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Rhoda Elison Hirsch

Publications and source records attributed to Rhoda Elison Hirsch.

9 recordsLinked to original sources
PubMed2003

Beta7E-beta132K salt bridge and sickle haemoglobin stability and conformation.

The liganded (R-state) form of sickle cell haemoglobin (HbS) is of particular relevance at non-polymerizing concentrations as oxy HbS exhibits unusual properties compared with oxy HbA: mechanical precipitability (resulting from surface denaturation), greater unfolding at an air-water interface and a tendency to oxidize more readily. In human haemoglobins, the beta7 (A4) Glu residue forms an intrachain salt bridge with beta132 (H10) Lys in both liganded and deoxy structures. In the present study, recombinant haemoglobins with substitutions in the beta7 and beta132 sites were studied in order to determine the role of the beta7-beta132 salt bridge on Hb conformational integrity and stability. The elimination of this interhelix bridge correlates with enhanced surface denaturation and conformational alterations in the central cavity 2,3-diphosphoglycerate (DPG) cleft and alpha1beta2 interface. The A-helix beta7 Ala substitution generates a class of conformational change at the DPG pocket and alpha1beta2 interface that is distinct from that dictated by the H-helix beta132 Ala substitution. These results are significant with regard to the communication pathway between the alpha1beta1 and alpha1beta2 interfaces, and the new understanding of Hb allostery dependent upon tertiary structural constraints caused by effector binding to the R-state.

Fluorometry↗
PubMed2002

Conformational changes in hemoglobin S (betaE6V) imposed by mutation of the beta Glu7-beta Lys132 salt bridge and detected by UV resonance Raman spectroscopy.

The impact upon molecular structure of an additional point mutation adjacent to the existing E6V mutation in sickle cell hemoglobin was probed spectroscopically. The UV resonance Raman results show that the conformational consequences of mutating the salt bridge pair, betaGlu(7)-betaLys(132), are dependent on which residue of the pair is modified. The betaK132A mutants exhibit the spectroscopic signatures of the R --> T state transition in both the "hinge" and "switch" regions of the alpha(1)beta(2) interface. Both singly and doubly mutated hemoglobin (Hb) betaepsilon7Alpha exhibit the switch region signature for the R --> T quaternary state transition but not the hinge signature. The absence of this hinge region-associated quaternary change is the likely origin of the observed increased oxygen binding affinity for the Hb betaepsilon7Alpha mutants. The observed large decrease in the W3 alpha14beta15 band intensity for doubly mutated Hb betaepsilon7Alpha is attributed to an enhanced separation in the A helix-E helix tertiary contact of the beta subunits. The results for the Hb A betaGlu(7)-betaLys(132) salt bridge mutants demonstrate that attaining the T state conformation at the hinge region of the alpha(1)beta(2) dimer interface can be achieved through different intraglobin pathways; these pathways are subject to subtle mutagenic manipulation at sites well removed from the dimer interface.

Dimerization↗
PubMed2002

Structure of mutant human carbonmonoxyhemoglobin C (betaE6K) at 2.0 A resolution.

Previous studies have demonstrated that in vitro crystallization of R-state liganded hemoglobin C (HbC), a naturally occurring mutant human hemoglobin (betaE6K), in high-phosphate buffer solutions provides a potential model system for the intracellular crystallization of HbC associated with chronic hemolytic anemia in CC disease. The first high-resolution crystal structure of liganded HbC is reported here. HbC was crystallized from high phosphate and the structure of the carbonmonoxy-liganded R-state form was refined at 2.0 A resolution. Crystals exhibit diffraction consistent with the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = 54.16, c = 195.30 A. The structure was solved by difference Fourier techniques and refinement by simulated annealing and restrained least-squares yielded a final R of 0.183 and an R(free) of 0.238 for all 19,382 unique reflections. The side chain of betaK6 exhibits very weak electron density consistent with significant mobility within the crystalline lattice. The highly dynamic nature of the side chain could potentially support a number of specific polar interactions that might reduce the barrier to crystallization and thus result in enhanced crystallization kinetics for HbC relative to HbA. Specifically, the NZ atom of the BK6 side chain could participate in an amino-aromatic hydrogen bond with the pi-electron cloud of betaH116 in a symmetry-related tetramer. BetaK6 NZ might also interact with the main-chain carbonyl O atom of betaH117 and the carboxylate group of betaE22 from a symmetry-related tetramer.

Carboxyhemoglobin↗
PubMed2002

Liquid-liquid separation in solutions of normal and sickle cell hemoglobin.

We show that in solutions of human hemoglobin (Hb)--oxy- and deoxy-Hb A or S--of near-physiological pH, ionic strength, and Hb concentration, liquid-liquid phase separation occurs reversibly and reproducibly at temperatures between 35 and 40 degrees C. In solutions of deoxy-HbS, we demonstrate that the dense liquid droplets facilitate the nucleation of HbS polymers, whose formation is the primary pathogenic event for sickle cell anemia. In view of recent results that shifts of the liquid-liquid separation phase boundary can be achieved by nontoxic additives at molar concentrations up to 30 times lower than the protein concentrations, these findings open new avenues for the inhibition of the HbS polymerization.

