MacGregor Ian K, Anderson Arthur L, Laue Thomas M
Center to Advance Molecular Interaction Science, University of New Hampshire, Durham, NH 03824, USA.
Biophys Chem. 2004 Mar 1;108(1-3):165-85. doi: 10.1016/j.bpc.2003.10.018.
Analytical ultracentrifugation (AUC) provides first-principle hydrodynamic and thermodynamic information concerning the size, shape and interactions of macromolecules. The fundamental measurement needed in AUC is the macromolecular concentration as a function of radial position and time. Currently, the Beckman Coulter XLI analytical ultracentrifuge may be equipped with absorbance and refractive detectors, which provide complementary concentration determinations. For detecting trace quantities of materials, fluorescence detection offers unique advantages over either absorbance or interference detection. A prototype fluorescence detector for the XLI analytical ultracentrifuge has been developed and its characteristics determined. An Ar(+) laser provides a continuous 488-nm excitation beam. Radial resolution is achieved by scanning the focused beam along a radial axis. Detection of the fluorescence signal uses a co-axial, front-face optical configuration to reduce inaccuracies in the concentration caused by inner filter effects. A high-speed A/D data acquisition system allows the fluorescence intensity to be monitored continuously and at a sufficiently high angular resolution so that at any radial position the intensities from all of the samples may be acquired at each revolution. The fluorescence detector is capable of detecting concentrations as low as 300 pM for fluorescein-like labels. The radial resolution of the fluorescence detector is comparable to that of the absorbance system. Both sedimentation velocity and sedimentation equilibrium measurements may be made with the fluorescence detector. Results are presented comparing data acquired using the fluorescence with those acquired using the absorbance detector.
分析超速离心法（AUC）可提供有关大分子的大小、形状和相互作用的第一性原理流体动力学和热力学信息。AUC所需的基本测量是大分子浓度作为径向位置和时间的函数。目前，贝克曼库尔特XLI分析超速离心机可配备吸光度和折射率检测器，它们可提供互补的浓度测定。对于检测痕量物质，荧光检测相对于吸光度或干涉检测具有独特优势。已开发出用于XLI分析超速离心机的原型荧光检测器并确定了其特性。氩离子激光器提供488纳米的连续激发光束。通过沿径向轴扫描聚焦光束实现径向分辨率。荧光信号的检测采用同轴、正面光学配置，以减少内滤光效应引起的浓度测量误差。高速A/D数据采集系统可连续监测荧光强度，并具有足够高的角分辨率，以便在任何径向位置，每次旋转时都能采集所有样品的强度。该荧光检测器能够检测低至300皮摩尔的荧光素类标记物浓度。荧光检测器的径向分辨率与吸光度系统相当。使用荧光检测器可进行沉降速度和沉降平衡测量。给出了使用荧光检测器和吸光度检测器采集的数据的比较结果。
