16 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



a mixture of three times the weight of concentrated sulphuric acid and six 

 times the weight of water under a reflux condenser for fourteen hours. 

 The exact amount of protein is then estimated by making the volume 

 up to i litre, 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. The acid solution is 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 and thoroughly washed out with boiling water. The filtrate and 

 washings are evaporated down and again made up to I litre. A de- 

 termination of the nitrogen in 5 or 10 c.c. of this solution gives the 

 amount of nitrogen contained in the melanin, which is carried down by 

 the barium sulphate. It is known as " humin nitrogen I.". 



In this liquid also two determinations are made of the amount of 

 nitrogen present as ammonia by distilling portions of 100 c.c. with 

 magnesium oxide. From the remainder the ammonia is removed by 

 evaporating with magnesia on the water bath. The three portions, free 

 from ammonia, are then combined, and made alkaline with baryta. 

 The precipitate of barium sulphate is filtered off and thoroughly washed 

 out, the excess of barium removed by dilute sulphuric acid and the 

 precipitate again filtered off and washed out. Filtrate and washings are 

 combined together, evaporated down and made up to I litre and a 

 Kjeldahl nitrogen determination again made. Allowing for the nitrogen 

 given off as ammonia, the difference between this and the previous 

 estimation gives the humin nitrogen II. contained in the alkaline barium 

 magnesia precipitate. 



III. The solution, now containing a small quantity of sulphuric acid, 

 is placed in a 5 -litre flask, made up to 3 litres and heated on the 

 water bath. Finely powdered silver sulphate is slowly added until the 

 solution contains sufficient to give a yellow, not a white, precipitate, 

 when a drop is removed and tested with baryta water in a watch-glass. 

 If, during the process, there be any undissolved silver sulphate at the 

 bottom of the flask it is dissolved by adding more water before a fresh 

 quantity is added, in order that a yellow precipitate be given in the test 

 drop with baryta. As soon as sufficient silver is present to combine 

 with all the arginine and histidine, it is allowed to cool to 40 C. and 

 then saturated with finely ' powdered baryta. The precipitate which is 

 thus formed, and which consists of the silver salts of arginine and histidine, 

 is filtered off, stirred up with baryta, again filtered off and washed with 

 baryta water. 



