42 Prof. W. B, Bottomley. The Isolation from 



During the process of hydrolysis a copious precipitate of humic acid was 

 formed, presumably from the carbohydrate radicle. The filtrate, however, 

 still gave a strong reducing reaction with FehKng's solution and responded to 

 Molisch's test for carbohydrates. It also gave positive results with the 

 phloroglucin and orcin tests for pentoses. 



The hydrolysed product also gave a strong reaction for phosphoric acid 

 with both magnesia mixture and ammonium molybdate. 



The material therefore contains phosphoric acid, a pentose sugar, one purine 

 and one pyrimidine base. This indicates that it is a dinucleotide — an adenine- 

 uracil dinucleotide — and not the typical tetranucleotide, nucleic acid. 



Examination of the Filtrate. 



(a) Separation of the Purine Base. — A preliminary test having shown that 

 the filtrate from the dinucleotide contained phosphoric acid, sugar and a 

 purine base, a further examination of the liquid was made. The alcohol was 

 distilled off and a small portion of the aqueous residue was made alkaline 

 with ammonia. A flocculent precipitate formed which settled as a crystalline 

 sediment. After standing for 24 hours this was filtered off, dissolved in a 

 small amount of boiling hydrochloric acid, its colour discharged with animal 

 charcoal, and the base, together with the phosphoric acid, reprecipitated with 

 ammonia at the boiling point. A small proportion of this precipitate, 

 evaporated on porcelain with a drop of nitric acid, left a muddy yellow spot 

 which gave a brownish-red colour with sodium hydroxide. This indicated 

 the presence of guanine. 



The whole of the filtrate was then treated in a similar way and the 

 precipitate dissolved in hydrochloric acid. From this solution of the chloride 

 a mixture of elongated tetrahedral and needle crystals was obtained on long 

 standing in a desiccator. This chloride was used for the preparation of the 

 following salts : the picrate, as a woolly mass of long, fine, thread-like needles, 

 which dried to a felt-like mass and on heating became orange-red, 

 decomposing without melting at 190° C. ; the bichromate, as bright orange 

 coloured prisms with truncated ends, which became dark violet when heated 

 at 100° C. These reactions, together with its solubility in ammonia and 

 hydrochloric acid and the formation of a gelatinous precipitate with 

 ammoniacal silver nitrate, identified the substance as guanine. 



Guanine was also isolated from the filtrate by precipitation with copper 

 sulphate and sodium bisulphite in the ordinary way. No other purine base 

 could be isolated from the filtrate. 



(b) Separation of the Pyrimidine Base. — A small portion of the filtrate after 



