86 METHODS FOR DETERMINING MOLECULAR SIZE AND SHAPE 



Proteins Cell debris and membranes 



I 



Fig. 41. A representation of the results of a centrifugation of a mixture of 

 crushed cells layered onto the inner edge of a sucrose gradient which increases 

 to the right. The fastest moving substances are cell membranes and internal 

 membranes, which are represented as just having reached the outer edge of the 

 tube. The next most rapidly moving parts are the nuclei, followed by the pro- 

 teins, with the lipid material still floating at the starting place. 



themselves to the left of the place where the density is 1.7 gm/ml will be 

 forced centrifugally outward until they reach that place. Particles find- 

 ing themselves to the right of that place will be less dense than the 

 solution, and will therefore be floated inward until they reach the same 

 place. Again, the density gradient and the particle band can be photo- 

 graphed by suitable optical methods, so that the particle density can be 

 measured. 



There is a further result from this density gradient sedimentation 

 method. Because of the thermal (Brownian) motion of the particles, they 

 will not be precisely in a thin band but will be in a band of some finite 

 thickness. Indeed, the smaller these particles, the faster and further they 

 will go at any given temperature. Thus it should be plausible that the 

 width of the band is related inversely to the size of the particles: the 

 bigger the particles, the smaller the band. This proposition can be 

 demonstrated mathematically to be true, and therefore the molecular 

 weight of the particles can also be obtained from the same photograph. 

 This method then gives both molecular weight and density from a single 

 photograph ! 



(e) Density gradient sedimentation can be used in another very fruit- 

 ful way. The density gradient is first established (more usually with 

 sugar than with salt, for technical reasons) and afterwards the solution 

 to be studied, containing a mixture of particles, is placed on top of (at the 

 inner edge of) the centrifuge tube. When the machine is then run for a 

 while, the various particles in the solution will have reached different 

 points in their travel towards the other end of the tube. If the centrifuge 

 is stopped, the different kinds of particles will be found to have been 

 separated into bands, as sketched in Fig. 41. In the sketch, we have 

 assumed that some cells have been broken and that the method has 

 separated the solution into the components indicated. By cutting the tube 

 carefully, the various components can be separated from each other. 



