Deoxy Sickle Cell Hemoglobin Research Articles

A coarse-grain molecular dynamics model for sickle hemoglobin fibers

The intracellular polymerization of deoxy-sickle cell hemoglobin (HbS) has been identified as the main cause of sickle cell disease. Therefore, the material properties and biomechanical behavior of polymerized HbS fibers is a topic of intense research interest. A solvent-free coarse-grain molecular dynamics (CGMD) model is developed to represent a single hemoglobin fiber as four tightly bonded chains. A finitely extensible nonlinear elastic (FENE) potential, a bending potential, a torsional potential, a truncated Lennard-Jones potential and a Lennard-Jones potential are implemented along with the Langevin thermostat to simulate the behavior of a polymerized HbS fiber in the cytoplasm. The parameters of the potentials are identified via comparison of the simulation results to the experimentally measured values of bending and torsional rigidity of single HbS fibers. After it is shown that the proposed model is able to very efficiently simulate the mechanical behavior of single HbS fibers, it is employed in the study of the interaction between HbS fibers. It is illustrated that frustrated fibers and fibers under compression require a much larger interaction force to zipper than free fibers resulting in partial unzippering of these fibers. Continuous polymerization of the unzippered fibers via heterogeneous nucleation and additional unzippering under compression can explain the formation of HbS fiber networks and consequently the wide variety of shapes of deoxygenated sickle cells.

Polymerization of Deoxy-Sickle Cell Hemoglobin in High-Phosphate Buffer

Deoxy-sicklecell hemoglobin (HbS) polymerizes in 0.05 M phosphate buffer to form long helical fibers. The reaction typically occurs when the concentration of HbS is about 165 mg/ml. Polymerization produces a variety of polymorphic forms. The structure of the fibers can be probed by using site-directed mutants to examine the effect of altering the residues involved in intermolecular interactions. Polymerization can also be induced in the presence of 1.5 M phosphate buffer. Under these conditions polymerization occurs at much lower concentrations (ca. 5 mg/ml), which is advantageous when site-directed mutants are being used because only small quantities of the mutants are available. We have characterized the structure of HbS polymers formed in 1.5 M phosphate to determine how their structures are related to the polymers formed under more physiological conditions. Under both sets of conditions fibers are the first species to form. At pHs between 6.7 and 7.3 fibers initially form bundles and then crystals. At lower pHs fibers form macrofibers and then crystals. Fourier transforms of micrographs of the polymers formed in 1.5 M phosphate display the 32- and 64-Å−1 periodicity characteristic of fibers formed in 0.05 M phosphate buffer. The 64-Å−1 layer line is less prominent in Fourier transforms of negatively stained fibers formed in 1.5 M phosphate possibly because salt interferes with staining of the fibers. However, micrographs and Fourier transforms of frozen hydrated fibers formed in high and low phosphate display the same periodicities. Under both sets of reaction conditions HbS polymers form crystals with the same unit cell parameters as Wishner–Love crystals (a = 64 Å, b = 185 Å, c = 53 Å). Some of the polymerization intermediates were examined in the frozen-hydrated state in order to determine whether their structures were significantly perturbed by negative staining. We have also carried out reconstructions of the frozen-hydrated fibers in high and low phosphate to compare their molecular coordinates. The helical projection of the reconstructions in low phosphate shows the expected 14-strand structure. In high phosphate the 14-strand fibers are also formed and their molecular coordinates are the same (within experimental error) as those of fibers formed in 0.05 M phosphate. In addition, the reconstructions of high-phosphate fibers reveal a new minor variant of fiber containing 10 strands. The polymerization products in 1.5 M phosphate buffer were generally indistinguishable from those formed in 0.05 M phosphate buffer. Micrographs of frozen hydrated specimens have facilitated the interpretation of previously published micrographs using negative staining.

Polymerization and Three Dimensional Reconstruction of Deoxy-Sickle Cell Hemoglobin Fibers in High and Low Phosphate Buffers

Abstract The amino acid substitution (B Glu → G6 Val) results in the conversion of Hemoglobin A (HbA) to sickle cell hemoglobin(HbS) which is responsible for sickle cell disease. Under physiological conditions this substitution causes a reduction of the solubility of HbA from about 340 mg/ml to 165 mg/ml (for HbS). One consequence of the reduction in solubility is that HbS polymerizes to form long fiber like structures about 240Å in diameter. The formation of these fibers causes sickle cell disease. The fibers fill the red cell and cause it to assume a characteristic sickle shape. More significantly the fibers cause the red cell to become rigid and, as a result, sickled cells can block blood flow in the capillaries. Understanding the polymerization process in detail is important for understanding the pathophysiology of sickle cell disease and for developing a specific therapy that could be used in its treatment.

