130 PHYSIOLOGICAL CHEMISTRY 



action of different portions of the solution with litmus, alizarin, and Congo red 

 solution. 



4. Boil a small portion of the nucleic acid with about 10 c.c. of 10 per cent 

 sulphuric acid for one to two minutes. Divide into three portions. 



(a) To one portion apply carbohydrate tests, e.g., the a-naphthol (Molisch) 

 reaction and Bial's test. What do these indicate? 



(b) To a second portion apply a test for purine bases. Add an excess of 

 ammonia and then a little silver nitrate solution. 



(c) To the third portion apply test for phosphate, adding ammonia in slight 

 excess, then making acid with nitric acid, adding molybdic solution and wanning. 



9. Preparation of Thymus Nucleic Acid. 1 "To a boiling mixture of 200 c.c. of 

 water, 10 grams of sodium acetate and 3.3 grams of NaOH, is added in small suc- 

 cessive portions 100 grams of trimmed and finely ground thymus gland. The tissue 

 usually dissolves completely forming a pale brown liquid, but any resistant portions 

 are either removed or gotten into solution by heating for a short time over a small 

 flame. The vessel containing the products is now immersed in a briskly boiling 

 water-bath where it is allowed to remain with occasional stirring for two hours, 

 when the product is diluted with one-third its volume of water and made faintly 

 but distinctly acid to litmus with 50 per cent acetic acid. The amount of acid 

 required is about 10 c.c. but the final additions must be made with care because the 

 fluid will not filter unless the proper condition of acidity is reached. Any difficulty 

 met at this point may be easily overcome by the alternate addition of acetic acid and 

 sodium hydroxide and testing a small portion of the material after each addition on 

 a small flat filter that has been heated with boiling water. When the acidity has 

 finally been obtained which is favorable to rapid nitration, the material is heated to 

 vigorous boiling and filtered with a hot water funnel. Under proper conditions the 

 filtration proceeds with considerable rapidity and continuously leaves a green slime 

 on the filter and gives a pale yellow filtrate which gelatinizes upon cooling. The 

 filtrate and washings are evaporated on a water-bath to about 75 c.c. and while warm 

 the concentrated solution is poured slowly into 100 c.c. of 95 per cent alcohol. On 

 standing over night the precipitated sodium nucleate settles sharply to a spongy 

 white mass from which the bulk of brown alcoholic fluid can be sharply decanted 

 and the remainder pressed out with a spatula leaving the material in one cohesive 

 mass. The substance is washed by decantation in turn with 80 per cent and 95 per 

 cent alcohol and, after pressing out the last wash fluid as far as possible is transferred 

 to a flask with 30 c.c. of hot water and heated on a water-bath. In half an hour or 

 less, insoluble phosphates will collect leaving a perfectly transparent interstitial 

 fluid which is treated with i c.c. of 20 per cent NaOH to lower the viscosity and 

 filtered with a hot water funnel. The perfectly transparent yellow filtrate is acidi- 

 fied with acetic acid and poured into 70 c.c. of 95 per cent alcohol when sodium 

 nucleate will be precipitated which can be washed by decantation as before with 

 alcohol of increasing strength and ground in a mortar with absolute alcohol until 

 it has crumbled to a fine white powder. If necessary the absolute alcohol may be 

 decanted and renewed once or twice but not oftener because the nucleate emulsifies 

 with alcohol after the last traces of acetic acid and sodium acetate have been washed 

 away. The material is finally washed on a filter with absolute alcohol and allowed 

 to dry in a sulphuric acid desiccator. The yield of nucleic acid is about 3.3 grams 

 from 100 grams of gland. The product is a fine white non-hygroscopic powder 



1 From Monograph on "Nucleic Acids" by Walter Jones: Longmans, Green & Co. 



