CHEMICAL COMPOSITION OF PROTEIN MOLECULE 29 



Separation of Leucine and Isoleucine. 



The mixed lead salts are dissolved in 15-20 parts of hot water + 

 J part of glacial acetic acid, and hydrogen sulphide is passed into the 

 solution. The filtrate from the lead sulphide is evaporated in vacuo to 

 dryness, and the residue is washed with a mixture of equal parts of 

 alcohol and ether to remove acetic acid. 



Since these isomers have been shown not to be racemised by heating 

 with acids, the composition of the mixture can be ascertained by de- 

 termining the rotation in 20 per cent, hydrochloric acid : 



Isoleucine has 



J-leucme [o]g, = + 15-6 

 hence percentage of d-isoleucine = 100 x 



/-leucine = 100 x 



difference = 2I . g o 



21'8 



From the weight of the mixed amino acids and these data the 

 amount of each isomer can be calculated. 



The mixed isomers are converted into their copper salts by boiling 

 with excess of copper oxide, which is thoroughly boiled out with water 

 to remove the last traces of the copper salt of leucine which is pale 

 blue in colour and very insoluble. 



The solution of the copper salts is evaporated to dryness in vacuo. 

 The dry and finely pulverised copper salts are then shaken in a shaking 

 machine with 94 per cent, methyl alcohol. The insoluble copper salt 

 of leucine is filtered off and washed with solvent. The soluble copper 

 salt of isoleucine may be contaminated with some of the copper salt 

 of leucine. It is therefore decomposed with hydrogen sulphide, re- 

 converted into copper salt and again extracted with methyl alcohol. 

 Both the leucine and isoleucine are obtained in the usual way from 

 the copper salt and recrystallised from water. 



They are identified by elementary analysis, rotation, and estimation 

 of copper in their copper salts. 



Separation of Glycine and Alanine. 



By the fractional crystallisation of the amino acids obtained from 

 the esters the following fractions may be obtained : 



1. Valine + leucines. These are treated as above described. 



2. Valine + alanine. These are separated by fractional crystal- 

 lisation and also by the fractional crystallisation of the copper salts. 



3. Alanine. This is purified by recrystallisation from dilute 

 alcohol. 



