Fluorescence lifetime characterization of magnesium probes: Improvement of Mg(2+) dynamic range and sensitivity using phase-modulation fluorometry.

We measured the Mg(2+)-dependent absorption spectra, emission spectra, quantum yields, and intensity decays of most presently available fluorescent magnesium probes. The lifetimes were found to be strongly Mg(2+) dependent for Mag-quin-1, Mag-quin-2, magnesium green, and magnesium orange and increased 2- to 10-fold upon binding of Mg(2+). The lifetimes of Mag-fura-2, Mag-fura-5, Mag-fura red, and Mag-indo-1 were similar in the presence and absence of Mg(2+). Detailed timeresolved measurements were carried out for Mag-quin-2 and magnesium green using phase-modulation fluorometry. Apparent dissociation constants (K d) were determined from the steady-state and time-resolved data. Their values were compared and discussed. Mg(2+) sensing is described using phase and modulation data measured at a single modulation frequency. Phase angle and modulation data showed the possibility of obtaining a wider Mg(2+)-sensitive range than available from intensity measurements. A significant expansion in the Mg(2+)-sensitive range was found for Mag-quin-2 using excitation wavelengths from 343 to 375 nm, where the apparentK d from the phase angle was found to vary from 0.3 to about 100 mM. Discrimination against Ca(2+) was also measured for Mag-quin-2 and magnesium green. Significant phototransformation and/or photode-composition, which affect the sensitivity to Mg(2+), were observed for Mag-quin-2 and magnesium green under intense and long illumination.