Walter Stafford, III, Ph.D., Senior Scientist
Analytical ultracentrifugation has been found invaluable in the elucidation of information about the mass, structure and interactions of macromolecules. In his book “The Molecular Biology of the Gene” J.D. Watson characterized analytical ultracentrifugation as perhaps the most striking contribution of physical chemistry to the study of biological macromolecules.
An analytical ultracentrifuge is a very high speed centrifuge with an optical system for viewing the sedimentation process. At its top speed of 60,000 rpm, it will develop an acceleration of 200,000 times the force of the earth’s gravity (g). At forces of this magnitude, very small particles and protein molecules can be sedimented. Sedimentation takes place in much the same way that a glass of freshly squeezed orange juice separates. The heaviest particles settle out first and the smallest ones last. The sedimentation in a glass of orange juice occurs at 1 x g, of course, so only the very largest particles of pulp will settle out. Protein molecules, on the other hand, are extremely small by comparison and, therefore, require much greater force to make them settle out. Most proteins will move only about 1/4 inch in several hours at 60,000 rpm. The optical system of the analytical ultracentrifuge allows one to measure the speed of movement of the protein molecules. By measuring the speed as well as the rate of spreading of the boundary between the water and the protein solution, one can calculate both the mass and the shape of the protein particles. For example, if two proteins have the same mass but different shapes, the more compact protein will sediment faster than a protein that is more extended. By measuring the sedimentation velocity of a protein of known mass, one can deduce its general shape. Interactions between molecules can also be studied because of the increase in mass of the complex that is formed when two proteins interact to form a new protein particle.
Sedimentation analysis gives us a tool for the quantitative characterization of individual proteins and their interactions. Examples of proteins and interactions that have been studied quantitatively by sedimentation analysis are smooth muscle myosin, transcription factor with DNA, and antigens with antibodies.
An analytical ultracentrifuge is a very high speed centrifuge with an optical system for viewing the sedimentation process.