134 PHYSIOLOGICAL CHEMISTRY 



solution. Now add an excess of an aqueous solution of barium hydroxide 

 and note the appearance of a purple color. 



Very dilute solutions do not give the test. Under these conditions 

 the solution should be evaporated to dryness, the residue dissolved in a 

 little bromine water and the excess of bromine removed. Then upon 

 adding an excess of barium hydroxide a decided bluish-pink or lavender 

 color will appear in the presence of as small an amount as o.ooi gram of 

 uracil. 



In testing solutions for cytosine it is preferable to warm or boil the 

 solution with bromine water, and after cooling the solution to apply 

 the test as suggested above, being careful to have a slight excess of 

 bromine present before adding barium hydroxide. 



14. Demonstration of Nucleases and Purinases in Tissues. 1 All 

 glandular tissues contain nucleic acids and enzymes capable of their 

 hydrolysis as well as the transformation of liberated purine bodies. 

 By allowing autolysis (self-digestion) to take place in such a tissue and 

 studying the products formed it is possible to determine what enzymes 

 were present in the tissue under examination. Typical results may be 

 obtained by using ox and pig spleens, 'which differ in the purine enzymes 

 which they contain. The two experiments should be run in parallel. 



A. Preparation of the Material for Digestion. Run the gland once or twice 

 through a meat chopper. Introduce about 650 grams into a two liter bottle, fill 

 about three-quarters full of water, add 20 c.c. of chloroform, and allow to remain 

 at room temperature for 12-36 hours with occasional agitation. Strain through 

 linen and replace the turbid extract in the bottle with 10 c.c. of chloroform. This 

 solution contains the enzymes and nucleic acid of the tissue. (Reserve 50 c.c. 

 of one of the extracts for the experiment on phosphonuclease (E), which should 

 also be started at this time.) The bottle is tightly closed and allowed to remain 

 in the thermostat 4-5 days. 



B. Separation of Purine Derivatives from Other Substances. Introduce 

 the products into a saucepan or large evaporating dish, heat to brisk boiling, 

 make f aintly alkaline with caustic soda, boil a few minutes, make f aintly acid with 

 acetic acid, boil vigorously and filter hot. (If desired one-half of the filtrate may 

 be treated with 100 c.c. of 20 per cent sulphuric acid per liter and boiled for one 

 hour, keeping at constant volume. At the end of the hydrolysis the sulphuric acid 

 is nearly neutralized with caustic soda and the purine bases of the solution 

 determined as in the other half of the filtrate. This will give information as to 

 the presence of deaminases acting upon the ammo purines remaining in combina- 

 tion). To the boiling filtrate add 100 c.c. of 10 per cent copper sulphate and small 

 successive portions (2-5 c.c.) of sodium bisulphite (commercial saturated solution) 

 until the precipitate takes on a decided yellow color due to precipitation of cuprous 

 oxide. Filter, wash the precipitate with boiling water, pierce the filter and wash 

 the precipitate into a flask. Add i per cent sodium sulphide solution to decom- 



1 From Monograph on "Nucleic Acids" and Laboratory Notes in Physiological Chem- 

 stry by Professor Walter Jones of Johns Hopkins University, with additions. 