The reaction of deoxy-sickle cell hemoglobin with hydroxyurea

In addition to its capacity to increase fetal hemoglobin levels, other mechanisms are implicated in hydroxyurea’s ability to provide beneficial effects to patients with sickle cell disease. We hypothesize that the reaction of hemoglobin with hydroxyurea may play a role. It is shown that hydroxyurea reacts with deoxy-sickle cell hemoglobin (Hb) to form methemoglobin (metHb) and nitrosyl hemoglobin (HbNO). The products of the reaction as well as the kinetics are followed by absorption spectroscopy and electron paramagnetic resonance (EPR) spectroscopy. Analysis of the kinetics shows that the reaction can be approximated by a pseudo-first order rate constant of 3.7×10 −4 (1/(s·M)) for the disappearance of deoxy-sickle cell hemoglobin. Further analysis shows that HbNO is formed at an observed average rate of 5.25×10 −5 (1/s), three to four times slower than the rate of formation of metHb. EPR spectroscopy is used to show that the formation of HbNO involves the specific transfer of NO from the NHOH group of hydroxyurea. The potential importance of this reaction is discussed in the context of metHb and HbNO being able to increase the delay time for sickle cell hemoglobin polymerization and HbNO’s vasodilating capabilities through conversion to S-nitrosohemoglobin.

Comparison of Polymerization of Deoxy-Sickle Cell Hemoglobin in High and Low Concentrated Phosphate Buffers

Substitution of Val for Glu-β6 in the β-globin chain of hemoglobin (Hb) results in deoxy-sickle hemoglobin (HbS) forming fibers which cause sickle cell anemia. We have previously determined the three-dimensional structure of the HbS fiber from electron micrographs and synthesized a model depicting the contacts stabilizing the fiber. In order to test the contacts predicated by the model we are preparing site directed mutants. However high concentrations of hemoglobin (ca. 160 mg/ml) are required for polymers to form. Quantities of the order of 10-20 mg are available from most site directed mutant preparations. Increasing the phosphate buffer concentration to 1.5 M can reduce the concentration of HbS required for polymerization. However the use of high concentrations of phosphate buffer to induce polymerization has been criticized because there is little data relating the structure of fibers formed in high phosphate to the structure of fibers formed under physiological conditions.

High-resolution crystal structure of deoxy sickle cell hemoglobin

High-resolution crystal structure of deoxy sickle cell hemoglobin

Computer models of a new deoxy-sickle cell hemoglobin fiber based on x-ray diffraction data

A new x-ray fiber diffraction pattern from deoxygenated sickle cell erythrocytes has been observed. It displays 14 layer lines with a 109 A periodicity compared with the 64 A periodicity of the "classic" sickle cell hemoglobin (HbS) fiber. These data and association energy calculations serve as a basis for computer model building. Systematic searches over four-dimensional parameter space yielded twelve protofilament models that satisfy the following constraints: (a) two HbS molecules be related by twofold screw symmetry with a translational repeat of 109 A; (b) at least one of the substituted residues in HbS, val beta 6, should participate in intermolecular contacts; and (c) the energy of intermolecular interaction be less than -24 kcal/mol. Each of the protofilament models is a zigzag mono-strand that stands in contrast to the double-stranded protofilament of the "classic" fiber. Fiber models were constructed with each of the 12 protofilament models, pseudo-hexagonally packed. Searches of variable packing parameters showed four fiber models with minimal protofilament association energies and minimal differences between calculated transforms and observed data. The R-factor was less than 0.24 for each of these four models. In three of the fiber models the protofilament association energy is between -(93 and 130) kcal, and in a fourth, the energy is -64 kcal. One protofilament model constituted three distinct fiber models of the lower energy class, and a second protofilament model packed with a higher association energy into a fourth fiber model. The selection of a unique fiber model from among these four cannot be made because of the limited available data. Fibers models constructed with any of the ten other protofilament models do not satisfy the conditions of minimal association energy and R-factor.

Dielectric constant of sickle cell hemoglobin: Dielectric properties of sickle cell hemoglobin in solution and gel

The dielectric constants of sickle cell hemoglobin were determined before and after gelation. The dielectric properties of oxy and deoxy sickle cell hemoglobin in solution are nearly identical to those of oxy and deoxy hemoglobin A. Only in the gel state did deoxy sickle cell hemoglobin display dielectric behavior different from that in solution. Upon gelation of deoxy sickle cell hemoglobin, the dielectric constant showed a marked decrease, and the relaxation frequency shifted towards higher frequencies. This result suggests that dielectric constant measurement can be used for the investigation of the kinetics of polymerization of sickle cell hemoglobin molecules. Despite the marked decrease in the dielectric constant, deoxy sickle cell hemoglobin still showed a well-defined dielectric dispersion even in the gel state. This indicates that individual molecules have considerable freedom of rotation in gels. It was observed that the dielectric properties of gelled deoxy sickle cell hemoglobin were affected by electrical fields at the level of 10 to 20 V/cm. This observation suggests that electrical fields of moderate strengths are able to perturb the gel structure if the system is near the transition region. The non-linear electrical behavior of gelled sickle cell hemoglobin will be discussed further in subsequent papers.