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Macromolecular Size-and-Shape Distributions by Sedimentation Velocity Analytical Ultracentrifugation

Protein Biophysics Resource, Division of Bioengineering and Physical Science, ORS, Office of the Director, National Institutes of Health, Bethesda, Maryland

Correspondence: Address reprint requests to Dr. Peter Schuck, National Institutes of Health, Bldg. 13, Rm. 3N17, 13 South Dr., Bethesda, MD 20892. Tel.: 301-435-1950; Fax: 301-480-1242; E-mail: pschuck{at}helix.nih.gov.

Sedimentation velocity analytical ultracentrifugation is an important tool in the characterization of macromolecules and nanoparticles in solution. The sedimentation coefficient distribution c(s) of Lamm equation solutions is based on the approximation of a single, weight-average frictional coefficient of all particles, determined from the experimental data, which scales the diffusion coefficient to the sedimentation coefficient consistent with the traditional s M^2/3 power law. It provides a high hydrodynamic resolution, where diffusional broadening of the sedimentation boundaries is deconvoluted from the sedimentation coefficient distribution. The approximation of a single weight-average frictional ratio is favored by several experimental factors, and usually gives good results for chemically not too dissimilar macromolecules, such as mixtures of folded proteins. In this communication, we examine an extension to a two-dimensional distribution of sedimentation coefficient and frictional ratio, c(s,f_r), which is representative of a more general set of size-and-shape distributions, including mass-Stokes radius distributions, c(M,R_S), and sedimentation coefficient-molar mass distributions c(s,M). We show that this can be used to determine average molar masses of macromolecules and characterize macromolecular distributions, without the approximation of any scaling relationship between hydrodynamic and thermodynamic parameters.

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