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Rapid injection linear dichroism for studying the kinetics of biological processes
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Hicks, Matthew R., Rodger, Alison, Lin, Yu-Pin, Jones, Nykola C., Hoffmann, Søren Vrønning and Dafforn, Tim (2012) Rapid injection linear dichroism for studying the kinetics of biological processes. Analytical Chemistry, Volume 84 (Number 15). pp. 6561-6566. doi:10.1021/ac300842h
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Official URL: http://dx.doi.org/10.1021/ac300842h
Abstract
Linear dichroism is defined as the differential absorbance of linearly polarized light oriented in two orthogonal directions by an aligned sample. The measurement of a linear dichroism (LD) spectrum of a sample provides two key pieces of structural information. First, that the sample and the chromophores within the sample are able to align. Second, given knowledge of the transition polarization directions of the chromophores, the orientation of the chromophores within the aligned sample can be resolved. It has been shown that LD can provide unique information on the structure of some of the more challenging biomolecular complexes. This has included macromolecular protein and peptide fibers such as actin, tubulin, and amyloids as well as protein–membrane complexes and DNA–protein complexes. Much of this work has been enabled by the development of a low volume Couette flow cell that efficiently aligns long molecules in solution. However, the current Couette system is inherently complex to assemble for each experiment and hence not suited to measurement of rapid reactions. In this paper we detail the development of the first rapid injection LD cell. The system utilizes a conventional stopped-flow injection system coupled to a modified low volume Couette cell, where a narrow bore capillary replaces the normal solid central rod. The system is shown to have similar optical characteristics to the conventional LD Couette flow cell but with the added benefit of a much shorter dead time (0.60 s compared to 60). The rapid injection Couette cell has been used to measure the degradation of DNA by DNA exonuclease I, providing data that would not be available using a conventional system.
| Item Type: | Journal Article | ||||
|---|---|---|---|---|---|
| Divisions: | Faculty of Science > Chemistry | ||||
| Journal or Publication Title: | Analytical Chemistry | ||||
| Publisher: | American Chemical Society | ||||
| ISSN: | 0003-2700 | ||||
| Official Date: | 7 August 2012 | ||||
| Dates: |
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| Volume: | Volume 84 | ||||
| Number: | Number 15 | ||||
| Page Range: | pp. 6561-6566 | ||||
| DOI: | 10.1021/ac300842h | ||||
| Status: | Peer Reviewed | ||||
| Publication Status: | Published | ||||
| Access rights to Published version: | Restricted or Subscription Access | ||||
| Funder: | European Community′s Seventh Framework Programme (FP7/2007−2013), Thee Lundbeck Foundation, The Danish Council for Independent Research | Natural Sciences (FNU), Engineering and Physical Sciences Research Council (EPSRC) | ||||
| Grant number: | 226716 (FP7/2007−2013); GR/T09224/01 (EPSRC) |
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