24 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



(5) The Separation and Characterisation of the Individual Mono- 



amino Acids. 



The separation and characterisation of each constituent contained 

 in an ester fraction has now to be carried out. A somewhat special 

 process has to be adopted for each individual product. 



(a) Fractions /., //., ///. 



The esters contained in these fractions are immediately reconverted 

 into the amino acids. This is effected by boiling the fractions with 

 5-10 volumes of water under a reflux condenser for six to seven hours, 

 until the alkaline reaction has disappeared. 



If leucine be present in considerable amount, as may be the case in 

 fraction III., the solution on cooling may deposit crystals of this sub- 

 stance. These are filtered off, washed with water, dried and weighed. 

 Identification of the product is carried out as described below. 



The three solutions are then evaporated to dryness in vacuo in 

 weighed flasks and the amount of residue ascertained for each fraction. 



Levene has found that leucine ester, like phenylalanine ester, is 

 readily dissolved by ether from water. The ester fractions may there- 

 fore, before hydrolysis, be mixed with 3 volumes of water and ex- 

 tracted with ether, and the ether extract washed three times with water. 

 The aqueous solution is then saponified by boiling and the extract, after 

 removal of the ether, is treated in the same way. 



Isolation and Characterisation of Proline. 



Proline is the only product contained in these fractions which is 

 soluble in alcohol ; it is also much more easily soluble in water than the 

 other products. 



The dry residues are therefore extracted several times with boiling ab- 

 solute alcohol ; these extracts on cooling frequently become turbid and on 

 standing deposit other amino acids which, though insoluble in alcohol, 

 are dissolved when proline is present. They are filtered off and re- 

 turned to the portion insoluble in alcohol. 



The combined alcoholic extracts of the three fractions are again evap- 

 orated to dryness in vacuo and the residue is treated several times with 

 cold absolute alcohol. A considerable amount is undissolved ; as be- 

 fore this is returned to the insoluble portion. 



The alcoholic solution is again evaporated to dryness in vacuo and 



