22 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



(3) Estimation by Bromi nation. 



It was shown by J. H. Millar [1903] that tyrosine was readily 

 brominated and converted into dibromotyrosine, and that the amount 

 of tyrosine in a simple mixture of amino acids could be accurately 

 estimated by means of this reaction. 



A. J. Brown and E. T. Millar [1906] using this reaction showed 

 that the tyr-sine in proteins was completely liberated at a very early 

 stage in the hydrolysis by trypsin. Their data gave the tyrosine con- 

 tent of edestin as 4^06 per cent, a figure which is considerably higher 

 than that obtained by direct isolation (2-1 per cent.). They made no 

 estimations of tyrosine in other proteins. 



Plimmer and Eaves [1913] studied this reaction more closely with 

 a view to the estimation of tyrosine in proteins. It was not found 

 possible to apply the reaction to the products of hydrolysis of proteins 

 by acids, since ,tryptophan, though almost entirely decomposed by 

 boiling with acids, gave rise to products which absorbed bromine, but 

 if the hydrolysis of the protein were effected by the action of trypsin, 

 and if the amount of bromine absorbed were determined after about 

 six hours, values for tyrosine were obtained which agreed closely with 

 those by the gravimetric method. If the amount of bromine absorbed 

 was determined after twenty-four to twenty-eight hours' hydrolysis, 

 the values for tyrosine were very similar to those by the colorimetric 

 method of Folin and Denis. 



The procedure was to digest a known weight of protein in faintly 

 alkaline solution with trypsin, to remove measured volumes of the 

 digest after the stated period, and to treat this volume with a 5 per 

 cent, solution of phosphotungstic acid in 5 per cent, sulphuric acid to 

 remove histidine, which absorbs bromine. A measured volume of the 

 filtrate was treated with a known volume of -2N sodium bromate solu- 

 tion and 10-20 c.c. of 20 per cent, sodium bromide solution. After 

 half an hour the excess of bromine was determined by titration with 

 thiosulphate solution, using starch and sodium iodide as indicator. 

 At the same time the same volume of trypsin solution was digested 

 by itself and treated in a similar way so as to deduct the amount of 

 tyrosine in this solution. 



The values of tyrosine by bromination are given in the following 

 table and are compared with the gravimetric and colorimetric : 



