Analytical ultracentrifugation

Analytical ultracentrifugation is an analytical technique which combines an ultracentrifuge with optical monitoring systems.

Analytical Ultracentrifuge. This appears to be a Spinco Model E. This is an early instrument, probably dating to the 1950s. The operator is seated in front of the sample chamber, with his left hand touching the rotor. In operation, the chamber would be sealed behind an armored shroud and pumped down to vacuum. The shroud has been lowered to allow access for loading or unloading the rotor.[1]

In an analytical ultracentrifuge (commonly abbreviated as AUC), a sample’s sedimentation profile is monitored in real time by an optical detection system. The sample is detected via ultraviolet light absorption and/or interference optical refractive index sensitive system. The operator can thus observe the evolution of sample concentration versus the axis of the rotation profile as a result of the applied centrifugal field. With modern instrumentation, these observations are electronically digitized and stored for further mathematical analysis.

The information that can be obtained from an analytical ultracentrifuge includes the gross shape of macromolecules, conformational changes in macromolecules, and size distributions of macromolecules. With AUC it is possible to gain information on the number and subunit stoichiometry of non-covalent complexes and equilibrium constant constants of macromolecules such as proteins, DNA, nanoparticles or other assemblies from different molecule classes.

Analytical ultracentrifugation has recently seen a rise in use because of increased ease of analysis with modern computers and the development of software, including a National Institutes of Health supported software package, SedFit.

History

Instrumentation

Rotors for an Analytical Ultracentrifuge for a maximal spinning velocity of 50'000 (left) and 60'000 (right) rpm

An analytical ultracentrifuge extends the ultracentrifuge by a light source and optical detectors. To allow the light pass through the analyte during the ultracentrifuge run, specialized cells are required which have to meet optical qualities as well as to resist the gravitational forces. Each cell consists of a housing, two windows from quartz glass, a center-piece with two sectors. These cell are placed into a rotor with continuous bore.

Theory

Types of experiments

By applying specific equipment and adapting measurement parameters several types of experiments can be performed. Most common AUC experiments are sedimentation velocity and sedimentation equilibrium experiments.

Sedimentation velocity

Sedimentation velocity experiments render the shape and molar mass of the analytes, as well as their size-distribution.[2] The size resolution of this method scales approximately with the square of the particle radii, and by adjusting the rotor speed of the experiment size-ranges from 100 Da to 10 GDa can be covered. Sedimentation velocity experiments can also be used to study reversible chemical equilibria between macromolecular species, by either monitoring the number and molar mass of macromolecular complexes, by gaining information about the complex composition from multi-signal analysis exploiting differences in each components spectroscopic signal, or by following the composition dependence of the sedimentation rates of the macromolecular system, as described in Gilbert-Jenkins theory.

The experiment aims to monitor the sedimentation behavior at a fixed angular speed.

Sedimentation equilibrium

Sedimentation equilibrium experiments reports on the molar mass of analytes and on chemical equilibrium constants. [3]

They are concerned with adjusting the rotor speed such that a steady-state concentration profile c(r) of the sample in the cell is formed, where sedimentation and diffusion cancel out each other.

Density gradient centrifugation

Data evaluation

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References

"Technical Manual, Spinco Ultracentrifuge Model E". Science History Institute Digital Collections. Retrieved 2018-12-18.
Perez-Ramirez, B. and Steckert, J.J. (2005). Therapeutic Proteins: Methods and Protocols. C.M. Smales and D.C. James, Eds. Volume 308: 301-318. Humana Press Inc, Totowa, NJ.
Ghirlando, R. (2011). "The analysis of macromolecular interactions by sedimentation equilibrium". Modern Analytical Ultracentrifugation: Methods. 58 (1): 145–156. doi:10.1016/j.ymeth.2010.12.005. PMC 3090454. PMID 21167941.