1 8 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



Tyrosine remained. It is better to dissolve the residue in hot water, 

 decolorise with charcoal, and allow the tyrosine to crystallise out. 



When large quantities of protein are under investigation, the 

 removal of the hydrochloric acid, after evaporation in vacuo, is effected 

 by treating the solution with cuprous oxide until it is green in colour, 

 filtering off and washing the cuprous chloride, and removing dissolved 

 copper by hydrogen sulphide. A current of air is then passed 

 through the solution to remove the hydrogen sulphide, and the re- 

 mainder of the hydrochloric acid is either neutralised with the calcu- 

 lated quantity of soda or is removed by treating with silver carbonate. 

 The solution on concentration deposits the tyrosine. 



Levene and van Slyke [1908, 2] prefer the use of hydrochloric 

 acid to that of sulphuric acid for separating tyrosine on account of the 

 difficulty of completely extracting it from the barium sulphate precipi- 

 tate and of obtaining it in a state of purity. Their procedure is the 

 following : The protein is hydrolysed with concentrated hydrochloric 

 acid ; the solution is concentrated and saturated with gaseous hydro- 

 chloric acid. Glutamic acid hydrochloride separates out. The filtrate 

 and washings from this precipitate are concentrated in vacuo to remove 

 the greater part of the hydrochloric acid. The solution is then 

 diluted to 7 litres (for 400 grams protein) and boiled with lead oxide 

 till its reaction is alkaline. 1 The lead oxide is prepared by precipita- 

 tion with baryta, washed by decantation and preserved in the form of 

 a paste. The precipitate of lead oxychloride is filtered off when the 

 solution has cooled. It retains the resinous matters and a nearly 

 colourless filtrate results. The remainder of tr>e chlorine, which is 

 estimated in an aliquot portion, is removed by means of the calculated 

 quantity of silver sulphate, the excess of lead by adding sulphuric acid 

 and passing in hydrogen sulphide, and of sulphuric acid by baryta. 

 On concentrating the solution to one-seventh almost pure tyrosine 

 separates out ; it is filtered off, washed, dried and weighed. A portion 

 of the other amino acids can be obtained by further concentration, 

 and treated for leucine and valine (see p. 45). The di-amino acids are 

 then precipitated (pp. 60, 98) and the filtrate is treated for the other 

 monoamino acids. 



If the solution be highly concentrated a mixture of tyrosine and 

 leucine may separate out. This mixture may be separated by treat- 

 ing with glacial acetic acid. Leucine is soluble, tyrosine is insoluble 

 [Habermann and Ehrenfeld, 1902]. 



1 Levene and Van Slyke [1910] state that excess of lead oxide should be avoided so as to 

 prevent the formation of the insoluble lead salt of tyrosine. 



