Carbon-13-Proton Nuclear Magnetic Double-Resonance Study of Deoxyhemoglobin S Gelation

Recently developed ¹³C-¹H nuclear magnetic double-resonance techniques have been used to study the gelation of deoxyhemoglobin S molecules, both in hemoglobin solutions and within sickle erythrocytes. Only isotropically mobile hemoglobin molecules (τ ;≲ 10^-6 s) are detected in a scalar-decoupled ¹³C NMR spectrum, whereas only the motionally restricted molecules of polymerized hemoglobin are detected in a proton-enhanced ¹³C spectrum. The latter spectrum is obtained with a matched Hartmann-Hahn contact to transfer polarization from protons to carbons (cross-polarization). Both isotropically mobile and polymerized hemoglobin molecules are detected in a dipolar-decoupled ¹³C spectrum. A comparison of integrated intensities obtained from the various types of spectra show that, at 37 °C, ap- proximately 0.4 of the molecules in a 28 g/dL preparation of deoxyhemoglobin S enters the polymer phase, while at 37 g/dL a significantly higher fraction (ca. 0.6) of the hemoglobin is in the polymer phase. As much as 0.8 of the hemoglobin is polymerized in deoxygenated sickled erythrocytes. The cell-free deoxyhemoglobin S preparation at 28 g/dL has the spectral characteristics of a two-phase system. The motionally narrowed signals observed in the scalar-decoupled spectrum indicate that one phase behaves as a low-viscosity solution of isotropically mobile molecules. In contrast, the width of the signal (SI50 ppm) observed in the carbonyl-aromatic region of the proton-enhanced ¹³C spectrum indicates that the second phase, containing the polymerized molecules, behaves as a crystalline solid.