CHEMICAL COMPOSITION OF PROTEIN MOLECULE 17 



IV. Separation of arginine and histidine. The above precipitate 

 of the silver salts of these compounds is suspended in water containing 

 sulphuric acid, and decomposed with hydrogen sulphide. The filtrate 

 from the silver sulphide, which is thoroughly extracted with boiling 

 water, is evaporated down to remove the hydrogen sulphide and again 

 made up to I litre ; a Kjeldahl nitrogen determination in 20 c.c. now 

 gives the amount of nitrogen in the substances precipitated by silver and 

 baryta. 



The liquid is now neutralised with baryta, and barium nitrate is 

 added, so long as a precipitate of barium sulphate is formed ; this is 

 filtered off and washed. The filtrate is concentrated to 300 c.c. and 

 treated with silver nitrate, as before, till a test drop gives a yellow 

 colour with baryta; when this occurs it is exactly neutralised with 

 baryta, and from a burette small quantities of baryta are added till the 

 silver salt of histidine is completely precipitated ; this is determined by 

 taking out a drop when the precipitate has settled and testing with 

 ammoniacal silver solution ; if a precipitate easily soluble in excess of 

 ammonia be formed, when the two liquids come together, histidine is 

 still present ; and more baryta water must be added, until it is completely 

 thrown out, when it is filtered off, stirred up with water, again filtered off 

 and washed out. 



The precipitate of the silver salt of histidine is then stirred up in a 

 measured quantity of 5 per cent, sulphuric acid and decomposed with 

 hydrogen sulphide. The filtrate and washings from the silver sulphide 

 are concentrated so that the solution contains 2.\ per cent, sulphuric 

 acid and then precipitated by not too large an excess of mercuric 

 sulphate. The precipitate is allowed to stand for twelve to twenty-four 

 hours, when it is filtered off and decomposed by sulphuretted hydrogen. 

 A nitrogen determination in this solution by Kjeldahl's method gives 

 the amount of nitrogen from which the amount of histidine can be 

 calculated ; the histidine itself is obtained by making alkaline with 

 baryta, filtering off the barium sulphate, removing excess of baryta by 

 carbon dioxide, evaporating to dryness, extracting the residue with boil- 

 ing water, filtering from barium carbonate, adding hydrochloric acid and 

 evaporating down, when histidine hydrochloride C 6 H 9 N 3 O 2 . 2HC1 is 

 obtained. 



V. The filtrate containing the arginine is saturated with baryta, and 

 the precipitate of the silver salt of arginine, so obtained, is stirred up 

 with baryta, filtered off and washed till free from nitric acid. It is then 

 suspended in water containing sulphuric acid and decomposed with 



hydrogen sulphide. The filtrate and washings from the precipitate 

 PT. i. 2 



