Micro-volume couette flow sample orientation for absorbance and fluorescence linear dichroism
Marrington, Rachel, Dafforn, Tim, Halsall, David J. and Rodger, Alison. (2004) Micro-volume couette flow sample orientation for absorbance and fluorescence linear dichroism. Biophysical Journal, Vol.87 (No.3). pp. 2002-2012. ISSN 0006-3495
WRAP_Marrington_Micro-volume.pdf - Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader
Official URL: http://dx.doi.org/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 μL (with concentrations such that the absorbance maximum of the sample in a 1-cm pathlength cuvette is not, vert, similar1). 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.
|Item Type:||Journal Article|
|Subjects:||Q Science > QD Chemistry|
|Divisions:||Faculty of Science > Chemistry|
|Library of Congress Subject Headings (LCSH):||Linear dichroism, Laminar flow, Polarization (Light), Fluorescence spectroscopy, Fluid dynamics|
|Journal or Publication Title:||Biophysical Journal|
|Official Date:||September 2004|
|Page Range:||pp. 2002-2012|
|Access rights to Published version:||Open Access|
|Funder:||Engineering and Physical Sciences Research Council (EPSRC), Syngenta Ltd.|
|Grant number:||GR/M91105 (EPSRC)|
Bloemendal and Vangrondelle, 1993 M. Bloemendal and R. Vangrondelle, Linear-dichroism spectroscopy for the study of structural properties of proteins, Mol. Biol. Rep. 18 (1993), pp. 49–69.
Dafforn et al., 2004 T.R. Dafforn, J. Rajendra, D.J. Halsall, L.C. Serpell and A. Rodger, Protein fibre linear dichroism for structure determination and kinetics in a low-volume, low-wavelength Couette flow cell, Biophys. J. 86 (2004), pp. 404–410
Wilson and Schellman, 1978 R. Wilson and J. Schellman, The flow linear dichroism of DNA: comparison with the bead-spring theory, Biopolymers 17 (1978), pp. 1235–1248.
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