NUCLEOPROTEINS, NUCLEIC ACIDS, RELATED SUBSTANCES 151 



Preparation 



Nueleoproteins are labile substances; hence to obtain them from cells 

 onl3^ mild reagents and low temperatures can be used. 



An example of present day methods is the procedure IVIirsky and Ris 

 used for the preparation of chromosomes from calf spleen. The tissue was 

 broken up in a Waring blender and soluble matter removed with 0.14M 

 sodium chloride, in which the chromosomes were insoluble. Unbroken 

 cells and other coarse materials were separated by filtering through finely 

 woven cloth and sedimenting the chromosomes in a centrifuge at 3500 

 rpm. The suspension, filtration through cloth, and centrifugation were 

 repeated several times until the phosphorus figure, which was taken 

 as a measure of the nucleic acid content, became constant. Under the 

 microscope the material showed the characteristic coiled pattern of 

 chromosomes. Differential centrifugation methods are rapid, and 10 to 

 15 g. of purified material can be obtained in the course of a morning's 

 work. 



Tobacco bushy stunt virus, which is destroyed by almost any chemical, 

 was separated by Stanley from the other constituents of ground tobacco 

 leaves by means of differential centrifugation and purified still further 

 by crystallization. 



Separation of components 



The nucleic acid-protein complex is usually a very loose one, and the 

 combination can be broken up and the two components separated in 

 various ways. Some of these procedures are as follows: 



The protein may be denatured either by heating or by treating with 

 urea to give an insoluble compound, this then being removed by filtration. 

 Alternatively, the protein may be obtained by extraction with chloroform 

 and octyl alcohol, leaving the nucleic acid in solution. A third method 

 is to destroy the nucleic acid with the enzyme, ribonuclease, and then 

 separate the unchanged protein from the digested material. 



If the nucleic acid is the component wanted, it may be obtained from 

 the solution after the protein has been removed by one of the methods 

 described above. The protein may also be destroyed by trypsin and 

 the nucleic acid recovered. If the protein is a histone, separation may 

 be accomplished by dialysis against IM sodium chloride. Histone passes 

 through the membrane and leaves the nucleic acid behind. 



Linkage between protein and nucleic acid 



The bond between protein and nucleic acid is in some cases electro- 

 static, or salt-like, since the two parts may be separated by the passage of 



