Early Stages in the Metabolism of Iron 197 



the solution was noted at various regions of the pH scale. The visible absorp- 

 tion spectrum of the ferric complex in the presence of excess itoic acid was 

 determined in 0-05 M-phosphate, pH 7-0. The method of continuous variation 

 was then applied, in conjunction with spectral analyses, in order to obtain 

 information on the composition of the ferric complex formed in neutral 

 solution. 



In an attempt to study the reaction with ferric ion quantitatively, 1 3 /<-moles 

 of ferric chloride and 39 /<-moles of itoic acid were mixed in 5 ml of water 

 and subjected to automatic electrometic titration with 1 N-NaOH. Similar 

 titrations were performed with 9-8 //-moles of cupric chloride and 19-6 

 /i-moles of itoic acid. An estimation of the relative stability of the 3:1 

 complex was obtained by equilibration of Fe+++ — ItoiCg with free ethylene- 

 diaminetetraacetic acid (EDTA). The surviving phenolic complex was 

 determined spectrophotometrically at 560 m//, a wavelength at which the 

 ferric chelate of EDTA exhibits insignificant hght absorption. The following 

 equilibria were employed : 



(i) Fe+++— Itoicg = Fe+++ + 3 Itoic; 



^ ^ (Fe+++-Itoic3) 

 ^ (Fe+++)(Itoic)=^ 



(ii) Fe+++ + EDTA = Fe+++— EDTA; 



_ (Fe+++— EDTA) 



" ~ (Fe+++)(EDTA) 



(iii) Fe+++— Itoic3 + EDTA = 3 Itoic + Fe^^-+— EDTA; 



_ ^ ,^ _ (Itoicf (Fe+++-EDTA) 

 Kui - KnIK, - ^_^-^_-_^^__^ 



The lability of both free itoic acid and the ferric complex was examined 

 under different laboratory conditions such as in the light and dark and at 

 different temperatures. 



Production of Itoic Acid as a Function of Iron Concentration. The usual 

 growth medium (Garibaldi and Neilands, 1956) was freed from iron by 

 treatment with 8-hydroxyquinoline followed by extraction with chloroform. 

 The itoic acid production by B. subtilis NRRL B-1471 as a function of added 

 iron and time of incubation was determined by use of the known extinction 

 coefficient of the 3:1 ferric complex in 0-1 m phosphate, pH 7-0. 



Utilization of Ferric-itoic^ by Bacillus Subtilis. In order to determine whether 

 or not iron complexed with itoic acid is nutritionally available to B. subtilis, 

 the following experiment was carried out. Eight 50 ml growth flasks fitted 

 with side-arms were charged with 8 ml of the usual, i.e. not oxine-extracted, 



