58 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



C 6 H 9 N 3 O 2 . 2 HC1, is obtained on evaporating down. The yield is 75-80 

 per cent of the histidine estimated by the KjeJdahl determination. 



2. As Picrolonate. The excess of sulphuric acid is removed by 

 treating the hot solution with excess of baryta, and excess of the 

 latter is removed by carbon dioxide ; it is evaporated down and filtered 

 from barium sulphate and carbonate, which are thoroughly washed. 

 The filtrate and washings are evaporated to about 10 c.c., if necessary 

 after the addition of a drop of sulphuric acid to remove the last traces 

 of barium. The necessary quantity of picrolonic acid (calculated from 

 the above Kjeldahl determination and dissolved in a small quantity of 

 alcohol) is added ; the precipitate of histidine picrolonate is filtered off 

 after three days, washed with water, dried and weighed. The amount 

 of histidine can be calculated from the formula C 6 H 9 N 3 O 2 . C 10 H 8 N 4 O 5 ; 

 it corresponds very closely with the amount calculated from the 

 Kjeldahl estimation. 



(b) Osborne, Leavenworth and Brautlecht find that it is better to 

 remove the greater portion of the histidine by precipitation with 

 mercuric sulphate. The solution is concentrated to about 250 c.c., 

 sulphuric acid is added till the solution contains 5 per cent, of this 

 acid, and it is treated with a slight excess of mercuric sulphate solution. 

 The precipitate of histidine mercury sulphate is allowed to stand for 

 twelve to twenty-four hours when it is filtered off, washed with 5 per 

 cent, sulphuric acid, suspended in water and decomposed with hydrogen 

 sulphide. The filtrate and washings from the mercuric sulphide which 

 contain the histidine are neutralised with baryta and barium nitrate 

 added until barium sulphate is no longer precipitated. The barium 

 sulphate is filtered off and thoroughly washed. The histidine is then 

 thrown down as silver compound and estimated as under (a). The 

 filtrate from the mercury precipitate is freed from mercury, neutralised 

 and treated as described under (a) so as to throw down the small 

 quantity of histidine not precipitated by mercuric sulphate. 



