CHEMICAL COMPOSITION OF PROTEIN MOLECULE 21 



pared, so as to make a 3 per cent, solution, is added. The sodium 

 chloride formed is filtered off. Its separation is greatly facilitated by the 

 addition of ether and cooling to O C. The alcohol is removed by evap- 

 oration in vacuo. A small quantity of the lower boiling esters of the 

 amino acids passes into the distillate with the alcohol, but is recovered 

 by acidifying with hydrochloric acid and evaporating when the amino 

 acid hydrochlorides are obtained. A dark-brown oil again results, 

 which is fractionally distilled in vacuo. 



Although this method prevents loss by the action of alkali, the 

 yield of the higher boiling fractions is not so great on account of the 

 more complex nature of the mixture of esters. The residue which 

 does not distil contains the tyrosine, the diamino acids and other sub- 

 stances. 



(c] Instead of employing caustic soda and potassium carbonate for 

 the liberation and salting out of the esters, Levene uses barium oxide 

 for which he claims the following advantages : 



(i) On account of its small solubility in water, a large excess of 

 alkali which causes saponification of the esters is avoided. 



(ii) In neutralising the free acid the rise in temperature is less than 

 with caustic soda. 



(iii) When the second esterification is required it is more easily 

 removed. 



His procedure is as follows : 



The concentrated solution of the ester hydrochlorides is poured 

 into a porcelain or enamelled vessel of capacity of I litre for every 

 125 to 150 grammes protein, and the flask is washed out with ice- 

 cold baryta solution. The vessel is placed in a freezing mixture. 

 When the contents are cold excess of crystallised baryta is added, and 

 the mixture is thoroughly stirred with a wooden or porcelain spatula. 

 The peculiar sticky mass in a few minutes becomes liquid and the 

 solution becomes alkaline in reaction. Several volumes of ether are 

 then poured over the solution. The ether is then poured off and a 

 fresh quantity added. More baryta is added and the stirring continued 

 The ether is replenished and more baryta added several times. During 

 the process the mixture becomes cloudy and pasty, but finally a light 

 dry mass of baryta is left. 



The ethereal solutions are treated as described in the (a) method. 



For the second esterification process the residue is stirred up several 

 times with water, filtered through asbestos and washed with water till 

 no more organic matter is extracted. Most of the baryta remains 



