316 ERWIN CHARGAFF 



employed for the preparation of nucleoprotamine^^-®''^ and nucleohis- 

 tone.^^'^^'*^'^" There exists, however, considerable evidence that, as regards 

 the physical intactness of the nucleoproteins,*^ this method is inferior to that 

 described in the preceding section (II. 3. a) since, at any rate in molar or 

 stronger sodium chloride solutions {M glycine is considered less harmfuP^), 

 a far-reaching separation between nucleic acid and protein takes place.^°"' 

 24,25,30,62,63 fhls cau bcst bc shown under experimental conditions that lead 

 to the removal of one of the partners. ^°'^^ On the other hand, an advantage 

 of this procedure may be seen in the suppression of the activity of deoxy- 

 ribonuclease in strong salt solutions.^'*" 



The preparation of a nucleoprotamine is given as an example. 



(/) Preparation of the Nucleoprotamine of Trout Sperm.''' The operations were car- 

 ried out in a cold room. Before extraction the freshly collected trout sperm were 

 washed with a solution containing in 1000 cc. : 7.8 g. NaCl, 0.664 g. KCl, and 0.687 g. 

 K2SO4." After stirring, the suspension was centrifuged at 5000 r.p.m. for 15 minutes. 

 The washed sperm were extracted with M NaCl (final concentration after mixing). 

 On adding salt solution, the sperm mass immediately became sticky and gelatinous, 

 so that it appeared at first as if the cells were merely swelling. It was necessary to add 

 a large volume of solution and to stir vigorously in a Waring mixer before it became 

 apparent that the cells were breaking up as their contents passed into solution. For a 

 mass of sperm with a dry weight of 900 mg. the volume of the extraction mixture 

 should be about 500 cc. Even so, the mixture was quite viscous. After vigorous stir- 

 ring the mixture was centrifuged at 12,000 r.p.m. for 60 minutes. A perfectly clear, 

 viscous supernatant and a scanty residue were obtained. The material extracted from 

 the sperm was precipitated by pouring the supernatant into 6 volumes of water.** The 



** I. Watanabe and K. Suzuki, J. Cheni. Soc. Japan, Pure Chem. Sect. 72, 578, 580, 

 604 (1951) ; Chem. Abstr. 46, 3666 (1952). 



" H. V. Euler and L. Hahn, Arkiv. Kemi, Mineral. Geol. 22A, No. 17 (1946) ; 23A, No. 

 5 (1946). 



" M. L. Petermann and C. M. Lamb, J. Biol. Chem. 176, 685 (1948). 



« G. Frick, Biochim. et Biophys. Acta 3, 103 (1949). 



" M. Fleming and D. O. Jordan, Discussions Faraday Soc. No. 13, 217 (1953). 



" E. J. Ambrose and J. A. V. Butler, Discussions Faraday Soc. No. 13, 261 (1953). 



«» J. M. Luck, D. W. Kupke, A. Rhein, and M. Hurd, J. Biol. Chem. 205, 235 (1953). 



" K. G. Stern and S. Davis, Federation Proc. 5, 156 (1946). 



" S. S. Cohen, J. Biol. Chem. 158, 255 (1945). 



" M. H. Bernstein and D. Mazia, Biochim. et Biophys. Acta 11, 59 (1953). 



" M. Laskowski, Arch. Biochem. 11, 41 (1946). 



" M. Kunitz, J. Gen. Physiol. 33, 363 (1950). 



** It may be of advantage to use a wash fluid containing sodium citrate. The solution 

 used by Chargaff et al." contained the following molarities: sodium chloride 0.123, 

 potassium chloride 0.009, potassium sulfate 0.004, sodium citraie 0.01. (Compare 

 also, Bernstein and Mazia. ^^) 



" E. Chargaff, R. Lipshitz, C. Green, and M. E. Hodes, J. Biol. Chem. 192, 223 (1951) . 



" This step is, in my experience, often accompanied by considerable losses, an ob- 

 servation also made by v. Euler and Hahn." If the nucleoprotein is to serve merely 

 as an intermediate in the preparation of deoxypentose nucleic acid, it is prefer- 

 able to employ precipitation with alcohol. (See below in Section III. 2.) 



