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.
Center for Fluorescence Spectroscopy and Medical Biotechnology Center, Department of Biological Chemistry, University of Maryland School of Medicine, 108 North Greene Street, 21201, Baltimore, MD.
H
H
Szmacinski
J R
JR
Lakowicz