The binding of nitrate to the human anion exchange protein (AE1) studied with 14N nuclear magnetic resonance.

The binding of [14N]nitrate to the human erythrocyte anion transport protein, AE1, was studied using 14N nuclear magnetic resonance spectroscopy (14N-NMR). The line-width at half-height of the 14NO3- resonance increased in direct proportion to the concentration of erythrocyte ghost protein. Addition of the AE1 specific inhibitor 4,4'-dinitrostilbene-2,2'-disulfonate markedly reduced this line-broadening, indicating that the broadening was predominantly due to a specific interaction between nitrate and AE1. The dependence of the AE1 specific line-broadening on nitrate concentration had a first-order dissociation constant KD of 6.9 +/- 0.9 mM. In contrast, Cl- interaction with AE1 studied by 35Cl-NMR showed a chloride concentration-dependent line-broadening with a KD of 74 +/- 10 mM, indicating that AE1 has a higher affinity for nitrate than for chloride. Bicarbonate and chloride were found to be competitive inhibitors of the AE1 specific 14NO3- line-broadening (94 +/- 6% and 101 +/- 3% inhibition, respectively). Based on the concentration dependence of inhibition and using a model of competitive inhibition, the KD of bicarbonate binding to AE1 was estimated to be 5.4 +/- 1.3 mM. Nitrate is a structural analog of bicarbonate, making the interaction of nitrate with AE1 a good model for the bicarbonate-AE1 interaction. The 14N-NMR nitrate binding assay, along with the 35Cl-NMR binding assay now in use, will provide a powerful tool for studying the structure of the AE1 binding site for both physiologic substrates, bicarbonate and chloride.