Immobilised metal affinity chromatography for the capture of hydroxamate-containing siderophores and other Fe(III)-binding metabolites directly from bacterial culture supernatants.

Nickel(II)-based immobilized metal affinity chromatography (IMAC) has been used to capture from standard samples the hydroxamate-containing siderophores, acetohydroxamic acid (ahaH), suberodihydroxamic acid (shaH(2)) or desferrioxamine B (DFOB) in recoveries ranging between 35-90%. The capacity of a 1 mL Ni(II)-charged IMAC column towards DFOB capture at the pH optima of 8.9 is approximately 3000 nmol. This method has been used for the direct capture of DFOB (approximately 65% recovery) from the untreated supernatant of iron-deprived cultures of Streptomyces pilosus, the soil bacterium from which DFOB was first discovered. In addition to selecting for DFOB and a related siderophore, two other Fe(III)-responsive species have been identified from RP-HPLC analysis of the IMAC-processed eluant from the S. pilosus supernatant. Since the characterisation of siderophores from natural systems is hampered by the low yields obtained from traditional purification methods, this IMAC-based affinity method offers significant potential for improving yields of this key class of bioligands and other small molecule metabolites with affinities to IMAC-compatible metal ions.