Measurement of the dissociation constant of MgATP at physiological nucleotide levels by a combination of 31P NMR and optical absorbance spectroscopy.

To resolve a controversy in the literature concerning the affinity of Mg++ for ATP to be used in our noninvasive 31P NMR procedure for the determination of free Mg++ in living cells, we have reinvestigated the apparent dissociation constant of MgATP under physiologic ionic conditions and over the cellular range of ATP concentrations by a combination of NMR and optical absorbance techniques. The new combination method utilizes 31P NMR chemical shifts to determine the degree of Mg++ chelation of ATP in a solution containing free ATP and MgATP, and uses a properly calibrated indicator dye, antipyrylazo III, for optical measurement of free Mg++ in the same solution. The data yield an average value of 50 +/- 10 microM for the apparent dissociation constant of MgATP which indicates low levels of free Mg++ (less than 1 mM) in several different types of tissues, including perfused heart muscle, contrary to a recent report in the literature.