SEPARATION BY PAPER CHROMATOGRAPHY 259 



c. Hydrolysis with formic acid 



The value of formic acid for liberating purine and pyrimidine bases from 

 nucleic acids without destruction was first shown by Vischer and Chargaff ,*^ 

 and this acid has proved very satisfactory for the quantitative hydrolysis 

 of DNA. The conditions used in Chargaff 's laboratory have been 98-100 % 

 formic acid at 175° for 2 hours. Shorter periods of heating have also been 

 employed. ^^' ^^ Recently," good results were obtained with DNA from bac- 

 teriophages and other sources using 88 % formic acid at 175° for 30 minutes, 

 and it was found that whether 88 % or 98 % formic acid is used, recoveries 

 of guanine and hydroxymethylcytosine are substantially increased by using 

 a relatively large volume of it (500 jul. per mg. DNA) and by avoiding ex- 

 cessive oxidizing atmosphere while heating. The hydrolysis is carried out 

 in sealed Pyrex glass bomb tubes, and decomposition of the formic acid 

 produces considerable pressure, which it is advisable to release by melting 

 the tips of the tubes in a flame before opening them. The hydrolysate is 

 evaporated under reduced pressure to dryness, then redissolved in a small 

 volume of A^ HCl, and samples are taken for chromatography and for esti- 

 mation of phosphorus. Because of the high recoveries and the absence of 

 insoluble residue in the hydrolysate, this is probably the method of choice 

 for accurate microanalysis of purified preparations of DNA. 



2. Hydrolysis of Pentose Nucleic Acids 



The ribosides of both purines and pyrimidines are split with more dif- 

 ficulty than their deoxyribosides, and the resistance of the pyrimidine ribo- 

 sides is such that it is very difficult to get quantitative yields of the free 

 bases from PNA. However, the ease with which mononucleotides are ob- 

 tained from PNA makes it possible to estimate the pyrimidines, or all of 

 the bases, as nucleotides. [Cf. Loring, Chapter 5, and Magasanik, Chapter 

 11.] 



a. Hydrolysis to purine and pyrimidine bases 



Concentrated formic acid at 175° for 2 hours has been used for liberation of pyrimi- 

 dines from PNA;'3 however, the yield of uracil is very low and the method has been 

 found unsatisfactory for quantitative hydrolysis of uridylic acid.^* Nearly quantita- 

 tive yields of all the bases can be obtained with 70-72% perchloric acid at 100° for 1 

 hour/'- '3 and this method has been used for analysis of PNA's.*^ 



h. Hydrolysis to pnrine bases and pyrimidine nucleotides 



The purine bases are liberated from PNA by relatively mild acid hydrolysis, e.g., 

 N HsSOi^' or N HC186 at 100° for 1 hour. The latter treatment was selected by Smith 



" E. Vischer and E. Chargaff, J. Biol. Chem. 176, 715 (1948). 

 «* A. Marshak, /. Biol. Chem. 189, 597 (1951). 



85 Smith and Markham" include a critical discussion of previously used methods of 

 hydrolysis. 



