Intracellular free Ca and Mg of human red blood cell ghosts measured with entrapped arsenazo III.

Arsenazo III in human red cell ghosts is calibrated to measure intracellular concentrations of free Ca and free Mg. This calibration was established by comparing the absorbance of arsenazo III in ghosts to its absorption in solution at 600, 630, and 655 nm as a function of buffered free Ca (0.4 microM to 70 microM), free Mg (0.05 to 5 mM), and free Ca (4 to 50 microM) at constant free Mg (1.2 mM) at three concentrations of total dye (1.09, 10.9, and 109 microM). In both ghosts and in solution the absorbance of the dye at all three wavelengths could be predicted from dissociation constants and molar extinction coefficients determined for a 1:1 complex with the dye and Mg, another with Ca, and a third complex consisting of two molecules of Ca and two of dye. The absorbance of the dye in ghosts at the same concentrations of free Ca, free Mg, and total dye is equal to that in solution multiplied by the percentage hematocrit and divided by 100, which demonstrates that arsenazo III responds the same inside ghosts as it does free in solution. The results of this paper show that arsenazo III can be used to measure quantitatively and to monitor continuously the concentration of intracellular Ca and Mg in red cell ghosts. Use of this method should facilitate the study of Ca-dependent mechanisms of red blood cells.