304 ZACHARIAS DISCHE 



pending on the species from which it was obtained [Cf. Chapters 10, 11], 

 rendered the determination of the content of these bases by simple and 

 sensitive reactions a matter of considerable interest. In the last few years 

 such methods were developed for adenine, thymine, cytosine, and uracil, 

 which are fairly characteristic for these substances and could perhaps be 

 applied for the analysis of nucleic acids. A micromethod for the deter- 

 mination of guanine in urine has not yet been applied to the determination 

 of this purine in nucleic acids and will not be discussed here. Unfortunately, 

 the preparations used by various authors for testing the applicability of 

 these methods for the determination of the bases in nucleic acids were not 

 completely pure and it is difficult, therefore, at present to evaluate how 

 far these methods could give completely correct values for the content of 

 individual purines and pyrimidines in nucleic acids and how far they can be 

 applied for the determination of these substances in tissue extracts without 

 interference from other cell constituents. 



1. Determination of Adenine 



The procedure of Woodhouse*^ is based on a color reaction of adenine 

 after its reduction with zinc dust and diazotization with NaN02 with N- 

 1-naphthylethylenediamine hydrochloride. The red color is still visible at a 

 concentration of 5 ng. per cc. of adenine and. is proportional to its concen- 

 tration up to 40 fxg. per cc. The determination of adenine in adenosine and 

 adenosine-3-phosphate gave correct values. In two preparations of nucleic 

 acids, the P content of which was about 20% below the theoretical, the 

 values were 10% lower than corresponded to the content in P. The reaction 

 is not specific for adenine, as it was originally proposed for the determina- 

 tion of folic acid. 



2. Determination of Thymine 



Two procedures by Woodhouse and by Pircio and Cerecedo for the de- 

 termination of thymine are both based on the Hunter'*^ reaction for this 

 substance. Thymine is coupled first with sulfanilic acid in Na2C03 solution, 

 and the reaction product then treated with hydroxy lamine in NaOH. A 

 red color is produced which is still visible at a concentration of 10 ^g- per 

 cc. of thymine. Cytosine and uracil do not interfere, but purines must be 

 removed either as silver salt or by palladium chloride. Woodhouse^^ pre- 

 cipitates thymine as a silver salt before its determination. Pircio and Cere- 

 cedo^* extract the evaporated hydrolysates of nucleotides or nucleic acids 



" D. L. Woodhouse, Arch. Biochem. 25, 347 (1950). 



^« G. Hunter, Biochem. J. 30, 745 (1936). 



" D. L. Woodhouse, Biochem. J. 44, 185 (1949). 



« A. Pircio and L. R. Cerecedo, Arch. Biochem. 26, 209 (1950). 



