60 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



VI. Estimation and Isolation of Lysine. 



The lysine is contained in the filtrate from the precipitate of the 

 silver salts of arginine and histidine. 



The solution is acidified with sulphuric acid and freed from silver 

 by hydrogen sulphide ; the filtrate and washings from the precipitate 

 of silver sulphide and barium sulphate, which is treated in the usual 

 manner, are evaporated down to 500 c.c. Sulphuric acid is then added 

 until the content is 5 per cent, and the lysine is precipitated by not 

 too large an excess of phosphotungstic acid. 1 This is added until a 

 portion of the clear liquid on the further addition of the reagent re- 

 mains clear for ten seconds. After twenty-four hours the precipitate 

 of lysine phosphotungstate is filtered off by suction and washed with 

 5 per cent, sulphuric acid by stirring up in a mortar. After making 

 up the filtrate and washings to a definite volume an estimation of the 

 substances not precipitated may be made in an aliquot part by the 

 Kjeldahl method. 



The lysine phosphotungstate is made into a uniform suspension 

 with water and poured into boiling water. A hot saturated solution 

 of baryta is added until the solution is strongly alkaline and contains 

 excess of baryta. The precipitate of barium phosphotungstate, which 

 is formed, is filtered off and boiled out several times with baryta and 

 then with water. The alkaline solution is freed from baryta by means 

 of carbon dioxide, concentrated, filtered, and evaporated on the water- 

 bath nearly to dryness. Water is then added, the barium carbonate 

 filtered off and washed, and the solution once more evaporated, after 

 which it is made up to a definite volume and the lysine estimated in 

 an aliquot portion by Kjeldahl's method. 



The lysine is separated from the remainder of the solution as 

 picrate. The solution is evaporated down in a porcelain basin to dry- 

 ness, and a small quantity of alcohol is added to the sticky residue. 

 It is then treated with a saturated solution of picric acid in alcohol 

 until no further precipitation of picrate occurs. After twenty-four 

 hours this precipitate is filtered off and washed with a small quantity 

 of absolute alcohol; it is then recrystallised by solution in boiling 

 water, filtering if necessary, and evaporating to a small volume, when 

 lysine picrate, C 6 H 4 N 2 O 2 . C 6 H 2 (NO 2 ) 3 OH, crystallises in needles on 



1 Schulze and Winterstein [1902] found that phenylalanine under certain conditions is 

 precipitated by phosphotungstic acid and they have been able to isolate phenylalanine in 

 this way. 



