Accounting for solvent signal offsets in the analysis of interferometric sedimentation velocity data.

ABSTRACT

Sedimentation velocity (SV) analytical ultracentrifugation has re-emerged as an important tool in the characterization of biological macromolecules and nanoparticles. The computational analysis of the evolution of the macromolecular concentration profile allows the characterization of many hydrodynamic and thermodynamic properties of the macromolecules and their interactions. The Rayleigh interference optical system is often the detection method of choice, for its usually superior data quality and the wide applicability of refractive index sensitive detection. However, the interference optical system is also sensitive to the redistribution of co-solvent molecules, which are not of primary experimental interest. In principle, their contribution can be eliminated by an exact geometric and compositional match of the sample solution and the reference solution, achieving the complete optical subtraction of unwanted buffer signals. Unfortunately, in practice, this can often not be perfectly achieved for various reasons, leading to signal offsets arising from unmatched sedimentation of solvent components. If unrecognized, this can lead to significant misfit, accompanied by significant errors in the macromolecular sedimentation parameters. In the present work, we describe an approach of computationally accounting for signals from sedimenting buffer components through explicitly modeling their redistribution with Lamm equation solutions, implemented in the software SEDFIT. We demonstrate how this can restore the SV analysis to yield a high quality fit of the data and to provide correct macromolecular sedimentation parameters.