The Location of Cytochromes in Escherichia Coli 



221 



sedimentation diagram even though they represent 5-10 % of the total fraction 

 (see below). 



Table I. DPNH oxidase activity, protein, RNA and relative amounts of 



RIBONUCLEOPROTEIN PARTICLES (aREA COVERED BY PEAKS ON SCHLIEREN DIAGRAMS) 

 IN PREPARATIONS ISOLATED BY CENTRIFUGATION 



4. Separation of Granules Containing Cytochromes from Ribonucleoprotein 

 particles 



(a) Centrifugation in Caesium Chloride Solution with a Density of 1-46- 

 4-3 ml of a 6% of 70 5 RNP preparation made as described previously 

 (Tissieres et al., 1959) was mixed with 5-7 ml of a 60% caesium chloride 

 solution. The density of the resultant solution was approximately 1-46. The 

 mixture was then centrifuged for 16 hr at 80,000 X ^ in a swinging bucket 

 rotor. A gelatinous, transparent brown layer, about 2 mm thick, formed at the 

 top of the centrifuge tube. This layer could be removed intact and spectro- 

 scopic examination showed strong absorption bands of cytochrome a2, 

 a^ and b^. The rest of the solution was colourless; at the bottom of the tube 

 there was a colourless pellet of RNP particles. No cytochrome could be 

 detected in either of these last two fractions. 



(b) Centrifugation in Sucrose Solution of Density 1-34. Sucrose (11-2 g) 

 was dissolved in 12 ml of a 2 % of 70 S preparation. The mixture was centri- 

 fuged for 12 hr at 100,000 X o^ in the swinging bucket rotor. A brown layer 

 formed at the top of the centrifuge tube, which showed strong cytochrome 

 absorption bands, as in the preceding experiment. The RNP particles had 

 sedimented to the bottom of the centrifuge tube: it was found that their 

 sedimentation coefficient (70 S) had not been modified by this treatment. 

 Before centrifugation in sucrose, the preparation contained 60% RNA and 

 40% protein, while after it, there was 65% RNA and 35% protein in the 

 pellet of RNP particles. The amount of protein in the layer containing 

 cytochromes, at the top of the tube, quantitatively accounted for this 



