HYDROLYSIS OF PROTEINS 317 



To this end Fischer, in 1 901, introduced his so-called 

 " Ester method," which consisted in converting the mixed 

 amino acids obtained by hydrolysis of proteins into their 

 corresponding esters, and then separating these by fractional 

 distillation. 



The method * is best illustrated by an example. Casein 

 was decomposed by hydrolysis with concentrated hydrochloric 

 acid, the hydrochloride of glutamic acid being separated by 

 filtration. The filtrate was then evaporated under reduced 

 pressure, taken up with alcohol and saturated with dry gaseous 

 hydrogen chloride ; in order to remove the water formed 

 by the reaction, the solution was once more evaporated down, 

 and the residue taken up with alcohol and again saturated with 

 hydrogen chloride. The esters were next liberated from 

 their hydrochlorides by evaporating the solution down to a 

 syrup in a vacuum, diluting with water and approximately 

 neutralizing by means of strong caustic soda solution while 

 keeping thoroughly cooled in a freezing mixture. Concen- 

 trated potassium carbonate was now added, and the esters of 

 aspartic and glutamic acid were extracted by ether ; after add- 

 ing more 33 per cent caustic soda and potassium carbonate 

 and extracting again with ether, the combined extracts were 

 dried with anhydrous sodium sulphate, evaporated and distilled 

 under 8-15 mm. pressure. The various fractions were then 

 separately hydrolysed, either by boiling with water or by 

 warming them on the water bath with 20 per cent baryta 

 water. 



This method, with slight modifications, has been applied 

 by several workers, more especially Abderhalden and Osborne, 

 to a considerable number of different proteins, with the result 

 that there are now more or less reliable data for comparing the 

 composition of proteins from various sources, both animal and 

 vegetable. 



A second method for gaining some insight into the com- 

 position of proteins consists in studying the distribution of 

 nitrogen in the molecule with a view to ascertaining whether 

 it is present in the form of mono- or di-amino acids, etc. A 

 method for distinguishing between the different types of nitro- 

 gen-linking occurring in the molecule was first suggested by 

 * Fischer: " Zeit. physiol. Chem.," igoi, 33, 151. 



