The total and free concentrations of Ca2+ and Mg2+ inside platelet secretory granules. Measurements employing a novel double null point technique.

The inorganic ion contents of platelet alpha-granules were determined by a combination of neutron activation analysis and flame photometry. Total concentrations were estimated using intragranular water spaces measured by isotope dilution. To measure the free concentrations of Ca2+ and Mg2+ we developed a novel double null point titration technique. The method requires independent determinations of transmembrane delta pH and the availability of two divalent cation-H+ exchange ionophores with different Ca2+/Mg2+ selectivity ratios. A23187 and the halogenated analog 4-bromo-A23187 were used for this purpose. Absolute delta pH was measured by methylamine distribution, while relative pH changes induced by the ionophores were monitored with 9-aminoacridine. The free concentrations of Ca2+ and Mg2+ were found to be 12 and 326 microM, respectively. These values are markedly lower than the calculated total concentrations of these cations, i.e. 32 mM for Ca2+ and 172 mM for Mg2+.