Experimental 31P NMR study of the influence of ionic strength on the apparent dissociation constant of MgATP

The classical method for31P NMR determination of intracellular free magnesium concentration ([Mg 2+free]) requires an accurate knowledge of the apparent dissociation constant (K D ) of MgATP. There is a large difference between the previously determined values ofK D . Although the value of 50 µM, determined by a31P NMR method, is now largely accepted, a value of 86 µM has more recently been measured with a fitting method derived from the original one, and with a different ionic strength. The purpose of our study was to assess if the cause of the difference between these two previously reportedK D values was due to the measuring method or to the ionic strength value used. Working at pH=7.2,T=37°C, and [KCl]=0.25 M, we performedK D measurements with the original31P NMR method and with the fitting method. The results (67±13 µM and 61±20 µM, respectively) were not significantly different. Then, with the first method, we measured KD at [KCl]=0.12 M and found a value of 19±5 µM. We conclude that the main cause of difference between theK D values measured by31P NMR reside in the disparity of ionic strength values used for their measurement. OurK D measurements at [KCl]=0.25 and 0.12 M demonstrate the importance of the ionic strength value used for imitating the intracellular medium on the absolute value of ([Mg 2+free]) measured by31P NMR spectroscopy.