The technique of analytical ultracentrifugation (AUC) was developed by Svedberg and Lysholm in 1927 (1). With AUC one is able to determine molecular weight, shape and stoichiometry of macromolecules and macromolecular complexes.
AUC is an absolute method not depending on standards for comparison. Though methods like gel electrophoresis or size exclusion chromatography replaced part of the applications of AUC, nowadays AUC experiences a renaissance based on the need for information about the oligomeric state or conformation of proteins under physiological conditions, which cannot be retrieved from the knowledge of the amino acid sequence of the single polypeptide chain alone. Additionally the utilisation of computer aided data acquisition and data evaluation disclosed new perspectives in the amount of information that can be retrieved from analytical ultracentrifugation experiments.
The method offers the opportunity to retrieve structural information about biological macromolecules in solution for a broad concentration range and under a wide variety of solvent conditions. The hydrodynamic behaviour of macromolecules under the influence of centrifugal forces is determined by their molecular size and molecular shape and the physical and chemical properties of the solvent.
Two different experimental protocols are used:
- Sedimentation equilibrium centrifugation (SEC) is utilized to determine the molecular weight of a molecule or a molecular complex. By prolonged centrifugation which leads to the establishment of an equilibrium between sedimentation and back diffusion of the particles a time-independent concentration profile is obtained from which the molecular weight can be extracted. AUC is an absolute method regarding the molecular weight determination because no calibration with compounds of known molecular weights is needed.
- Sedimentation velocity centrifugation (SVC) or moving boundary sedimentation is utilized to determine sedimentation coefficients or sedimentation coefficient distributions by observation of the particle behaviour during the sedimentation process. Under the influence of the applied centrifugational field in an initially uniformly filled sample cell a boundary (Fig. 1, right) is formed between an area depleted of the sedimenting molecules near the rotor axis and the remaining solution. The motion of this boundary over time is a measure for the velocity of the sedimenting molecules. From the sedimentation velocity molecular properties like size and shape of a particle can be infered.
The instrument, an Optima XL-A from Beckman-Coulter (Fig1, left), allows for sedimentational speeds from 3000 to 60,000 rpm generating up to 250,000 fold acceleration of gravity in the centre of the sample cell. The instrument is equipped with an absorption optics, allowing measurements in the spectral range between 190 and 600 nm with a sensitivity of 0.3 to 0.9 OD for SVC and 0.1 to 0.3 OD for SEC.
(1) An Ultracentrifuge of oil turbine type for the determination of molecular weights : Contribution from the laboratory of physical chemistry of the University of Upsala / The. Svedberg ; Alf Lysholm: Nova Acta Regiae Societatis scientiarum Upsaliensis ; Ser. 4, vol. extra ord. 1927,