28 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



(2) Colorimetric Estimations. 



Attempts have been made by several investigators to estimate the 

 amount of tryptophan in proteins by colorimetric methods. 



1. Levene and Rouiller [1906-07] suggested a procedure depend- 

 ing upon the bromine absorption of tryptophan. The protein was 

 digested by trypsin and the tryptophan precipitated by mercuric 

 sulphate as described above. This precipitate was decomposed by 

 hydrogen sulphide in 1-2 per cent, sulphuric acid solution and the 

 filtrate from the mercuric sulphide made up to a definite volume. To 

 15 c.c. of this solution 2 c.c. of amyl alcohol were added and then 

 gradually bromine water with vigorous shaking. The end point was 

 reached when the purple colour of the amyl alcohol disappeared and 

 became yellow. 



The absorption of bromine is influenced by the presence of tyrosine 

 and cystine, which are precipitated by mercuric sulphate with trypto- 

 phan. The amount of tyrosine thrown down is negligible, if the 

 mercuric sulphate reagent is added only as long as the solution gives 

 a reaction for tryptophan with bromine water and if the precipitate is 

 washed until the washings give no reaction for tyrosine. A correction 

 is made for the absorption by cystine, the amount of cystine in the 

 solution being determined by a sulphur estimation. The bromine 

 water used is standardised against solutions of tryptophan and cystine. 



Up to the present no values of the amount of tryptophan in pro- 

 teins have appeared, the further work of these authors [1907] being 

 concerned with the hydrolysis of the protein by baryta and the nature 

 of the bromine derivatives of tryptophan. They found that tryptophan 

 was not completely separated from protein by the hydrolysis with 

 baryta unless the hydrolysis was prolonged and that under these con- 

 ditions the colour reaction was not satisfactory. By a short hydrolysis 

 polypeptides containing tryptophan are present, which also absorb 

 bromine and affect the estimation. At the stage when a violet colour 

 is produced by bromine water they consider that a mixture of mono- 

 and dibromo-tryptophan is present and that the dibromide is present 

 when excess of bromine has been added. 



2. Proteins containing tryptophan give a reddish-violet to blue 

 colour reaction with glyoxylic acid and sulphuric acid the Adamkie- 

 wicz-Hopkins reaction. It is positive for a concentration of tryptophan 

 of I in 100,000. This reaction has been made use of by Fasal [1912] 

 for estimating tryptophan in proteins and is carried out in the follow- 



