13C NMR quantitation of polymer in deoxyhemoglobin S gels

Abstract

13C/1H magnetic double-resonance spectroscopy has been used to quantitate the amount of polymerized hemoglobin S in deoxygenated gels at 30 degrees C, for samples whose hemoglobin concentration range from 21 to 32 g/dl. Scalar- and dipolar-decoupled spectra and a 13C proton-enhanced dipolar-decoupled spectrum were recorded for each sample as was a scalar-decoupled spectrum for a matching oxyhemoglobin S control. The difference between the oxyhemoglobin S and deoxyhemoglobin S scalar-decoupled spectra was used to determine the polymer fraction, and this value was compared with the polymer fraction determined by using ultracentrifugation sedimentation on the same sample (assuming a two-phase model). The polymer fraction value determined by uncorrected sedimentation averaged 0.15 more than the value obtained from NMR. The discrepancy between the two techniques was largely removed when the analysis of the sedimentation data included a correction for depletion of hemoglobin in the supernatant or sol phase due to sedimentation of free molecules. The best fit to both the sedimentation and NMR data was obtained by using a solubility of deoxyhemoglobin S at 30 degrees C of 17.3 +/- 1 g/dl. These results indicate that the NMR techniques, which do not require separation of the sample into a sol phase and a pellet phase, provide quantitative information about the deoxyhemoglobin S polymer and will be useful for studies of sickle erythrocytes.