196 J. B. Neilands 



(iii) The new substances produced in iron deficiency are intended to serve, 

 either as such or as the ferric complex, as a by-pass for electron trans- 

 port around the normal cytochrome system. 



The isolation, characterization and chemical synthesis of itoic acid (iron- 

 transferring-orthophenol ; 2 : 3-dihydroxybenzoylglycine ; 2 : 3-dihydroxyhip- 

 puric acid) has been described elsewhere (Neilands, 1958; Ito and Neilands, 

 1958). The following experiments relate to the iron-complexing activity, 

 production and metabolism of the new compound. 



The isolation and general properties of the ferrichrome compounds have 

 been reviewed previously (Neilands, 1957). The present report will describe 

 recent experiments leading to the identification of the iron-binding centre of 

 the ferrichromes as a polyhydroxamic acid (Emery and Neilands, 1959; 

 Emery, 1960). 



EXPERIMENTAL 



Materials 



Itoic acid was synthesized from 2:3-dihydroxybenzoic acid and ethyl 

 glycinate in the manner previously described (Ito and Neilands, 1958). The 

 preparation, after recrystallization, was chromatographically homogeneous 

 in the following solvent systems {Rf in brackets): «-butanol, 4, acetic acid, 1, 

 water, 5 (0-80); benzene, 2, acetic acid, 2, water, 1 (0-29); methanol, 20, 

 water, 5, pyridine, 1 (0-67); tert. -bwiyX alcohol, 10, methyl ethyl ketone, 10, 

 water, 5, diethylamine, 1 (0-39). The chromatograms were analyzed by 

 ultraviolet illumination or by spraying with either 1 % aqueous ferric chloride 

 alone or acidic sodium nitrite solution followed by dilute NaOH. 



Ferrichrome and ferrichrome A were prepared by 'low-iron' fermentation 

 with U. sphaerogena and recrystallized by published methods (Neilands, 

 1952; Garibaldi and Neilands, 1955). Both preparations gave single spots in 

 «-butanol, 4, acetic acid, 1, water, 5 (0-40 and 0-51, respectively) and methanol, 

 4, water, 1 (0-68 and 0-52, respectively). The ferrichromes can generally be 

 detected visually on paper as tea-coloured spots without the application of a 

 developing spray; alternatively, when only very small amounts of material 

 are present, the chromatograms may be sprayed first with dilute sodium 

 sulphite solution and then with a very dilute solution of 1 : 10-phenanthroline. 

 Under these conditions iron is instantly released from the ferrichromes and 

 appears as the intensely red-coloured ferrous-phenanthroline complex. 



Methods 



Ferric Complex of Itoic Acid. The colour reaction of itoic acid with ferric 

 chloride was found to be markedly dependent on pH. A slight excess of 

 itoic acid was added to a dilute solution of ferric chloride. The pH of the 

 solution, initially about 2, was then raised in the automatic titration apparatus 

 (Neilands and Cannon, 1955) by the addition of dilute alkali. The colour of 



