Document Detail


Validation of new microvolume Couette flow linear dichroism cells.
MedLine Citation:
PMID:  16284659     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Long molecules such as fibrous proteins are particularly difficult to characterise structurally. We have recently designed a microvolume Couette flow linear dichroism (LD) cell whose sample volume is only 20-40 microL in contrast to previous cells where the volume of sample required has typically been of the order of 1000-2000 microL. This brings the sample requirements of LD to a level where it can be used for biological samples. Since LD is the difference in absorption of light polarised parallel to an orientation direction and perpendicular to that direction, it is the ideal technique for determining relative orientations of subunits of e.g. fibrous proteins, DNA-drug systems, etc. For solution phase samples, Couette flow orientation, whereby the sample is sandwiched between two cylinders, one of which rotates, has proved to be the optimal technique for LD experiments in many laboratories. Our capillary microvolume LD cell has been designed using extruded quartz rods and capillaries and focusing and collecting lenses. We have developed applications with PCR products, fibrous proteins, liposome-bound membrane proteins, as well as DNA-dye systems. Despite this range of applications, to date there is nothing reported in the literature to enable one to validate the performance of Couette flow LD cells. In this paper we establish validation criteria and show that the data from the microvolume cells are reproducible, vary by less than 1% with sample reloading, follow the Beer-Lambert law, and have signals linear in voltage over a wide voltage range. The microvolume cell data are consistent with those from the large-volume cells for DNA samples. Surprisingly, upon extending the wavelength range by adding the intercalator ethidium bromide, the spectra in the microvolume and large-volume cells differ by a wavelength dependent orientation parameter. This wavelength variation was concluded to be the result of Taylor-vortices in the large-volume cells which have inner rotating cylinders in our laboratory. Thus the microvolume LD cells can be concluded to provide better data than our large-volume LD cells, though the latter are still to be preferred for titration series as it is extremely difficult to add sample to the capillary cells without introducing artefacts.
Authors:
Rachel Marrington; Timothy R Dafforn; David J Halsall; James I MacDonald; Matthew Hicks; Alison Rodger
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Validation Studies     Date:  2005-10-14
Journal Detail:
Title:  The Analyst     Volume:  130     ISSN:  0003-2654     ISO Abbreviation:  Analyst     Publication Date:  2005 Dec 
Date Detail:
Created Date:  2005-11-14     Completed Date:  2006-05-04     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0372652     Medline TA:  Analyst     Country:  England    
Other Details:
Languages:  eng     Pagination:  1608-16     Citation Subset:  IM    
Affiliation:
Department of Chemistry, University of Warwick, Coventry, UK CV4 7AL.
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MeSH Terms
Descriptor/Qualifier:
DNA / analysis*
Microchemistry / instrumentation*,  methods
Spectrum Analysis / instrumentation,  methods
Chemical
Reg. No./Substance:
9007-49-2/DNA

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