ISOLATION AND COMPOSITION OF DEOXYPENTOSE NUCLEIC ACIDS 313 



a. Extraction with Solutions of Low Ionic Strength 



This procedure, which has been used repeatedly for the isolation of 

 nucleoproteins,'"^-"^^ in particular of calf thymus nucleohistone, has the 

 advantage of avoiding the exposure of the conjugated protein to high salt 

 concentrations, and therefore to dissociating conditions, in the course of its 

 preparation. There exists, however, the danger of a partial enzymic degrada- 

 tion of the nucleic acid brought about by the release of nucleases. Attempts 

 are usually made, based on the behavior of the pancreatic deoxyribonuclease 

 (see Chapter 15), to suppress the enzymic attack by the use of arsenate, 

 citrate, or such chelating agents as sodium ethylenediamine tetraacetate. 

 There is, however, some doubt as to the role of these complex-forming 

 agents, owing to the existence of nucleases that differ in their requirements 

 from the pancreatic enzyme,""" and their efficacy may have to be explained 

 in a different manner.'* 



A preparation, at a low electrolyte concentration, of calf thymus nucleo- 

 histone is given as the first example, and the nucleoprotein of tubercle 

 bacilli as the second. 



(1) Prefaration of Calf Thymus Nucleohistone.^" Trimmed calf thymus was ob- 

 tained fresh at the slaughter-house, chilled immediately, and processed without de- 

 lay. All subsequent operations were performed at 4-6°. Fifty-gram portions of tissue 

 were triturated for 30 seconds in a high-speed mixer equipped with cutting blades 

 with 50 cc. of an ice-cold mixture of aqueous 0.1 M NaCl and 0.05 M sodium citrate 

 (previously adjusted to pH 7). The supernatant fluid resulting from centrifugation at 

 2000 X g for 30 minutes was discarded and the suspension of the sediment in 100 cc. 

 of saline-citrate once more centrifuged. The sediment was washed three times by 

 thorough resuspension and centrifugation, each time with 50 cc. of distilled water 

 (previously adjusted to pH 7 by being made about 0.0004 M with respect to NaHCOs) , 

 in order to remove electrolytes. During the final washing the sediment swelled, but 

 yielded less than 0.5% of its total phosphorus to the supernatant fluid. The gelatinous 

 sediment then was blended (15 seconds in the high-speed mixer) with 250 cc. of dis- 

 tilled water (pH 7) and shaken overnight. The extremely viscous mixture was again 

 briefly stirred in the high-speed mixer and centrifuged for 30 minutes at 2000 X (j- 

 The P contents of the very viscous, opalescent supernatant fluids averaged 430 ^g. 



'' K. G. Stern, G. Goldstein, J. Wagman, and J. Schryver, Federation P roc. 6, 296 



(1947). 

 " D. C. Gajdusek, Biochim. et Biophys. Ada 5, 397 (1950). 

 ^* K. G. Stern, G. Goldstein, and H. G. Albaum, J. Biol. Chem. 188, 273 (1951). 

 " M. H. Bernstein and D. Mazia, Biuchim. et. Biophys. Acta 10, 600 (1953). 

 •i« J. A, V. Butler, P. F. Davison, D. W. F. James, and K. V. Shooter, Biochim. et 



Biophys. Acta 13, 224 (1954). 

 " S. Zamenhof and E. Chargaff, J. Biol. Chem. 180, 727 (1949). 

 38 M. E. Maver and A. E. Greco, J. Biol. Chem. 181, 861 (1949). 

 " M. Webb, Nature 169, 417 (1952). 

 « M. Webb, Exptl. Cell Research 5, 27 (1953). 



