Hemoglobin Tetramer Research Articles

Development of Zero-Link Polymers of Hemoglobin, Which do not Extravasate and do not Induce Pressure Increases upon Infusion

Intramolecular crosslink of hemoglobin tetramers solved the problem of urine elimination and short intravascular retention time of cell free hemoglobin infusion. It also produced a family of crosslinked hemoglobins with P50 between 18 and 30 mmHg. However, it did not solve the problem of MAP increases in infused animals. It was proven that extravasation of hemoglobin into interstitial fluid was responsible for MAP increases. Extravasation and the MAP increase was avoided using a hemoglobin polymer with average size near 25 MDa. In spite of a very high oxygen affinity, this polymer delivered oxygen to tissues, producing either vasodilation or vasoconstriction according to oxygen needs. It was also proven that cell free hemoglobins are more efficient than red cells in delivering oxygen to tissues.

Pulmonary Hypertension in Thalassemia: Association with Hemolysis, Arginine Metabolism Dysregulation, and a Hypercoagulable State

Pulmonary Hypertension in Thalassemia: Association with Hemolysis, Arginine Metabolism Dysregulation, and a Hypercoagulable State

Assembly of Recently Translated Human γ-Globin Chains with a Pool of α-Globin Chains To Form Hb F in a Cell-Free System.

A pool of free α chains as well as hemin appears compatible with cellular homeostasis and important for ensuring efficient assembly with non-α globin chains to form functional hemoglobin tetramers in erythroid cells. Furthermore, globin-chain assembly with partner chains and efficient assembly of subunits to form functional hemoglobin is critical to prevent accumulation of unstable single globin chains which could lead to cell destruction. Efficient assembly also is critical for optimizing gene therapy for the treatment of hemoglobinopathies, since one therapeutic scenario involves expression of excess β-chain variants or γ-globin chains. We studied determinants for efficient assembly of αγ hetero-dimers followed by tetrameric Hb F formation. Indeed, γ-globin chains in Hb F are negatively charged; and, therefore, they would be expected to assemble efficiently with positively charged α chains. However, Hb F formation in vitro using purified γ- and α-globin chains is very slow compared to Hb A formation from purified β- and α-globin chains. This slow Hb F formation in vitro is to some extent due to stable γ2-dimer formation. Differences in amino acids at α1β1 and α1γ1 interaction sites and differences in surface charge also lead to different in vitro assembly rates for Hb A and Hb F. In contrast, expressed radiolabeled γ- and β-globin chains made in a wheat germ, coupled cell-free transcription/translation system using γ- and β-globin cDNA expression vectors with excess unlabeled α chains added prior to translation initiation showed similar formation rates for radiolabeled Hb A and Hb F. These results suggested in the absence of free α chains the newly translated monomeric γ chains form stable homo-dimers in erythroid cells, which leads to lower levels of αγ dimers. Therefore, we assessed formation of Hb A and Hb F after expression of radiolabeled β- or γ-globin chains in a wheat germ, coupled cell-free transcription/translation system. Results indicate that assembly of Hb F, but not Hb A, is enhanced by early addition of excess free α chains that act by inhibiting the formation of stable γ2 homo-dimers. Mutational analysis of truncated γ-globin chains showed that removal of more than 11 but not 2 amino acids from the carboxy terminus of γ chains inhibits assembly with α chains. Polysome profile studies showed that all carboxy-terminal deleted γ-chain mutants retained on polysomes due to removal of the translation termination codon in the cDNA expression vector do not assemble with unlabeled holo α chains. However, the two amino acid-deleted γ chains coded from vectors lacking a stop codon only assembled with α chains after puromycin-induced polysome release. In addition, small amounts of radiolabeled α γ dimers were detected on polysomes in reactions using wild type γ-chain templates only when excess unlabeled α chains were present. Together, our results suggest that Hb F formation is enhanced by a stable pool of free α chains that interact with newly synthesized γ chains, either before or soon after their release from the ribosome and prior to formation of stable γ2 homo-dimers.

