AMINO ACIDS, PEPTIDES AND PROTEIN'S 



221 



R 



R 



HO2C - CH - NH3+ + HNO2 - HO2C - CH - OH + N2 + H3O+ 



There are, however, complications. Imino acids, for example, proline and azetidine 

 carboxylic acid do not contain primary amino groups and, therefore, yield no gaseous 

 nitrogen on reaction with nitrous acid. On the other hand, glutamine yields almost two 

 moles of nitrogen per mole. 



Formaldehye reacts with amino acids to give a mixture of compounds. Since in these 

 substances the acidity of the ammonium group is increased. 



NH, 



O H2N+CH2OH HN+(CH20H)2 



RCHCO2- + HCH - RCHCO2- + RCHCO2- + others 



the addition of formaldehyde to amino acids permits the quantitative determination of 

 these substances by titration with standardized base to the phenolphthalein endpoint (58). 



The reaction of primary and secondary amines with ninhydrin is used very extensively 

 for the detection of amino acids and for their quantitative determination (59). Amides and 

 tertiary amines do not react. With ammonia and most primary and secondary amines in- 

 tensely colored violet, blue, brown or yellow pigments are obtained. 



In tiie reaction of most alpha amino acids with ninhydrin the violet color formation 

 is accompanied by carbon dioxide formation. Therefore, the combined determination of 

 carbon dioxide and color makes this reaction fairly specific for alpha amino acids. The 

 reaction is believed to involve several steps (59). Ninhydrin (I) reacts with an alpha 

 amino acid to produce the amine II plus carbon dioxide and an aldehyde with one carbon 

 atom less than the original amino acid. At the appropriate pH, compound II condenses 



(1 





OH 



NH 



+ 



+ R 



'3 



CH co; 







11 



,c 



OH 



C-NH, 



II 







I 



OH 



CO2 + 





 II 



RC-H 



with a molecule of ninhydrin to yield III, a violet pigment. The reaction of a given alpha 

 amino acid with ninhydrin does not necessarily yield compound III in stoichiometric 



N = 



n 



amounts. However, a procedure has been described in which equivalent amodnts of color 

 formation is claimed for all common amino acids except tryptophan, 80%, and lysine, 

 110%, (60). Another advantage of this procedure is its low sensitivity to ammonia. 



