Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling.
A new method for the size-distribution analysis of polymers by sedimentation velocity analytical ultracentrifugation is described. It exploits the ability of Lamm equation modeling to discriminate between the spreading of the sedimentation boundary arising from sample heterogeneity and from diffusion. Finite element solutions of the Lamm equation for a large number of discrete noninteracting species are combined with maximum entropy regularization to represent a continuous size-distribution. As in the program CONTIN, the parameter governing the regularization constraint is adjusted by variance analysis to a predefined confidence level. Estimates of the partial specific volume and the frictional ratio of the macromolecules are used to calculate the diffusion coefficients, resulting in relatively high-resolution sedimentation coefficient distributions c(s) or molar mass distributions c(M). It can be applied to interference optical data that exhibit systematic noise components, and it does not require solution or solvent plateaus to be established. More details on the size-distribution can be obtained than from van Holde-Weischet analysis. The sensitivity to the values of the regularization parameter and to the shape parameters is explored with the help of simulated sedimentation data of discrete and continuous model size distributions, and by applications to experimental data of continuous and discrete protein mixtures.
Schuck
P
P
Molecular Interactions Resource, Bioengineering and Physical Science Program, ORS, National Institutes of Health, Bethesda, Maryland 20892, USA. pschuck@helix.nih.gov
D016001:Confidence Intervals; D004058:Diffusion; D008962:Models, Theoretical; D008970:Molecular Weight; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D012984:Software; D014461:Ultracentrifugation / Q000379:methods*
D011506:Proteins