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Fluorescence of pyoverdin in response to iron and other common well water metals
Michael F Yoder1, William S Kisaalita
1Biological and Agricultural Engineering Department, Driftmier Engineering Center, University of Georgia, Athens, Georgia, USA.
Abstract:
When the fluorescent siderophore pyoverdin (produced by Pseudomonas aeruginosa) binds to a metal ion the fluorescence changes. A pyoverdin solution (in 0.1 M acetate buffer, pH 5.0) was placed in the microwells of a 96-well plate and varying concentrations of the metal cations Al(3+), Ca(2+), Cu(2+), Fe(2+), Fe(3+), Mn(2+), Mg(2+), and Zn(2+) were added. The fluorescence of pyoverdin 60 sec after the addition of an equimolar concentration of metal indicated: (1) no change for Ca(2+), Fe(2+), Mn(2+), Mg(2+), and Zn(2+); (2) a small increase (109%) for Al(3+); (3) decreases in fluorescence for Cu(2+) (83%) and for Fe(3+) (66%). The fluorescence of pyoverdin 24 hr after the addition of equimolar metal indicated: 1) very little change for Ca(2+), Mn(2+), Mg(2+), and Zn(2+); 2) a very large (270%) increase in fluorescence due to Al(3+); 3) an increase (113%) due to Cu(2+); 4) large decreases in fluorescence for both Fe(2+) (15%) and Fe(3+) (0%). Thus, for an iron assay using a free solution of pyoverdin, even with a short (60 sec.) reaction time there can be interference due to Cu(2+), and interference due to high levels of Al(3+).
Insights
Pyoverdin fluorescence changes upon metal binding. Copper and aluminum ions interfere with pyoverdin assays, affecting iron quantification. This highlights the need for careful assay design.
Area of Science:
- Biochemistry
- Analytical Chemistry
Background:
- Pyoverdin, a fluorescent siderophore produced by Pseudomonas aeruginosa, is utilized in metal ion detection.
- Changes in pyoverdin's fluorescence intensity correlate with its binding to specific metal cations.
Purpose of the Study:
- To investigate the effect of various metal cations on the fluorescence of pyoverdin.
- To assess potential interferences in pyoverdin-based assays, particularly for iron quantification.
Main Methods:
- A pyoverdin solution was prepared in acetate buffer (pH 5.0).
- Microwells containing pyoverdin were treated with equimolar concentrations of Al(3+), Ca(2+), Cu(2+), Fe(2+), Fe(3+), Mn(2+), Mg(2+), and Zn(2+).
- Pyoverdin fluorescence was measured at 60 seconds and 24 hours post-metal addition.
Main Results:
- Short-term (60 sec) measurements showed fluorescence decreases with Cu(2+) and Fe(3+), and a slight increase with Al(3+).
- Long-term (24 hr) measurements revealed significant fluorescence increases with Al(3+) and Cu(2+), and decreases with Fe(2+) and Fe(3+).
- Ca(2+), Mn(2+), Mg(2+), and Zn(2+) showed minimal fluorescence changes at both time points.
Conclusions:
- Copper and aluminum ions can interfere with pyoverdin-based assays, even with short reaction times.
- Accurate iron quantification using free pyoverdin requires consideration of potential interferences from other metal ions.
- Assay conditions, including reaction time and the presence of competing cations, are critical for reliable results.
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