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

Calculated and measured [Ca2+] in buffers used to calibrate Ca2+ macroelectrodes

“…The two solutions are then mixed in the appropriate ratios to give the 10 buffer solutions (see Ref. [3]) in which the total calcium ([Ca] T ) is known but the ionized [Ca 2+ ] needs to be determined. Sufficient NaOH was substituted for NaCl to compensate for the acidity of the ligands so that the pH of both the ligand solution and the Ca-ligand solution was just slightly more alkaline than pH 7.4.…”

“…It has been shown that such calculations can be seriously misleading [1], and this has major repercussions for fields in which precise buffering of [Ca 2+ ] is essential such as in patch clamping, measurement of intracellular [Ca 2+ ], and molecular biology. Given the crucial role that [Ca 2+ ] and [Mg 2+ ] play in determining the behavior of numerous physiologically important proteins [2], the lack of precision in Ca 2+ and Mg 2+ buffering may well rank high on the list of factors contributing to the variability of experimental outcomes in physiology and biology.In a review of the various methods to measure the ionized concentrations in buffer solutions [3], the ligand optimization method[4] based on Ca 2+ /Mg 2+ macroelectrodes was shown to be the most accurate. Using this method, it was possible to determine both ligand purity and the [Ca 2+ ] and [Mg 2+ ] in Ca 2+ -EGTA [ethylene glycol-bis(b-aminoethylether)-N,N,N|,N|-tetraacetic acid], Mg 2+ -ATP (adenosine-5 0 -triphosphate), and Mg 2+ -EDTA [2,2 0 ,2 00 ,2 000 -(ethane-1,2-diyldinitrilo)tetraacetic acid] buffers.…”

“…In a review of the various methods to measure the ionized concentrations in buffer solutions [3], the ligand optimization method…”

“…In a review of the various methods to measure the ionized concentrations in buffer solutions [3], the ligand optimization method [4] based on Ca 2+ /Mg 2+ macroelectrodes was shown to be the most accurate. Using this method, it was possible to determine both ligand purity and the [Ca 2+ ] and [Mg 2+ ] in Ca 2+ -EGTA [ethylene glycol-bis(b-aminoethylether)-N,N,N|,N|-tetraacetic acid], Mg 2+ -ATP (adenosine-5 0 -triphosphate), and Mg 2+ -EDTA [2,2 0 ,2 00 ,2 000 -(ethane-1,2-diyldinitrilo)tetraacetic acid] buffers.…”

“…In this study, the reliability of the ligand optimization method was further scrutinized by (i) measuring the pCa of buffer solutions using not only EGTA as previously [1] but also BAPTA [1,2- [3]. The excellent correlation among the results obtained by the four buffers and the agreement of the EGTA purity estimated by the ligand optimization method and the pH method underpins the accuracy of this method for the determination of the [Ca 2+ ] and/or [Mg 2+ ] in the buffers.…”