Effects of monovalent cations, pH and temperature on the dissociation constant (KD) for the fluorescent indicator mag-fura-2 at different excitation wavelengths.

The use of fluorescent indicators to quantify intracellular magnesium ([Mg(2+)]i) requires accurate determination of the dissociation constant (K(D)). Ideally, the K(D) should be determined intracellularly; however, in practice this is difficult to achieve. An alternative method is to measure the K(D) in solutions which attempt to mimic the intracellular milieu. This study investigated the effect of monovalent cations, pH and temperature on the Mg(2+)-mag-fura-2 K(D) determined at individual excitation wavelength intensities of either 340 nm or 380 nm, with emission fluorescence set at 510 nm. Monovalent cation concentration had little effect on K(D). Temperatures in the range 25 degrees C to 37 degrees C had significant effects on K(D) measured at 340 nm and 380 nm. At 340 nm the K(D) values at 25 degrees C and 37 degrees C were 2.02 and 1.15 mM, respectively and at 380 nm 2.84 to 1.68 mM, respectively. Changes of pH in the range 6.5 to 8.5 had variable effects on the K(D) depending on the wavelength at which it was measured. The results illustrate not only how estimation of K(D) can vary with environmental conditions, including temperature and pH. but also the choice of wavelength.