HALL AND DOTY 29 



NaHC0 3 containing 4 mM MgCl 2 (pH 8.5) yielded 81 for i° 2 o,w. The ratio of 

 optical densities at 260 and 280 mp was 1.80 in this solvent. The ratio at 260 

 and 230 mu was 1.32. Various mild treatments such as heating to 37° C cause 

 the appearance of 50 S and 5 S components accompanied by increases in the 

 optical density at 260 mp (resembling the denaturation of DNA) and conse- 

 quently increases in the values of the optical density ratios reported above. 



Preparation of RNA from the Microsomal Particles. In order to obtain RNA 

 of high purity from microsomal particles, two principal steps must of neces- 

 sity be included in the procedure: (1) dissociation of the RNA from the pro- 

 tein; (2) separation of denatured proteins and other contaminating substances 

 from the nucleic acid. This procedure makes use of one anionic detergent, 

 sodium lauryl sulfate, to disrupt the protein-nucleic acid complex (by denatur- 

 ing the protein and displacing the nucleic acid from cationic groups on the 

 protein), and another, sodium xylene sulfonate (Naxonate), to remove the 

 denatured protein from solution [5]. 



The pellet of microsomal particles was suspended in 0.01 M versene, pH 7.0. 

 In a typical preparation, beginning with 200 g of liver, the volume of the sus- 

 pension was 100 ml. The suspension was brought to 20° C, and sufficient solid 

 sodium lauryl sulfate was added (with stirring) to bring the concentration to 

 4 per cent. Stirring was continued until a clear solution resulted; this was 

 allowed to stand for 12 hours at 20° C. At the end of this time, the solution 

 was cooled to 5° C, and to it were added 3 volumes of an ice-cold solution of 

 0.2 M KCl, 0.01 M versene pll 7.0 containing 12 g sodium xylene sulfonate per 

 100 cc. The pll of this mixture was reduced to 4.3 by dropwise addition of 6 TV 

 acetic acid. After standing 15 minutes at 0°, the suspension was centrifuged 

 for 30 minutes at 1500g- in the cold. The supernatant solution was decanted 

 and brought to pH 7.0 by addition of 6 N NH 4 OH. After the solution was 

 warmed to 20° C, RNA was precipitated with 2 volumes isopropyl alcohol. 

 The precipitate was allowed to settle for 2 hours; then it was centrifuged down. 

 The liquid was decanted, and the RNA was dissolved in 0.03 M sodium acetate 

 solution. This solution was treated with Naxonate to complete the removal of 

 protein. To it were added 3 volumes 40 per cent Naxonate; then the solution 

 was stirred for 30 minutes. After cooling to 0° C, the pH was brought to 4.3 

 and the solution was filtered through celite and sintered glass. 1 This treatment 

 removed the protein-Naxonate complex, which is insoluble, leaving glycogen 

 as the only nondialyzable impurity in the RNA. Glycogen may conveniently 

 be removed by centrifugation for 20 minutes at 30,000^ (it forms a pellet). 

 After the removal of glycogen, the RNA solution was dialyzed against 0.01 M 

 KH2PO4-K2HPO4 (1:1) in order to remove ultraviolet-absorbing impurities. 



1 It has recently been found that a substantial part of the RNA (up to 85 per cent of the 

 total) is lost in this step because of adsorption on the celite. Besides lowering the yield, 

 this may have led to fractionation of the RNA, if the adsorption was selective. This step 

 may be omitted, for the protein-Naxonate aggregates can be removed along with glycogen 

 in the centrifugation at 30,000^. 



