16 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

 The Isolation and Estimation of Tryptophane. 



Tryptophane is not obtained in any large amount by the hydrolysis 

 of proteins by acids and is best prepared by the action of trypsin. 

 According to Hopkins and Cole, the protein is digested in alkaline 

 solution by trypsin, until the solution gives a maximal coloration when 

 tested with bromine water ; the solution is then 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 mercuric sulphate dissolved 

 in 5 per cent, sulphuric acid is added as long as a precipitate, which 

 contains tryptophane, cystine and tyrosine, is formed. The precipi- 

 tate 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. It is then decomposed by 

 sulphuretted hydrogen, and the solution containing cystine and trypto- 

 phane is again acidified with sulphuric acid to 5 per cent, and frac- 

 tionally precipitated with the mercuric sulphate reagent. The cystine 

 is thrown down first, and filtered off, and then the tryptophane is pre- 

 cipitated. The precipitate is again decomposed by hydrogen sulphide, 

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

 alcohol being continually added to hasten the evaporation and prevent 

 decomposition of the tryptophane, which is estimated by weighing. 



Neuberg and Popowsky, as also Abderhalden and Kempe, have 

 introduced a few alterations in the procedure, such as evaporation in 

 vacuo, and Levene and Rouiller suggested in 1 907 that the tryptophane, 

 on account of its proneness to decompose on evaporation of its solution 

 with consequent loss, be estimated colorimetrically ; the mercury sul- 

 phate precipitate is decomposed, and the solution, freed from hydro- 

 gen sulphide, is titrated with bromine water in presence of amyl 

 alcohol. Both cystine and tyrosine react with bromine water; the 

 latter can, however, be removed, but for the former a correction has 

 to be made. Up to the present no values concerning the amount of 

 tryptophane in various proteins by this method have appeared, and it 

 will be of interest to see if the values so obtained are very much 

 higher than those obtained by crystallisation of the tryptophane. 



