Magnetic resonance studies of the binding of ATP and cations to human hemoglobin.

Abstract

Reversible interactions of ATP, MgATP, and three cations (Mg”‘, Mn”+, and Zn’+) with human oxyand deoxyhemoglobins have been studied at physiological pH and salt concentration by 31P NMR, EPR, and water proton relaxation techniques. In the absence of hemoglobin a value of 45 f 8 pM was obtained for the dissociation constant of MgATP from the 31P chemical shifts of solutions containing sufficiently low levels of Mg2+ and ATP (0.5 to 5 mM) in the presence of 0.15 M KC1 (pH 7.2, T = 25°C). Although hemoglobin does not change the positions of the 31P NMR peaks of ATP or its Mg”+ complex at pH 7.2, the spectra do provide evidence of interactions under aerobic and anaerobic conditions. The presence of deoxyhemoglobin was found to result in appreciable displacement of the apparent equilibrium, Mg”+ + ATP4+ MgATP2-, as measured by phosphorus chemical shifts in favor of ATP thus showing competition between deoxyhemoglobin and Mg2+ for binding ATP. The presence of oxyhemoglobin produces only small changes in the apparent equilibrium as measured by ‘I’P NMR. Both oxyand deoxyhemoglobin significantly broaden the 31P resonances of ATP. A larger broadening of the 31P resonances is observed in the deoxyhemoglobin solutions compared to that in the oxyhemoglobin solutions under similar conditions and reflects the higher affinity of ATP for deoxyhemoglobin. While the affinities of ATP and MgATP for deoxyhemoglobin differ by more than an order of magnitude, our data indicate that their affinities for oxyhemoglobin are comparable. The NMR spectral data are generally consistent with the dissociation constants of Hamasaki and Rose ((1974) J. Biol. Chem. 249,7896). The binding of Mg’+ to human hemoglobin was examined by its effects on the EPR spectra and water proton relaxation enhancement (eg = 9.5) of a solution containing Mn2+ and hemoglobin. It is found that Mg’+ shows little affinity (KDM” = 0.4 -+ 0.1 M) for the four divalent cation sites per tetramer of oxyor deoxyhemoglobin detected here whereas Mn2+ and Zn2+ bind to an appreciable extent (Kn”” = 5 +1 mM; KD~” = 1.3 + 0.4 mM). These results argue against any significant binding of Mg2+ to hemoglobin in red blood cells.