Effect of Magnesium Chelation on the 31P Spectra of ATP-An Undergraduate Biochemistry Lab Experiment

³¹ P NMR spectra of 100 mM solutions of ATP in 100 mM Tris buffer, pH 7.4, were measured with various concentrations of magnesium ion in order to develop an undergraduate biochemistry laboratory illustrating how magnesium binding effects the ATP phosphates. A downfield shift of 95.55 ppm was observed for the b-phosphate peak at 200 mM magnesium when compared to the spectrum of ATP without added magnesium, as well as a shift of 52.4 ppm for the g-phosphate, and a small upfield shift of 15.4 ppm for the a-phosphate. A ³¹ P NMR spectrum of ATP under the same conditions with 50 mM calcium ion instead of magnesium ion showed b-, g-, and a-phosphate downfield shifts of 69.36, 30.83, and 6.16 ppm, respectively. The significance of the chelations on the role of Mg ⁺² in ATP enzymology is discussed.