Hemoglobin Oxygen Binding Research Articles

Soluble adenylyl cyclase is an acid-base sensor in rainbow trout red blood cells that regulates intracellular pH and haemoglobin-oxygen binding.

To identify the physiological role of the acid-base sensing enzyme, soluble adenylyl cyclase (sAC), in red blood cells (RBC) of the model teleost fish, rainbow trout. We used: (i) super-resolution microscopy to determine the subcellular location of sAC protein; (ii) live-cell imaging of RBC intracellular pH (pHi) with specific sAC inhibition (KH7 or LRE1) to determine its role in cellular acid-base regulation; (iii) spectrophotometric measurements of haemoglobin-oxygen (Hb-O2) binding in steady-state conditions; and (iv) during simulated arterial-venous transit, to determine the role of sAC in systemic O2 transport. Distinct pools of sAC protein were detected in the RBC cytoplasm, at the plasma membrane and within the nucleus. Inhibition of sAC decreased the setpoint for RBC pHi regulation by ~0.25 pH units compared to controls, and slowed the rates of RBC pHi recovery after an acid-base disturbance. RBC pHi recovery was entirely through the anion exchanger (AE) that was in part regulated by HCO3 --dependent sAC signaling. Inhibition of sAC decreased Hb-O2 affinity during a respiratory acidosis compared to controls and reduced the cooperativity of O2 binding. During invitro simulations of arterial-venous transit, sAC inhibition decreased the amount of O2 that is unloaded by ~11%. sAC represents a novel acid-base sensor in the RBCs of rainbow trout, where it participates in the modulation of RBC pHi and blood O2 transport though the regulation of AE activity. If substantiated in other species, these findings may have broad implications for our understanding of cardiovascular physiology in vertebrates.

Abstract PO4-14-05: Germline and acquired genetic variants and long-term cancer-related fatigue among survivors of early-stage breast cancer (BC)

Abstract BACKGROUND: Previous studies suggested that bio-behavioral models explain part of the variability of cancer-related fatigue, and pro-inflammatory and age-related processes emerged as contributors to persistent fatigue. However, the biological underpinnings of this complex symptom, including its relevant genomic correlates, are still poorly understood. We aimed to comprehensively explore the association of genetic variants with long-term cancer-related fatigue. METHODS: We used the prospective, multicenter, longitudinal CANcer TOxicity cohort, enrolling patients at diagnosis of stage I-III BC (NCT01993498). Outcomes included severe (≥40/100) global (EORTC QLQ-C30) as well as physical, emotional, and cognitive (FA12) fatigue at year (Y)1, Y2, and Y4 after BC diagnosis. Applying a hypothesis-driven approach, we investigated (i) germline genetic variants in a pre-specified Genetic Risk Index (GRI) assaying expression-regulating single nucleotide polymorphisms (SNPs) in the promoter regions of 3 pro-inflammatory cytokine genes (IL1B [rs16944], IL6 [rs1800795], and TNF [rs1800629]; Bower J, JCO 2013; N=9035), and (ii) age-related expansions of hematopoietic clones carrying recurrent acquired mutations, commonly defined as clonal hematopoiesis of indeterminate potential (CHIP; NGS with unique molecular identifiers for 17 genes [including DNMT3A, TET2, ASXL1, PPM1D, ATM, JAK2, and TP53]; N=1219). The contribution of GRI and CHIP to fatigue was assessed by multivariable logistic regression models. Taking an agnostic approach, we then performed a genome-wide association study (GWAS) of fatigue and 1,894,511 germline SNPs (Illumina InfiniumExome24/Illumina GSA24; N=9056). All genetic variants were assessed at BC diagnosis. Tested associations were adjusted by validated clinical predictors (Di Meglio A, JCO 2022). RESULTS: In the overall cohort (N=9056) mean age was 56.5 years (SD 11.2), 51.0% had stage II or III BC, 52.6% received chemotherapy, and 82.1% endocrine therapy. The prevalence of severe fatigue was 26.1% at diagnosis and increased to 36.5%, 34.3%, and 32.6% at Y1, Y2, and Y4 after diagnosis, respectively. Most patients (77.4%) had 3-6 high-frequency alleles across the 3 SNPs in the pro-inflammatory GRI. Among patients with available CHIP data, 15.2% and 3.7% had a Variant Allele Frequency (VAF) >2% and >10%, respectively. The pro-inflammatory GRI was not associated with fatigue outcomes. We found significant associations between CHIP and severe long-term global fatigue at Y4 (adjusted OR [95%CI]: VAF ≥2% vs < 2%, 1.64 [1.08-2.51], p=0.021; VAF ≥10% vs < 10%, 2.22 [1.00-4.94], p=0.050). The GWAS identified several regions (each including ≥2 SNPs) as associated with severe fatigue outcomes (suggestive p-value cut-off < 5 × 10−5; see Table for number of identified regions by outcome and respective nearby genes). Overlap in biological pathways was observed in some of the gene regions associated with global and physical fatigue, but not for emotional or cognitive fatigue. Common biological processes that were associated (FDR < 0.05) with such genes included synaptic transmission, hemoglobin-oxygen binding, and response to stress. CONCLUSIONS: This study provides suggestive association data between gene variants and long-term cancer-related fatigue. While previously reported associations with pro-inflammatory GRI were not confirmed, some biological processes that may inform the mechanistic understanding of this symptom emerged, including associations with aging and response to stress that warrant further exploration. ///: marks different regions. All association analyses were adjusted by age, body mass index, smoke behavior, pre-treatment fatigue, pain, insomnia, and anxiety, and by the first ten axes of principal component analysis of the genetic data to control for population stratification. There was no overlap in biological pathways for the gene regions associated with emotional or cognitive fatigue (gene regions not shown). Table. Genes in proximity of regions associated with severe fatigue outcomes Citation Format: Antonio Di Meglio, Emilie Thomas, Youenn Drouet, Jean-Baptiste Micol, Davide Soldato, Maria Alice Franzoi, Martina Pagliuca, Julie Havas, Anne-Laure Martin, Sibille Everhard, Celine Besse, Anne Boland, Christophe Marzac, Nathalie Droin, Sandrine Boyault, Marina Rousseau, Olivier Trédan, Paul Cottu, Christelle Jouannaud, Marion Fournier, Laurence Vanlemmens, Charles Coutant, Asma Dhaini Merimeche, Baptiste Sauterey, Florence Joly, Mario Campone, Florence Lerebours, Marie-Ange Mouret-Reynier, Olivier Rigal, Thierry Petit, Sophie Guillermet, Antoine Arnaud, Mahmoud Ibrahim, Sylvie Giacchetti, Florence Dalenc, Johanna Wassermann, Olivier Arsène, Ariane Darut-Jouve, Aurélie Bertaut, Fabrice André, Jean-Francois Deleuze, Alain Viari, Ines Vaz Luis. Germline and acquired genetic variants and long-term cancer-related fatigue among survivors of early-stage breast cancer (BC) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-14-05.

