34 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



B. THE DIAMINO ACIDS. 



The isolation and estimation of the three compounds arginine, 

 histidine, lysine is carried out by the method described by Kossel and 

 Kutscher in 1900, which was slightly modified in 1903 by Kossel and 

 Patten. Further modifications and improvements have been added 

 by Steudel, Weiss, and Osborne and his associates Leaven worth and 

 Brautlecht. 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 precipitation of lysine 

 from the filtrate by phosphotungstic acid, and then by picric acid. 



It is carried out as follows : 



I. Hydrolysis and Estimation of Protein. 



About 25-50 grammes of protein are hydrolysed by boiling 

 with a mixture of three times the weight of concentrated sulphuric 

 acid and six times the weight of water under a reflux condenser firstly for 

 one to one and a half hours on a water-bath until frothing has ceased and 

 then in an oil-bath at 105 for fourteen, or better twenty- four, hours. 

 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 



