On the Use of Fluorescent Probes to Estimate Free Mg2+in the Matrix of Heart Mitochondria

The fluorescent probe furaptra shows increases and decreases in the concentration of free magnesium ion, [Mg2+], in the mitochondrial matrix with changes in total Mg2+and ligand availability. The factors involved in the calibration of these fluorescence changes in terms of absolute [Mg2+] have been investigated. The affinity of furaptra for Mg2+is highly dependent on both temperature and ionic strength. TheKdfor Mg–furaptra in solution in 100 mMKCl was found to be 2.1 ± 0.1 mMat 25°C. The use of thisKdto calculate matrix [Mg2+] is more reliable thanin situ Kdmeasurements because ionophores, such as BrA23187 and ionomycin, do not equilibrate external Mg2+with the matrix in an acceptable way. Furaptra is present at high concentrations (up to 500 μM) in the matrix when introduced by hydrolysis of the acetoxymethyl ester. However, absorbance spectra of aqueous solutions show no evidence of dimerization of the probe or other changes in properties at these concentrations. Fluorescence intensity at 340 nmexis strongly attenuated for matrix-sequestered furaptra, mag-fura-5, and mag-indo-1. This appears to result in part from preferential binding of the Mg-probe to mitochondrial proteins. The fluorescence of uncomplexed furaptra at 375–380 nmexseems unaffected by protein binding, however, and changes in intensity in this region of the spectrum can be used in conjunction with theKdfound in aqueous solution to estimate matrix [Mg2+]. The presence of secondary equilibria, such as protein binding, and possible changes in ionic strength may undermine exact quantitation by this method. However, values for matrix [Mg2+] obtained in this way (0.5 to 0.7 mM) correspond well to estimates by other available methods and each of these methods suffers from comparable uncertainties.