Conformational dynamics of the tetrameric hemoglobin molecule as revealed by hydrogen exchange: III. The influence of heme removal

Two main types of conformational fluctuations, local and global, are characteristic of the native protein structure and are detectable by hydrogen exchange. The probability of such fluctuations changes to a different degree during hemoglobin (Hb) oxygenation, changes in pH, and splitting of the intersubunit contracts. For comparison with the effect of heme removal, the rate of the hydrogen-deuterium (H-D) exchange of peptide H atoms (PHs) of human apoHb was studied by IR spectroscopy at pH 5.5–9.0 and temperatures of 10–38°C. The removal of heme increased the H-D exchange rate for 80% of Hb PHs with the exchange retardation factor P ∼ 102-108. For the majority of PHs, the probability of local fluctuations depended weakly on the temperature; changes in enthalpy upon such local conformational transitions were ΔH op o = 0–15 kcal/M. Global fluctuations, displaying a stronger temperature dependence, did not arise with an increase in temperature to 38°C at pH 7.0, although apoHb began slowly denaturing and aggregating under these conditions. Destabilization of the apoHb structure with a concurrent decrease in pH to 5.5 and temperature to 10°C intensified global fluctuations in the native protein structure with ΔH op o < 0. The mechanism underlying the overall intensification of local fluctuations upon the heme removal, the specific features of apoHb heat denaturation under conditions close to those of in vivo Hb self-assembly, and the analogies between low-temperature global fluctuations and cold denaturation of globular proteins are discussed.

P128. Congenital nitric oxide synthase 3 (NOS3) deficiency prevents the systemic hypertensive reaction to tetrameric hemoglobin infusion

P128. Congenital nitric oxide synthase 3 (NOS3) deficiency prevents the systemic hypertensive reaction to tetrameric hemoglobin infusion

Conformational dynamics of the tetrameric hemoglobin molecule as revealed by hydrogen exchange: 2. Effect of intersubunit contact cleavage

IR spectroscopy was used to study the rate of hydrogen-deuterium (H-D) exchange of peptide NH atoms in isolated α and β subunits of human hemoglobin (Hb) at pH 5.5–9.0 and 20°C. The H-D exchange occurs by the EX2 mechanism. The retardation factor of subunit exchange rate (P) is in a range of approximately 102–107. Compared to tetrameric Hb, the probability of local fluctuations (1/P) increases to a slightly greater extent in monomeric α subunits than in tetrameric β subunits. Unlike in the whole Hb molecule, oxygenation of its subunits has no effect on the probability of local fluctuations, and the subunits show no pH-dependent changes in 1/P values (observed for liganded Hb). Probable mechanisms accounting for overall intensification of local fluctuations upon the cleavage of contacts between subunits of the tetrameric Hb molecule are discussed with regard to structural crystallographic data.

Mass Spectrometry in the Study of Hemoglobin: from Covalent Structure to Higher Order Assembly

Hemoglobins are dioxygen transport proteins, which are universally present in higher vertebrates as a tetrameric protein. Recently there has been much interest in mutations and posttranslational modifications to hemoglobins as these may be directly implicated in disease states like thalassemia and diabetes mellitius. The analysis of covalent adducts to hemoglobin provides information on the extent of exposure to many carcinogenic compounds. Studies of the assembly of hemoglobins from mammalian sources are being pursued for their clinical use as blood transfusion products. Over the past several years, numerous developments in mass spectrometry (MS) methods and instrumentation have revolutionized the analysis of proteins from their primary sequences to the quaternary structures of large oligomeric complexes. This review provides a summary of the uses of mass spectrometry in the analysis of hemoglobins, in particular, tetrameric hemoglobins. It presents some results of novel mass spectrometric studies on hemoglobins on all levels of protein structure and assembly, and interaction with other biomolecules. Full and partial sequencing by MS in the analysis of hemoglobin mutations, posttranslational modifications and chemical modifications; analyses of hemoglobin structures, dynamics and assembly; and analyses of hemoglobin interactions with other biomolecules of clinical importance are discussed. The review will conclude with a discussion of the utility of MS techniques in hemoglobin analyses, where the field is headed, and possible areas for improvement.

