136 PHYSIOLOGICAL CHEMISTRY 



What does the finding of guanine and hypoxanthine but not adenine or xan- 

 thine indicate as to the type of purine deaminase present in the pig's spleen? 



D. Treatment of Filtrate from Ox Spleen. The filtrate from the copper sul- 

 phide should be evaporated to dryness on the water-bath. Extract with cold 

 water. Test a part of this aqueous solution with picric acid. A lack of precipi- 

 tate indicates the absence of adenine. To another portion add ammonia (a 

 lack of precipitate indicates the absence of guanine), and then a little ammo- 

 niacal silver nitrate solution (lack of appreciable precipitate indicates absence of 

 purines of any kind in more than traces). Dissolve half of the residue, which 

 should consist mainly of uric acid and xanthine, in as few drops of concentrated 

 sulphuric acid as possible and dilute with 4 volumes of water. Stir until the 

 uric acid begins to separate and then let stand for about three hours. The uric 

 acid is completely precipitated. Apply the murexide test. To the remainder 

 of the xanthine-uric acid residue add a little 4 per cent potassium hydroxide solu- 

 tion. Warm and add an equal volume of 30 per cent nitric acid. Allow to cool. 



FIG. 40. HYPOXANTHINE CHLORIDE. 1 

 (Reproduced from crystals furnished by Professor Walter Jones.) 



Xanthine nitrate separates out in a granular form, showing characteristic crystals 

 under the microscope. Apply the nitric acid test. 



What does the presence of uric acid and xanthine and the absence of guanine 

 and adenine indicate as to the purine enzymes of ox spleen? 



E. Demonstration of Nucleotidase (Phosphonuclease). In this experiment 

 use the 50 c.c. portion of enzyme solution retained from Experiment A preceding. 

 Prepare a 2 per cent solution of yeast nucleic acid aiding the solution by the slow 

 addition of KOH solution until the reaction, as indicated by a few drops of litmus 

 added, is neutral. Prepare a series of three large test-tubes as follows : In No. i 

 place 10 c.c. of enzyme solution and 5 c.c. of nucleic acid. In No. 2 place 10 c.c. 

 of enzyme solution and 5 c.c. of water. In No. 3 place 10 c.c. of enzyme solution, 

 boiled and 5 c.c. of nucleic acid. To each tube add 2-3 c.c. each of CHC1 3 and 

 toluol as preservatives. Place the tubes at 37C. for 24 hours. Add i c.c. of 

 litmus solution to each tube and note whether any changes in reaction have taken 

 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. 



1 Hypoxanthine nitrate crystallizes in similar form. 