A novel perspective on the evolutionary loss of plasma-accessible carbonic anhydrase at the teleost gill.

The gills of most teleost fishes lack plasma-accessible carbonic anhydrase (paCA) that could participate in CO2 excretion. We tested the prevailing hypothesis that paCA would interfere with red blood cell (RBC) intracellular pH regulation by β-adrenergic sodium-proton exchangers (β-NHE) that protect pH-sensitive haemoglobin-oxygen (Hb-O2) binding during an acidosis. In an open system that mimics the gills, β-NHE activity increased Hb-O2 saturation during a respiratory acidosis in the presence or absence of paCA, whereas the effect was abolished by NHE inhibition. However, in a closed system that mimics the tissue capillaries, paCA disrupted the protective effects of β-NHE activity on Hb-O2 binding. The gills are an open system, where CO2 generated by paCA can diffuse out and is not available to acidifying the RBCs. Therefore, branchial paCA in teleosts may not interfere with RBC pH regulation by β-NHEs, and other explanations for the evolutionary loss of the enzyme must be considered.

The effect of temperature on haemoglobin-oxygen binding affinity in regionally endothermic and ectothermic sharks.

Haemoglobin (Hb)-O2 binding affinity typically decreases with increasing temperature, but several species of ectothermic and regionally endothermic fishes exhibit reduced Hb thermal-sensitivity. Regionally endothermic sharks, including the common thresher shark (Alopias vulpinus) and lamnid sharks such as the shortfin mako shark (Isurus oxyrinchus), can maintain select tissues and organs warmer than ambient temperature by retaining metabolic heat with vascular heat exchangers. In the ectothermic bigeye thresher shark (Alopias superciliosus), diurnal movements above and below the thermocline subject the tissues, including the blood, to a wide range of operating temperatures. Therefore, blood-O2 transport must occur across internal temperature gradients in regionally endothermic species, and over the range of environmental temperatures encountered by the ectothermic bigeye thresher shark. While previous studies have shown temperature-independent Hb-O2 affinity in lamnid sharks, including shortfin mako, the Hb-O2 affinity of the common and bigeye thresher sharks is unknown. Therefore, we examined the effect of temperature on whole blood Hb-O2 affinity in common thresher shark and bigeye thresher shark. For comparison, analyses were also conducted on the shortfin mako shark and two ectothermic species, blue shark (Prionace glauca) and spiny dogfish (Squalus acanthias). Blood-O2 binding affinity was temperature-independent for common thresher shark and shortfin mako shark, which should prevent internal temperature gradients from negatively affecting blood-O2 transport. Blue shark and spiny dogfish blood-O2 affinity decreased with increasing temperature, as expected, but bigeye thresher shark blood exhibited both a reduced temperature-dependence and a high Hb-O2 affinity, which likely prevents large changes in environment temperature and low environmental oxygen from affecting O2 uptake.

