32 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



(3) Estimation by Bromination. 



The bromination of tryptophan with a view to its estimation in 

 solution has been studied by Homer [1915], Solutions of tryptophan 

 were treated with excess of (a] nascent bromine liberated from 10 

 c.c. of a sodium bromate solution (15*1 grams per 1000 c.c.) and 10 

 c.c. of sodium bromide solution (51*5 grams per 1000 c.c.) by 5 c.c. 

 of concentrated hydrochloric acid ; (b] saturated bromine water. 

 After varying periods of time, from thirty minutes to eight hours, the 

 excess was titrated with -iN thiosulphate solution after adding 

 potassium iodide. Under these conditions tryptophan was found to 

 absorb eight atoms of bromine. The first reaction is identical with 

 that used by Plimmer and Eaves in their estimation of tyrosine; these 

 workers found that tryptophan absorbed six atoms of bromine. 



In carrying out the estimation in proteins the protein was hydro- 

 lysed by boiling 100 grams with 350 grams barium hydrate dissolved 

 in 2500 c.c. of water for 20 to 120 hours. In the absence of metallic 

 salts tryptophan was found to be stable to the action of baryta (see 

 above, 3). The baryta was removed with sulphuric acid and the 

 solution acidified to 5 per cent. On the addition of the mercuric sul- 

 phate reagent the tryptophan is precipitated. The precipitate was 

 filtered off after forty-eight hours and washed until free from tyrosine ; it 

 was then suspended in 2 percent, sulphuric acid and decomposed with 

 hydrogen sulphide. The filtrate from the mercuric sulphide was freed 

 from hydrogen sulphide and treated with phosphotungstic acid to 

 remove polypeptides. The filtrate containing the tryptophan was 

 treated with baryta to remove phosphotungstic acid, and with sul- 

 phuric acid to remove baryta, and made up to a definite volume (usually 

 IOOO c.c.). 50 c.c. of this solution were treated with sodium bromate 

 and bromide solutions, and at the same time a tryptophan solution was 

 treated in the same way. The excess of bromine was titrated with 

 iN thiosulphate. From the bromine absorption of the tryptophan 

 the amount of tryptophan in the solution was calculated and hence the 

 amount in the protein. 



The tryptophan content of caseinogen was determined in this way 

 and was found to be from 0-99-1-59 per cent. No other determina- 

 tions were made. 



