CHEMICAL COMPOSITION OF PROTEIN MOLECULE 35 



with phosphotungstic acid. The filtrate and washings are evaporated 

 down best in vacuo at 70 C. 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 

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

 magnesium oxide. 



The ammonia is removed from the remainder by evaporating with 

 magnesia, or better barium carbonate, on the water-bath. 



The two portions, freed from ammonia, are then combined, and 

 made alkaline with baryta, or barium carbonate. 



The separate solutions are now combined, the precipitate of barium 

 carbonate and barium sulphate is filtered off and washed by boiling 

 with water three times ; the excess of barium is removed from the 

 filtrate 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 de- 

 termination 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. Precipitation of Arginine and Histidine. 



The solution, which now contains a small quantity of sulphuric 

 acid, is placed in a 5 litre flask, and treated with a hot saturated solu- 

 tion of silver sulphate, 1 which is slowly added, until the solution con- 

 tains 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, i.e. until some remains undis- 

 solved after repeated shaking. The precipitate, which is thus formed 



1 Osborne prefers to use silver nitrate. 



3* 



