324 ERWIN CHARGAFF 



this stage the viscosity of the solution increased considerably. After extraction at 0° 

 for 48 hours, the insoluble material was removed by centrifuging (6300 r.p.m.) and 

 the deoxypentose nucleoprotein precipitated from the resultant solution (pH 6.5) by 

 the addition of an equal volume of industrial methanol. The precipitated solid was 

 washed with 70%, then 100% industrial methanol and dried in a vacuum at room 

 temperature. Yield, 1.69 kg. of a very slightly yellow fibrous solid. 



The nucleoprotein (500 g.) was powdered to assist solution and dissolved in 10% 

 sodium chloride (45.5 1.) with vigorous mechanical stirring. The solution, which was 

 viscous, was clarified by centrifuging (6300 r.p.m.). To the clear solution was added 

 an equal volume of a mixture of chloroform (35 parts) and amyl alcohol (10 parts), 

 and the mixture was emulsified by rapid mechanical stirring. The emulsion was then 

 separated by centrifuging (DeLaval, model "500", disc -type bowl, 5500 r.p.m.) into 

 three parts: (a) the chloroform - amyl alcohol mixture; (b) a solution containing the 

 sodium salt of deoxypentose nucleic acid and nucleoprotein; (c) a gel of protein 

 hydrochloride and the chloroform - amyl alcohol mixture. The protein gel remained 

 in the bowl of the centrifuge whereas the chloroform - amyl alcohol mixture and the 

 solution of nucleic acid and nucleoprotein were discharged from separate outlets. 

 The last-mentioned solution was again emulsified with the chloroform - amyl alcohol 

 mixture and the process repeated until no gel was formed on emulsification; this re- 

 quired nine emulsifications. The sodium salt of the deoxypentose nucleic acid was 

 precipitated by the addition of an equal volume of methanol, washed free from chlo- 

 ride with 70% methanol, then 100% ethanol, and finally ether, and dried in a vacuum 

 at room temperature. Yields from two 500-g. quantities of nucleoprotein were 130 g. 

 and 150 g. of a white fibrous solid giving negative biuret and Sakaguchi tests. 



(a) Small-scale preparation. A tj'pical preparation from our laboratory is briefly 

 described here. All operations were carried out at about 4°. Two hundred and twenty 

 grams of freshly obtained, well-trimmed calf thymus were cut into small pieces and 

 triturated for 3 minutes in a "Waring Blendor" in 400 cc. of a mixture of 0.1 M NaCl 

 and 0.05 M sodium citrate (adjusted to pH 7). The sediment resulting from centri- 

 fugation at 1900 X g for 45 minutes was washed twice more with 350-cc. portions of 

 the same fluid and stirred in a high-speed mixer with 1250 cc. of 10% NaCl. The mix- 

 ture was kept overnight and centrifuged at 20,000 X g for 45 minutes. The sediment 

 was reextracted for several hours with 800 cc. of 10% NaCl and the mixture centri- 

 fuged as above. The combined extracts (about 1600 cc.) were injected in a slow stream 

 into 2 vol. of 95% ethanol. The white strands of nucleohistone were lifted by spooling, 

 drained, and washed with 70% and 80% ethanol. The material was dissolved with the 

 aid of rapid stirring in 2500 cc. of 10% NaCl and the solution freed of protein by being 

 stirred eight times, each time for 150 seconds and being followed by centrifugation at 

 1900 X g, with one-third volume of chloroform - amyl alcohol (3:1). The supernatant 

 aqueous solution was finally injected into 2 vol. of 95% ethanol and the fibers were 

 spooled, lifted, drained, and washed with 70%, 80%, and 100% ethanol. They were 

 then dried in air or recovered by the lyophilization of their dialyzed aqueous solution. 

 The yield of sodium nucleate amounted to 1.4. to 1.9% of the fresh tissue. 



(2) Sodium Deoxyribonucleate of Yeast^^ The isolation of this substance 

 may be regarded as an example of an extreme case, as yeast cells contain 

 almost 50 times as much pentose as deoxypentose nucleic acid. 



One kilo of fresh bakers' yeast was washed with 1 1. of 0.1 M sodium citrate (pH 

 7.3). The 3'east cells, recovered by centrifugation, were suspended in 180 cc. of the 

 sodium citrate solution and the thick suspension was passed through an ice-cooled 

 wet crushing mill for bacteria. Each 50-cc. portion was ground for 30 minutes. Fol- 



