CHEMICAL COMPOSITION OF PROTEIN MOLECULE 25 



extracted with cold absolute alcohol, and this operation is continued 

 until all the insoluble amino acids are removed. 



The final alcoholic solution is evaporated to dryness and the residue 

 weighed. As thus obtained, the proline is a mixture of the optically 

 active and the racemic forms. These are separated by conversion into 

 their copper salts by boiling with freshly precipitated copper oxide. 

 The resulting dark-blue solution is evaporated to dryness, and the re- 

 sidue is treated with absolute alcohol which dissolves the copper salt 

 of the optically active proline. This solution on concentration yields 

 the greater part of the compound in a crystalline state, but the remain- 

 der is amorphous. The copper salt of the racemic proline, which is 

 insoluble in alcohol, is purified by crystallisation from water. 



The identity of the compounds is established by a determination 

 of the water of crystallisation and of the copper. The racemic copper 

 salt contains 2 molecules of water of crystallisation, and in this state 

 it is dark blue in colour ; in the anhydrous state its colour is violet. 

 Further characterisation is obtained by preparing proline from it. The 

 copper salt is dissolved in water and decomposed with hydrogen sul- 

 phide. The filtrate is concentrated to a small volume and precipitated 

 with alcohol. The product, crystallised from alcohol, is obtained in flat 

 needles, /-proline has a sweet taste, melts at 206-209 C. and has a 

 rotation of [a] 2 D = -77'4. 



The amount of proline in the protein is given by the yield of the 

 two copper salts obtained in a pure state. The actual proline content 

 of the protein is considerably larger than the figures given in the tables 

 (pp. 53-62). 



The real proline content can be accurately determined, as Van 

 Slyke has shown by his nitrous acid method (p. 69), by a determina- 

 tion of the total and amino-nitrogen of the product soluble in absolute 

 alcohol. The alcoholic solution is made up to a definite volume and 

 aliquot portions are taken for these estimations. The difference gives 

 the amount of nitrogen present as proline from which the amount of 

 proline can be calculated. In the case of caseinogen the proline con- 

 tent was found to be 67 per cent, a figure which is twice that found 

 by Abderhalden. It agrees with that found by Engeland by his 

 method of methylation (p. 43). 



