^"UCLEIC ACID IX SAKCOMA SLICES 773 



PURIFICATION OF THE NUCLEIC ACID 



Since in most cases the activity of the deoxyribonucleic acid P ialls 

 far behind the activity of the remaining phosphorus-containing fractions, 

 the nucleic acid should always be carefully purified, even in in vivo 

 experiments, from phosphorus-containing impurities. In experiments 

 in which a study is made of nucleic acid formation in tissue slices, there 

 is a still greater demand for purification. Various studies have been de- 

 signed as checks on the purification. 



Active sodium phosphate (50 ingm) (2 x 10^ units of activity) was added 

 to the solution containing 236 mgm of crude nucleic acid and the latter 

 was accordingly precipitated. Tw^o further purifications were performed 

 after the addition of 20 and 10 mgm of sodium phosphate, respectively. 

 Further purification was obtained by precipitating twice with methanol 

 containing hydrochloric acid. The pure nucleic acid thus obtained 

 (64.4 mgm) showed an activity of 6.8 relative units (pulses/min in the 

 Geiger tube); 30 mgm of this nucleic acid was reprecipitated yet again 

 with methanol and hydrochloric acid, and the 19.5 mgm finally obtained 

 had an activity of 0.6 units. The above result shows that our purification 

 procedure is effective, since we have found about 100 units in purified 

 nucleic acid samples of similar weight in the experiments showing positive 

 results. 



The purification of nucleic acid with respect to free P can accordingly 

 be made quite substantially. Apart from active free P, however, our 

 original fractions also contain active acid-soluble phosphorus com- 

 pounds, and active phosphatides. We have, therefore, made a study of 

 the completeness with which these impurities may be removed. 



The active acid-soluble phosphorus compounds were obtained in the 

 following way: 30 //c of ^sp -^^^8 injected into a rat. The animal was 

 killed 6 hr later and the acid-soluble P compounds of the Jensen sarcoma 

 were extracted with 10% trichloroacetic acid. One-half of the neutralized 

 active trichloracetic acid extract was added to the crude nucleic acid 

 which had been exlracted^^^ from an inactive Jensen sarcoma (9.94 gni). 

 A fraction of the crude nucleic acid was analysed (No. I) and the main 

 part was purified by precipitation with methanol and hydrochloric acid, 

 adding 20 mgm of inactive NagHPOg to the solution before precipitation. 

 A fraction was again used for analysis (No. II) and the major part of 

 the specimen, dissolved in alkali, was treated with 10 mgm sodium phos- 

 phate and precipitated with methanol and hydrochloric acid. Further 

 purifications, the results of which are to be seen in Table 1, were carried 

 out with additions of sodium phosphate. 



(1) cf. H. ExjLER and G. Hevesy, Kgl. Dan-sk. Vid. Akad. Biol. Medd. 17, No. 8 

 (1942). 



