Evaluation of mag-fura-5, the new fluorescent indicator for free magnesium measurements.

The new fluorescent indicator, mag-fura-5, was evaluated for its ability to measure accurately physiological changes in cytosolic free magnesium. The apparent dissociation constants (Kd) of the fluorochrome for Mg2+, Mg2+/EGTA and Ca2+/EGTA solutions were 14.7 mM, 15.4 mM, and 1.8 mM respectively. The calculated difference in the fluorescence ratios and in the resulting pMg between the standards with low-Ca2+ or low H+ backgrounds and the corresponding samples with approximately physiological levels were not significant. In contrast, the changes due to an increased Ca2+ or H+ content were statistically significant, with mean pMg differences of 0.10 +/- 0.09 (P < 0.02) and 0.33 +/- 0.26 (P < 0.01) respectively. Repetitive measurements on 3 consecutive days yielded comparable data with differences not exceeding 4%. Because of the good reproducibility, it is suggested that the new fluorescent probe may be suitable for free cytosolic magnesium determinations in isolated cells.