Yoshida T M, Zarrin F, Barisas B G
Department of Chemistry, Colorado State University, Fort Collins 80523.
Biophys J. 1988 Aug;54(2):277-88. doi: 10.1016/S0006-3495(88)82957-6.
Polarized fluorescence depletion (PFD) methods (Yoshida, T. M. and B. G. Barisas. Biophys. J. 1986. 50:41-53) are approximately 10(3)-10(4) fold more sensitive than other techniques for measuring protein rotational motions in cell membranes and other viscous environments. Proteins labeled with fluorophores having a high quantum yield for triplet formation are examined anaerobically in a fluorescence microscope. In time domain PFD experiments a several-microsecond pulse of linearly polarized light produces an orientationally-asymmetric depletion of ground state fluorescence in the sample. Monitoring the decay of ground state depletion with a probe beam alternatively polarized, parallel, and perpendicular to the depletion pulse permits the triplet lifetime and rotational correlation time to be resolved and evaluated. We have now explored fluorescence depletion methods in the frequency domain to see whether such measurements could provide simpler and more efficient routine measurements of protein rotational relaxation than previous time domain PFD methods. An acousto-optic modulator (AOM) modulates the intensity of a 514.5 nm argon ion laser beam and a Pockels cell (PC) rotates its plane of polarization. These devices are driven by sinusoidal or square waves in fixed frequency relation, and rigidly phase locked, one to another. The fluorescence emitted from a sample then contains various overtones and combinations of the AOM and PC frequencies. The magnitude and phase of individual fluorescence signal frequencies are measured by a lock-in amplifier using a reference also phase-locked to both the AOM and PC. Specific frequencies permit evaluation of the rotational correlation time of the macromolecule and of the fluorophore triplet state lifetime, respectively. Measurement of bovine serum albumin rotation in glycerol solutions by this method is described.
偏振荧光耗尽（PFD）方法（吉田，T.M.和B.G.巴里萨斯。《生物物理杂志》。1986年。50:41 - 53）比其他用于测量细胞膜及其他粘性环境中蛋白质旋转运动的技术灵敏度高约10³ - 10⁴倍。用对三重态形成具有高量子产率的荧光团标记的蛋白质在荧光显微镜下进行厌氧检测。在时域PFD实验中，一个几微秒的线偏振光脉冲会使样品中基态荧光产生取向不对称的耗尽。用与耗尽脉冲交替偏振、平行和垂直的探测光束监测基态耗尽的衰减，可分辨和评估三重态寿命及旋转相关时间。我们现在探索了频域中的荧光耗尽方法，以了解这种测量是否能比以前的时域PFD方法提供更简单、更高效的蛋白质旋转弛豫常规测量。一个声光调制器（AOM）调制514.5nm氩离子激光束的强度，一个普克尔盒（PC）旋转其偏振平面。这些装置由具有固定频率关系的正弦波或方波驱动，并彼此严格锁相。然后从样品发射的荧光包含AOM和PC频率的各种谐波和组合。通过一个与AOM和PC都锁相的参考信号，用锁相放大器测量各个荧光信号频率的幅度和相位。特定频率分别允许评估大分子的旋转相关时间和荧光团三重态寿命。描述了用这种方法测量甘油溶液中牛血清白蛋白的旋转。
