ISOLATION AND ESTIMATION OF TRYPTOPHAN 27 



Tryptophan. 



(i) Isolation and Gravimetric Estimation. 



Tryptophan is not obtained by the hydrolysis of proteins by acids 

 owing to its decomposition and is prepared by the action of trypsin. 

 By the method of Hopkins and Cole [1901, 2] the protein is digested 

 in alkaline solution by trypsin, until the solution gives a maximal 

 coloration when tested with bromine water ; the solution is acidified, 

 boiled and filtered. The clear solution (better after concentrating in 

 vacuo and filtering off tyrosine, which crystallises out) is acidified with 

 sulphuric acid until it contains 5 per cent, and then 10 per cent, 

 mercuric sulphate in 5 per cent, sulphuric acid solution is added as 

 long as a precipitate, which contains tryptophan, cystine and tyrosine, 

 is formed. The yellow precipitate is freed from tyrosine by washing 

 with 5 per cent, sulphuric acid in which the tyrosine compound is 

 soluble, that is, until the washings no longer react with Millon's 

 reagent. The precipitate is decomposed by sulphuretted hydrogen, 

 and the solution containing cystine and tryptophan after removing the 

 hydrogen sulphide is again acidified with sulphuric acid to 5 per cent, 

 and fractionally precipitated with the mercuric sulphate reagent. The 

 cystine is thrown down first, filtered off, and then the tryptophan is 

 precipitated. The precipitate is again decomposed by hydrogen sul- 

 phide, and the solution, freed from sulphuric acid, is evaporated down, 

 alcohol being continually added to hasten the evaporation and prevent 

 decomposition of the tryptophan, which crystallises out. It is purified 

 by recrystallisation from water. The yield represents its amount in 

 the protein. 



Neuberg and Popowsky [1907] introduced a few alterations in the 

 procedure, such as evaporation to one quarter and filtration from tyro- 

 sine before precipitating with mercuric sulphate ; in decomposing the 

 precipitate with hydrogen sulphide they make the solution faintly 

 alkaline with baryta and treat with the gas for twenty-four hours, 

 warming several times. The removal of sulphuric acid in the last 

 operation is carried out with lead carbonate in ammoniacal solution, the 

 lead being subsequently removed with hydrogen sulphide. 



Abderhalden and Kempe [1907, i] made very little alteration in 

 the procedure of Hopkins and Cole beyond evaporation of the solutions 

 in vacuo and removal of hydrogen sulphide with a current of carbon 

 dioxide. The yield of tryptophan obtained by these workers (0-53 

 per cent.) is less than the yield mentioned by Hopkins and Cole in the 

 case of caseinogen (1-5 per cent). Usually about I per cent is ob- 

 tained from caseinogen. 



