Nuclear Magnetic Resonance Studies of Hemoglobin and Red Blood Cell Function

Pulse and Fourier transform nuclear magnetic resonance techniques have been employed to study various aspects of hemoglobin and red blood cell function. The binding of 13C-enriched carbon monoxide to myoglobins and hemoglobins from a variety of animal species has been studied by 13C NMR. The environments experienced by 13CO bound to α or β subunits, the relative facility with which oxygen displaces 13CO, and the relative thermodynamic affinities of the unliganded subunits for 13CO are found to differ. These results clearly demonstrate the various degrees of nonequivalence that may exist between the subunits of normally occurring hemoglobins which must be accounted for in any physical model of hemoglobin ligation if it is to accurately describe hemoglobin function. The chemical shifts of 13CO bound to reconstituted myoglobins and hemoglobins containing synthetically modified hemes have been studied and show that the degree of electronic interaction between the heme and the bound ligand may be modulated by the nature of the surrounding protein. T1 and NOE studies indicate that the bound 13CO ligand interacts strongly with the valine residue (E11) and forms a hydrogen bond with the distal histidine residue (E7) in the ligand binding pocket. These results provide new information about the manner in which the protein environment controls the chemistry and reactivity of the heme. Observation of the 31P resonances of inorganic phosphate and 2,3-diphosphoglycerate in whole blood has led to the development of a noninvasive technique for the determination of intracellular pH. This technique is generally applicable for the study of intracellular pH in a large number of different cell and vesicle systems. The reactions of 13C-enriched cyanate with human adult and sickle cell hemoglobins have also been studied by 13C NMR. Results were obtained for the pH dependence of carbamylation for the N-terminal amino groups and lysine residues of these hemoglobins, both in purified hemoglobin solutions and in whole blood. These studies suggest that maximal effects of cyanate in clinical in vitro treatments will be realized when cyanate concentrations are kept below 10-20 mM and reaction pH is in the range 6.5-7.0. Natural abundance 13C NMR has been used to study the solution conformations of human and rabbit hemoglobins. The results suggest that average hemoglobin quaternary conformations are ligand dependent. Methemoglobin is found to have a solution quaternary conformation roughly intermediate between those of oxyhemoglobin and deoxy hemoglobin at neutral pH.