²⁵Mg NMR Studies of magnesium binding to erythrocyte constituents

The binding of Mg²⁺ ion to ATP, ADP, AMP, 2,3-bisphosphyoglycerate (DPG), and hemoglobin has been studied by ²⁵Mg NMR spectroscopy at 9.4 T. Addition of any of these ligands to a solution of 2 mM²⁵MgCl at pH 7.2 caused a progressive increase in linewidth, with no discernible chemical shift. ATP and ADP, which form tight 1:1 complexes with Mg²⁺, did not cause maximal broadening until present in several-fold excess, implying that bis(nucleotide) complexes also form. The studies showed progressively weaker Mg²⁺ binding to ATP, ADP, DPG, and AMP, consistent with published binding constants. Hemoglobin cause fairly little broadening, consistent with its known weak affinity for Mg²⁺. Competition studies determined ATP affinities for Ca²⁺ and H⁺ that were also in good agreement with published values. ²⁵Mg NMR spectra of 2 mM bound ²⁵Mg²⁺ were obtained with good signal to noise in less than 1 hr. The technique may now be a practical means for studying the binding of Mg²⁺ within erythrocytes and other cells.