III. CENTRIFUGATION 71 



average follow diverging radial paths and since their rate of migration 

 increases with the distance from the axis of rotation, the concentra- 

 tion in the plateau region steadily decreases. However, the true 

 boundary position at any time is still very nearly the level at which the 

 concentration is half that of the plateau region at that time. Further- 

 more, there is a definite mathematical relationship (see Sec. C) be- 

 tween the concentration of the plateau region and the displacement of 

 the boundary, so that the initial concentration of any differentiated 

 component can still be determined by applying a correction factor. 



The curves in Figure 1 correspond to behavior of a typical "globular" 

 protein of 18,000 molecular weight in average centrifugal field of 300,000 

 times gravity. To first approximation, a similarly shaped larger protein 

 sedimented to corresponding positions by the same force would show re- 

 duced boundary spreading inversely proportional to the two-thirds power of 

 the molecular weight. The time required for sedimentation would be lowered 

 in the same ratio. 



3. Significance of Ultracentrifugal Method 



Although sedimentation rate is not of itself an indication of par- 

 ticle size, shape, or density, it is for a given preparation under ap- 

 propriate conditions a characteristic constant that may be combined 

 with other experimental data to investigate these properties. Al- 

 though average sedimentation rates may be determined from the 

 proportional amount of material sedimented to the bottom of or 

 across a certain level of the containing vessel within a known time, 

 the moving-boundary method is unique in that through a single ex- 

 periment one can, with proper recording equipment (usually photo- 

 graphic), study the distribution of sedimentation rates and thus the 

 number, centrifugal homogeneity, and respective concentrations of 

 the resolvable components. For the perfection of this method in the 

 form of the analytical ultracentrifuge, and for numerous demonstra- 

 tions of its practicability in the study of macromolecules, science is 

 indebted to the pioneering research of Svedberg and associates 

 (1,9,13) begun about 1923. 



As already indicated, no measurable sedimentation boundary is 

 established ^vith diffusible materials in centrifuge cells of ordinary 

 size when the centrifugal force applied is below a certain value usually 

 dependent primarily on the particle size. However, if steady centri- 

 fugation is continued for a sufficient length of time (at least one day 

 for most proteins), an equilibrium is established between sedimenta- 



