ISOLATION AND ESTIMATION OF CYSTINE 25 



(2) Separation of Cystine and Tyrosine. 



The mixture of cystine and tyrosine was separated by Morner by 

 fractional crystallisation from ammonia ; if much tyrosine was present 

 it separated out first, but if cystine exceeded tyrosine in quantity this 

 compound crystallised out first ; the remainder was only separated 

 with difficulty. 



The method adopted by Embden and followed by Friedmann was 

 the solution of the tyrosine from the mixture, suspended in water, by 

 the addition of very dilute nitric acid. The separation could be 

 checked by microscopic observation of the crystals, the needle-shaped 

 crystals of tyrosine dissolving whilst the plates of cystine remained. 

 After washing free from acid with water, the cystine was recrystallised 

 from ammonia. 



Separation of the mixture was effected by Abderhalden [1903] 

 and by Abderhalden and Pregl [1905, 2] by dissolving in ammonia 

 and adding glacial acetic acid keeping the reaction alkaline ; tyrosine 

 was precipitated : on acidifying with glacial acetic acid the cystine 

 separated. 



The separation of cystine from hydrolysed Bence-Jones protein by 

 Hopkins and Savory [1911] was carried out by precipitation of a 

 tryptic digest with mercuric sulphate in acid solution (see under 

 tryptophan). Before the second precipitation the solution was con- 

 centrated after removal of the acid ; cystine crystallised out. 



The precipitation of cystine by mercuric sulphate in 5 per cent, 

 sulphuric acid solution in which the mercury compound of tyrosine is 

 soluble was used by Osborne and Clapp in the cases of squash seed 

 globulin [1907, 6] , and wheat gliadin [1906] respectively. 



Winterstein [1901] showed that cystine was precipitated by phos- 

 photungstic acid, but the observation seems to have been overlooked. 

 Cystine and tyrosine can be separated by means of this reagent. The 

 mixture is dissolved in 5 per cent, sulphuric acid and treated with 

 excess of phosphotungstic acid solution. Cystine phosphotungstate 

 generally separates out in a crystalline condition. From this precipi- 

 tate the cystine can be obtained by the usual method (see under 

 di-amino acids), but a large excess of baryta must be avoided as 

 cystine is readily decomposed by alkalies. Cystine crystallises out on 

 neutralising the filtrate from the barium phosphotungstate. Winter- 

 stein decomposed the phosphotungstate with hydrochloric acid. The 

 precipitate is made into a paste with water, placed in a separating 



