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-che late 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 o f 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 absorbanc e spectrum so that it exhibited a single peak at 410 nm but did not gi ve rise to a new absorbance maximum at approximately 460 nm, which app eared when Fe3+ was added, Fluorescence quenching experiments revealed that the forward reaction rate constant with pyoverdines was much hig her 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 dissociate d by EDTA at a low concentration (0.1 mM), while the level of Fe3+-pyo verdine complex dissociation at the same EDTA concentration was relati vely low. The dissociation of Fe3+-pyoverdine complexes was EDTA conce ntration dependent. Formation of free pyoverdine was observed when the three types of Fe3+-pyoverdine complexes were incubated separately wi th P. fluorescens 2-79 cells, thus demonstrating that pyoverdines PEA, FEB, and PGC mediate iron transport.