362 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



present are in most cases greater than is given. The total sum of the 

 isolated amino acids is generally about 50 per cent. ; it is 70 per cent, 

 in the case of haemoglobin and nearly 90 per cent, has been obtained 

 from a vegetable protein. 



The analytical data of all the proteins which have been analysed 

 will be found in the "Chemical Constitution of the Proteins," Part I. 

 In this book is also given a fujl account of the methods of analysis 

 of the proteins. Here it is not possible to give more than a very 

 brief outline of the method of separating the amino acids. 



Hydrolysis of Proteins and Separation of the Amino Acids. 



The hydrolysis of proteins is generally effected according to the par- 

 ticular amino acid or group of amino acids required. The preparation of 

 cystine, tyrosine and tryptophan is given on pp. 143, 267, 348. 



The other amino acids are divided into two groups monoamino acids 

 and diamino acids. The latter is separated from the former by precipitation 

 with phosphotungstic acid. 



It the diamino acids are required, the protein is hydrolysed by boiling for 

 12-24 hours with 6 times its weight of 33 per cent, sulphuric acid. 



If the monoamino acids are required, the protein is hydrolysed by boil- 

 ing for 6-12 hours with 3 times its weight of concentrated hydrochloric acid. 



If both groups are required, the protein is hydrolysed with sulphuric acid, 

 the diamino acids are removed with phosphotungstic acid, the filtrate is freed 

 from sulphuric acid and treated according to the procedure for monoamino 

 acids. 



If all amino acids are required, tryptophan is first separated; the solution 

 after removing mercury is hydrolysed with sulphuric acid, the diamino acids 

 are precipitated with phosphotungstic acid and the filtrate treated for mono- 

 amino acids as mentioned below. 



The separation of the diamino acids depends upon the following re- 

 actions : 



The phosphotungstate precipitate is decomposed with baryta ; the solu- 

 tion is treated with silver sulphate and saturated with baryta. Arginine and 

 histidine are precipitated. The lysine in the filtrate is finally precipitated 

 by picric acid. Arginine and histidine are separated by a second precipita- 

 tion with silver nitrate in neutral solution ; the histidine is thrown down ; the 

 arginine is thrown down by saturating the filtrate with baryta. The diamino 

 acids are then recovered from the precipitates. 



The separation of the monoamino acids is effected thus : 



The hydrolysed solution is evaporated and saturated with dry hydrogen 

 chloride ; glutamic acid hydrochloride is precipitated. 



The filtrate is esterified by mixing with 3 volumes of absolute alcohol and 

 saturating it with dry hydrogen chloride; glycine ester hydrochloride is pre- 

 cipitated. 



The esters of the other amino acids are separated by extracting with ether 

 after liberating them from their hydrochlorides with caustic soda and saturat- 

 ing the solution with potassium carbonate. The esters, after distilling off the 

 ether which has been dried with sodium sulphate, are separated by fractional 

 distillation in vacua. 



The fractions of esters are hydrolysed by water, or by baryta, and the 

 mixture of two or three amino acids separated by fractional crystallisation, or 

 by the fractional crystallisation of their copper and other salts. Proline is 

 contained in the lower boiling fractions. It is soluble in alcohol and is thus 

 separated from the amino acids which are insoluble. 



