HYDROLYSIS — ESTIMATION OF BASES IN PNA 



203 



TABLE I 

 Per Cent Recovery of Purine Bases and Pyrimidine Nucleotides After 



Various Treatments 



Treatment 



Acid hydrolysis N H2SO4 , 1 hr., 



100° 

 Precipitation as silver salts 

 Acid hydrolysis and precipitation 



as silver salts 



Adenine 



262- 

 280 mix 



99 



99 

 98 



262- 

 240 mM 



99 



98 

 98 



Guanine 



262- 

 280 m/x 



101.5 



99 

 99 



262- 

 240 mju 



101.5 



100 

 99 



Total purine 



252 mM 



100.5 



98.5 

 99 



276 mM 



100 



98 

 98 



Treatment 



Dephosphorylation by acid phosphatase 

 Acid hydrolysis and dephosphorylation 

 Acid hydrolysis, Ag"*" added and removed, 



and dephosphorylation 

 Dephosphorylation and Dowex 1 column 



(amino acids present) 



Cytidine 



260-278 

 m/i 



100 

 97 

 96 



100 



295 mM 



102 

 96 

 96 



98 



Uridine 



260-278 

 mM 



102 

 104 

 102 



99 



Total 

 pyrimi- 

 dine 



267 mM 



100 



100 



99 



99 



A 150-mg. sample" in about 30 ml. of 1 N sulfuric acid in a test tube capped with a 

 small beaker is heated for 1 hour in a boiling water bath. The solution is filtered 

 through a sintered glass funnel to remove a few particles of material presumed to be 

 coagulated protein, and the filtrate and several washings are diluted to 50 ml. in a 

 volumetric flask. Portions are analyzed directly for total nitrogen and, after suitable 

 dilution, for total phosphorus. 



For the determination of purine and pyrimidine content, 10-ml. portions of the 

 hydrolysate, in triplicate, are placed in 15-ml. centrifuge tubes. The following pro- 

 cedures are carried out in an identical manner on each of these aliquots as well as on 

 a 10-ml. sample of 1 A'' H2SO4, which provides a reagent blank for all subsequent op- 

 tical density measurements. The pH of the solution is brought to about 1.0 by the 

 addition of 11 A^ KOH, and, after warming to 90°, 1 ml. of a 20% AgNOs solution is 

 added to precipitate the adenine and guanine as silver salts. The suspension, after 

 standing overnight in the refrigerator, is centrifuged in the cold to pack the precipi- 

 tate of the silver-purines and a small amount of Ag2S()4 . The precipitate is washed 

 four times in the centrifuge tube with 3-ml. portions of ice-cold 0.1 A'^ H2SO4 . The 

 original supernatant solution and the washings are fiiltered through a sintered glass 



" This amount of nucleic acid diluted as described is convenient when micro-Kjel- 

 dahl analyses for nitrogen are to be performed. If the sample is analyzed for pur- 

 ine, pyrimidine, and phosphorus only, the amount used can be decreased to about 

 2 mg. by decreasing the volumes of reagents used proportionally throughout.* 