Hemoglobin Is Expressed by Alveolar Epithelial Cells

Hemoglobin gene expression in non-erythroid cells has been previously reported in activated macrophages from adult mice and lens cells, and recent studies indicate that alveolar epithelial cells can be derived from hematopoietic stem cells. Our laboratory has now produced strong evidence that hemoglobin is expressed by alveolar type II (ATII) cells and Clara cells, the primary producers of pulmonary surfactant. ATII cells are also closely involved in innate immunity within the lung and are stem cells that differentiate into alveolar type I cells. Reverse transcriptase-PCR was used to measure the expression of transcripts from the alpha- and beta-globin gene clusters in several human and rodent pulmonary epithelial cells. Surprisingly, the two major globin mRNAs characteristic of adult erythroid precursor cells were clearly expressed in human A549 and H441 cell lines, mouse MLE-15 cells, and primary ATII cells isolated from normal rat and mouse lungs. DNA sequencing verified that these PCR products were indeed the result of specific amplification of globin gene cDNAs. These alveolar epithelial cells also expressed the corresponding hemoglobin protein subunits as determined by Western blotting, and tandem mass spectrometry sequencing was used to verify the presence of both alpha- and beta-globin polypeptides in rat primary ATII cells. The function of hemoglobin expression by cells of the pulmonary epithelium will be determined by future studies, but this novel finding could potentially have important implications for the physiology and pathology of the lung.

Cooperativity inScapharca dimeric hemoglobin: Simulation of binding intermediates and elucidation of the role of interfacial water

Cooperative binding of ligands to proteins can serve to increase their efficiency and to regulate their activity. Thus, understanding of the mechanism of cooperativity is one of the central concerns of molecular biology. For the tetrameric human hemoglobin (HbA), the cooperative mechanism involves a reasonably well understood combination of tertiary and quaternary changes that occur during the binding process. The dimeric hemoglobin ofScapharca (HbI), which is composed of subunits with a similar fold, is also highly cooperative but the structural changes on ligand binding are small. A reorientation of Phe 97 in the binding pocket and changes in the number of interfacial waters have been implicated in the cooperative mechanism. To explore the role of these factors, we have investigated partially liganded intermediate states of HbI with molecular dynamics simulation methods. Since, unlike HbA, no structures for intermediates are available, they were constructed by combining subunits from the unliganded and liganded dimers. Two structurally distinct intermediates were examined, and it was shown that the transition between the two intermediates is directly coupled to the number of interfacial water molecules. Further, it was found that there is a well-defined water channel that connects the interface between the subunits to bulk water. The bottleneck (gate) of the channel is made of hydrophilic residues that can open or close. The implication of the present results for the cooperative mechanism of HbI is discussed.

Conformational dynamics of the tetrameric hemoglobin molecule as revealed by hydrogen exchange: I. Effects of pH, temperature, and ligand binding

Transcriptional regulation depends on the appropriate set of positive and negative signals that provide correct gene expression. Studies in eukaryotic gene expression have shown NC2 to be a general repressor of transcription which blocks the interaction between TFIIB and TBP. However, during the last few years NC2 has been found to bind the transcriptionally active promoters and interact with several positive transcription factors. These data suggested a controversial role of NC2 in transcription. Using in vitro transcription on minimal LTR promoter of HIV-1, we show that removal of NC2 from HeLa nuclear extract increases the yield of transcripts as well as unspecific transcription initiation in a template amount dependent manner. Fractions of HeLa nuclear extract containing NC2 can restore basal transcription repression and precise selection of transcription initiation point. This points to a new role for NC2 as a repressor of inaccurate transcription initiation that allows specific transcription to take place.

