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

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, monitored by light-sensitive diode array or by film in the older machines. The operator can thus observe the change of sample concentration versus the axis of the rotation profile with time 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 A covalent bond is a chemical bond that involves the sharing of electrons to form electron pairs between atoms. These electron pairs are known as shared pairs or bonding pairs. The stable balance of attractive and repulsive forces between atoms ...

complexes and equilibrium constants of macromolecules such as

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...

s, DNA,

nanoparticle A nanoparticle or ultrafine particle is usually defined as a particle of matter that is between 1 and 100 nanometres (nm) in diameter. The term is sometimes used for larger particles, up to 500 nm, or fibers and tubes that are less than 10 ...

s or other assemblies from different molecule classes. The simplest measurement to be obtained is the

sedimentation coefficient The sedimentation coefficient () of a particle characterizes its sedimentation during centrifugation. It is defined as the ratio of a particle's sedimentation velocity to the applied acceleration causing the sedimentation. : s = \frac The sedime ...

, which depends upon the size of the molecules being sedimented. This is the ratio of a particle's sedimentation velocity to the applied acceleration causing the sedimentation. 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 The National Institutes of Health, commonly referred to as NIH (with each letter pronounced individually), is the primary agency of the United States government responsible for biomedical and public health research. It was founded in the late ...

supported software package, SedFit.

History

Instrumentation

An analytical ultracentrifuge has a light source and optical detectors. To allow the light to pass through the analyte during the ultracentrifuge run, specialized cells are required which have to meet high optical standards as well as to resist the centrifugal forces. Each cell consists of a housing, two windows made from optically pure quartz glass, and a centrepiece with one or two sectors and filling holes for the sector(s), closed with a screw plug in the housing. These cell are placed into a rotor cavity with a continuous bore, with a collar at the bottom to retain the cell.

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. 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 the molar mass of analytes and their

chemical equilibrium In a chemical reaction, chemical equilibrium is the state in which both the Reagent, reactants and Product (chemistry), products are present in concentrations which have no further tendency to change with time, so that there is no observable chan ...

constants. The rotor speed is adjusted 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

References

External links

Reversible Associations in Structural and Molecular Biology (RASMB -an Analytical Ultracentrifugation Forum)
* ttp://www.biophysj.org/cgi/content/full/89/1/651 Gilbert-Jenkins theorybr>Report on an ultracentrifuge explosion.