3.1. Metal-Binding Experiments

Metal–anthocyanin binding experiments were carried out according to previously reported procedures [13]. Fresh 5 mM solutions of Fe²⁺ and Al³⁺ were respectively prepared from FeSO₄–7H₂O and AlCl₃–6H₂O (Sigma-Aldrich, St-Quentin Fallavier, France) in 1 mM aqueous HCl. Concentrated stock solutions of pigment (5 mM) were prepared in 50 mM aqueous HCl. Absorption spectra were recorded on an Agilent 8453 diode-array spectrometer in thermostated and magnetically stirred quartz cuvettes (pathlength = 1 cm). The following solutions were directly added to the cuvette in this order: 2 mL of 10 mM phosphate buffer (pH 7 or 8), 20 μL of anthocyanin stock solution and, after a few seconds (negligible formation of colorless forms), a small volume of the 5 mM Fe²⁺ or Al³⁺ solution (final iron/anthocyanin molar ratio = 1 or 2). The full UV-VIS spectra were recorded in kinetic mode for 1 to 2 min. For an optimal sensitivity, the detection in the visible range was set at 550 or 610 nm for Al³⁺ (close to the complex’s λ_max) and at 670 nm for Fe²⁺ (charge-transfer contribution of the Fe³⁺ complexes). The hyperchromic and bathochromic shifts were calculated from the initial (free ligand) and final (metal complex) spectra as (A_max,f − A_max,0)/A_max,0 and λ_max,f − λ_max,0, respectively.

Binding experiments were also carried out in the pH range 2–6 (50 mM acetate buffer) to determine the pH for the onset of metal binding and investigate the competition with water addition to the flavylium ion.

The anthocyanin isolates were diluted to a 50 µM concentration in buffers of pH 6 (0.1 M sodium acetate), 7, and 8 (0.25 M TRIS). Small volumes of concentrated Al₂(SO₄)₃ solutions were then added to reach metal/anthocyanin ratios of 0.5, 1, and 5. Samples were equilibrated for 30 min at room temperature in the dark prior to analysis (in triplicates). UV-VIS spectra were collected from 380 to 700 nm using 300 µL samples in poly-D-lysine-coated polystyrene 96-well plates with a SpectraMax 190 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).