Humanized β-Thalassemia Mouse Model Containing the Common IVSI-110 Splicing Mutation

Splicing mutations are common causes of beta-thalassemia. Some splicing mutations permit normal splicing as well as aberrant splicing, which can give a reduced level of normal beta-globin synthesis causing mild disease (thalassemia intermedia). For other mutations, normal splicing is reduced to low levels, and patients are transfusion-dependent when homozygous for the disease. The development of therapies for beta-thalassemia will require suitable mouse models for preclinical studies. In this study, we report the generation of a humanized mouse model carrying the common IVSI-110 splicing mutation on a BAC including the human beta-globin ((hu)beta-globin) locus. We examined heterozygous murine beta-globin knock-out mice ((mu)beta(th-3/+)) carrying either the IVSI-110 or the normal (hu)beta-globin locus. Our results show a 90% decrease in (hu)beta-globin chain synthesis in the IVSI-110 mouse model compared with the mouse model carrying the normal (hu)beta-globin locus. This notable difference is attributed to aberrant splicing. The humanized IVSI-110 mouse model accurately recapitulates the splicing defect found in comparable beta-thalassemia patients. This mouse model is available as a platform for testing strategies for the restoration of normal splicing.

Pulsed Hydrogen/Deuterium Exchange MS/MS for Studying the Relationship between Noncovalent Protein Complexes in Solution and in the Gas Phase after Electrospray Ionization

Electrospray ionization mass spectrometry (ESI-MS) has become a standard method for monitoring noncovalent protein-protein interactions. Studies employing this approach tend to operate on the premise that the ionic species observed in the mass spectrum directly reflect the corresponding solution-phase protein quaternary structures. However, dissociation or clustering events taking place during ESI may lead to disparities between the ions observed in the mass spectrum and the protein binding state in bulk solution. Recognizing the occurrence of dissociation or clustering artifacts is not straightforward, leading to possible ambiguities in the interpretation of ESI-MS data. This work employs on-line pulsed hydrogen-deuterium exchange (HDX) for probing the origin of various species in the ESI mass spectrum of hemoglobin. In addition to the canonical hemoglobin tetramer, ESI-MS reveals the presence of monomers, dimers, hexamers, and octamers. Tandem mass spectrometry (MS/MS) is used for extracting HDX levels in a subunit-specific manner. Dimeric species exhibit exchange levels that are significantly above those of the tetramer. Monomeric hemoglobin subunits are labeled to an even greater extent. This HDX pattern implies that monomers and dimers do not represent dissociation artifacts generated during ESI. Instead, they are derived from preexisting solution-phase structures. In contrast, hexamers and octamers exhibit HDX levels that resemble those of the tetramer, thus identifying these larger species as nonspecific clustering artifacts. Overall, it appears that the pulsed HDX MS/MS approach introduced in this work represents a widely applicable tool for deciphering the relationship between ESI mass spectra and protein quaternary structures in solution.

Polyhemoglobin with Different Percentage of Tetrameric Hemoglobin and Effects on Vasoactivity and Electrocardiogram

There has been considerable discussions on why some types of haemoglobin-based blood substitutes increase vasoactivity whereas a very few others do not. In this study, we prepare four different types of PolyHb each containing different percentage of tetrameric hemoglobin using glutaraldehyde crosslinking and characterized to ensure that they all have the same oxygen affinity. Thus the preparations are prepared from the same chemical method and have the same oxygen affinity. We infused these in the form of 1/6 volume toploading into anesthetized rats to simulate the use of blood substitutes in surgery. Mean arterial pressure (MAP) increased immediately after injection of PolyHb containing 38% or 78% of tetrameric hemoglobin. However, there was no significant increase in blood pressure with the injection of PolyHb containing 16% or 0.4% tetrameric hemoglobin. In electrocardiogram (ECG) study, we observe that high percentage (78%) of tetrameric hemoglobin causes marked changes in ECG immediately after infusion. Injection of PolyHb containing 16% or 38% of tetrameric hemoglobin resulted in minimal elevation of the ST segment. Infusion of PolyHb containing 0.4% of tetrameric hemoglobin did not result in any changes.

