Automatic determination of ligand purity and apparent dissociation constant (Kapp) in Ca2+/Mg2+ buffer solutions and the Kapp for Ca2+/Mg2+ anion binding in physiological solutions from Ca2+/Mg2+-macroelectrode measurements

Calibration of Ca2+/Mg2+ macroelectrodes and flurochromes in the nmolar and mu molar range, respectively, require the use of buffer solutions. In these buffers the apparent dissociation constant (K-app) has to be measured since calculation based on tabulated constants gives variable results. The ligand concentration [Ligand](T) has also to be estimated. The most accurate and general method for measuring both is the ligand optimisation method based on macroelectrode potential measurements, but this iterative method is time consuming, thus limiting its application. This paper describes an automatic program based on the method, which on entering the measured macroelectrode data calculates K-app, [Ligand](T) and the ionised concentration [X2+] within minutes. This optimisation method cannot be used at K-app values greater than 0.1 mM, but can be extended into this region if the anion concentration is known. The program has been modified to cover this eventuality. Ca2+/Mg2+ macroelectrodes in conjunction with these programs offer an accurate, routine method for determining K-app and [Ligand](T) in buffer solutions at the appropriate ionic strength, temperature and pH and the K-app for divalent cations binding to physiological anions under experimental conditions. (C) 2007 Elsevier Ltd. All rights reserved.