Continuous-channel flow linear dichroism

Abstract

Linear dichroism (LD) is the difference in absorbance of light polarized parallel to a sample orientation axis and polarized perpendicular to it. Flow LD provides information about samples with high enough aspect ratios to be oriented in a flow stream. It is particularly useful for studying DNA–ligand systems, protein fibres and membrane assemblies and is the ideal technique for monitoring growth or destruction of particles. Standard Couette flow cells are limited to a dead time in kinetic processes of ∼40 s (the assembly time). Recently an injection Couette flow cell has reduced the dead time to ∼600 ms. In this paper we report an alternative system, based on syringe drives, 3 alternative flow-through cells, and the optical system of a Biologic MOS-450 spectrometer that has been adapted for LD. This system can reduce the dead time to 25 ms. The sample requirement is ∼100 μL per time point. Less sample is required for longer dead times. The system has been applied to measure the kinetics of DNase digestion of DNA and GTP-induced polymerization of the bacterial cell division protein FtsZ.