Validation of new microvolume Couette flow linear dichroism cells

Abstract

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.

The Synergistic Action of Melittin and Phospholipase A2 with Lipid Membranes: Development of Linear Dichroism for Membrane-Insertion Kinetics

“…LD spectra were collected using a Jasco J-815 spectropolarimeter adapted for LD measurements in a microvolume Couette flow cell manufactured by Crystal Precision Optics, Rugby, UK and now available via Kromatek Ltd., (Great Dunmow, UK). 7,[10][11][12][22][23][24][25][26] Far UV CD and LD spectra (190-320 nm) were collected using a data pitch of 0.2 nm, continuous scanning mode, scanning speed of 100 nm/min, response of 1 s, band width of 1 or 2 nm. The Couette cell rotation speed was ~3000 rpm (setting: 3.0 V).…”

Apparent circular dichroism signature of stirring-oriented DNA and drug–DNA complexes

“…Since LD signals tend to be orders of magnitude larger than the CD ones, it can be supposed that the LD effect makes a chief contribution to the CD spectrum of stirred solutions [10,11]. Prevalence of the LD contribution is supported by close 6 resemblance of the anomalous CD curves presented in this work (see further spectra below) to the flow LD spectra of DNA [5] and drug-DNA complexes [8,12] 1C). The DNA molecules are now aligned along the long axis of the cuvette, so they exhibit an inverted, positive LD effect (Fig.…”

Redox-active and DNA-binding coordination complexes of clotrimazole

“…All UV-Vis and CD measurements were collected in 1 cm pathlength quartz cuvettes (Starna UK). The LD measurements were performed using a quartz capillary LD Couette flow cell built by Crystal Precision Optics spinning at 3000 RPM [18,19]. CD and LD spectra were measured over the desired wavelength range using a bandwidth of 2 nm, a data pitch of 0.2 nm, scanning speed of 100 nm/min, response time of 1 s and 2 (LD) or 4 (CD) accumulations.…”