132 PHYSIOLOGICAL CHEMISTRY 



(d) Guanine. i. Dissolve a little substance in 20-25 times its weight of 

 boiling 5 per cent alcohol. Allow to cool and examine crystals microscopically. 



2. For other tests oh uric acid see Chapter XXIII on Urine. 



3. Dissolve a little guanine in 20-25 times its weight of boiling 5 per cent 

 hydrochloric acid. Allow to cool and examine crystals under microscope. 

 (See Fig. 39, page 134.) 



4. Perform Experiment 3 under Xanthine. 



(e) Uric Acid. i. On a small amount of uric acid try the test as given under 

 Xanthine number 3. This test on uric acid is called the Murexide test. 



12. Isolation of Guanine and Adenine from Nucleic Acid (Method of Wal- 

 ter Jones). 1 The amino purines may be isolated from yeast or thymus nucleic acid 

 or from glandular tissue (such as the pancreas) after hydrolysis of the material with 

 sulphuric acid. 



In the case of yeast nucleic acid, heat 10 grams of the substance with 50 c.c. of 

 to per cent sulphuric acid on a boiling water-bath for about two hours, replacing 

 any water lost, or using a condenser tube. To the hot solution add concentrated 

 ammonia slowly until approximately neutral. Then add enough excess of ammonia 

 to make about a 2 per cent solution. Filter off the precipitate of guanine and wash 

 it with i per cent ammonia. Dissolve in as small an amount of 20 per cent sul- 

 phuric acid as possible, add a little animal charcoal and boil Filter, heat to boiling 

 and precipitate with excess of ammonia. Filter, dry the precipitate at 4oC. and 

 dissolve it in about 20 parts of boiling 5 per cent hydrochloric acid. As the solu- 

 tion cools guanine chloride separates out as needle-shaped crystals. Filter off, 

 wash with very dilute hydrochloric acid and dry in the air (do not put in desiccator). 

 Perform the nitric acid test on the product. 



Combine the ammoniacal nitrates obtained in the isolation and purification of 

 guanine. Filter if necessary. The ammonia may then be boiled off and an excess 

 of picric acid added in which case a yellow precipitate of adenine picrate is produced 

 which is filtered off and dried. It is better, however, to neutralize the ammonia of 

 the combined filtrates and make faintly acid with sulphuric acid. Then precipitate 

 the adenine as its copper compound (see directions under experiment on Demonstra- 

 tion of Nucleases B) decomposing this with hydrogen sulphide and evaporating the 

 filtrate from the copper sulphide to dryness on the water-bath. Dissolve the residue 

 in hot 5 per cent sulphuric acid and allow to crystallize out. If necessary dissolve 

 in hot water decolorize with a little charcoal and allow to crystallize out again. 

 The compound has the formula (CsHsNs^.H^SO^E^O. Apply the picric acid 

 and nitric acid tests as given under adenine (page 131). 



13. The Pyrimidine Derivatives. The pyrimidine derivatives, 

 cytosine, thymine, and uracil, are separated from nucleic acid with 

 some difficulty. The following test may be made on a solution of 

 cytosine or uracil. Thymine does not give the test. 



Wheeler- Johnson Reaction for Uracil and Cytosine. To about 5 c.c. of the 

 solution under examination add bromine water until the color is permanent. 

 Avoid the addition of a large excess as this will interfere with the test. In 

 case the solution contains only small quantities of cytosine or uracil it is advis- 

 able to remove any excess of bromine by passing a stream of ah- through the 



1 See Walter Jones: Monograph on "Nucleic Acids," 1914, Longmans, Green & Co. 



