314 ERWIN CHARGAFF 



per cc. (about 90% of total P in the mixture) . The reextraction of the insoluble residue 

 with water or M NaCl yielded negligible amounts of P. 



For the precipitation of the nucleohistone the aqueous solution was made 0.15 M 

 with respect to NaCl by the addition of 5.66 vol. of 0.177 M NaCl. The resulting 

 precipitate was collected 30 minutes later by centrifugation and washed on the centri- 

 fuge with 0.15 M NaCl and then, very briefly, with a very small amount of distilled 

 water. The weight ratios of protein to P in such preparations were around 12. 



The extinction, expressed as eCP),"^ of nucleohistone thus prepared is 

 around 6600, regardless of the presence or absence of salt and close to the 

 figure found for most specimens of sodium deoxypentose nucleate. [See 

 below, Section IV. 1, and also Beaven, Holiday, and Johnson, Chapter 14.] 

 This is of interest, since it would have been conceivable that the extinction 

 of a nucleoprotein would be less than that of the free nucleic acid, if the 

 purines and pyrimidines were involved in the architecture of the intact 

 conjugated protein in such a manner as to bring about the suppression of 

 some of the chromophores.*^ 



Nucleohistone is soluble at a very low ionic strength. With increasing 

 salt concentration, the solubility first decreases steeply, reaching a mini- 

 mum in isotonic solutions, and then rises gradually. Though variations 

 occur, freshly prepared nucleohistone is, in general, soluble in NaCl solu- 

 tions stronger than 0.7 or 0.8 M. Small amounts of phosphorylated com- 

 pounds that can be extracted from nucleohistone by 0.02 to 0.15 M NaCl 

 solutions usually lack the typical nucleic acid spectrum and represent 

 impurities. 



(2) Preparation of the Nucleoprotein of Avian Tubercle Bacilli.^^ A mixture of 25 g. 

 of ether-washed dry bacilli and 100 g. of washed, very fine, Pyrex glass powder (di- 

 ameter 3 n) was moistened with 0.1 M borate buffer (pH 8.3) and divided into eight 

 portions, each of which was ground for 30 minutes in a mortar. The ground cells were 

 united, shaken in a refrigerator with 500 cc. of the borate buffer for 2 days, and centri- 

 fuged for 30 minutes at 1900 X g. The strongly opalescent, slightly yellow supernatant 

 was decanted through a filter. The centrifugation residue was washed with 500 cc. of 

 borate buffer which then served for the extraction of a second 25-g. portion of disin- 

 tegrated bacilli. In this manner a total of 100 g. of organisms was processed. The ex- 

 tracts were dialyzed against running water for 48 hours, concentrated by pervapora- 

 tion to about one-third of the original volume, and again dialyzed against ice-cold 

 distilled water for 72 hours. Ethyl mercurithiosalicylate was added (0.01%) and the 

 bacterial glycogen" removed by sedimentation at 31,000 X g. The supernatants were 

 once more dialyzed, and the crude nucleoprotein fraction was recovered by evapora- 

 tion of the frozen solution in a vacuum (yield 2.7 g.). The yields varied for different 

 preparations between 2.4 and 3.4% of the starting material. The crude nucleoprotein 

 fractions, which could easily be dispersed in water or buffer solutions, gave positive 

 reactions for deoxypentose with diphenylamine and cysteine (see Chapter 9) and also 



" E. ChargafT and S. Zamenhof, J. Biol. Chem. 173, 327 (1948). 



« B. Magasanik and E. ChargafT, Biochim. etBiophys. Acta 7, 396 (1951). 



« E. Chargaff and D. H. Moore, J. Biol. Chem. 156, 493 (1944). 



