30 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



4. Alanine + glycine. There are two methods of separating these 

 compounds : 



(a) By reconverting the mixture into esters, separating the glycine 



as ester hydrochloride and distilling the alanine ester, which 

 is then decomposed by boiling with water and the alanine ob- 

 tained by crystallisation. 



(b) By precipitating the glycine as picrate (Levene). The mixture 



is dissolved in a small quantity of hot water and an equal 

 quantity of picric acid, dissolved in alcohol, is added. Glycine 

 picrate crystallises out on cooling. The pure compound 

 melts at 190. The alanine may be obtained from the filtrate 

 by acidifying, extracting the excess of picric acid with ether, 

 removing the acid and then crystallising out. 



The glycine is identified by the melting-point and analysis of its 

 ester hydrochloride and picrate ; its amount in the protein is given by 

 the yield. 



Alanine is identified by elementary analysis and its rotation in 

 hydrochloric acid solution. The amount in the protein is also given by 

 the yield. 



(b) Fraction IV. 



(a) Phenylalanine. 



The ester of phenylalanine differs from the esters of aspartic acid, 

 glutamic acid and serine, which are present with it in this fraction, by 

 being only slightly soluble in water. 



The mixed esters are dissolved in 5 volumes of water. If a 

 large amount of phenylalanine be present, it may separate in the form 

 of oily drops. The aqueous solution is extracted with an equal volume 

 of ether. The ether extract is then washed several times with water 

 to remove the last traces of any of the other esters which may have 

 been dissolved by the ether. The ether is removed by distillation and 

 the ester is hydrolysed by evaporation with concentrated hydrochloric 

 acid. The resulting phenylalanine hydrochloride is purified by 

 crystallisation from hydrochloric acid, and can be identified by an 

 estimation of its content in chlorine. 



The free amino acid is obtained from the hydrochloride by treating 

 with sodium acetate, or ammonia, and precipitating from hot aqueous 

 solution with alcohol. A determination of the melting-point of 283 C 

 and rotation of [a] Q = ~35'i in aqueous solution characterises the 

 compound 



