An improvement to the ligand optimisation method (LOM) for measuring the apparent dissociation constant and ligand purity in Ca²⁺ and Mg²⁺ buffer solutions

McGuigan, J. A.S., Kay, J. W. and Elder, H. Y. (2014) An improvement to the ligand optimisation method (LOM) for measuring the apparent dissociation constant and ligand purity in Ca²⁺ and Mg²⁺ buffer solutions. Progress in Biophysics and Molecular Biology, 116, 203 - 211. (doi: 10.1016/j.pbiomolbio.2014.04.001) (PMID:24836054)

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Abstract

In Ca2+/Mg2+ buffers the calculated ionised concentrations ([X2+]) can vary by up to a factor of seven. Since there are no defined standards it is impossible to check calculated [X2+], making measurement essential. The ligand optimisation method (LOM) is an accurate method to measure [X2+] in Ca2+/Mg2+ 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 Ca2+-EGTA and Mg2+-ATP buffers, to calculate electrode and buffer characteristics as a function of Σ. Ca2+-electrodes have a Σ < 10−6 mol/l and there was no difference amongst the three methods. The Σ for Mg2+-electrodes lies between 10−5 and 1.5 ∗ 10−5 mol/l and calculated [Mg2+] with ALE were around 3% less than the true value. SALE and AEC correctly predicted [Mg2+]. SALE was used to recalculate K/ and pK/ on measured data for Ca2+-EGTA and Mg2+-EDTA buffers. These results demonstrated that it is pK/ that is normally distributed. Until defined standards are available, [X2+] in Ca2+/Mg2+ buffers have to be measured. The most appropriate method is to use Ca2+/Mg2 electrodes combined with the Excel programs SALE or AEC.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:McGuigan, Prof John and Elder, Dr Hugh and Kay, Dr James
Authors: McGuigan, J. A.S., Kay, J. W., and Elder, H. Y.
College/School:College of Medical Veterinary and Life Sciences > School of Life Sciences
College of Science and Engineering > School of Mathematics and Statistics > Statistics
Journal Name:Progress in Biophysics and Molecular Biology
Publisher:Elsevier
ISSN:0079-6107
ISSN (Online):1873-1732
Published Online:15 May 2014

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