300 R. B. ROBERTS, R. J. BRITTEN, AND B. J. MCCARTHY 



processing of ribosomes. The buffer most frequently used is tris(hy- 

 droxymethyl)aminomethane 0.01 il/ pH 7.4 with magnesium added as 

 the chloride at the desired concentration (usually 10- i\/). 



The cells may then be broken by grinding the pellet with twice its 

 weight of alumina, by repeated freezing and thawing with lysozyme, or 

 by forcing them through a small orifice in the French pressure cell. The 

 pressure cell is preferable for the preparation of ribosomes as it is highly 

 efficient in breaking most cells and the DNA is degraded so that the cell 

 juice is not viscous. 



The cell juice is then diluted to less than 100 mg wet w-eight/ml and 

 the cell walls, membranes, and any unbroken cells are removed by 

 centrifugation for 1 minute at 40,000 rpm in the angle head rotor of the 

 Spinco Model L ultracentrifuge. The supernatant fluid (40K I'S) is 

 decanted and the pellet is either discarded or resuspended to recover its 

 content of ribosomes (about 10-20%). 



The crude ribosome pellet is then obtained by centrifugation of the 

 40K I'S. If complete recoveiy of ribosomes as small as 308 is required, 

 2-3 hours of centrifugation is needed. Alternatively, if only a 90% 

 recovery of 70-lOOS ribosomes is desired, 30 minutes' centrifugation will 

 suffice. 



The first ribosome pellet is highly contaminated, partly by small bits 

 of cell wall and membrane and partly by soluble protein and RNA. 

 jMucli of the contaminating material will not resuspend readily and can 

 be removed by a brief centrifugation (40K 1'). Alternate short and long 

 cycles of centrifugation helps to reduce the contamination and the 

 ribosome pellet becomes more colorless and transparent. By appropriate 

 choice of the times of centrifugation, pellets rich in one or another class 

 of ribosome can be obtained. However, this technique is not adequate 

 for measurements of the protein or enzyme content of ribosomes. Further 

 purification of the pellet by sedimentation through sucrose gradients or 

 by column chromatography shows that after three cycles of sedimenta- 

 tion in the angle head rotor as much as half the protein content of the 

 pellet may be due to contamination. The best use of differential centrifu- 

 gation is to prepare partially ])urified ribosomes as the starting material 

 for other techniques of separation. 



B. protein/nucleic acid RATIO 



Various values reported for the protein and nucleic acid content of 

 ribosomes are listed in Table I. As many of these experiments were 

 carried out before the difficulties of removing protein contaminants were 

 fully appreciated, the protein content is likely to be overestimated. 

 There is no certainty that the protein/nucleic acid ratio varies in 



