128 PHYSIOLOGICAL CHEMISTRY 



minutes. Pour the thick liquid into a bottle aiding the transfer with enough 

 0.4 per cent NaOH to make a final volume of about i25.c.c. The alkali extracts 

 the nucleoprotein along with the water-soluble proteins of the yeast. Add a little 

 toluene and allow to stand with frequent shaking for 12-24 hours. Filter through 

 a wet, fluted filter. While thoroughly stirring add i drop at a time of 10 per cent 

 HC1 cautiously continuing the addition as long as the milkiness of the mixture can 

 be increased. Continue until the protein completely separates and the liquid is 

 practically clear. Note that the solution is now acid in reaction. Excess of acid 

 causes resolution. Filter on a wet, fluted filter. Retain the precipitate on the 

 filter for nucleoprotein tests. 



2. Tests on Nucleoprotein. Try the following tests on the nucleoprotein 

 prepared as above. 



(a) Try the xanthoproteic and Millon's tests. 



(b) Test the solubility in water, 10 per cent NaCl, 10 per cent HC1, dilute 

 KOH, and alcohol. 



(c) Test for organically combined phosphorus by one of the following 

 methods. 



Tests for Phosphorus in Organic Matter. i. Fusion Test. To a small 

 amount of the substance in a crucible add about five times its bulk of fusion mix- 

 ture (2 parts of sodium carbonate to i of potassium nitrate). Heat carefully until 

 the resulting mixture is colorless. Cool, dissolve the mass in a little warm water, 

 acidify with nitric acid, heat to about 6sC. and add a few cubic centimeters of 

 molybdate solution. In the presence of phosphorus a yellow precipitate of am- 

 monium phosphomolybdate is formed. 



Instead of acidifying with nitric acid, the aqueous solution may be approxi- 

 mately neutralized with hydrochloric acid, a few cubic centimeters of magnesia 

 mixture added and then excess of ammonium hydroxide solution. A white pre- 

 cipitate of magnesium ammonium phosphate is formed. 



2. Moist Ashing Procedure. Treat a small amount of the substance in a 

 large test-tube with about i c.c. of concentrated sulphuric acid. Then add drop by 

 drop an equal volume of concentrated nitric acid, and warm gently until a clear 

 solution is obtained. A few more drops of nitric acid may be added if necessary. 

 This treatment with sulphuric and nitric acids must be carried out with the 

 greatest caution particularly when fatty substances are present; otherwise an 

 explosive reaction may take place. Dilute the acid solution with a little water, 

 make slightly alkaline with ammonia and then acid with nitric acid. Add 

 molybdate solution and warm. A yellow precipitate is formed. 



(d) Dissolve a little of the precipitate in very dilute KOH and then make 

 slightly acid with acetic acid. 



(e) Mix a small portion of the nucleoprotein with 10 c.c. of alcohol. Filter 

 and wash free from HC1 with more alcohol. (Freedom from HC1 is indicated by 

 absence of AgNO 3 -chloride reaction hi the filtrate.) Wash free from alcohol 

 with a little water. Transfer small particles of the precipitate to moistened red 

 and blue litmus paper on a microscopic slide. What is the reaction of nucleo- 

 protein thus freed from adherent acid? 



3. To Show the Presence of Purine Base Radicals hi Nucleoprotein. The 

 nucleic acid portion of the protein molecule contains phosphoric acid, carbohy- 

 drate, and purin base radicals (see page 125). Hence on the complete acid hydro- 

 lysis of nucleoprotein material these substances will be liberated as well as the 

 decomposition products of the protein part of the molecule. To show v their pres- 



