Organic Substances 89 



both for the specificity of at least some of the nucleases and 

 for the quantitative eiBFect of enzymes (for instance, diastase 

 and ribonuclease ) . 



The preparation of highly active ribonuclease of excellent 

 specificity is simple and v^ill be given here (Brachet's 

 method ).^^^ The step of boiling the crude enzyme solution 

 destroys all enzymes except the heat-resistant ribonuclease. 



Method 



Grind beef pancreas to a smooth pulp and suspend it for 

 24 hours at 37° C. in 1-2 volumes of 0.1 N acetic acid. Boil 

 it for 10 minutes and filter. Neutralize the filtrate to about 

 pH 6.9-7.5 and filter it once more. This solution can be kept 

 in the icebox, with some camphor or thymol added, for sev- 

 eral months. Brachet recommends dialysis as a last step, but 

 it is not necessary. 



Incubate sections in the enzyme solution for 1 hour at 

 65°-70° C. Treat a control section with a buffer of the same 

 pH. The enzyme-treated section will show a complete loss of 

 all basophiha due to RNA^^^ (but not of that due to muco- 

 polysaccharides ) . The Feulgen reaction of the nuclei is not 

 affected. 



Of the desoxyribonuclease preparations, McCarty's^^* ap- 

 pears to be the most specific. Its preparation is not easy; in 

 most cases it will be simpler to purchase a commercial prep- 

 aration. Desoxyribonuclease is not resistant to heat; it should 

 be used around 37° C. A good preparation will abolish the 

 Feulgen reaction of the nuclei but leave cytoplasmic baso- 

 philia intact. 



According to Mazia,^^^ intestinal phosphatase is also effec- 

 tive in removing RNA. Acid phosphatase has not been used 

 histochemically. 



142. Brachet, J.: Compt. rend. Soc. de biol., 133:88, 1940. 



143. Brachet, J., and Shaver, J. R.: Stain Technol., 23:177, 1948; Deane, 

 H. W.: Am. J. Anat, 78:227, 1946. 



144. McCarty, M.: J. Gen. Physiol., 29:123, 1946. 



145. Mazia, D.: Cold Spring Harbor Symp. Quant. Biol, 9:40, 1941. 



