The different components of the homogenate must then be separated. Such cell fractionations became possible only after the commercial development in the early 1940s of an instrument known as the preparative ultracentrifuge, which rotates extracts of broken cells at high speeds (Figure 8–5). This treatment separates cell components by size and density: in general, the largest objects experience the largest centrifugal force and move the most rapidly. At relatively low speed, large components such as nuclei sediment to form a pellet at the bottom of the centrifuge tube; at slightly higher speed, a pellet of mitochondria is deposited; and at even higher speeds and with longer periods of centrifugation, first the small closed vesicles and then the ribosomes can be collected (Figure 8–6). All of these fractions are impure, but many of the contaminants can be removed by resuspending the pellet and repeating the centrifugation procedure several times.
Figure 8–5 The preparative ultracentrifuge. (A) The sample is contained in tubes that are inserted into a ring of angled cylindrical holes in a metal rotor. Rapid rotation of the rotor generates enormous centrifugal forces, which cause particles in the sample to sediment against the bottom sides of the sample tubes, as shown here. The vacuum reduces friction, preventing heating of the rotor and allowing the refrigeration system to maintain the sample at 4°C. (B) Some fractionation methods require a different type of rotor called a swinging-bucket rotor. In this case, the sample tubes are placed in metal tubes on hinges that allow the tubes to swing outward when the rotor spins. Sample tubes are therefore horizontal during spinning, and samples are sedimented toward the bottom, not the sides, of the tube, providing better separation of differently sized components (see Figures 8–6 and 8–7).
Centrifugation is the first step in most fractionations, but it separates only components that differ greatly in size. A finer degree of separation can be achieved by layering the homogenate in a thin band on top of a salt solution that fills a centrifuge tube. When centrifuged, the various components in the mixture move as a series of distinct bands through the solution, each at a different rate, in a process called velocity sedimentation (Figure 8–7A). For the procedure to work effectively, the bands must be protected from convective mixing, which would normally occur whenever a denser solution (for example, one containing organelles) finds itself on top of a lighter one (the salt solution). This is achieved by augmenting the solution in the tube with a shallow gradient of sucrose prepared by a special mixing device. The resulting density gradient—with the dense end at the bottom of the tube—keeps each region of the solution denser than any solution above it, and it thereby prevents convective mixing from distorting the separation.
When sedimented through sucrose gradients, different cell components separate into distinct bands that can be collected individually. The relative rate at which each component sediments depends primarily on its size and shape—normally being described in terms of its sedimentation coefficient, or S value. Present-day ultracentrifuges rotate at speeds of up to 80,000 rpm and produce forces as high as 500,000 times gravity. These enormous forces drive even small macromolecules, such as tRNA molecules and simple enzymes, to sediment at an appreciable rate and allow them to be separated from one another by size.
The ultracentrifuge is also used to separate cell components on the basis of their buoyant density, independently of their size and shape. In this case, the