van Holde - Weischet Tutorial

Applications

The van Holde - Weischet analysis (vHWA) is a versatile analysis method that can be applied to a wide range of different systems. Its biggest strength is its ability to separate diffusional boundary spreading from the boundary spreading caused by sedimentation alone, which is not possible with difference/derivative based methods, which have to rely on fitting of sums of gaussian distributions for the identification of heterogeneity, an inherently ill-conditioned approach. Hence, the ultimate utility of this analysis lies in the unambiguous detection of homo- and heterogeneity.

For the characterization of unknown samples, a sedimentation velocity experiment analyzed with the vHWA goes a long way towards the determination of sample composition and sample properties. Unlike finite element and other whole boundary fitting methods, the vHWA allows for a completely model-independent analysis of the sedimentation profile, which is not subject to degenerate and non-unique solutions possible with whole boundary fitting methods (this is not to say that your interpretations are always unique! But there are ways to guard against this, see the section on experimental design for more information).

Next, the vHWA provides several important diagnostics for velocity experiments. These include its ability to detect, and to a certain degree, even quantify, concentration dependency of S. In addition, the vHWA provides an indication for loss of material due to pelleting, even if the sample is very heterogeneous. It is possible with the vHWA to identify those scans of an experiment where the total plateau concentration has decreased more than expected by radial dilution alone, hence, for scans, where material has been lost due to pelleting. This feature provides then a method for selecting scans that contain a stable plateau. Should time dependent changes occur such as aggregation or degradation, the vHWA provides a clear indication for this occurence, although a correct analysis of the sample may be compromised by this behaviour. Please see the section on diagnostics for further details.

The vHWA can be successfully applied to the following systems:

• homogeneous systems
• heterogeneous systems
• self-associating systems
• hetero-associating systems
• aggregating systems
• isodesmic associations
• degrading systems
• floating experiments
• systems exhibiting concentration dependency of S

In general, the following questions can be answered with the vHWA:

• Is the sample homo- or heterogeneous?
• How many different components are present in my sample?
• Does the sample exhibit significant concentration dependency in S for the concentration range under which I investigate?
• Does the sample self-associate?
• Does the sample aggregate, and if so, by how much?
• what are the relative and partial concentrations of each component?
• Is the aggregation/association reversible?
• Do two different components in the system interact and bind?
• What is the stoichiometry of this interaction?
• What is the range of S-values?
• For homogeneous solutions: what is the approximate diffusion coefficient?
• For paucidisperse soutions: while exact determination of diffusion coefficients becomes increasingly more difficult with additional components in the system, for some well-conditioned cases this is possible, at least qualitatively.
• Does the sample change composition over time, i.e., does the sample undergo degradation or aggregation during the sedimentation process?
• Does the sample undergo conformational changes?

In conjunction with equilibrium experiments, the additional questions below can be answered:

• Does the (pure, single-component) sample isomerize into a two-state isomerization equilibrium?
• Is my sample's heterogeneity in S due to heterogeneity in molecular weight, heterogeneity in shape, or both?

For experiments where small changes in the sample's properties need to be detected, an overlay of van Holde - Weischet distribution plots from each experiment provides a very sensitive method for comparing samples under two different conditions. It is advised that all samples to be compared are measured in the same experimental run to with identical loading volumes to assure that temperature, sedimentation behavior, sample age, etc. do not introduce additional variables into the experiment. See the section on experimental design for further details.