Buffers↗
PubMed2002

Intermolecular interactions, nucleation, and thermodynamics of crystallization of hemoglobin C.

The mutated hemoglobin HbC (beta 6 Glu-->Lys), in the oxygenated (R) liganded state, forms crystals inside red blood cells of patients with CC and SC diseases. Static and dynamic light scattering characterization of the interactions between the R-state (CO) HbC, HbA, and HbS molecules in low-ionic-strength solutions showed that electrostatics is unimportant and that the interactions are dominated by the specific binding of solutions' ions to the proteins. Microscopic observations and determinations of the nucleation statistics showed that the crystals of HbC nucleate and grow by the attachment of native molecules from the solution and that concurrent amorphous phases, spherulites, and microfibers are not building blocks for the crystal. Using a novel miniaturized light-scintillation technique, we quantified a strong retrograde solubility dependence on temperature. Thermodynamic analyses of HbC crystallization yielded a high positive enthalpy of 155 kJ mol(-1), i.e., the specific interactions favor HbC molecules in the solute state. Then, HbC crystallization is only possible because of the huge entropy gain of 610 J mol(-1) K(-1), likely stemming from the release of up to 10 water molecules per protein intermolecular contact-hydrophobic interaction. Thus, the higher crystallization propensity of R-state HbC is attributable to increased hydrophobicity resulting from the conformational changes that accompany the HbC beta 6 mutation.

Algorithms↗
PubMed2002

Role of redox potential of hemoglobin-based oxygen carriers on methemoglobin reduction by plasma components.

A functional requirement for all hemoglobin-based oxygen carriers (HBOCs) is the maintenance of the heme-iron in the reduced state. This is necessary for the reversible binding/release of molecular oxygen and minimization of methemoglobin (Fe+3) formation. Acellular hemoglobins are especially susceptible to oxidation and denaturation. In the absence of the intrinsic reducing systems of the red blood cell, the reduced heme-Fe+2 can be oxidized to form increasing levels of methemoglobin that can give rise to free radicals and oxidative cellular damage. If acellular HBOCs are to be utilized as red cell substitutes for oxygen delivery, these carriers must be stabilized in the plasma, the carrier medium. Normal plasma contains reducing components, such as ascorbic acid and glutathione, that can afford protection to these acellular HBOCs through electron-transfer mediated processes. For these components to provide effective reduction to an HBOC, a favorable reduction potential difference must exist between the reducing agent and the HBOC. Using a modified thin-layer spectroelectrochemical method, a determination of the formal reduction potential (vs. Ag/AgCl) of several oxygen carriers, including monomeric myoglobin, tetrameric HbA and HbS, chemically cross-linked HbXL99alpha, polymerized oxyglobin (FDA approved for canine anemia), and the natural cross-linked polymeric Lumbricus hemoglobin, have been determined. In contrast to the negative formal reduction potentials (-155 to -50 mV) obtained for Mb, HbA, HbS, HbXL99alpha, and oxyglobin, Lumbricus hemoglobin exhibited a positive formal reduction potential (approximately 100 mV). These results may help explain the greater effectiveness of the tested reducing agents to reduce met Lumbricus hemoglobin, compared to the other HBOCs, back to the required reduced form necessary for physiological binding/release of oxygen. Each reducing agent was capable of reducing met Lumbricus hemoglobin to the fully reduced state, although the kinetics of these reactions were different. HbA, HbXL99alpha, and oxyglobin were only partially reduced (10 to 37%) by glutathione, beta-NADH, and ascorbic acid under similar conditions.

Animals↗
PubMedDate not supplied

Distinct domain responses of R-state human hemoglobins A, C, and S to anions.

Anionic regulation of hemoglobin (Hb) is of increasing interest for the design of Hb-based oxygen carriers. Even "external" amino-acid substitutions can alter the nature and extent of anionic control. This was shown by evaluation of the anion sensitivities of liganded, R-state, forms of HbA, HbC (beta6 Glu --> Lys) and HbS (beta6 Glu --> Val). The beta6 mutants differ in the anion-sensitivity of their central cavities, alpha1beta2 interfaces, and heme and beta93 Cys environments. The mutant Hbs also exhibit increased anion-dependent oxidation and surface denaturation. Moreover, differential chloride effects on oxygen binding by Hbs C, S compared to HbA occur after R-state stabilization by fluoresceination of beta93 Cys. It is concluded that the "external" substitutions in the mutant Hbs have structural consequences that are propagated to varying extents to other domains as a result of anion binding, and that these anion-dependent changes may underlie mechanisms leading to the observed increase in oxidation propensity and surface denaturation.

Anions↗