ISOLATION AND ESTIMATION OF DI-AMINO ACIDS 55 



B. THE DI-AMINO ACIDS. 



The isolation and estimation of the three compounds arginine, his- 

 tidine, lysine are carried out by the method described by Kossel and 

 Kutscher [1900-1] which was slightly modified by Kossel and Patten 

 [1903]. Further modifications and improvements have been added by 

 Steudel [1903], Kossel and Pringle [1906] and Osborne, Leavenworth 

 and Brautlecht [1908]. The method is based upon the earlier work of 

 Drechsel, Hedin, and Kossel, and depends upon the precipitation of 

 arginine and histidine as their silver salts, their separation by difference 

 in solubility in water and in strongly alkaline solution, and the pre- 

 cipitation of lysine from the filtrate by phosphotungstic acid, and then 

 by picric acid. 



The method has been described in full by Weiss [1907] and by 

 Steudel [1910] and is carried out as follows : 



I. Hydrolysis and Estimation of Protein. 



About 25-50 grams of protein are hydrolysed by boiling with 

 dilute sulphuric as stated on p. 1 1. The exact amount of protein is 

 then estimated by making the volume up to I litre with water and 

 determining the nitrogen in 5 or 10 c.c. by Kjeldahl's method; from 

 this figure the amount of protein can be calculated, if the amount of 

 nitrogen in it be known. 



II. Removal of Sulphuric Acid. Estimation of Ammonia and 



Humin Nitrogen. 



The acid solution is heated to boiling and treated with a hot 

 concentrated solution of baryta until the reaction is only faintly acid 

 and almost the whole of the sulphuric acid is precipitated as barium 

 sulphate, which is filtered off by suction and thoroughly washed 

 with boiling water, by stirring up and raising to the boiling-point. 

 This should be repeated twice or until the filtrate gives no precipitate 

 with phosphotungstic acid. The filtrate and washings are evaporated 

 down best in vacuo at 70 and again made up to I litre. A deter- 

 mination of the nitrogen in 5 or 10 c.c. of this solution gives by differ- 

 ence the amount of nitrogen contained in the melanin, which is 

 carried down by the barium sulphate. It is known as " humin nitro- 

 gen I. " 



In this liquid two determinations are made of the amount of 



