CHEMICAL COMPOSITION OF PROTEIN MOLECULE 27 



fraction, Levene and Van Slyke have worked out a method which de- 

 pends upon the precipitation of leucine and isoleucine as their lead 

 salts from ammoniacal solution and the subsequent separation of these 

 two amino acids by means of the different solubility of their copper 

 salts in methyl alcohol. The details were published in 1909 and are 

 as follows : 



The mixture is carefully analysed and the amount of leucine + 

 isoleucine is calculated from the carbon content : 



Leucine and isoleucine contain 54-92 per cent. C. ^ ,. 



Valine contains . . .51-24 } difference = 3-68 per cent. 



Per cent, carbon - 51^24 



^g = per cent, of leucine isomers in the mixture. 



The mixture is pulverised and suspended in 7 parts of water and 

 the water is raised to the boiling-point : for each gramme of sub- 

 stance 1-5 c.c. of concentrated ammonia solution is then added. The 

 flask is stoppered and shaken so as to dissolve the amino acids; if 

 necessary the solution may be again heated. 



Four c.c. of 1*1 M lead acetate solution (sp. gr. 1*254 at 20) 

 for each gramme of leucine and isoleucine are slowly added to the hot 

 solution, which is thoroughly stirred during the addition. The solu- 

 tion is then cooled in ice-water and after one to two hours is filtered 

 through a Buchner funnel, or a Gooch crucible, according to the 

 amount of precipitate. The solid matter is pressed down so as to 

 remove the mother liquor as completely as possible, and it is washed 

 firstly with 90 per cent, alcohol, then with ether, and dried in vacuo 

 over sulphuric acid. 



It is curious that the presence of valirie facilitates the separation of 

 the lead salts of the leucine isomers. 



If the proportion of isomers : valine be less than 2 : I the pre- 

 cipitation is not so complete. In these cases less lead acetate solution 

 (37 c.c.) should be taken and the filtrate concentrated in vacuo till the 

 percentage of valine reaches 10. Ammonia is again added and the 

 precipitate treated as before. It is preferable to treat the filtrate once 

 more in the above manner after the valine has been separated. 



