34 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



[1912] states that glutamic acid is most rapidly obtained from the 

 hydrochloride by dissolving in water, passing in ammonia, evaporat- 

 ing to dry ness, and recrystallising from water. The remaining glutamic 

 acid is obtained from the mother liquor by adding alcohol. 



(ia) Isolation of Glutamic Acid as Zinc Salt. 



In view of the sometimes incomplete separation of glutamic acid as hydrochloride, 

 especially in the cases of the proteins containing less than 10 per cent, of this unit and in 

 view of its incomplete isolation as ester at a later stage, the observation of Kutscher [1903] 

 that glutamic acid forms a very insoluble zinc salt might be made use of to isolate it. The 

 mixture of amino acids, after removal of the greater part of the hydrochloric acid by 

 evaporation in vacuo, is diluted with water and boiled with excess of zinc oxide and allowed 

 to cool. The precipitate consisting of the excess of zinc oxide and zinc glutamate is dis- 

 solved in hot dilute acetic acid and treated with hydrogen sulphide. The filtrate on 

 evaporation yields glutamic acid. 



A disadvantage of this method is the fact that leucine also forms a zinc salt which is 

 soluble with difficulty. The separation of glutamic acid and leucine may be effected by the 

 method of Osborne and Liddle [1910, 2], i.e., neutralising the acidity of the glutamic acid 

 and crystallising out the leucine. The glutamic acid is then obtained on acidifying and 

 crystallising, or as hydrochloride. 



(ib) Isolation of Glutamic and Aspartic Acids as Silver Salts. 



Glutamic acid and aspartic acid form silver salts which are not easily soluble in water 

 [Habermann, 1875]. Siegfried [1891] was able to isolate aspartic acid as silver salt and 

 Kutscher [1903] has shown that the solubility of the silver salts of these amino acids in 

 water is very slight. Kutscher devised a method for isolating these units depending upon 

 the insolubility of the silver salts. The mixture of amino acids after hydrolysis with 

 sulphuric acid and removal of the di-amino acids, or similarly after hydrolysis with hydro- 

 chloric and removal of di-amino acids, is evaporated in vacuo to remove as much as possible 

 of the hydrochloric acid ; its removal is completed as silver chloride. The solution is 

 treated with silver nitrate or better another soluble silver salt and baryta water carefully 

 added, avoiding excess which decomposes the silver salts. This may be tested by with- 

 drawing a drop of the solution and placing it in contact with a drop of ammoniacal silver 

 nitrate; a turbidity indicates that more baryta is required. The silver salts are decom- 

 posed and the mixture is separated, either by preparing glutamic acid hydrochloride, or by 

 preparing the zinc salts ; the zinc salt of aspartic acid is easily soluble in water. 



(ic) Isolation of Glutamic and Aspartic Acids as Calcium Salts. 



Foreman [1914, i] has found that the calcium salts of these dibasic amino acids 

 are insoluble in alcohol and has described a method for isolating them based upon 

 this property. 



The hydrochloric acid solution is evaporated to a syrup in vacuo ; the syrup is dis- 

 solved in 200-400 c.c. of water per 20-40 grams of protein and 0-5 gram of slaked 

 calcium oxide per gram of protein is added. The mixture is shaken well and filtered from 

 excess of lime and humin substances. The filtrate is evaporated in vacuo at 40-45 to a 

 volume of 3*5-4 c.c. per gram of protein ; ammonia is thus removed. The temperature 

 at which evaporation is carried out should not exceed 45 so as to prevent the transforma- 

 tion of glutamic acid into pyrrolidone carboxylic acid. Rectified spirit is now added in 

 small quantities at a time with vigorous shaking so as to prevent clumping of the pre- 