Effect of intravenous laser irradiation on the molecular structure of blood and blood components

We present the results of a spectral study of the effect of low-intensity laser radiation on the molecular structure of blood and blood components. Analysis of the Fourier transform IR absorption spectra of blood confirmed the changes we observed previously in the oxygen transport characteristics of blood with intravenous exposure to the emission from a He-Ne laser. We show that structural and conformational changes in the hemoglobin tetramer, initiated by laser-induced photoreactions between Hb and oxygen, lead to characteristic changes in the shape and intensity of the IR bands for NH stretching vibrations, and also the amide I and amide II absorption bands. In the IR spectra of irradiated blood samples, we note increased absorption in the bands for stretching vibrations of the phosphate groups (945–1280 cm−1), which is evidence for an increase in the nucleic acid content (DNA, RNA). In the spectra of plasma and erythrocytes prepared from irradiated blood, there are no changes in this region of the IR spectrum. At the same time, in the IR spectra of samples of irradiated plasma, the intensity of the bands for stretching vibrations of the CH2 groups increases substantially.

Soret Spectral and Bioinformatic Approaches Provide Evidence for a Critical Role of the α -Subunit in Assembly of Tetrameric Hemoglobin

Soret spectral contributions of the alpha-subunit heme pocket have been evaluated by performing static titrations of apohemoglobin A with CNProtohemin under varied experimental conditions. Increasing the temperature from 5 to 30 degrees C in 0.05 M potassium phosphate buffer, pH 7, resulted in a decreasingly prominent hypsochromic shifts reflecting altered the vinyl-globin interactions. Studies at 10 degrees C in over pH range of 6.7-8.0 revealed a profile for the spectral shifts approximating the side chain pK value (7.4) a histidyl residue. These overall spectral changes correspond to DeltaE of < or = 7 kJ/mol indicative of electrostatic noncovalent interactions. Further our current molecular modeling studies indicate that the spatial arrangement and critical noncovalent interactions of tyrosine 42 and histidine 45 (aromatic residues unique to the alpha-subunit) make significant contribution to the Soret spectra. Most interestingly, phylogenetic analyses have revealed the presence of a histidyl triad in the alpha-chain of all vertebrates that form heterotetramers.

Recombinant hemoglobin βG83C‐F41Y

We have engineered a stable octameric hemoglobin (Hb) of molecular mass 129 kDa, a dimer of recombinant hemoglobin (rHb βG83C‐F41Y) tetramers joined by disulfide bonds at the β83 position. One of the major problems with oxygen carriers based on acellular hemoglobin solutions is vasoactivity, a limitation which may be overcome by increasing the molecular size of the carrier. The oxygen equilibrium curves showed that the octameric rHb βG83C‐F41Y exhibited an increased oxygen affinity and a decreased cooperativity. The CO rebinding kinetics, auto‐oxidation kinetics, and size exclusion chromatography did not show the usual dependence on protein concentration, indicating that this octamer was stable and did not dissociate easily into tetramers or dimers at low concentration. These results were corroborated by the experiments with haptoglobin showing no interaction between octameric rHb βG83C‐F41Y and haptoglobin, a plasma glycoprotein that binds the Hb dimers and permits their elimination from blood circulation. The lack of dimers could be explained if there are two disulfide bridges per octamer, which would be in agreement with the lack of reactivity of the additional cysteine residues. The kinetics of reduction of the disulfide bridge by reduced glutathione showed a rate of 1000 m−1·h−1 (observed time coefficient of 1 h at 1 mm glutathione) at 25 °C. Under air, the cysteines are oxidized and the disulfide bridge forms spontaneously; the kinetics of the tetramer to octamer reaction displayed a bimolecular reaction of time coefficient of 2 h at 11 µm Hb and 25 °C. In addition, the octameric rHb βG83C‐F41Y was resistant to potential reducing agents present in fresh plasma.

Mutual effects of proton and sodium chloride on oxygenation of liganded human hemoglobin

The different effects of pH and NaCl on individual O2-binding properties of alpha and beta subunits within liganded tetramer and dimer of human hemoglobin (HbA) were examined in a number of laser time-resolved spectroscopic measurements. A previously proposed approach [Dzhagarov BM & Lepeshkevich SV (2004) Chem Phys Lett390, 59-64] was used to determine the extent of subunit dissociation rate constant difference and subunit affinity difference from a single flash photolysis experiment. To investigate the effect of NaCl concentration on the association and dissociation rate constants we carried out a series of experiments at four different concentrations (0.1, 0.5, 1.0 and 2.0 m NaCl) over the pH range of the alkaline Bohr effect. As the data suggest, the individual properties of the alpha and beta subunits within the completely liganded tetrameric hemoglobin did not depend on pH under salt-free conditions. However, different effects NaCl on the individual kinetic properties of the alpha and beta subunits were revealed. Regulation of the O2-binding properties of the alpha and beta subunits within the liganded tetramer is proposed to be attained in two quite different ways.

