Robin W. Briehl

Effects of pH, 2,3-diphosphoglycerate and salts on gelation of sickle cell deoxyhemoglobin.

Abstract Gelation of fully deoxygenated sickle cell hemoglobin was assayed by (1) determination of the temperature at which viscosity increased sharply and (2) a high-speed sedimentation equilibrium method in which three zones are seen. These are a pre-gelation zone, a narrow transition zone exhibiting aggregation, followed by a phase change and a zone of gelation. Only the first zone is seen with deoxyhemoglobin A and CO hemoglobins A and S up to about 0·35 g protein/ml. Minimal gelling temperatures by the viscosity method and, by ultracentrifugation, minimal gelling concentrations determined at the onset of aggregation and at the phase change showed: (a) lowering the pH toward 6·7 favors gelation; (b) deoxyhemoglobin S gels more readily in 6 m m -2,3-diphosphoglycerate than in its total absence; (c) 1 m -NaCl and l m -KCl inhibit gelation. The known favoring of gelation by warming is confirmed by the equilibrium method and is about 2% change in minimal gelling concentration per degree. The effects of pH and high ionic strengths are consistent with contributions of specific polar interactions to gel structure. The effect of 2,3-diphosphoglycerate probably depends on known structural changes which this cofactor induces rather than on alteration of the allosteric quaternary structure equilibrium.

Nonideality and the Nucleation of Sickle Hemoglobin

The homogeneous and heterogeneous nucleation kinetics of sickle hemoglobin (HbS) have been studied for various degrees of solution crowding by substitution of cross-linked hemoglobin A, amounting to 50% of the total hemoglobin. By cross-linking hemoglobin A, hybrid formation between hemoglobin A and hemoglobin S was prevented, thus simplifying the analysis of the results. Polymerization was induced by laser photolysis, and homogeneous nucleation kinetics were determined by observation of the stochastic behavior of the onset of light scattering. Heterogeneous nucleation was determined by observing the exponential growth of the progress curves, monitored by light scattering. At concentrations between 4 and 5 mM tetramer (i.e., approximately 30 g/dl), the substitution of 50% HbA for HbS slows the reaction by a factor of 10(3) to 10(4). Using scaled particle theory to account for the crowding of HbA, the observed decrease in the homogeneous nucleation rate was accurately predicted, with no variation of parameters required. Heterogeneous nucleation, on the other hand, is not well described in the present formulation, and the theory for this process appears to require modification of the way in which nonideality is introduced. Nonetheless, the accuracy of the homogeneous nucleation description suggests that such an approach may be useful for other assembly processes that occur in a crowded intracellular milieu.

Gelation of sickle cell haemoglobin: II. Methaemoglobin☆

Abstract Sickle cell methaemoglobin was assayed for gel formation by an equilibrium ultracentrifugation method previously described. A phase change from sol to gel, indicative of gelation, occurred, depending on conditions, at concentrations between 0.35 and 0.5 g/ml, considerably higher than concentrations observed previously for gelation of deoxyhaemoglobin S. Inositol hexaphosphate favours gelation, but gelation is seen also in its absence. Lowering pH toward 6 favours gelation. If gelation is assumed to require molecules in the T quaternary conformation, these results provide further evidence that methaemoglobin exists in R-T equilibrium in solution and that this equilibrium lies between the extremes exhibited by deoxyhaemoglobin (T-state) and carbon monoxide or oxyhaemoglobin (R-state).

Gelation of sickle hemoglobin. III. Nitrosyl hemoglobin

Abstract Sickle cell nitrosyl hemoglobin was examined for gelation by an ultracentrifugal method previously described (Briehl & Ewert, 1973) and by birefringence. In the presence of inositol hexaphosphate gelation which exhibited the endothermic temperature dependence seen in gels of deoxyhemoglobin S was observed by both techniques. In the absence of inositol hexaphosphate no gelation was observed, nor did nitrosyl hemoglobin A exhibit gelation. On the assumption that gelation is dependent on the deoxy or T (low ligand affinity) as opposed to the oxy or R (high ligand affinity) quaternary structure this supports the conclusion that nitrosyl hemoglobin S in inositol hexaphosphate assumes the T structure, in contrast to the other liganded ferrohemoglobin derivatives oxy and carbon monoxide hemoglobin. Assuming further that the quaternary structures and isomerizations are the same in hemoglobins A and S it can also be concluded that nitrosyl hemoglobin A in inositol hexaphosphate assumes the T state. Since no gelation was seen in stripped nitrosyl hemoglobin S, inositol hexaphosphate serves to effect an R to T switch in this derivative. Thus R-T isomerization in nitrosyl hemoglobin occurs without change in ligand binding at the sixth position of the heme group confirming the conclusion of Salhany (1974) and Salhany et al. (1974). Lowering of the pH toward 6 favors gelation of NO hemoglobin S as it does of deoxy and aquomethemoglobin S (Briehl & Ewert, 1973,1974), consistent with a favoring of the T structure due to strengthening of the interchain salt bridges and the binding of inositol hexaphosphate and/or changes in site-to-site interactions on which gelation depends.

