CHEMICAL COMPOSITION OF PROTEIN MOLECULE $7 



until a test portion remains clear. The precipitate of silver salt of 

 histidine is then filtered off and added to the main portion which has 

 been treated in the same way. 



Instead of adding excess of baryta Steudel directs that a suspen- 

 sion of barium carbonate be added to the neutral solution, the solution 

 warmed on the water-bath and then raised to the boiling-point. After 

 cooling, the histidine silver salt is filtered off and washed with baryta till 

 free from nitric acid. The filtrate and washings are treated as in V. 

 for arginine. 



The two precipitates of the silver salt of histidine are suspended 

 and heated in water to which sulphuric acid is added until the reaction 

 is acid and decomposed with hydrogen sulphide. Excess of hydrogen 

 sulphide is removed by boiling and the silver sulphide is filtered off 

 and washed. The solution and washings are concentrated and made 

 up to 250 c.c. A nitrogen estimation in 20-25 c.c. by Kjeldahl's 

 method gives the amount of histidine. 



The estimation of histidine can be carried out entirely as described 

 under (#). 



From the remainder of the solution the histidine may be isolated as 

 hydrochloride or as picrolonate : 



1. As Hydrochloride. The solution is made alkaline with baryta, 

 the barium sulphate formed is filtered off, excess of baryta is removed 

 by carbon dioxide, and the whole is evaporated to dryness. The resi- 

 due is extracted with boiling water and to the solution, filtered from 

 barium carbonate, hydrochloric acid is added. Histidine dichloride, 

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

 per cent, of the histidine estimated by the Kjeldahl 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 then 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. 



