ISOLATION AND ESTIMATION OF UNITS 15 



II. THE ISOLATION AND ESTIMATION OF THE UNITS. 



The units composing the protein molecule belong to four different 

 classes of organic compounds, but are divided into two main groups for 

 the purpose of isolation and estimation : 



A. The mono-amino acids, including proline and oxyproline. 

 Tyrosine and cystine differ from the other mono-amino acids by 



their extremely slight solubility in neutral aqueous solutions. They 

 are therefore easily obtained after hydrolysis by acids by neutralising 

 and concentrating the solution, when they crystallise out. 



B. The di-amino acids, including histidine. The three compounds 

 in this group were formerly called the hexone bases on account of 

 their basic properties and the fact that each of them contains six 

 carbon atoms. 



The remaining unit, tryptophan, is almost completely destroyed 

 by hydrolysis by acids ; it is usually isolated after hydrolysis by trypsin. 



Ammonia (amide nitrogen) is estimated by hydrolysing the protein 

 with concentrated hydrochloric acid, removing the great excess of acid 

 by evaporation under reduced pressure, adding excess of magnesia, 

 distilling off the ammonia in vacua and collecting it in excess of 

 standard acid. This operation is usually carried out in the determina- 

 tion of the distribution of the total nitrogen amongst the two main 

 groups (p. 97). The estimation of ammonia is frequently combined 

 with the estimation of the di-amino acids (p. 55). 



The separation and estimation of the two main groups of amino 

 acids is generally not carried out in one experiment, but only when 

 the amount of protein available is small, as very different quantities 

 of material are required. Thus, the di-amino acids can be determined 

 in 25-50 grams of protein with considerable accuracy, whereas the 

 mono-amino acids can only be determined with fair accuracy when 

 250-500 grams of protein can be used. The two processes, of 

 which the details are given under the two sections, may be combined 

 as follows : 



The protein is hydrolysed by boiling for fifteen to twenty-four hours 

 with six times its quantity of 25-30 per cent, sulphuric acid. The 

 solution is neutralised with baryta and the filtrate and washings from 

 the barium sulphate are evaporated down to a small volume. Tyrosine 

 (and cystine) crystallise out. The filtrate is diluted with water and 

 sulphuric acid added till the content of acid is 5 per cent. The 



