Sedimentation velocity analysis of highly heterogeneous systems

Abstract

This article discusses several improvements to the van Holde–Weischet (vHW) method [Biopolymers 17 (1978) 1387] that address its capability to deal with sedimentation coefficient distributions spanning a large range of s values. The method presented here allows the inclusion of scans early and late in the experiment that ordinarily would need to be excluded from the analysis due to ultracentrifuge cell end effects. Scans late in the experiment are compromised by the loss of a defined plateau region and by back-diffusion from the bottom of the cell. Early scans involve partial boundaries that have not fully cleared the meniscus. In addition, a major refinement of the algorithm for determining the boundary fractions is introduced, taking into account different degrees of radial dilution for different species in the system. The method retains its desirable model-independent properties (the analysis of sedimentation data does not require prior knowledge of a user-imposed model or range of sedimentation coefficients) and reports diffusion-corrected s value distributions, which can be presented either in a histogram format or the traditional integral distribution format. Data analyzed with the traditional vHW method are compared with those of the improved method to demonstrate the benefit from the added information in the analysis.