136 PHYSIOLOGICAL CHEMISTRY 



place. Put the tubes in boiling water for a few minutes to coagulate proteins. 

 Then add 5 c.c. of 5 per cent HC1 and let stand for an hour. This precipitates 

 any unaltered nucleic acid which may be present. Filter and take an aliquot 

 portion of each filtrate (15 c.c.). Add magnesia mixture and a few cubic centi- 

 meters of strong ammonia. Let stand over night. Any phosphoric acid present 

 will be precipitated as magnesium ammonium phosphate. Observe the relative 

 amounts of phosphate in each case. Has any phosphate been set free from the 

 nucleic acid added? From the nucleic acid of the gland extract? 



15. Experiments on Uncase (Uricolytic Enzyme). A. Preparation of Extract 

 Containing Uricase. Extract about 50 grams of pulped kidney tissue of the ox 

 with 200 c.c. of toluene or chloroform water at 38C. for 24 hours, with occasional 

 shaking. Filter and use the filtrate in the following experiment. 



B. Demonstration of Uricase. Add about o.i gram of uric acid to 10 c.c. 

 of water and bring the uric acid into solution by the addition of the minimal quan- 

 tity of KOH. To 5 c.c. of this uric acid solution in a test-tube add 50 c.c. of the 

 uricolytic enzyme extract prepared as described above. Prepare a second tube 

 containing a like amount of the uric acid solution but boil the extract before 

 it is introduced. Place the two tubes at 38C. for two days. The vessels should 

 be open to the air and the contents stirred occasionally, or much better, a con- 

 tinuous current of air which has gone through a chloroform wash bottle is passed 

 through the mixture. Make both mixtures faintly acid with acetic acid and 

 boil. Filter and take an aliquot of each filtrate. Evaporate to low volume, make 

 faintly alkaline with ammonia and filter. Add a few cubic centimeters of 

 ammoniacal silver nitrate solution. Any uric acid will be precipitated as silver 

 urate. The control should give a heavy precipitate while the test should show 

 no precipitate or one much lighter than the control, due to uric acid destruction hi 

 the latter case. 



If it is desired to separate out the pure uric acid the silver -purine precipitate 

 may then be filtered off. It is washed with water and transferred to a beaker with 

 the aid of a little water. To the mixture add a few cubic centimeters of hydrogen 

 sulphide solution and a few drops of HC1 and allow to stand over night. The uric 

 acid should separate out hi crystalline form and should be found in less amount 

 in the test than hi the control experiment. The uric acid may also be titrated 

 with permanganate as in the Folin-Shaffer method for uric acid in urine. (See 

 Chapter XXVII on Quantitative Analysis of Urine.) This will enable us to 

 determine exactly how much of the uric acid was destroyed through the action 

 of the enzyme extract. 



