ISOLATION AND COMPOSITION OF DEOXYPENTOSE NUCLEIC ACIDS 327 



traded with water, and the nucleohistone was precipitated at 0.15 M NaCl. It was 

 collected in the Sharpies centrifuge, dissolved i water, brought to a 2.6 M NaCl 

 concentration, and the sodium nucleate precipitated with alcohol. The mother liquor 

 served for the isolation of histone. The sodium nucleate was dissolved in water, treated 

 once with sodium dodecyl sulfate (see below) and recovered as described in the next 

 section. The sodium deoxyribonucleate, dried in air, weighed 23.1 g. (2.6% of the 

 tissue) and contained only 0.65% of protein. 



d. Extraction with the Aid of Anionic Detergents 



In the recent past, deoxypentose nucleic acid preparations have often 

 been made with the aid of sodium dodec}^ sulfate'"^ or of detergents of the 

 Duponol type.'"' The use of sodium xylene sulfonate has also been pro- 

 posed.'"* As in all preparative methods, the type of source material will 

 not be without influence on the success of the isolation. In our own experi- 

 ence, a nucleic acid preparation from sea urchin sperm could not be freed 

 entirely of protein by treatment with a detergent. '"■* 



(/) Sodium Deoxijribonucleale of Calf Thyrnus.^"^ Fifty grams of frozen calf thymus 

 were minced in a "Waring Blendor" for 3 minutes'"* in 200 cc. of ice-cold 0.9% NaCl 

 solution containing 0.01 M sodium citrate. The sediment resulting from centrifuga- 

 tion at 0° and 2500 r.p.m. for 30 minutes was three more times suspended in citrate - 

 saline, centrifuged as described before, and treated in the "Waring Blendor" for 3 

 minutes with 1 1. of ice-cold physiol. saline.'"^ The mixture was transferred to a large 

 beaker and 90 cc. of the detergent solution (5 g. of sodium dodecyl sulfate or purified 

 Duponol made up to lOOcc. with45%ethanol) were added. The resulting gel wasstirred 

 vigorously at room temperature for 3 hours during which time it turned gradually 

 into a very viscous solution. The NaCl concentration then was brought to 1 M by 

 the addition of 55 g. of salt,'" stirring was continued for 10 minutes, and the mixture 

 was centrifuged at 0° and 2500 r.p.m. (or at a higher speed) for 3 hours .'°* The crude 

 sodium nucleate was precipitated by the addition to the supernatant fluid of an equal 

 volume of 95% ethanol, the fibrous precipitate was lifted by spooling, pressed out, 



><" A. M. Marko and G. C. Butler, J. Biol. Chr.m.. 190, 165 (1951). 

 '•"iE. R.M.Kay, N.S.Simmons, and A. L.Dounce, J. ^7n. Chcm.Soc. 74,1724 (1952). 

 "" N. S. Simmons, S. Chavos, and H. K. Orbach, Federation Proc. 11, 390 (1952). 

 10^ E. Chargaff, R. Lipshitz, and C. Green, /. Biol. Chem. 195, 155 (1952). 



105 ^ grinding period of 30 to 45 seconds is sufficient in our experience. Excessively 

 long treatment in the "Waring Blendor" should, in general, be avoided. 



106 Yor the preparation of large quantities we have found it advantageous to extract 

 the nucleohistone at this stage with 10% NaCl solution and to precipitate with 

 alcohol. The nucleohistone can be stored in the cold und^r aqueous alcohol and then 

 be processed as described in the following. 



"" Care must be taken not to exceed this NaCl concentration, in order toprevent the 

 precipitation of the detergent. 



'"* The mixture must not be stored in the cold at this stage, since it may jellify. We 

 centrifuge in the cold for 30 minutes at 13,000 r.p.m. in a rotor that has not been 

 precooled or, if large quantities are to be processed, in a Sharpies supercentrifuge 

 at 37,000 r.p.m. with a jet delivering 1 1 in 15 minutes. Filtration of the mixture"" 

 is not recommended; it is accompanied by losses and reijuires a disproportionate 

 amount of patience. 



