Comparison between measured and calculated ionised concentrations in Mg2+ /ATP, Mg2+ /EDTA and Ca2+ /EGTA buffers; influence of changes in temperature, pH and pipetting errors on the ionised concentrations.
The apparent dissociation constants (Kapp) and total ligand concentrations ([Ligand]T) from extensive published and unpublished macroelectrode measurements for Mg2+/ATP, Mg2+/EDTA and Ca2+/EGTA buffers have been recalculated. These calculations were made feasible by the introduction of an Excel program which reduced the time of calculation for Kapp and [Ligand]T from over an hour to under five minutes. These estimations of Kapp and [Ligand]T allowed, not only a comparison between measured and calculated ionised magnesium and calcium concentrations ([Mg2+] and [Ca2+]) for Mg2+/ATP, Mg2+/EDTA and Ca2+/EGTA buffers but also a comparison amongst calculated values. Calculated [X2]1 values always differed from measured, and calculated values differed amongst themselves by factors of at least 2. These variations cast doubts on the published absolute values for intracellular [Mg2+] estimated by 31P-NMR and the resting values for [Ca2+] in cells. The allowable range for [X2+] in the buffers and consequently for Kapp and [Ligand]T has not been defined, which introduces uncertainties into published absolute values for [X2+]. This paper shows that an upper limit of +/- 10% deviation from the mean value for [X2+] is attainable. This requires the temperature to be maintained within +/- 0.5 degrees C, pH within +/- 0.01 units and pipetting errors of less than 0.25%. Until internationally defined buffer standards are available, the lack of correlation between measured and calculated [X2+] means that measurement of Kapp and [Ligand]T and hence [X2+] is more reliable than calculation.
McGuigan
John A S
JA
Institute of Biomedical and Life Sciences, West Medical Building, University of Glasgow, G12 8QQ, UK. jasmcguigan@yahoo.com
Kay
James W
JW
Elder
Hugh Y
HY
Lüthi
Daniel
D
D000255:Adenosine Triphosphate / Q000737:chemistry*; D002021:Buffers; D002118:Calcium / Q000737:chemistry*; D002138:Calibration; D002413:Cations, Divalent; D002614:Chelating Agents / Q000737:chemistry*; D004492:Edetic Acid / Q000737:chemistry*; D004533:Egtazic Acid / Q000737:chemistry*; D004563:Electrochemistry / Q000379:methods; D004566:Electrodes; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000737:chemistry*; D008956:Models, Chemical; D009994:Osmolar Concentration; D012015:Reference Standards; D012107:Research Design; D013696:Temperature
D002021:Buffers; D002413:Cations, Divalent; D002614:Chelating Agents; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D008274:Magnesium; D002118:Calcium