Reassessment of Fura-2 and the ratio method for determination of intracellular Ca2+ concentrations.

To determine intracellular Ca2+ concentrations more accurately, we examined Kd of Fura-2 for Ca2+ in conditions which were systemically changed. In a solution comprising of 150 mM KCI, 20 mM MOPS-KOH, pH 6.94, 60-100 microM EGTA and 1 microM Fura-2, Kd at 20 degrees C was 0.266 +/- 0.016 microM (mean +/- SEM) (21 determinations). The ionic strength (I) of the solution strongly affected Kd: the relation of -log Kd versus 2 square root of I/(1 + square root of I) - 0.4.I was 3.6 times as steep as that of EGTA. Kd was moderately changed by pH higher than 7.1, while it was very slightly changed by pH between 6.7 and 7.1. Kd was minimally affected by temperature. The apparent Kd values for Ca2+ in the presence of various concentrations of Mg2+ gave an estimate of the Kd for Mg2+ of about 100 mM, which is about 10 times as great as the estimated value by Grynkiewicz et al. [1]. This estimation assumes competitive binding between Ca2+ and Mg2+ for Fura-2. However, the possibility that Mg2+ may bind Fura-2 in a more complicated way is also suggested. Co-existing proteins in the solution dose-dependently increased an apparent Kd, independent of the type of proteins used, up to a limiting value of about 1.0 microM. With the ratio method, the Ca2+ concentration which gives (Rmin + Rmax)/2 is Kd.beta. The range of Ca2+ concentrations on which R values show steep dependence is determined not only by Kd but also by beta. This means that the excitation spectra and pair of excitation wavelengths selected as well as Kd are critical factors.(ABSTRACT TRUNCATED AT 250 WORDS)