200 



J. B. Neilands 



10^^ to 10^^. These data are not considered as highly accurate since con- 

 siderable 'drift' was observed and since the equilibrium was not approached 

 from the reverse direction. Nonetheless it is evident that itoic acid exhibits a 

 very high avidity for ferric ion under approximately physiological conditions. 

 Inasmuch as there was no pH effect on mixing solutions of itoic acid with 

 cupric ion, the complexing of the latter metal is presumably weaker than for 

 ferric ion. 



Table 4 illustrates the lability of both free itoic acid and the ferric complex 

 under various laboratory treatments. Under the conditions tested, the free 

 substance was stable in both light and dark and was only slightly decomposed, 

 concomitant with browning of the solution, on autoclaving. Solutions which 

 were allowed to stand in dilute alkah, on the other hand, rapidly assumed a 

 coffee-colour that could not be discharged by re-acidiiication. The ferric 

 complex was stable for at least one day at room temperature but was largely 

 decomposed on autoclaving or on standing over a period of several weeks at 

 room temperature. 



Table 4. Lability of itoic acid and the ferric complex under 

 various conditions 



The stability of the free substance was determined by use of the absorption maxima at 314 m/t 

 (.^mm — 3 0) and the surviving ferric complex was measured at 560 m/t. The samples in light and 

 dark were allowed to stand at room temperature; autoclaving was carried out at 15 lb for 15 min. 

 The solvent was 0-1 m phosphate buffer, pH 70, and measurements were made 6 hr after mixing 



the solutions 



Relationship Between Iron Supply and Itoic Acid Production 



The correlation between the initial iron level of the medium and the amount 

 of itoic acid produced by B. subtilis is shown in Table 5. The cell yield was 

 very low and very high at zero and 100 /<g of added iron/1., respectively. It is 

 apparent that intermediate levels of added iron, i.e. 20 /tg, are the most 

 conducive for the accumulation of itoic acid. It should be stressed that the 

 application of the ferric chloride reaction to the crude, cell-free culture 

 supernatant of B. subtilis will measure the total phenolic acid content. That 

 the latter is essentially equivalent to itoic acid is indicated both from paper- 

 chromatographic analyses and from the fact that 50 mg of crystalline itoic 

 acid can be obtained per liter of medium. The yield of isolated itoic acid 

 would thus be about 20%, an entirely logical figure in view of the several 

 steps required in obtaining the pure compound from B. subtilis cultures. 



