HYDROLYSIS — ESTIMATION OF BASES IN PNA 197 



b. Liberation of Pyrimidine Nucleotides and Free Bases 



The above-mentioned experiments show the relative ease with which the 

 purine bases are removed from DNA. In quantitative estimations alcoholic 

 HCl (methanol saturated with HCl for 3-5 hours at 50° ■* or 1.5 % metha- 

 nolic HCl for 20 hours at 37°) "^ has been used. If DNA is heated in 2 % 

 H2SO4 for 2 hours under reflux conditions, the remaining polynucleotide 

 structure after removal of the purine bases is further degraded to mixtures 

 of thymine deoxyribodiphosphoric acid and cytosine deoxyribodiphosphoric 

 acid, which may be fractionated and obtained as crystalline compounds. 

 [Cf. Baddiley, Chapter 4.] The extent to which other pyrimidine derivatives 

 also may be formed has not been determined. 



The hydrolysis of DNA with concentrated formic acid at 175°,^ with 6 

 N HCl at 120° ^° or with 12 N perchloric acid^^ leades to a relatively quanti- 

 tative formation of the free pyrimidine bases. The merits of the different 

 hydrolysis procedures are discussed in Chapters 7 and 10 as regards the 

 recovery of individual bases. 



3. Alkaline Hydrolysis of PNA 



a. Formation of Nvcleosides 



The acid instability of the purine ribosidic linkages, in contrast to their 

 alkali stability, was recognized when guanosine (then called vernine) and 

 inosine were discovered.^^' ^* This property of nucleosides has played an 

 important role in establishing the structures of the purine and pyrimidine 

 mononucleotides, since when alkaline hydrolysis is applied to yeast nucleic 

 acid (4-5 % NH3 for 3.5 hours at about 145°) ^^ or to mononucleotides, it is 

 the phosphate ester linkage which is hydrolyzed with the formation of in- 

 organic phosphate and purine and pyrimidine nucleosides. The conversion 

 of inosinic acid to inosine by alkaline hydrolysis or to hypoxanthine and 

 ribose phosphate by acid hydrolysis as carried out by Levene and Jacobs^^ 

 thus laid the groundwork for the structure of all the nucleotides as phos- 

 phate esters of nucleosides linked through a hydroxyl group of the sugar as 

 shown below. 



HO HO 



^ H+ . OH- 

 purine + 0=P — O — sugar «- 0=P — O — sugar-punne > 



/ / 



HO HO 



sugar-purine + HP04~ 



32 E. Schulze and E. Bosshard, Z. physiol. Chem. 10, 80 (1886). 



33 F. Haiser and F. Wenzel, Monatsh. 29, 157 (1908). 



34 P. A. Levene and W. A. Jacobs, Ber. 43, 3150 (1910). 



35 P. A. Levene and W. A. Jacobs, Ber. 41, 2703 (1908) ; 42, 335, (1909) ; 42, 1198 (1909) ; 

 44,746 (1911). 



