CHEMICAL COMPOSITION OF PROTEIN MOLECULE 41 



ceived special attention from Osborne and Jones, who are confirmed 

 in their observations by Abderhalden. 



1. Hydrolysis. In many cases the hydrolysis of the proteins may 

 not have been complete. Some proteins are very difficult to bring 

 into solution in the concentrated hydrochloric acid, and portions may 

 adhere to the sides of the flask and may therefore not be hydrolysed. 

 Even if there be apparent total solution a small amount may escape 

 hydrolysis by becoming enclosed in the " humin " which is formed to 

 a greater or lesser extent. The insoluble material should be filtered 

 off, washed, and tested with the biuret reaction. The absence of the 

 biuret reaction is not an absolute criterion that hydrolysis is com- 

 plete, for many polypeptides do not show it and are very resistant 

 to hydrolysis. The residue, if large, should therefore be hydrolysed 

 again. Complete hydrolysis should be tested for by Van Slyke's 

 amino-group method. Osborne has found that hydrolysis is some- 

 times only complete after boiling for two to five days. 



Sulphur-containing substances and sometimes sulphur have been 

 found in the reflux condenser, and the smell of iodoform has been 

 noticed in the hydrolysis of spongin. 



2. Formation of Humin. Nearly all proteins on hydrolysis yield 

 an insoluble brownish-black residue. This is probably a mixture of 

 secondary products formed from tryptophane, histidine, and carbohy- 

 drate ; since zein, which contains no tryptophane, no carbohydrate and 

 only a small quantity of histidine, yields very little humin. The loss of 

 products is considerably greater than the quantity of " humin," which 

 generally amounts to 1-2 per cent, of the protein. A loss, which can- 

 not be estimated, is represented by the soluble brown pigment which 

 colours the solution. 



3. Separation of Glutamic acid Hydro chloride. The quantity of 

 glutamic acid precipitated as hydrochloride never represents the total 

 quantity present in the protein. The amount precipitated depends 

 very largely on conditions. The precipitate usually contains am- 

 monium chloride; this is removed by boiling with baryta, and if 

 the baryta be removed with carbon dioxide the barium carbonate may 

 contain barium glutamate, which is very insoluble, and consequently 

 there is loss of this constituent. The remainder of the glutamic acid 

 is recovered with the esters. 



4. Esterification. The loss in this process is not great, especially 

 if it be repeated. Loss is chiefly due to hydrolysis of the esters 



