| Fluorescent tetracarboxylate indicator dyes are used to measure intracellular calcium fluxes that occur in response to biological stimuli. This is hugely accomplished with steady-state fluorescence techniques, e.g., spectral shifts and/or intensity enhancements. Implicit is the assumption that intracellular probe behavior mimics that in solution and that changes in observed signals are due solely to calcium binding. Steady-state fluorescence methods have several limitations such as the ability to resolve multiple cation-specific changes and distinguish cation-binding from other environmental effects. In these studies, steady-state, phase-resolved, and time-resolved fluorescence techniques were used to characterize the behavior of calcium-chelating probes, e.g., Quin2 and Calcium Green, in response to environmental perturbations, multiple divalent cations, and biopolymers. A fluorescence technique for characterizing ligand binding is evaluated and applied to calcium binding by Quin2 and Calcium Green. It is shown that calcium, cadmium, zinc, and magnesium complexes of Quin2 display unique emission spectra, anisotropy values, and/or fluorescence decay times. The combination of steady-state and time-resolved parameters provide sufficient information to resolve complex mixtures, generate decay associated spectra, and evaluate the effect of magnesium on Quin2 binding of calcium. Steady-state emission spectra and anisotropy, gel permeation chromatography, and resonance energy transfer served as qualitative and quantitative measures of probe-protein interactions. Global analysis programs were applied to the study of probe-protein interactions and their effects on calcium binding as well as to unravel complexities in solution, in artificial membrane systems, and in cells. Kinetic phase-resolved and time-resolved fluorescence measurements were made on Quin2-1oaded A431 cells and were used to study ionomycin-mediated calcium transport into these cells. |
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