664 ADVENTURES IN RADIOISOTOPE RESEARCH 



is composed of acid soluble P present as an impurity, a grossly erroneous 

 value will be found for the specific activity of the nucleic acid P, viz. 

 12 • 10~* instead of 2 • 10"*. This example illustrates the necessity of 

 an exceedingly careful purification of the nucleic acid fraction from all 

 non-nucleic acid phosphorus. In our work, we are not faced with the great 

 difficulties which were surmounted by Hammahsten" in his experiments 

 which lead to the preparation of non-depolymerized nucleic acid. On the 

 other hand, w^e have to avoid the presence of even minimal amounts 

 of non-nucleoprotein P, the presence of which in any other but the 

 radioactive investigations would certainly not be found disturbing. 



EXPERIMENTAL PROCEDURE 



We applied the method of extraction and purification described by Klein 

 and Beck (1935) adapted to work with tissue containing radioactive phosphorus, 

 as previously used by H. von Euler and one of the present writers (1942) in their 

 work on the rate of formation of nucleic acid in the Jensen sarcoma of the rat. 

 The washed tissue is stirred with an equal weight of 5 per cent sodium chloride 

 solution brought to boihng. Acetic acid is added until the major part of the pro- 

 teins present is precipitated. Sodium acetate and sodium hydroxyde are then 

 added and the alkaline solution is heated until the tissue is dissolved. 



The next operation is carried out in a slightly acid solution. This is obtained 

 by adding acetic acid. From this solution, the protein present is removed by adding 

 a dialysed colloidal iron hydroxide solution containing 5 per cent FgOg. An excess 

 of acetic acid is added and the hot solution is filtered. By adding an equal volume 

 of methylalcohol to the filtrate, the crude nucleic acid precipitates. 



The crude nucleic acid is dissolved in sodium hydroxide and is precipitated 

 with hydrochloric acid and methylalcohol. Before re-precipitating the nucleic acid, 

 we added about 10 mgm (NH4)2HP04 for each mgm nucleic acid. By doing so, we 

 diluted the free radioactive phosphate possibly present in the nucleic acid. If the 

 crude nucleic acid carried before and after the precipitation 1 mgm free phosphate, 

 the free phosphate will be but 1/100 as active after precipitation as previously. 

 This procedure is repeated several times, and each time inactive (NH^)2HP0^ 

 is added to the alkaline solution. 



The purification process entails a substantial loss of nucleic acid. However, 

 it is not the total desoxyribose nucleic acid content of the organs in which we 

 are interested, but the percentage of the desoxyribose nucleic acid content which 

 is built up during the experiment, i. e. the rate of renewal of the nucleic acid 

 molecules. We are interested in the activity of 1 mgm nucleic acid P and not in the 

 activity of the total nucleic acid present in the organs. 



The purified nucleic acid is brought into solution by wet ashing, i/g is reserved 

 for colorimetric P determination, while ^/^ are precipitated as ammonium magne- 

 sium phosphate; the activity of the precipitate is determined. The interpretation 

 of the activities of which are to be compared, have the same weight. To obtain 

 this, a suitable amount (about 80 mgm) of NagHPO^ is added to the solution before 

 precipitating the ammonium magnesium compound. 



An aliquot of the solution administered by subcutaneous injection is treated 

 in the same way. If this "standard preparation" has, for example, 1/1000 of the 

 activity administered and the nucleic acid fraction containing 1 mgm P has 1/100 



