Intermolecular interactions of oxygenated sickle hemoglobin molecules in cells and cell-free solutions.

We have measured the intermolecular interactions of oxygenated sickle hemoglobin molecules in cells and in cell-free solutions, and have compared the results with similar data for liganded normal adult hemoglobin. The experiments involve the measurement of the spin-lattice relaxation time T1 of protons of solvent water molecules, as a function of an externally applied static magnetic field. From such data, one can derive a correlation time tauc, for each sample, which is a measure of the time taken for a hemoglobin molecule to randomize its orientation due to Brownian motion. Thus tauc is a measure of the freedom of rotational motion, on a molecular or microscopic level, of hemoglobin molecules. Intermolecular interactions will reduce this freedom of motion and lengthen tauc. We find that oxygenated sickle hemoglobin molecules have an additional intermolecular interaction not found for normal hemoglobin. This extra interaction is increased by the presence of either inorganic phosphate or diphosphoglycerate, and is greater for sickle hemoglobin within cells than in cell-free solutions. By comparing the present results with published data on the viscosity of oxygenated sickle and normal hemoglobin, we conclude that, at concentrations comparable to intracellular values, oxygenated sickle hemoglobin molecules form aggregates several tetramers in size. The possibility exists that these aggregates are the earliest stage of fiber formation itself, the physical basis of the sickling phenomena.