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Determination of free Mg2+ in isolated heart mitochondria using fluorescent probes
1Department of Medical Biochemistry, Ohio State Univesity Medical Center, Columbus.
Abstract:
The concentration of free Mg2+ in the matrix of isolated heart mitochondria has been monitored using the fluorescent probe furaptra. The techniques used for loading, calibrating and using furaptra fluorescence to monitor matrix free Mg2+ are described. Furaptra is loaded as the membrane-permeable acetoxymethyl ester which is hydrolyzed to the impermeant acid form by nonspecific esterases. Loading is sufficient in a 20-min period at 25 degrees C to give signals approximately 5-fold greater than nonloaded mitochondria. Uncertainties concerning the apparent dissociation constant value for Mg2+ for entrapped furaptra and the ability of ionophores to equilibrate Mg2+ across the mitochondrial membrane are discussed. We conclude that our best estimate for matrix Mg2+ in isolated mitochondria is 0.5 mM and that it can change significantly with changing Mg2+ ligand availability in the matrix.
Insights
Researchers monitored free Mg2+ concentration in heart mitochondria using the fluorescent probe furaptra. The study estimates matrix Mg2+ at 0.5 mM, highlighting its variability with ligand availability.
Area of Science:
- Biochemistry
- Cell Biology
- Mitochondrial Physiology
Background:
- Mitochondrial function is critically dependent on intracellular magnesium ion (Mg2+) concentration.
- Accurate measurement of matrix free Mg2+ is essential for understanding mitochondrial bioenergetics.
- Previous methods for monitoring matrix Mg2+ have limitations in precision and applicability.
Purpose of the Study:
- To describe and validate the use of the fluorescent probe furaptra for monitoring free Mg2+ concentration in isolated heart mitochondria.
- To establish a reliable method for loading, calibrating, and utilizing furaptra fluorescence.
- To determine the physiological concentration of free Mg2+ within the mitochondrial matrix.
Main Methods:
- Isolated heart mitochondria were loaded with the acetoxymethyl ester form of furaptra.
- The membrane-permeable ester was hydrolyzed to the impermeant acid form by endogenous esterases.
- Fluorescence signals were calibrated and analyzed to quantify matrix free Mg2+ concentration.
- Potential uncertainties regarding furaptra's dissociation constant and ionophore efficacy were addressed.
Main Results:
- Furaptra loading was efficient within 20 minutes at 25°C, yielding a 5-fold increase in signal compared to nonloaded mitochondria.
- The study determined the best estimate for matrix free Mg2+ concentration in isolated mitochondria to be 0.5 mM.
- Significant fluctuations in matrix Mg2+ were observed, correlating with changes in Mg2+ ligand availability.
Conclusions:
- Furaptra is a suitable fluorescent probe for accurately measuring matrix free Mg2+ in isolated heart mitochondria.
- The physiological concentration of matrix free Mg2+ is approximately 0.5 mM.
- Matrix free Mg2+ levels are dynamic and responsive to the availability of intracellular Mg2+ ligands, impacting mitochondrial function.