8 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



of specific gravity rip, when it gradually passes into solution, if the flask 

 in which it is contained be occasionally shaken and slightly warmed, 

 and then boiling under a reflux condenser for five to ten hours, depend- 

 ing on the particular protein, and until the biuret reaction has completely 

 disappeared. The solution, which may become at first violet, becomes 

 finally of a dark brown colour and a portion of the hydrochloric acid is 

 evolved as gas ; it is boiled with charcoal, filtered from humin substances 

 (secondary products probably arising frt>m carbohydrate) and fatty 

 material and consists of a solution of amino acids in 25 per cent, hydro- 

 chloric acid. 



It is concentrated in vacuo to a small volume, and glutamic acid, if 

 present in any large amount, is removed as its hydrochloride by saturat- 

 ing the solution with dry gaseous hydrochloric acid and allowing to 

 stand at o C. for some days, when glutamic acid hydrochloride crystal- 

 lises out. This occurs in the case of caseinogen and certain vegetable 

 proteins, which contain from 10 to 40 per cent, of this amino acid. The 

 glutamic acid hydrochloride is filtered off after adding an equal volume 

 of ice-cold alcohol, redissolved in water, boiled with charcoal and again 

 precipitated as hydrochloride by saturating the solution with gaseous 

 hydrogen chloride, and weighed. 



The solution thus freed from the greater part of the glutamic acid is 

 again concentrated in vacuo at a low temperature to a thick syrup ; this 

 is dissolved in absolute alcohol (3 litres to I kilo, protein) and the 

 amino acids are esterified by saturating the alcohol with dry gaseous 

 hydrochloric acid at the ordinary temperature and then warming on the 

 water bath for half an hour. In the process of esterification a large 

 amount of water is formed, which prevents complete esterification ; the 

 alcohol is therefore evaporated off in vacuo at a temperature below 50 C., 

 and the resulting syrup again dissolved in absolute alcohol and saturated 

 with dry gaseous hydrochloric acid. In some cases it may be necessary 

 to repeat this operation once more. At this stage, glycine, if it occurs 

 in the protein, e.g., in gelatin, in any considerable amount, is separated as 

 glycine ester hydrochloride by seeding the solution with a crystal of 

 this compound and allowing to stand for twenty-four hours at o C. It 

 is filtered off while still cold, and the mother-liquor, on further con- 

 centration and saturation again with hydrochloric acid, may give 

 another crop of glycine ester hydrochloride, so that almost the whole 

 of the glycine may be isolated in this way. One recrystallisation from 

 alcohol suffices to purify it and it is characterised by its melting point 

 of 144 C. and analysis. 



The filtrate containing the esters of the hydrochlorides of the other 



