Water, Ions, and Hemoglobin: Effects on Allostery and Polymerization.

Proteins that function in aqueous solution can be perturbed by the solvent. Here we present experimental studies on two such interactions in the hemoglobin molecule. (1) Hemoglobin's oxygen binding is altered by introduction of crowding species or osmoticants, such as sucrose, through the linked binding of ions such as Cl or CO, but not otherwise.

Calibrating Sickle Cell Disease.

Sickle cell disease is fundamentally a kinetic disorder, in which cells containing the mutated hemoglobin (hemoglobin S; HbS) will cause occlusion if they sickle in the microvasculature, but have minimal (or no) consequences if they sickle in the venous return. Physiologically, sickling always occurs when some ligands are present; nonetheless, the kinetics in the presence of ligands are virtually unstudied. Sickling arises from nucleation-controlled polymer formation, triggered when the HbS loses ligands (e.

Nucleation of sickle hemoglobin mixed with hemoglobin A: experimental and theoretical studies of hybrid-forming mixtures.

Sickle hemoglobin (HbS) is a point mutation of the two β subunits in normal Hb (HbA) that leads to nucleated polymerization and accompanying pathology. We measured the rates of homogeneous and heterogeneous nucleation of HbS in the presence of up to 50% HbA under conditions in which hybrid HbAS molecules will also form. The replacement of 50% of HbS by HbA slows polymerization by factors of ∼100 in the physiological range, which is substantially less than previously thought.

Band 3 catalyzes sickle hemoglobin polymerization.

We have measured homogeneous and heterogeneous nucleation rates of sickle hemoglobin (HbS) in the presence of a strongly binding deletion mutant of the cytoplasmic domain of band 3 (cdb3), a membrane protein known to form dimers and to bind 2 HbS molecules to such a dimer, and we find that it accelerated both rates by a factor of 2. A weakly binding mutant, in contrast showed no impact on nucleation rates, contrary to naïve expectations of a slight enhancement based on the molecular crowding of the solution by the mutant. We find we can explain these phenomena by a model of HbS-cdb3 interaction in which the strong binding mutant, by stabilizing an HbS dimer, catalyzes the nucleation process, while the weak mutant binds only 1 HbS molecule, effectively inactivating it and thereby compensating for the crowding of the solution by the cdb3.

Metastable polymerization of sickle hemoglobin in droplets.

Sickle cell disease arises from a genetic mutation of one amino acid in each of the two hemoglobin beta chains, leading to the polymerization of hemoglobin in the red cell upon deoxygenation, and is characterized by vascular crises and tissue damage due to the obstruction of small vessels by sickled cells. It has been an untested assumption that, in red cells that sickle, the growing polymer mass would consume monomers until the thermodynamically well-described monomer solubility was reached. By photolysing droplets of sickle hemoglobin suspended in oil we find that polymerization does not exhaust the available store of monomers, but stops prematurely, leaving the solutions in a supersaturated, metastable state typically 20% above solubility at 37 degrees C, though the particular values depend on the details of the experiment.

The Hb A variant (beta73 Asp-->Leu) disrupts Hb S polymerization by a novel mechanism.

Polymerization of a 1:1 mixture of hemoglobin S (Hb S) and the artificial mutant HbAbeta73Leu produces a dramatic morphological change in the polymer domains in 1.0 M phosphate buffer that are a characteristic feature of polymer formation. Instead of feathery domains with quasi 2-fold symmetry that characterize polymerization of Hb S and all previously known mixtures such as Hb A/S and Hb F/S mixtures, these domains are compact structures of quasi-spherical symmetry.

Heterogeneous nucleation in sickle hemoglobin: experimental validation of a structural mechanism.

Sickle hemoglobin polymerizes by two types of nucleation: homogeneous nucleation of aggregates in solution, and heterogeneous nucleation on preexisting polymers. It has been proposed that the same contact that is made in the interior of the polymer between the mutant site beta6 and its receptor pocket on an adjacent molecule is the primary contact site for the heterogeneous nucleus. We have constructed cross-linked hybrid molecules in which one beta-subunit is from HbA with Glu at beta6, and the other is from HbS with a Val at beta6.

Molecular crowding limits the role of fetal hemoglobin in therapy for sickle cell disease.

The dominant assumption central to most treatments for sickle cell anemia has been that replacement of sickle hemoglobin (HbS) by fetal hemoglobin (HbF) would have major clinical benefit. Using laser photolysis, we have measured polymerization kinetics including rates of homogeneous and heterogeneous nucleation on mixtures of 20% and 30% HbF with HbS. We find that the present model for polymerization, including molecular crowding, can accurately predict the rates of such mixtures, by using the single assumption that no significant amount of HbF enters the polymer.

The effects of erythrocyte membranes on the nucleation of sickle hemoglobin.

Pathology in sickle cell disease begins with nucleation-dependent polymerization of deoxyhemoglobin S into stiff, rodlike fibers that deform and rigidify red cells. We have measured the effect of erythrocyte membranes on the rate of homogeneous nucleation in sickle hemoglobin, using preparations of open ghosts (OGs) with intact cytoskeletons from sickle (SS) and normal adult (AA) red cells. Nucleation rates were measured by inducing polymerization by laser photolysis of carboxy sickle hemoglobin and observing stochastic variation of replicate experiments of the time for the scattering signals to reach 10% of their respective maxima.

Crowding and the polymerization of sickle hemoglobin.

Under physiological conditions, sickle hemoglobin, a natural mutant of human hemoglobin A with a surface hydrophobic valine in place of a negatively charged glutamic acid, polymerizes at high volume occupancy. Equilibrium solubility of sickle hemoglobin entails activity coefficients that can approach 10(3) at high concentrations. Polymerization occurs by homogeneous and heterogeneous nucleation mechanisms, which are both profoundly sensitive to crowding; homogeneous nucleation rates for example are enhanced by 10(10) when the initial concentration is augmented by 50% non-polymerizing hemoglobin.