Marrington Rachel, Dafforn Timothy R, Halsall David J, Rodger Alison
Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK.
Biophys J. 2004 Sep;87(3):2002-12. doi: 10.1529/biophysj.103.035022.
Linear dichroism (LD) can be used to study the alignment of absorbing chromophores within long molecules. In particular, Couette flow LD has been used to good effect in probing ligand binding to DNA and to fibrous proteins. This technique has been previously limited by large sample requirements. Here we report the design and application of a new micro-volume Couette flow cell that significantly enhances the potential applications of flow LD spectroscopy by reducing the sample requirements for flow linear dichroism to 25 microL (with concentrations such that the absorbance maximum of the sample in a 1-cm pathlength cuvette is approximately 1). The micro-volume Couette cell has also enabled the measurement of fluorescence-detected Couette flow linear dichroism. This new technique enables the orientation of fluorescent ligands to be probed even when their electronic transitions overlap with those of the macromolecule and conversely. The potential of flow-oriented fluorescence dichroism and application of the micro-volume Couette LD cell are illustrated by the collection of data for DNA with minor groove and intercalating ligands: DAPI, Hoechst, and ethidium bromide. As with conventional fluorescence, improved sensitivity compared with absorbance LD is to be expected after instrumentation optimization.
线性二色性（LD）可用于研究长分子内吸收发色团的排列。特别是，库埃特流LD在探测配体与DNA及纤维状蛋白质的结合方面已取得良好效果。该技术此前受到大样本需求的限制。在此，我们报告一种新型微体积库埃特流动池的设计与应用，通过将流动线性二色性的样本需求降低至25微升（样本在1厘米光程比色皿中的最大吸光度约为1时的浓度），显著增强了流动LD光谱的潜在应用。微体积库埃特流动池还实现了荧光检测的库埃特流线性二色性的测量。这项新技术即使在荧光配体的电子跃迁与大分子的电子跃迁相互重叠时，也能探测荧光配体的取向，反之亦然。通过收集与小沟和嵌入配体（DAPI、Hoechst和溴化乙锭）结合的DNA的数据，展示了流动取向荧光二色性的潜力以及微体积库埃特LD流动池的应用。与传统荧光一样，在仪器优化后，预计与吸光度LD相比灵敏度会有所提高。
