Critical review of the methods used to measure the apparent dissociation constant and ligand purity in Ca2+ and Mg2+...

Using simulated Ca2+ and Mg2+ buffers, methods proposed to measure both ligand purity and the apparent dissociation constant (Kapp) were investigated regarding (1) predicted accuracy of both parameters and (2) generality of the solution. The Bers' Ca2+ macroelectrode method [Bers, D. M., 1982 A simple method for the determination of free [Ca] in Ca-EGTA solutions Am. J. Physiol. 242, C404-C408] cannot be used with Mg2+ -macroelectrodes and is partly arbitrary since the linear part of the Scatchard plot is judged subjectively. Iterative methods have therefore been introduced. Iteration based on Bers' method or the lumped interference in the Nicolsky-Eisenman equation also failed with Mg2+ macroelectrodes. The Oiki et al., method [Oiki, S., Yomamoto, T., Okada, Y., 1994. Apparent stability constants and purity of Ca-chelating agents evaluated using Ca-sensitive electrodes by the double-log optimization method Cell Calcium 15, 209-46.] cannot be applied to Mg2+ macroelectrodes. The pH titration method of Moisescu and Pusch (Pflügers, Arch., 355, R122, 1975) predicted EGTA purity and Ca2+ contamination, but Kapp values for EGTA were approximate. It cannot be applied to Mg2+ binding. The partition method [Godt, R.E., 1974. Calcium-activated tension of skinned muscle fibres of the frog. Dependence on magnesium adenosine triphosphate concentration J. Gen. Physiol. 63, 722-739.] only approximately estimated the K(app). Calibration, maintaining contaminating [Ca2+]/[Mg2+] at < 1micromol l(-1), and setting standards by dilution, is the ultimate check of calculated ionised concentrations, although technically difficult. The macroelectrode method of Lüthi et al. [1997. Calibration of Mg2+ -selective macromolecules down to 1 micromol l(-1) in intracellular and Ca+ - containing extracellular solutions. Exp. Physiol. 82, 453-467] accurately predicted purity and Kapp at pKapp values > 4 and was independent of electrode characteristics. It is considered the method of choice. Macroelectrode primary calibration should be carried out in solutions varying from 0.5 to 10 mmol l(-1) combined with either Ca-EGTA or Mg-EDTA buffers; the [Ca2+] and [Mg2+] in other buffer ligands can be measured in a secondary calibration.