328 ERWIN CHARGAFF 



and washed 3 times with 95% ethanol and then with acetone until the washings were 

 no longer cloudy. It was dried in air (yield about 2 g.)-^"' The crude product was sus- 

 pended in 700 CO. of distilled water and brought into solution by being stirred rapidly 

 at room temperature; 63 cc. of the detergent solution was added and the mixture was 

 stirred for 1 hour. The mixture was made 1 Af by the addition of 45 g. of NaCl, centri- 

 fuged at 13,000 r.p.m. for 1 hour, and the nucleate precipitated with ethanol from the 

 supernatant and washed as before. It was dissolved once more in water and the solu- 

 tion adjusted to 0.14 M NaCl and centrifuged at 13,000 r.p.m. for 1 hour. The NaCl 

 concentration of the supernatant fluid was increased to 1 M and the sodium deoxy- 

 ribonucleate precipitated by the slow addition, with stirring, of an equal volume of 

 95% ethanol. The fibers, washed with ethanol and acetone and dried in air, weighed 

 about 1.3 to 1.4 g. 



e. Comparison of Different Isolation Procedures 



A systematic investigation of the merits of the several procedures still 

 remains to be carried out, except for some preliminary information on 

 relative yields which has recently been provided by Frick^^^ and a few 

 findings published by Schwander and Signer.^"'' Frick compared the methods 

 of Gulland et al.^^ (see Section III.2.a.(l)), of Hammarsten^" (compare the 

 modification by Schwander and Signer^"" described in Section 111.2.6.(1)), 

 and of Kay et al}°^ (see Section III.2.c?.(l)). His conclusion, only partially 

 borne out by the experience of this laboratory, was that the Gulland pro- 

 cedure gave yields amounting to onlv 5 to 10 % of the quantity of nucleic 

 acid present in the nucleohistone serving as the starting material; that the 

 Hammarsten procedure led to a product in good yield, but of a relatively 

 high protein content; and that the method of Kay et al. afforded the 

 greatest yield, but produced some degradation, as judged from ultraviolet 

 absorption. 



It is quite clear that yield is only one of several criteria. The constancy 

 of physical and chemical characteristics of preparations isolated by differ- 

 ent procedures has been investigated only to a very limited extent; but the 

 rather extensive experience of our laboratory has failed to reveal divergen- 

 ces in composition or extinction that were due to the preparative methods, 

 if the precautions outlined in the preceding sections were observed. (Com- 

 pare, for instance, the study on deoxypentose nucleic acids of mammalian 

 origin."^) As regards biological properties it is worth mientioning that treat- 

 ment with neither sodium deoxycholate and chloroform^^ nor an anionic 

 detergent^^ appeared to destroy the transforming activity. 



The content in deoxyribonucleic acid of fresh calf thymus may be esti- 

 mated as being in the neighborhood of 2.5%. [See Leslie, Chapter 16.] 

 Gulland et al.^^ report an average yield of 0.87% for a preparation made 



i"' If the freshly precipitated and washed fibers are cut into very small pieces before 

 being suspended in distilled water, rapid stirring for 1 to 2 hours will be suflficient 

 to bring them into solution. 



I'o G. Frick, Biochim. et Biophys. Acta 13, 41, 374 (1954). 



1" E. Chargaff and R. Lipshitz, J. Am. Chem. Soc. 75, 3658 (1953). 



