The determination of the free magnesium level in the human red blood cell by 31P NMR.

The phosphorus NMR spectra of intracellular ATP in glycolyzing human red blood cells maintained at 37 degrees C in an atmosphere containing 5% CO2 have been obtained and quantitated under completely aerobic and anaerobic conditions. A comparison of the separation between the alphaP and betaP resonances and the alpha-beta and beta-gamma 31P spin-spin coupling constants of intracellular ATP with appropriate controls directly reveals that 84 +/- 4% and 78 +/- 4% of the total ATP are complexed to Mg2+ in the aerobic and anaerobic states of the cell, respectively. This determination does not differentiate between molecules free in solution and those bound to hemoglobin due to rapid exchange on the NMR time scale of free and complexed ATP. From these data and the prior knowledge of the interactions of various phosphorylated metabolites with hemoglobin and Mg2+, the intracellular concentration of free Mg2+ is determined to be 0.25 +/- 0.07 mM in the aerobic and 0.67 +/- 0.15 mM in the anaerobic state in a sample of normal red blood cells. Knowledge of free Mg2+ permitted a calculation of the distribution of ATP, ADP, glycerate-2,3-P2, and hemoglobin among their free and complexed forms. The results indicate that approximately 40% of the glycerate-2,3-P2 and ATP are complexed to hemoglobin even in aerobic cells and approximately 90% and approximately 60%, respectively, in completely anaerobic cells. The level of free ATP decreases 3-fold anaerobically whereas the MgATP changes very little. The intracellular level of MgADP increases about 2-fold upon deoxygenation. Since the Mg2+ in the red cell is largely complexed, the 3-fold increase in free Mg2+ under fully anaerobic conditions would significantly affect the rates of enzymatic reactions.