized parallel [||(r)] and perpen-dicular [_L (i)] to the polarizedexcitation light: