An improvement to the ligand optimisation method (LOM) for measuring the apparent dissociation constant and ligand...

In Ca(2+)/Mg(2+) buffers the calculated ionised concentrations ([X(2+)]) can vary by up to a factor of seven. Since there are no defined standards it is impossible to check calculated [X(2+)], making measurement essential. The ligand optimisation method (LOM) is an accurate method to measure [X(2+)] in Ca(2+)/Mg(2+) buffers; independent estimation of ligand purity extends the method to pK(/) < 4. To simplify calculation, Excel programs ALE and AEC were compiled for LOM and its extension. This paper demonstrates that the slope of the electrode in the pX range 2.000-3.301 deviates from Nernstian behaviour as it depends on the value of the lumped interference, Σ. ALE was modified to include this effect; this modified program SALE, and the programs ALE and AEC were used on simulated data for Ca(2+)-EGTA and Mg(2+)-ATP buffers, to calculate electrode and buffer characteristics as a function of Σ. Ca(2+)-electrodes have a Σ < 10(-6) mol/l and there was no difference amongst the three methods. The Σ for Mg(2+)-electrodes lies between 10(-5) and 1.5 (∗) 10(-5) mol/l and calculated [Mg(2+)] with ALE were around 3% less than the true value. SALE and AEC correctly predicted [Mg(2+)]. SALE was used to recalculate K(/) and pK(/) on measured data for Ca(2+)-EGTA and Mg(2+)-EDTA buffers. These results demonstrated that it is pK(/) that is normally distributed. Until defined standards are available, [X(2+)] in Ca(2+)/Mg(2+) buffers have to be measured. The most appropriate method is to use Ca(2+)/Mg(2) electrodes combined with the Excel programs SALE or AEC.