Measurement of the hemoglobin concentration in deoxyhemoglobin S polymers and characterization of the polymer water compartment.

Biological polymers contain freely exchangeable water within intermolecular crevices with restricted access to large extrapolymer solutes. Our recent studies highlighted large osmotic effects of such polymer water compartments (PWCs), and their substantial physiological and pathophysiological relevance. The size and accessibility of the PWC are critical parameters determining the polymers' osmotic properties. We report here a new experimental approach to investigate these parameters in deoxyhemoglobin S polymers. The size of the PWC is inversely related to the deoxyhemoglobin S concentration in the polymer (CP). Only an approximation of CP (approximately 69 g/dl) was previously available. By analyzing the distributions of soluble hemoglobin and a large molecular weight (MW) marker (14C-dextran, MW approximately 70kDa) in the supernatant and pellet of centrifuged gels, we obtained a reproducible value of CP, 54.7 (+/- 0.7)g/dl. This indicates that 60% of the polymer is composed of a water compartment inaccessible to soluble Hb and other non-interactive macromolecules. The accessibility properties of this PWC to smaller molecules were explored with markers of different MW. Non-interactive markers with MW < 200 kDa diffused freely in the PWC, whereas those with 300 kDa < MW < 1000 kDa showed partial exclusion. Higher MW markers were generally excluded, except molecules with elongated (rather than spherical) shapes or possible interactivity with hemoglobin. These results predict that dense sickle cells would significantly dehydrate on deoxygenation, generating a PWC of up to 60% to 80% of the cell water. Soluble enzymes would concentrate in the residual cytosol. For osmotic equilibrium, most of the ions and low MW substrates would concentrate in the PWC. Oxygenation-deoxygenation would thus cause dynamic oscillations in cell hydration and between states of single and double cytoplasmic water phases, the latter with a substantially altered internal environment. The relevance of such oscillations to the membrane and metabolic abnormalities of dense sickle cells requires further investigation.