(The figures are clickable for separate printing on transparencies.)
In the early 1950's days of molecular biology scientists had to have been trained in other disciplines since the field of "molecular biology" didn't exist when they were students. During that time a number of techniques were picked up from other fields. One such "picked-up" method was boundary sedimentation which was begotten in geology to study the sizes of sediments. Svedberg got sought to use the technique on viruses - critters of constant size for each species. He then honed his formulas on such things as the various pieces of ribosomes, which resulted in things being appended with "S-values" (take your pick: sedimentation values or Svedberg values). At any rate Svedberg determined that something sediments at a rate proportional to the square of its mass. See if you can "see" this knowing that a "16-S" piece of ribosome is about half the mass of a "23-S" piece.
mass1/mass2 = 162/232 = approx 0.5
One of the first problems was getting microscopic particles to sediment - a battle against Brownian motion. To amplify gravity, a centrifuge was needed. Very quickly Svedberg found that he needed a very fast centrifuge, one that ran so fast that its rotor heated up in the air in which it was revolving. So an "ultracentrifuge" was developed by Beckman Instruments in which the rotor revolved in a vacuum. Then a new problem was found: the rotor still heated up from the minor amount of friction in the bearings that was transmitted via the drive shaft to the rotor, which would hold the experimental samples. That problem was quickly solved by painting the rotor and the inside of the vacuum chamber black. Black-body radiation stipulates that any black thing will slowly radiate infrared and thus cool. The infrared was then absorbed by the black walls of the chamber. And those walls were refrigerated!
What the Beckman Company invented was a disk of titanium that would revolve in a vacuum at about 60,000 rpm (which is about 30-times faster than any electric motor you have at home!). In that disk (top view), they placed a small cuvette made of half-inch thick quartz windows between which a sample of solution was placed.
Since most materials will absorb ultraviolet light, the strong quartz windows were perfect as quartz does not absorb UV. What was then done, when the centrifuge was running at top speed, was that UV light was focused through the window and projected onto film on the other side. Each time the rotor rotated, the window would flash by and the light would go through - sort of like looking through a picket fence as you drive by).
In the top view picture above, the cuvette is filled with a mix of two sedimentable components in water. What you see on the UV photograph are the top boundaries of each component as each is making its way to the bottom of the whirling cuvette under forces of greater than 100,000xG.
Let's look at a macroscopic device to get a better idea of what Svedberg was doing. Suppose we had made a slurry of a mix of fine sand, silt and clay. We poured this into a graduated cylinder and waited to see how everything settled out. At first the whole water column looks homogeneously turbid, but as time goes on, the bigger sand particles settle a little faster than does the silt, and the silt is a bit heavier than the particles of clay, which settles the slowest. Awhile later we see that the water column consists of several layers. The top one is pure water; the next consists of water AND slowly settling clay particles; the next layer consists of water, clay AND silt particles; and them comes a layer of water, clay, silt AND sand. Finally, at the bottom, whatever has reached that far down has settled out.
The thing to note is that the top boundary of each layer started out at the top of the cylinder at time zero. Thus Svedberg was interested in the relative distances each boundary was from the top. This is thus a very simple system: dump the mix in, and watch. No density gradients were needed.
What we have discussed above is a dynamic system - it is an active, on-going process. If we wait to long before making our observations, all the boundaries will have reached the bottom.
Svedberg also used this process to assess the homogeneity of his particles. If they were all identical, then the top boundary would be expected to be sharp. However, if there was considerable variation in sizes of the clay particles, the boundary would be blurred as it was really a composite of many boundaries each settling at slightly different rates. Another scientist, Schlieren, came on scene now to help. He had invented an optical system that could be incorporated into the projection lens of the centrifuge. Using a cylindrical lens, he found that he could focus the light as a line on the cuvette. However, whenever there was a change in the liquid - a boundary - the line would suffer some refraction and be knocked off of the straight line. The camera would see a sigmoidal squiggle in the line. To the right are shown two such pictures using Schlieren optics. The left one is of a homogeneous population of particles and the other is less homogeneous. Later, Svedberg determined an equation that related the displacement between the two major swings of the line to the molecular weight of the particle that was sedimenting. The smaller the particle, the more Brownian motion would affect the individual particles at the boundary - some would appear moving too slowly and others too fast - thus the smaller the particles, the broader the boundary.
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