Analytical Ultracentrifugation in Biochemistry

Analytical Ultracentrifugation (AUC) is a classical method of biochemistry – just think of the 80 S ribosome, where S stands for “Svedbergs”, the unit in which the sedimentation coefficient is usually given. It is the “gold-standard” for measuring protein-protein interactions and can give invaluable and often unique information about proteins, nucleic acids, viruses, lipids, carbohydrates and their complexes.

Major benefits of using Analytical Ultracentrifugation for characterising your proteins:

  • No standards are required
  • Analytical Ultracentrifugation is sensitive, precise, accurate and robust (yes – all at a time!)
  • No interactions with a stationary phase or matrix
  • Wide range of molar masses can be investigated in the same experiment – works well for peptides to viruses.
  • No protein modifications required
  • No change in mobile phase – your protein is examined in exactly the same solvent in which you want to study it
  • Theory of sedimentation is based on first principles and well understood
  • All auxiliary parameters can be independently determined
  • Analytical Ultracentrifugation is a true orthogonal method to SEC and/or FFF and can help validate protocols for these methods

Examples for real-world scenarios:

  • Aggregate content of a protein
  • Self-association of a protein

Aggregate content of a protein:

Here’s an example of BSA at 1 mg/mL in PBS-buffer examined with Sedimentation Velocity (SV).
Aggregate content 1

There is a dominant peak for the monomeric BSA (72%) and dimer (19%) which are well resolved. If we zoom in to see more clearly the smaller components, the excellent resolution of Analytical Ultracentrifugation is capable of becomes even clearer.
Aggregate content 2

There are about 3% of a potential proteolytic fragment, 4% of tetrameric BSA and 2% of higher order polymers, probably hexa- and octamers.
This type of analysis can be used for stability studies (stressed vs. non-stressed sample), to validate an SEC and/or FFF analysis or to understand the influence protein manipulation has on aggregation. It is also useful to show that a protein produced in different cell is indeed identical.

Self-association of a protein:

Here’s a case where a protein is self-associating. This is an intrinsic property of the molecule and is often important for its proper functioning.
Self-association 1

There’s an apparent new peak at lower sedimentation coefficients at lower concentrations. The average sedimentation coefficient is also decreasing with concentration.
Self-association 2

A qualitative and quantitative description of the self-association is obtained by Sedimentation Equilibrium (SE).
Self-association 3

Here, the experimental curves (dots, only every 3rd point is shown) are described by a theoretical model (solid lines) for a monomer-dimer equilibrium of KD 1 µM. The fact that the theoretical lines closely follow the experimental data indicates that the description of the data is correct. This experiment is useful to demonstrate biosimilarity or when characterizing binding interfaces.