Short-sweep capillary electrophoresis with a selective zinc fluorescence imaging reagent FluoZin-3 for determination of free and metalothionein-2a-bound Zn2+ ions

A capillary electrophoretic (CE) method using a short-sweep approach and laser-induced fluorescence (LIF) detection (ShortSweepCE-LIF) was developed for determination of Zn²⁺ and Cd²⁺ as complexes with highly selective and sensitive fluorescent probe FluoZin-3. The ShortSweepCE-LIF method, established in this work, can be used for examining competitive Zn²⁺ and Cd²⁺ binding properties of metalloproteins or peptides. The parameters including background electrolyte composition, injection pressure and time as well as separation voltage were investigated. Under the optimized conditions, 80 mM HEPES, pH 7.4, with 1.5 μM FluoZin-3 was used as an electrolyte, hydrodynamic injection was performed at 50 mbar for 5 s, and separation voltage of 25 kV. Limits of detection for Zn²⁺ and Cd²⁺ were 4 and 125 nM, respectively. The developed method was demonstrated in a study of interactions between metalothionein-2a isoform and metal ions Zn²⁺, Co²⁺ and Cd²⁺. It was found that FluoZin-3 was able to extract a single Zn²⁺ ion, while added Co²⁺ (in surplus) extracted only 2.4 Zn²⁺ ions, and Cd²⁺ extracted all 7 Zn²⁺ ions present in the metalothionein molecule.