Informationen zur Analytischen Ultrazentrifugation (AUZ)

Abstract: K. Schilling, H. Cölfen, Progr. Coll. Polym. Sci., im Druck (1999)

 

Application of the Solvent Density Variation Method to Sedimentation Velocity Experiments on Biological Systems

 
Kristian Schilling, Helmut Cölfen

Max Planck Institute of Colloids and Interfaces, Colloid Chemistry, Am Mühlenberg, D-14476 Golm, Germany
Author for correspondence: Dr. Helmut Cölfen
Tel: 0331-567-9513, Fax: 0331-567-9502
E-Mail: coelfen@mpikg-golm.mpg.de

Abstract:
The solvent density variation method has proved its use for the simultaneous determination of the partial specific volume of biopolymers and the molar mass / hydrodynamic size applying smallest sample quantities in the mg range. For this purpose, the equilibrium method suggested by Schachman turned out to be best. If more complex multicomponent or impure systems need to be characterized, the fractionating sedimentation velocity experiments are far better suited. Problems can occur, if the sedimenting boundaries show extensive diffusion broadening. Therefore, an extra term has been introduced into the underlying equation taking diffusion broadening into account. The so improved solvent density variation method for sedimentation velocity experiments is capable to analyse not only monodisperse samples but furthermore multimodal or polydisperse samples both with or without significant diffusion broadening. Even if the boundary shows only one apparent step for a bimodal mixture due to diffusion broadening, the improved method is well able to resolve the density as well as the hydrodynamic radii of both species. This method has been tested on a number of simulated data files as well as on an impure apoferritin preparation. The partial specific volume for apoferritin was found in good agreement with literature data as well as the protein hydration if the shape is provided. This result suggests that the solvent density variation method is a fast and convenient method for the determination of the frictional coefficient and thus protein hydration exclusively from sedimentation velocity experiments.

Key words:
Sedimentation velocity, Density variation method, partial specific volume, complex mixtures, protein hydration

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