Experimental³¹p nmr study of the influence of ionic strength on the apparent dissociation constant of mgatp

Abstract

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 K_D 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.