FIXATION OF NITROGEN 53 



nitrogen-fixation, one which can be regarded both as the 

 product of the fixation mechanism and as the substrate for 

 the reactions by which inorganic nitrogen is incorporated 

 into organic compounds. During the last decade, the eluci- 

 dation of the nature of this substance has occupied the 

 attention of two groups of workers, one associated with 

 Virtanen in Finland [36] and the other with Burris and 

 Wilson in the U.S.A. By using the isotope N^^, the American 

 workers have obtained convincing evidence of the key role 

 of NH3 in the fixation of N2 , a concept first advanced by 

 Winogradsky [49]. In the isotope experiments, the period 

 of exposure to the substance enriched with N^^ was not 

 long enough for equilibrium to be established, and prior to 

 isotopic analysis, the nitrogenous components of the experi- 

 mental system were separated into fractions to facilitate 

 location of the compounds with the highest concentration 

 of N^^. For example, after acid hydrolysis, various amino- 

 acids were isolated by classical precipitation procedures, or 

 more recently, by the use of columns of ion exchange resins 

 or starch [51, 43]. It was argued that if the assimilation of 

 nitrogen involved a number of intermediates, then, before 

 equilibrium was established, the concentration of the iso- 

 tope would be the greater the nearer the intermediate to the 

 substance initially enriched with N^^. Moreover, if a sub- 

 stance on the fixation pathway was supplied instead of, or 

 in addition to, molecular Ng , it should be utilized not only 

 as a source of nitrogen, but also in preference to its pre- 

 cursors (cf. simultaneous adaptation, p. 13) [11, 44]. 



The resultant distribution of the isotope amongst the 

 constituents of Az. vinelandii was the same irrespective of 

 whether nitrogen was derived from Ng^^ or N^^Ht, and of 

 the cells' amino-acids, glutamic acid followed by aspartic 

 acid, contained the highest concentration of N^^ [12]. The 

 establishment of such a distribution, even when the period 

 of contact with N^^Ht was short (3 min.), indicated that the 

 enzymes responsible for the assimilation of NHg already 

 existed in the bacteria. Moreover, the fact that NH3 gave 

 rise to the same distribution of N^^ as N2 ^ implied that it 

 was either itself on the pathway of Ng-fixation or at least 



