Structural Biochemistry/Analytical Ultracentrifugation

ANALYTICAL ULTRACENTRIFUGATION

In principle, analytical centrifugation is similar to differential centrifugation in that both techniques apply the principles of centrifugal acceleration to separate components of a sample based on shape and mass differences. They both require a rotor capable of spinning samples at speeds enough to generate forces up to tens of thousand times greater than the force of gravity. However, analytical centrifugation is able to perform analysis of the concentration of the sample during centrifugation through the incorporation of light detection devices into the system, and this is the key point that differentiates the two techniques.

Analytical centrifuges can perform at least two different types of hydrodynamic analysis: (1) sedimentation velocity; and (2) sedimentation equilibrium. These two techniques, along with some of their advantages and disadvantages, are discussed below.



1)	Sedimentation Velocity This test is sensitive to both the mass and the shape of the molecules. To perform this test, a uniform sample is first loaded into the sample slots and subjected to high acceleration spinning.  A typical velocity is anywhere between 40,000 and 60,000 rpm's.  Due to the difference in force applied to the components caused largely by mass and shape differences, the components will separate out in layers, forming boundaries in solution.  The boundary is basically a concentration gradient that forms as a result of the movement of the particles.  Although the velocity of the individual particles resulting from the centrifugal force cannot be determined, a series of scans (such as absorbance or refractive index detection) is performed on the sample as it spins to record the movement of particle boundaries over time.

More specifically, the rate of movement of the boundary can be used to calculate the sedimentation coefficient (s). The sedimentation coefficient can be affected by at least the following factors:

•	Molecular weight—heavier particles tend to sediment faster;

•	Density—more dense particles tend to sediment faster;

•	Molecular shape—unfolded proteins or a more highly elongated shape will experience more friction from solvent, so will tend to sediment slower;

•	Solute concentration—higher solute concentration tends to lower the rate of sedimentation;

•	Solvent concentration/viscosity—higher solvent concentration and viscosity will tend to increase friction and lead to a lower sedimentation coeffient; and

•	Charge of the protein and how it interacts with polarity of the solvent—for example, a charged particle will travel more quickly through a polar solvent.

In addition to analyzing the rate at which the boundary moves (i.e., the sedimentation coefficient), the characteristics of the boundary itself can also provide information regarding the sample. The diffusion coefficient (D) can be determined by measurement of the spreading of a boundary. A homogeneous product will often produce a boundary that is sharper. In contrast, a heterogeneous sample can produce multiple boundaries or a very broad boundary. However, these are only general rules of thumb because characteristics of the sample can produce contradictory results. For example, a single boundary is not necessarily indicative of a homogeneous sample where it includes two molecules that have similar sedimentation coefficients that would result in a what appears to be a single boundary. Likewise, multiple boundaries do not necessarily result from a heterogeneous sample because a homogeneous sample can have several stable aggregation states that can produce multiple boundaries depending on how rapid the states introconvert.

An additional factor that can create complications in analyzing the characteristics of the boundary is a phenomenon known as self-sharpening. Self-sharpening occurs where the molecules at the "front" end of the boundary move in a higher concentration of solvent and are restricted, whereas molecules at the "back" end of the boundary are in a less concentrated portion of the solvent and move more quickly. This causes an artificial narrowing of the boundary.

Sedimentation velocity is a useful technique for a variety of analyses, including: (1) determining whether a sample is homogeneous; (2) determining whether a protein is a monomer, dimer or other multimer in its native state; (3) determining the overall shape of a protein (for example: is it spherical or more extended); and (4) quantifying the distribution of sizes of proteins in a sample that includes a range of sizes. A critical advantage of a sedimentation velocity procedure is that it can be performed in a relatively short amount of time (often as low as 3–5 hours), as opposed to sedimentation equilibrium (which can often take days). Another important advantage of sedimentation velocity is that it can be used to analyze samples over a broader range of pH, ionic strength, and temperature conditions. One disadvantage is that interacting systems (such as proteins that reversibly self-associate—see discussion above) can lead to data that is difficult to interpret if those systems change during the course of the testing.

2)	Sedimentation Equilibrium

This type of analysis is sensitive only to the mass of a particle (not its shape), and is performed at slower velocities than those for sedimentation velocity. As the sample spins, the components separate out due to acceleration from the spinning while diffusion simultaneously provides an opposing force. Eventually, these forces balance each other out and the components in solution reach an equilibrium point. A series of scans (such as absorbance or refractive index detection) monitors the sample for this equilibrium point, which provides information on the molar weight of the component in sedimentation.

Sedimentation is still regarded by many as the best method to determine the molecular weights of macromolecules in a sample. Although sedimentation equilibrium is conducted at a lower velocity than sedimentation velocity, it must be conducted at higher velocities when analyzing lower molecular weight samples. Sedimentation can also be used to separate heterogeneous samples of different molecular weights. Higher molecular weight particles will move further toward the bottom of the cell, whereas lower molecular weight particles will collect near the top of the cell.

In combination, these tests are able to provide details on the purity of samples and information on molecular weights quite accurately. In particular, analytical ultracentrifugation becomes extremely useful for the analysis of molecular weights for large macromolecules which wouldn’t be able to undergo sequencing tests, such as polysaccharides. Additionally, sedimentation equilibrium is able to provide information on the attractive forces between components of a sample in solution without disturbing the solution, which makes this method very reliable and accurate. Although analytical ultracentrifugation techniques can be used in isolation, they are also used in combination with other analytical techniques to provide more clear and complete conclusions. For example, these techniques are often used in combination with cheaper techniques such as gel electrophoresis and other chromatographic techniques. In addition, they are often used in combination with other analytical techniques such as mass spectrometry, x-ray crystallography, and multidimensional nuclear magnetic resonance (NMR).

Sedimentation Velocity Patterns:

Ultracentrifugation studies of ATCase have shown two different graphs of Protein concentration versus migration distance. Native ATCase has one peak and the 6 catalytic and 6 regulatory subunits are gound together. When the enzyme is treadted with p-hydroxymercuribenzoate, the enzyme is dissociated into two subunits. A 2 regulatory subunit and a 3 catalytic subunit. These experiments have helped show that the interaction of the subunits in the native enzyme produce its regulatory and catalytic properties.

The origin of ultracentrifugation Ultracentrigugation is one of the powerful techniques to determine structure proteins because this method can be used as preparative and analytical. Thus, it is common use in biology, biochemistry and polymer area. In 1923, the analytical ultracentrifuge was invented by Theodor Svedberg and three years later, he won a Nobel Prize in Chemistry for his research on using the ultracentrifuge on separating the collides and proteins. In 1946, Pickel designed the first model preparative ultracentrifuge that can reach the velocity of 40,000 rpm.

Analytical ultracentrifuge

Preparative ultracentrifuge

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