J Biol Chem, Vol. 274, Issue 19, 13375-13383, May 7, 1999

Calcein as a Fluorescent Probe for Ferric Iron
APPLICATION TO IRON NUTRITION IN PLANT CELLS

Fabrice Thomas, Guy Serratrice, Claude Béguin, Eric Saint Aman^§, J. Louis Pierre, Marc Fontecave^¶, and J. Pierre Laulhère^¶

From the  Laboratoire de Chimie Biomimétique, UMR CNRS 5616, Université Joseph Fourier, Grenoble, France B.P.53 38041, Grenoble cedex 9 France, the ^§ Laboratoire d'Electrochimie Organique et de Photochimie Rédox, UMR 5630, Université Joseph Fourier, Grenoble, France B.P.53 38041, Grenoble cedex 9, France, the ^¶ Laboratoire de Chimie et Biochimie des Centres Rédox Biologiques, CEA Grenoble/EP 1087 CNRS/Université Joseph Fourier, 17 rue des Martyrs, 38054 Grenoble cedex 9, France

The recent use of calcein (CA) as a fluorescent probe for cellular iron has been shown to reflect the nutritional status of iron in mammalian cells (Breuer, W., Epsztejn, S., and Cabantchik, Z. I. (1995) J. Biol. Chem. 270, 24209-24215). CA was claimed to be a chemosensor for iron(II), to measure the labile iron pool and the concentration of cellular free iron(II). We first study here the thermodynamic and kinetic properties of iron binding by CA. Chelation of a first iron(III) involves one aminodiacetic arm and a phenol. The overall stability constant log ₁₁₁ of Fe^IIICAH is 33.9. The free metal ion concentration is pFe^III = 20.3. A (Fe^III)₂ CA complex can be formed. A reversible iron(III) exchange from Fe^IIICAH to citrate and nitrilotriacetic acid is evidenced when these ligands are present in large excess. The kinetics of iron(III) exchange by CA is compatible with metabolic studies. The low reduction potential of Fe^IIICAH shows that the ferric form is highly stabilized. CA fluorescence is quenched by 85% after Fe^III chelation but by only 20% using Fe^II. Real time iron nutrition by Arabidopsis thaliana cells has been measured by fluorimetry, and the iron buffer Fe^IIICAH + CA was used as source of iron. As a siderophore, Fe^IIICAH promotes cell growth and regreening of iron-deficient cells more rapidly than Fe^IIIEDTA. We conclude that CA is a good chemosensor for iron(III) in cells and biological fluids, but not for Fe(II). We discuss the interest of quantifying iron buffers in biochemical studies of iron, in vitro as well as in cells.