Breaking the 200 nm limit for routine flow linear dichroism measurements using UV synchrotron radiation.
Dicko C., Hicks MR., Dafforn TR., Vollrath F., Rodger A., Hoffmann SV.
The first synchrotron radiation flow linear dichroism spectra are reported. High-quality spectral data can be collected from 450 nm down to 180 nm in contrast to the practical cutoff of approximately 200 nm on benchtop instruments. State-of-the-art microvolume capillary Couette flow linear dichroism was successfully ported to a synchrotron radiation source. The sample volume required is < 50 microL. A characterization of the synchrotron radiation linear dichroism with known DNA and DNA-ligand systems is presented and the viability of the setup confirmed. Typically, wavelengths down to 180 nm are now routinely accessible with a high signal/noise ratio with little limitation from the sample concentration. The 180 nm cutoff is due to the quartz of the Couette cell rather than the beamline itself. We show the application of the simultaneous determination of the sample absorption spectrum to calculate the reduced linear dichroism signal. Spectra for calf thymus DNA, DNA/ethidium bromide, and DNA/4',6-diamidino-2-phenylindole systems illustrate the quality of data that can be obtained.