MEASUREMENT OF THE HEMOGLOBIN CONCENTRATION IN DEOXYHEMOGLOBIN-S POLYMERS AND CHARACTERIZATION OF THE POLYMER WATER COMPARTMENT

Biological polymers contain freely exchangeable water within intermole cular crevices with restricted access to large extrapolymer solutes. O ur recent studies highlighted large osmotic effects of such polymer wa ter compartments (PWCs), and their substantial physiological and patho physiological relevance. The size and accessibility of the PWC are cri tical parameters determining the polymers' osmotic properties. We repo rt 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 (C-P). Only an a pproximation of C-P (approximate to 69 g/dl) was previously available. By analyzing the distribution a of soluble hemoglobin and a large mol ecular weight (MW) marker (C-14-dextran, MW approximate to 70kDa) in t he supernatant and pellet of centrifuged gels, we obtained a reproduci ble value of C-P. 54.7 (+/-0.7)g/dl. This indicates that 60% of the po lymer is composed of a water compartment inaccessible to soluble Hb an d other non-interactive macromolecules. The accessibility properties o f this PWC to smaller molecules were explored with markers of differen t MW. Non-interactive markers with MW < 200 kDa diffused freely in the PWC, whereas those with 300 kDa < MW < 1000 kDa showed partial exclus ion. Higher MW markers were generally excluded, except molecules with elongated (rather than spherical) shapes or possible interactivity wit h hemoglobin. These results predict that dense sickle cells would sign ificantly dehydrate on deoxygenation, generating a PWC of up to 60% to 80% of the cell water. Soluble enzymes would concentrate in the resid ual cytosol. For osmotic equilibrium, most of the ions and low MW subs trates would concentrate in the PWC. Oxygenation-deoxygenation would t hus cause dynamic oscillations in cell hydration and between states of single and double cytoplasmic water phases, the latter with a substan tially altered internal environment. The relevance of such oscillation s to the membrane and metabolic abnormalities of dense sickle cells re quires further investigation.