Chemical exchange in tissue extracts revisited: Bicarbonate and deuterium isotope effects on ³¹P resonances of phosphoethanolamine and phosphocreatine

It is not sufficiently appreciated that chemical exchange can markedly affect the appearance of ³¹P tissue extract NMR spectra. In addition to the commonly recognized ³¹P chemical shift effects of divalent metal cation (e.g. Mg²⁺) binding upon ATP resonances, multiple resonances for phosphoethanolamine (PE) and phosphocreatine (PCr) are observed under certain conditions of pH, temperature, and D₂O and bicarbonate concentrations. In the presence of bicarbonate ion (commonly used to neutralize acidic extractions) carbamate formation causes a second ³¹P resonance for PE to appear. This effect has been described previously for ¹³C and ¹H amino acid resonances in tissue extracts [Sherry et al. J. Magn. Reson. 89, 391––398 (1990)]. The observation of a splitting of the PCr ³¹P resonance in aqueous solutions containing D₂O has been recently ascribed to proton scalar coupling but was described earlier in an underappreciated report [Kupriyanov et al. Biochem. Biophys. Res. Comm. 114, 1117–1125 (1983)] as due to a deuterium isotope effect. These effects, carbamate formation and deuterium isotope shift, are verified herein to cause marked shifts in PE and PCr ³¹P resonances. The dependence upon experimental parameters is explored.