Sickle hemoglobin fibers: mechanisms of depolymerization.

We examined the depolymerization of hemoglobin (Hb) S fibers in the presence of CO by using photolysis of COHbS to create and isolate individual fibers, then removing photolysis to induce depolymerization. Depolymerization occurs at two sites, fiber ends and fiber sides, with different kinetics and by different mechanisms. At low partial pressure of CO (pCO), end-depolymerization is dominant, proceeding at approximately 1 microm s(-1), whereas at high pCO fibers vanish very rapidly, in much less than one second, by side-depolymerization. Each kind of depolymerization could occur by a ligand-independent path, in which deoxyHb depolymerizes and then is prevented from returning to the polymer by liganding with CO, or by a ligand-dependent path in which CO binds to the polymer inducing dissociation of the newly liganded molecules from it. We find that ligand-independent depolymerization is the dominant path for end-depolymerization and ligand-dependent depolymerization dominates, at least at high pCO, for side-depolymerization. On the basis of our kinetic results and electron micrographs of depolymerizing fibers, we propose a model for side-depolymerization in which a hole is nucleated by cooperative loss of a few molecules from fiber sides, followed by rapid depolymerization from the newly created fiber ends abutting the hole. Potential significance of these results for the pathophysiology of sickle cell disease is discussed.

Length distributions of hemoglobin S fibers

Electron microscopy of sickle cell hemoglobin fibers fixed at different times during gelation shows an exponential distribution of fiber lengths, with many short fibers and few long ones. The distribution does not change significantly with time as polymerization progresses. If this distribution of lengths reflects kinetic mechanism of fiber assembly, it complements information from studies of the progress of average properties of the polymers and, as has been done for other rod-like polymerizing systems, permits testing of models for the mechanism of fiber assembly. In this case, the results are consistent with the double nucleation model of Ferrone et al. or with a related alternative model based on fiber breakage. However, other possible causes of this microheterogeneity exist, including: breakage due to solution shearing of the long, rod-like, fibers; the presence of residual nuclei; equilibrium relations governing polymerization; and breakage of solid-like but weak gels that develop early and adhere to the grid. The arguments against the first three of these possibilities suggest that they are not responsible. However, breakage of entanglements or cross-links in a solid-like and adherent gel is consistent with the distributions.

Universal Metastability of Sickle Hemoglobin Polymerization

Sickle hemoglobin (HbS) polymerization occurs when the concentration of deoxyHbS exceeds a well-defined solubility. In experiments using sickle hemoglobin droplets suspended in oil, it has been shown that when polymerization ceases the monomer concentration is above equilibrium solubility. We find that the final concentration in uniform bulk solutions (i.e., with negligible boundaries) agrees with the droplet measurements, and both exceed the expected solubility. To measure hemoglobin in uniform solutions, we used modulated excitation of trace amounts of CO in gels of HbS. In this method, a small amount of CO is introduced to a spatially uniform deoxyHb sample, so that less than 2% of the sample is liganded. The liganded fraction is photolyzed repeatedly and the rate of recombination allows the concentration of deoxyHbS in the solution phase to be determined, even if polymers have formed. Both uniform and droplet samples exhibit the same quantitative behavior, exceeding solubility by an amount that depends on the initial concentration of the sample, as well as conditions under which the gel was formed. We hypothesize that the early termination of polymerization is due to the obstruction in polymer growth, which is consistent with the observation that pressing on slides lowers the final monomer concentration, making it closer to solubility. The thermodynamic solubility in free solution is thus achieved only in conditions with low polymer density or under external forces (such as found in sedimentation) that disrupt polymers. Since we find that only about 67% of the expected polymer mass forms, this result will impact any analysis predicated on predicting the polymer fraction in a given experiment.

Fiber Depolymerization: Fracture, Fragments, Vanishing Times, and Stochastics in Sickle Hemoglobin

The well-characterized rates, mechanisms, and stochastics of nucleation-dependent polymerization of deoxyhemoglobin S (HbS) are important in governing whether or not vaso-occlusive sickle cell crises will occur. The less well studied kinetics of depolymerization may also be important, for example in achieving full dissolution of polymers in the lungs, in resolution of crises and/or in minimizing gelation-induced cellular damage. We examine depolymerization by microscopic observations on depolymerizing HbS fibers, by Monte Carlo simulations and by analytical characterization of the mechanisms. We show that fibers fracture. Experimental scatter of rates is consistent with stochastic features of the analytical model and Monte Carlo results. We derive a model for the distribution of vanishing times and also show the distribution of fracture-dependent fiber fragment lengths and its time dependence. We describe differences between depolymerization of single fibers and bundles and propose models for bundle dissolution. Our basic model can be extended to dissolution of gels containing many fibers and is also applicable to other reversible linear polymers that dissolve by random fracture and end-depolymerization. Under the model, conditions in which residual HbS polymers exist and facilitate repolymerization and thus pathology can be defined; whereas for normal polymers requiring cyclic polymerization and depolymerization for function, conditions for rapid cycling due to residual aggregates can be identified.