Density Contrast Sedimentation Velocity for the Determination of Protein Partial-Specific Volumes (original) (raw)

Assessing sedimentation equilibrium profiles in analytical ultracentrifugation experiments on macromolecules: from simple average molecular weight analysis to molecular weight distribution and interaction analysis

Biophysical reviews, 2016

Molecular weights (molar masses), molecular weight distributions, dissociation constants and other interaction parameters are fundamental characteristics of proteins, nucleic acids, polysaccharides and glycoconjugates in solution. Sedimentation equilibrium analytical ultracentrifugation provides a powerful method with no supplementary immobilization, columns or membranes required. It is a particularly powerful tool when used in conjunction with its sister technique, namely sedimentation velocity. Here, we describe key approaches now available and their application to the characterization of antibodies, polysaccharides and glycoconjugates. We indicate how major complications, such as thermodynamic non-ideality, can now be routinely dealt with, thanks to a great extent to the extensive contribution of Professor Don Winzor over several decades of research.

Different Levels of Self-Sufficiency of the Velocity Sedimentation Method in the Study of Linear Macromolecules

Analytical Ultracentrifugation, 2016

The general principles for the interpretation of the sedimentation velocity for linear macromolecules are discussed in combination with the characteristics obtained by other hydrodynamic methods. The several levels of self-sufficiency of the sedimentation velocity method are demonstrated. They include scaling correlation between velocity sedimentation coefficient s0 and concentration coefficient \( {k}_s={K}_{sks}{s_0}^{bks} \), application of the sedimentation parameter β s for molar mass estimation, and qualitative conformation zoning of linear macromolecules using the normalized double logarithmic plots (k s M L vs [s]/M L ). The last example of self-sufficiency of the method is the study of the concentration dependence of the sedimentation velocity in combination with continuous c(s) distribution using general scaling law model of Sedfit software. This model allows to establish the relationship \( {s}_0={K}_s{M}^{bs} \) for flexible polymers and perform further gross conformational analysis and analysis of the molar mass distribution. It is shown that the K s coefficient is strongly dependent on the polymer concentration.

Detection of protein aggregates by sedimentation velocity analytical ultracentrifugation (SV-AUC): Sources of variability and their relative importance

Journal of Pharmaceutical Sciences, 2009

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has found application in the biopharmaceutical industry as a method of detecting and quantifying protein aggregates. While the technique offers several advantages (i.e., matrix-free separation and minimal sample handling), its results exhibit a high degree of variability relative to orthogonal size-sensitive separation techniques such as size exclusion chromatography (SEC). The goal of this work is to characterize and quantify the sources of variability that affect SV-AUC results, particularly size distributions for a monoclonal antibody monomer/dimer system. Contributions of individual factors to the overall variability are examined. Results demonstrate that alignment of sample cells to the center of rotation is the most significant contributing factor to overall variability. The relative importance of other factors (e.g., temperature equilibration, time-invariant noise, meniscus misplacement, etc.) are quantified and discussed. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3522–3539, 2009