PURIFICATION OF PYOVERDINES OF PSEUDOMONAS-FLUORESCENS-2-79 BY COPPER-CHELATE CHROMATOGRAPHY

Three pyoverdines, Pf-A, Pf-B, and Pf-C, were purified with copper-chelate Sepharose and Sephadex G-15 columns from Pseudomonas fluorescens 2-79, and the yields (per 100 ml of culture supernatant) were 2.8, 21.6, and 3.2 mg, respectively. The absorption and fluorescence spectra of these pyoverdines were strongly pH dependent. Characteristic changes in the maximal absorbance wavelengths were observed when Fe3+ or Cu2+ was added, The addition of Cu2+ shifted the pyoverdine Pf-B absorbance spectrum so that it exhibited a single peak at 410 nm but did not give rise to a new absorbance maximum at approximately 460 nm, which appeared when Fe3+ was added, Fluorescence quenching experiments revealed that the forward reaction rate constant with pyoverdines was much higher with Cu2+ (10(4) to 10(5) M(-1) s(-1)) than with Fe3+ (10(2) M(-1) s(-1)), However, Cu2+-pyoverdine complexes were completely dissociated by EDTA at a low concentration (0.1 mM), while the level of Fe3+-pyoverdine complex dissociation at the same EDTA concentration was relatively low. The dissociation of Fe3+-pyoverdine complexes was EDTA concentration dependent. Formation of free pyoverdine was observed when the three types of Fe3+-pyoverdine complexes were incubated separately with P. fluorescens 2-79 cells, thus demonstrating that pyoverdines PEA, FEB, and PGC mediate iron transport.