Radiophosphorus and Nucleic Acids 179 



truly protein-bound tissue constituent and not merely a 

 residue of acid soluble inorganic phosphate which has failed 

 to be extracted with TCA. 



Investigations of this type have, however, raised the 

 question of the number of washings which are necessary to 

 remove all traces of acid-soluble phosphate from a tissue 

 homogenate. It is generally considered that three or four 

 extractions are adequate, but in dealing with ^^p it should be 

 emphasized that the exceedingly minute amounts of phos- 

 phorus removed in the later stages of repeated washing with 

 TCA may nevertheless be significantly radioactive, since the 

 acid soluble inorganic phosphate in a tissue has a much 

 greater activity than have any of the protein-bound phos- 

 phorus fractions. Frazer and Hutchison have investigated 

 this matter by adding ^^p along with TCA to a homogenate of 

 liver tissue from a non-radioactive animal. Such artificially 

 added inorganic radiophosphate might be expected to be 

 completely removed by subsequent extractions with TCA, 

 leaving the protein-bound phosphorus fractions inactive, 

 but this has not proved to be the case. In one experiment 

 as many as 57 washings with 10 per cent TCA in a refrigerated 

 centrifuge failed to remove all traces of radioactivity from the 

 protein, and the protein-bound phosphorus fractions showed 

 significant activity. Repeated washings with TCA containing 

 15 per cent non-radioactive NaH2P04.2H20 also failed to 

 remove contaminating radioactivity (Davidson, Frazer and 

 Hutchison, 1951). It should be emphasized that in our 

 experience the activity of the protein-bound phosphorus 

 fractions obtained in this type of experiment show a much 

 greater variation than those from animals which had received 

 the ^^P by injection. 



These experiments serve to emphasize the need for rigid 

 purification of a tissue constituent before its specific activity 

 can be determined. 



It is clear from our results that before any reliable informa- 

 tion can be forthcoming about the specific activities of the ribo- 

 nucleotides obtained in a Schmidt-Thannhauser separation, 