The Features of Mössbauer Spectra of Hemoglobins: Approximation by Superposition of Quadrupole Doublets or by Quadrupole Splitting Distribution?

Mossbauer spectra of hemoglobins have some features in the range of liquid nitrogen temperature: a non-Lorentzian asymmetric line shape for oxyhemoglobins and symmetric Lorentzian line shape for deoxyhemoglobins. A comparison of the approximation of the hemoglobin Mossbauer spectra by a superposition of two quadrupole doublets and by a distribution of the quadrupole splitting demonstrates that a superposition of two quadrupole doublets is more reliable and may reflect the non-equivalent iron electronic structure and the stereochemistry in the α- and β-subunits of hemoglobin tetramers.

The Reaction between Nitrite and Deoxyhemoglobin: REASSESSMENT OF REACTION KINETICS AND STOICHIOMETRY

Recent evidence suggests that the reaction between nitrite and deoxygenated hemoglobin provides a mechanism by which nitric oxide is synthesized in vivo. This reaction has been previously defined to follow second order kinetics, although variable product stoichiometry has been reported. In this study we have re-examined this reaction and found that under fully deoxygenated conditions the product stoichiometry is 1:1 (methemoglobin:nitrosylhemoglobin), and unexpectedly, the kinetics deviate substantially from a simple second order reaction and exhibit a sigmoidal profile. The kinetics of this reaction are consistent with an increase in reaction rate elicited by heme oxidation and iron-nitrosylation. In addition, conditions that favor the "R" conformation show an increased rate over conditions that favor the "T" conformation. The reactivity of nitrite with heme is clearly more complex than has been previously realized and is dependent upon the conformational state of the hemoglobin tetramer, suggesting that the nitrite reductase activity of hemoglobin is under allosteric control.

Topical Benzocaine (Hurricaine®) Induced Methemoglobinemia during Endoscopic Procedures in Gastric Bypass Patients

Methemoglobinemia results from oxidation of ferrous to ferric iron in hemoglobin. In addition to a functional anemia, methhemoglobinemia causes the O2-binding affinity of the remaining O2 sites in the hemoglobin tetramer to increase; essentially shifting the oxyhemoglobin dissociation curve to the left and decreasing O2 delivery. Patients develop profound cyanosis unresponsive to O2 when methemoglobin (MHb) levels exceed 10%. It can be lethal if levels exceed 70%. Benzocaine 20% (Hurricaine) spray, commonly used in endoscopy (EGD) can cause methemoglobinemia. We report our experience. Two patients out of >1,000 EGDs in 4 yrs developed methemoglobinemia. Patient 1: 34 F, BMI 46, open distal gastric bypass. Patient 2: 26 F, BMI 49, laparoscopic proximal gastric bypass. Both had nausea and vomiting from stomal stenosis requiring EGD for which benzocaine 20% spray was used. Severe cyanosis (despite pulse oximetry readings of 86% and 89%), dyspnea and tachycardia, were seen within 13 and 7 minutes. They were unresponsive to O2, despite being awake and conversant after complete reversal of sedatives. MHb levels were 35.6% and 18.8% (normal <1%). Patients dramatically improved after 1% methylene blue at 1-2 mg/kg IV over 5 minutes. MHb levels dropped to 2.3% and 0.8 % within 150 and 110 minutes. Neither patient had any evidence of pulmonary embolism or DVT or G6PD deficiency. Topical benzocaine 20% (Hurricaine) spray used in EGDs gets absorbed and can cause methemoglobinemia. Sprays should be limited to 1 second. Prompt treatment with 1% methylene blue IV can be life-saving.