184 A. I. VIRTANEN, J. K. MIETTINEN VOL. 12 (1953) 



pression is that the increase of free amino acids during the darkness is due chiefly to 

 protein breakdown. It may also be possible that the synthesis of amino acids continues 

 still in the dark although the synthesis of peptides and proteins is inhibited, whereby just 

 such amino acids are concentrated that normally are used for the protein synthesis as 

 they are formed. 



The amino acid composition of the "peptide'' fraction 



The nitrogen of the free amino acids constitute about 30-70% of the soluble 

 nitrogen of pea at different ages. Thus, the soluble nitrogen fraction contains besides 

 free amino acids a number of other nitrogen compounds, e.g. purines, pyrimidines, free 

 bases and also compounds from which amino acids are liberated by hydrolysis. These 

 last mentioned compounds, in particular, have been subjects of our interest. 



In the acid hydrolysis of the ethanol extract especially of young pea plants we were 

 able to demonstrate for certain a heavy increase of glutamic acid. Likewise threonine, 

 glycine, lysine, and proline were increased. The amount of certain amino acids, as 

 serine, alanine and valine, was decreased by hydrolysis in some samples. Since in the 

 total hydrolysis (6 N HCl for 20 h at 108° C in closed tube) only serine and threonine are 

 decomposed to any greater extent in the absence of carbohydrates, the decrease of 

 alanine, valine, and some other amino acids in the ethanol extract must be due to the 

 reactions between amino acids and carbohydrates and even other non-nitrogen com- 

 pounds present in the extract. Copious formation of brown decomposition products in 

 the hydrolysis (10-20% of the total N of the extract was found in the brown precipitate) 

 indicates that such reactions really take place. 



In order to obtain more information of the compounds from which amino acids 

 are liberated by hydrolysis we have tried to isolate the fraction in question. By using 

 ion-exchange resin Amberlite IR-105 (H+) of the sulphonic acid type, we obtained from 

 the ethanol extract of pea a fraction which contained no free amino acids. The nitrogen 

 of this fraction amounted to 10-30% of the nitrogen of the extract. On hydrolysis 

 of the "peptide" fraction the same amino acids were formed as are generally met in free 

 state in the pea extract with a voluminous brown precipitate, containing ca. 20 % of 

 the nitrogen of this fraction. Abundant formation of glutamic acid, aspartic acid, alanine, 

 valine, leucine, woleucine, moderate of glycine, serine, threonine, proline, homoserine, 

 lysine, slight of tyrosine, phenylalanine, y-AB, ^-alanine, and two unknown compounds 

 and traces of a-AB and arginine occurred in total hydrolysis of the fraction. 



On dialysis of the fraction 98.5% of its nitrogen passed through the dialysis mem- 

 brane. The amino acid composition of the "non-dialysable" did not essentially differ 

 from that of the "dialysable". About 30% of the nitrogen of the dialysate was absorbed 

 in the active carbon. All of the bound tyrosine and a major part of the bound ^-alanine 

 were retained by the carbon but otherwise the adsorbed fraction did not differ essentially 

 from the unadsorbed one. About half of the adsorbed nitrogen compounds correspond- 

 ing to II % of the nitrogen of the peptide fraction, could be displaced again from the 

 carbon by phenol, the rest remaining irreversibly adsorbed. 



In order to ascertain whether the amino acids bound in the "peptide" fraction are 

 linked together by peptide bonds or whether they are attached to sugars or appear as 

 acyl-amino acids etc., a hydrolysis was carried out on the dialysed fraction in three 

 different ways: a. i iV HCl i h 100° C, b. i A" HCl 3 h 100 '^ C, and c. total hydrolysis 

 6 N HCl 24 h 108° C. 



References p. 18 j. 



