Maria Rotter

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. The catalytic behavior we observe could play a role in intracellular processes.

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.g., oxygen). Thus, understanding how nucleation responds to the presence of oxygen is the key to understanding how sickling proceeds in a physiological context. We have measured the rate of nucleus formation in HbS partially liganded with NO or CO, which we find have equivalent effects in reducing the nucleation rates. We find that hemoglobin must be in the T (tense) quaternary structure for nucleation, but the presence of ligands inhibits nucleus formation even when the correct quaternary structure is present. From these results, we can predict the fraction of cells that will sickle at any given partial ligand saturations. The ability to make such predictions may prove especially useful in designing future therapies, particularly those where the oxygen affinity is perturbed.

Nucleation of Hybrid Polymers in Sickle Cell Disease

Upon deoxygenation, sickle hemoglobin (HbS) can polymerize into complex, 14 stranded polymers via a double nucleation process. While sickle cell hemoglobin nucleation has been well described, the description has focused on nucleation of a single species, deoxyHbS, with perhaps the presence of a crowding non-polymerizing species. However, there are three important cases of polymerization where hybrid polymers are created, and thus the nucleation process in such situations needs to be characterized and tested. First in vivo, polymerization always occurs in the presence of ligands. Second, polymerization is also possible in the presence of normal hemoglobin (HbA, eg. in sickle trait). Finally, antisickling drugs may not bind to all molecules and thus create a heterogeneous population with the opportunity for hybrid polymerization. Thus we have studied polymerization in the presence of HbA, as well as under cases of partial ligation (with CO, NO and O2). Homogeneous nucleation rates have been measured by laser photolysis of the CO-derivative and analysis of the stochastic fluctuations of onset-times. Heterogeneous nucleation is determined by following the exponential growth of light scattering. Existing models for nucleation have been successfully modified to account for the copolymerization probability of hybrid species, and revised models will also be presented. Incorporation of ligands is particularly challenging since it appears necessary to account for tertiary as well as quaternary effects, and these appear to differ depending on the ligands.

Do Different Ligands Produce Different Effects in Sickle Hemoglobin Polymer Growth

Sickle Hemoglobin (HbS) is a variant of human hemoglobin with a point mutation on two subunits. This mutation causes HbS molecules to grow into polymers when the ligands it transports are released it and changes conformation from an R (relaxed) state to a T (tense) state. The polymer mass that grows inside a red blood cell can cause it to become too rigid to deform to pass through tight capillaries. This causes vaso occlusion and is one of many side effects of sickle cell disease. Polymer growth can be measured by fully photolyzing an HbS sample with a laser, thereby causing the solution molecules to release all their ligands and switch into a T-state. However, in vivo, the partial pressure of oxygen rarely falls below 50% which makes the Hb a combination of fully, partially and un-liganded species. Equilibrium and kinetic measurements were done previously on fractional O2, CO and NO species, although a complete systematic comparison has never been conducted to quantify all of the differing data. A comparison of previous data along with new kinetic results will be presented. Partially ligated crystal protein structures will also be employed to rationalize the results.