26 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



funnel and treated with small quantities of concentrated hydrochloric 

 acid. Ether is added and the mixture well shaken. At first an 

 emulsion is formed, but on adding more ether and acid and thoroughly 

 shaking a clear ethereal solution of phosphotungstic acid settles to the 

 bottom ; if this does not occur, decomposition is not complete and 

 more acid must be added and the shaking repeated. The middle layer 

 containing the cystine hydrochloride is separated ; the other layers are 

 treated once more in the same way and the middle layers are combined. 

 Ether is removed from the acid solution by warming on the water- 

 bath and the cystine is separated by exactly neutralising with soda. 



The separation of cystine and tyrosine has been examined by 

 Plimmer [1913]. Neither the phosphotungstic acid method nor the 

 mercuric sulphate method was found to be satisfactory, as loss of 

 both substances occurred. In the case of phosphotungstic acid some 

 tyrosine was carried down with the precipitate and was only removed 

 by prolonged washing, and some of the cystine was decomposed in 

 recovering it from its phosphotungstate. In the case of mercuric 

 sulphate, the cystine is not completely precipitated. A quantitative 

 separation of the two compounds was effected by means of absolute 

 alcohol saturated with hydrogen chloride. Tyrosine is converted into 

 its ethyl ester hydrochloride; cystine is not acted upon. The mixture 

 of the two compounds is covered with the alcoholic hydrochloric acid 

 and warmed upon the water-bath to dissolve the tyrosine. An equal 

 volume of absolute alcohol is now added to precipitate any cystine 

 which may dissolve. The undissolved cystine is filtered off and 

 washed with absolute alcohol till it is free from acid and recrystallised 

 from ammonia in the usual way. The filtrate containing the tyrosine 

 ester is diluted with two volumes of water and boiled for eight hours, 

 water being added when necessary. The ester is thus hydrolysed and 

 on neutralising the tyrosine is precipitated. 



The usefulness of this method is shown by the treatment of pre- 

 sumably pure tyrosine prepared from wool by Folin's method for ob- 

 taining cystine ; one specimen of 5 grams contained 0*05 gram of 

 cystine, another specimen of 4 grams yielded O'2 gram of cystine. 



