258 G. R. WTATT 



virtually quantitative yield by appropriate acid hydrolysis. Good yields of 

 nucleosides or nucleotides have not been obtained from DNA by any means 

 of hydrolysis with acid or alkali. Nucleotides may be obtained enzymically/* 

 but DNA containing 5-hydroxymethylcytosine resists this treatment''® and 

 chemical methods are preferable for routine work. [Cf. Chargaff, Chapter 10.] 



a. Hydrolysis with hydrochloric acid 



The purines are completely released from DNA by very mild acid treatment (pH 

 1 .6 at 37° for 24 hr . , or pH 2.8 at 100° for 1 hour") . More drastic hydrolysis with hydro- 

 chloric acid (10 mg. nucleic acid in 2 ml. 6 A' HCl at 120° for 2 hours''*- '') gives a good 

 yield of pyrimidines but causes some destruction of purines. Nearly quantitative 

 yields of all the bases (including 5-hydroxymethylcytosine but with evidence for 

 slight destruction of adenine) have been obtained by Hershey et al.^'' by subjecting 

 the partially hydrolyzed nucleic acid extracted from bacteriophage T2 with hot tri- 

 chloroacetic acid to further hydrolysis with 3 ml. redistilled 6 A' HCl under CO2 in 

 sealed tubes at 100° for 3 hours. 



h. Hydrolysis with 'perchloric acid 



The use of concentrated perchloric acid for liberation of bases from 

 nucleic acids was introduced by Marshak and Vogel.^^-^" With DNA, 

 either 7.5 N or 12 N (70 %) HCIO4 may be used, and maximal yields are 

 obtained in 1 hour at 100°. Loss of a small percentage of the thymine has 

 been noted during hydrolysis with 70 % HCIO4 , which may be minimized 

 by using not more than 15 yul. HCIO4 per mg. DNA, although if much pro- 

 tein is present the amount maybe increased.^' 5-Hydroxymethylcytosine is 

 extensively destroyed during hydrolysis of bacteriophage DNA with per- 

 chloric acid, although the isolated pyrimidine is apparently not attacked by 

 this acid."' " By hydrolyzing with HCIO4 it is possible to estimate the total 

 purine and pyrimidine bases from a mixture containing both DNA and 

 PNA, and to estimate the bases without isolation of nucleic acid from some 

 biological materials. Non-nucleic acid components are degraded to products 

 which interfere very little on the chromatograms. Perchloric acid hydroly- 

 sates may be diluted with water and applied directly to paper for chroma- 

 tography. If phosphorus is to be estimated in samples of the hydrolysate, 

 the insoluble residue from the nucleic acid carbohydrate, which tends to ad- 

 sorb phosphate, should first be brought into suspension. ^^ 



75 R. O. Hurst, J. A. Little, and G. C. Butler, /. Biol. Chem. 188, 705 (1951). 



'« S. S. Cohen and G. R. Wyatt, unpublished results, 1953 



" C. Tamm, M. E. Hodes, and E. Chargaff, /. Biol. Chem. 195, 49 (1952). 



'8 M. M. Daly, V. G. Allfrey, and A. E. Mirsky, J. Gen. Physiol. 33, 497 (1950). 



79 N. I. Gold and S. H. Sturgis, J. Biol. Chem. 196, 143 (1952). 



«" A. Marshak and H. J. Vogel, Federation Proc. 9, 85 (1950). 



" G. R. Wyatt, J. Gen. Physiol. 36, 201 (1952). 



82 Levenbook** has obtained evidence for differential adsorption of purine and 



pyrimidine bases also to the charcoal from nucleic acid samples contaminated with 



carbohydrates. 



