Dw. Jung et al., ON THE USE OF FLUORESCENT-PROBES TO ESTIMATE FREE MG2-MITOCHONDRIA( IN THE MATRIX OF HEART), Archives of biochemistry and biophysics, 332(1), 1996, pp. 19-29
The fluorescent probe furaptra shows increases and decreases in the co
ncentration of free magnesium ion, [Mg2+], in the mitochondrial matrix
with changes in total Mg2+ and ligand availability. The factors invol
ved in the calibration of these fluorescence changes in terms of absol
ute [Mg2+] have been investigated, The affinity of furaptra for Mg2+ i
s highly dependent on both temperature and ionic strength. The K-d for
Mg-furaptra in solution in 100 mM KCl was found to be 2.1 +/- 0.1 mM
at 25 degrees C. The use of this K-d to calculate matrix [Mg2+] is mor
e reliable than in situ K-d measurements because ionophores, such as B
rA23187 and ionomycin, do not equilibrate external Mg2+ with the matri
x in an acceptable way. Furaptra is present at high concentrations (up
to 500 mu M) in the matrix when introduced by hydrolysis of the aceto
xymethyl ester. However, absorbance spectra of aqueous solutions show
no evidence of dimerization of the probe or other changes in propertie
s at these concentrations, Fluorescence intensity at 340 nm(ex) is str
ongly attenuated for matrix-sequestered furaptra, mag-fura-5, and mag-
indo-1. This appears to result in part from preferential binding of th
e Mg-probe to mitochondrial proteins, The fluorescence of uncomplexed
furaptra at 375-380 nm(ex) seems unaffected by protein binding, howeve
r, and changes in intensity in this region of the spectrum can be used
in conjunction with the K-d found in aqueous solution to estimate mat
rix [Mg2+]. The presence of secondary equilibria, such as protein bind
ing, and possible changes in ionic strength may undermine exact quanti
tation by this method, However, values for matrix [Mg2+] obtained in t
his way (0.5 to 0.7 mM) correspond well to estimates by other availabl
e methods and each of these methods suffers from comparable uncertaint
ies. (C) 1996 Academic Press, Inc.