ISOLATION AND COMPOSITION OF DEOXYPENTOSE NUCLEIC ACIDS 325 



lowing dilution with 590 cc. of sodium citrate, the crushed suspension was centrifuged 

 for 2 hours at 4000 r.p.m. Two solid layers sedimented underneath a very opalescent 

 supernatant (915 cc), a bottom layer of intact cells and an upper layer of cellular 

 fragments. A separate determination showed that about 55% of the cells had actually 

 been crushed. 



The upper solid layer consisting of cellular fragments (485 cc.) was suspended in 

 850 cc. of ice-cold M sodium chloride solution of pH 6.3. The slimy mixture was kept 

 in the refrigerator for 72 hours and then centrifuged at 4000 r.p.m. for 2 hours. The 

 rapid addition of 2 vol. of chilled absolute ethanol to the very viscous supernatant 

 resulted in the precipitation of white threads that could easily be wound on a glass 

 rod and thereby separated from a granular precipitate suspended in the mother 

 liquor. The threads were washed thoroughly by successive immersion in three por- 

 tions of 73% ethanol, drained, and redissolved in 300 cc. of M sodium chloride with 

 the use of a high-speed mixer. 



The turbid solution was freed of protein by being stirred in a high-speed mixer 

 with one-third of its volume of a 9:1 mixture of chloroform- octyl alcohol (for 5 

 minutes), followed by centrifugation at 4000 r.p.m. for 1 hour. After eight treatments 

 the solution was free of protein and gave no biuret test. At this stage, it was found to 

 contain 0.6 mg. of deoxypentose nucleic acid per cubic centimeter, corresponding to a 

 total of 180 mg. in the original 300 cc. of solution. 



The addition of 2 vol. of ethanol to the clear protein-free solution again produced 

 white threads that were spooled on a rod, as described before. An additional amount 

 of fibrous nucleic acid could be recovered by reworking the granular precipitate re- 

 maining in the mother liquor. 



The threads obtained by the above procedure were found to contain only 19% of 

 deoxypentose nucleic acid; the remainder consisted of ribonucleic acid (64%) and of 

 a polysaccharide. The well-drained threads were taken up in 20 cc. of neutral 10% 

 aqueous calcium chloride and the viscous milky solution was clarified by centrifuga- 

 tion at 20,000 r.p.m. for 2 hours. The sediment was washed twice, under the same 

 centrifugal conditions, with 6-cc. portions of 10% CaCh . The slow addition of 0.2 

 to 0.3 vol. of cold absolute ethanol to the combined clear supernat ants (32 cc.) brought 

 about the separation of white fibers that were lifted in the usual manner and washed 

 twice with 10% calcium chloride solution containing 0.3 vol. of ethanol. 



This fraction was contaminated with about 20% of ribonucleic acid which could be 

 removed by enzymic digestion. To a solution of the precipitate in 45 cc. of 0.2 M so- 

 dium borate buffer of pH 7.8, 1.5 mg. of crystalline ribonuclease was added. The solu- 

 tion was subjected to dialysis at room temperature against two changes of 2-1. por- 

 tions of the borate buffer for 14 hours, against running tap water for 17 hours, and, 

 finally, against several changes of ice-cold distilled water for 26 hours. Then it was 

 again deproteinized, as described before, and evaporated in the frozen state in a 

 vacuum or precipitated with ethanol in the presence of sodium acetate. The sodium 

 salt of deoxypentose nucleic acid thus obtained weighed 105.5 mg. It formed a white 

 fluff which was readily soluble in water, giving a clear viscous solution. The biuret 

 reaction was negative. 



6. Extraction with Strong Salt Solution, Dcproteinization by Saturation with 

 Sodium Chloride 



(/) Sodium Deoxyribonucleate of Calf Thymus. ^^■^'"' All operations were carried 

 out at 0° with solutions that were 0.01 M with respect to sodium citrate. 450 g. of 



95 R. Signer and H. Schwander, Helv. Chim. Acta 32, 853 (1949) . 

 !«« H. Schwander and R. Signer, Helv. Chim. Acta 33, 1521 (1950). 



