Variable Field Analytical Ultracentrifugation: II. Gravitational Sweep Sedimentation Velocity

Abstract

Sedimentation velocity (SV) analytical ultracentrifugation is a classical biophysical technique for the determination of the size-distribution of macromolecules, macromolecular complexes, and nanoparticles. SV has traditionally been carried out at a constant rotor speed, which limits the range of sedimentation coefficients that can be detected in a single experiment. Recently we have introduced methods to implement experiments with variable rotor speeds, in combination with variable field solutions to the Lamm equation, with the application to expedite the approach to sedimentation equilibrium. Here, we describe the use of variable-field sedimentation analysis to increase the size-range covered in SV experiments by ∼100-fold with a quasi-continuous increase of rotor speed during the experiment. Such a gravitational-sweep sedimentation approach has previously been shown to be very effective in the study of nanoparticles with large size ranges. In the past, diffusion processes were not accounted for, thereby posing a lower limit of particle sizes and limiting the accuracy of the size distribution. In this work, we combine variable field solutions to the Lamm equation with diffusion-deconvoluted sedimentation coefficient distributions c(s), which further extend the macromolecular size range that can be observed in a single SV experiment while maintaining accuracy and resolution. In this way, approximately five orders of magnitude of sedimentation coefficients, or eight orders of magnitude of particle mass, can be probed in a single experiment. This can be useful, for example, in the study of proteins forming large assemblies, as in fibrillation process or capsid self-assembly, in studies of the interaction between very dissimilar-sized macromolecular species, or in the study of broadly distributed nanoparticles.