Fluorescence Lifetime Measurement of Prefibrillar Sickle Hemoglobin Oligomers as a Platform for Drug Discovery in Sickle Cell Disease.

The molecular origin of sickle cell disease (SCD) has been known since 1949, but treatments remain limited. We present the first high-throughput screening (HTS) platform for discovering small molecules that directly inhibit sickle hemoglobin (HbS) oligomerization and improve blood flow, potentially overcoming a long-standing bottleneck in SCD drug discovery. We show that at concentrations far below the threshold for nucleation and rapid polymerization, deoxygenated HbS forms small assemblies of multiple α2β2 tetramers. Our HTS platform leverages high-sensitivity fluorescence lifetime measurements that monitor these temporally stable prefibrillar HbS oligomers. We show that this approach is sensitive to compounds that inhibit HbS polymerization with or without modulating hemoglobin oxygen binding affinity. We also report the results of a pilot small-molecule screen in which we discovered and validated several novel inhibitors of HbS oligomerization.

Coarse snapshots of oxygen-dissociation intermediates of a giant hemoglobin elucidated by determining the oxygen saturation in individual subunits in the crystalline state.

Cooperative oxygen binding of hemoglobin (Hb) has been studied for over half a century as a representative example of the allostericity of proteins. The most important problem remaining to be solved is the lack of structural information on the intermediates between the oxygenated and deoxygenated forms. In order to characterize the intermediate structures, it is necessary to obtain intermediate-state crystals, determine their oxygen saturations and then determine the oxygen saturations of each of their constituent subunits, all of which are challenging issues even now. Here, intermediate forms of the 400 kDa giant Hb from the tubeworm Oligobrachia mashikoi are reported. To overcome the above problems without any artificial modifications to the protein or prosthetic groups, intermediate crystals of the giant Hb were prepared from fully oxygenated crystals by a soaking method. The oxygen saturation of the crystals was measured by in situ observation with a microspectrophotometer using thin plate crystals processed by an ultraviolet laser to avoid saturation of absorption. The oxygen saturation of each subunit was determined by occupancy refinement of the bound oxygen based on ambient temperature factors. The obtained structures reveal the detailed relationship between the structural transition and oxygen dissociation. The dimer subassembly of the giant Hb shows strong correlation with the local structural changes at the heme pockets. Although some local ternary-structural changes occur in the early stages of the structural transition, the associated global ternary-structural and quaternary-structural changes might arise at about 50% oxygen saturation. The models based on coarse snapshots of the allosteric transition support the conventional two-state model of Hbs and provide the missing pieces of the intermediate structures that are required for full understanding of the allosteric nature of Hbs in detail.

MWC allosteric model explains unusual hemoglobin-oxygen binding curves from sickle cell drug binding

An oxygen-affinity-modifying drug, voxelotor, has very recently been approved by the FDA for treatment of sickle cell disease. The proposed mechanism of action is by preferential binding of the drug to the R quaternary conformation, which cannot copolymerize with the T conformation to form sickle fibers. Here, we report widely different oxygen dissociation and oxygen association curves for normal blood in the presence of voxelotor and interpret the results in terms of the allosteric model of Monod, Wyman, and Changeux with the addition of drug binding. The model does remarkably well in quantitatively explaining a complex data set with just the addition of drug binding and dissociation rates for the R and T conformations. Whereas slow dissociation of the drug from R results in time-independent dissociation curves, the changing association curves result from slow dissociation of the drug from T, as well as extremely slow binding of the drug to T. By calculating true equilibrium curves from the model parameters, we show that there would be a smaller decrease in oxygen delivery from the left shift in the dissociation curve caused by drug binding if drug binding and dissociation for both R and T were rapid. Our application of the Monod, Wyman, and Changeux model demonstrates once more its enormous power in explaining many different kinds of experimental results for hemoglobin. It should also be helpful in analyzing oxygen binding and in vivo delivery in future investigations of oxygen-affinity-modifying drugs for sickle cell disease.

Engineering hemoglobin to enable homogenous PEGylation without modifying protein functionality.

In order to infuse hemoglobin into the vasculature as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the molecule to enhance vascular retention. This aim can be achieved by PEGylation. However, using non-specific conjugation methods creates heterogenous mixtures and alters protein function. Site-specific PEGylation at the naturally reactive thiol on human hemoglobin (βCys93) alters hemoglobin oxygen binding affinity and increases its autooxidation rate. In order to avoid this issue, new reactive thiol residues were therefore engineered at sites distant to the heme group and the α/β dimer/dimer interface. The two mutants were βCys93Ala/αAla19Cys and βCys93Ala/βAla13Cys. Gel electrophoresis, size exclusion chromatography and mass spectrometry revealed efficient PEGylation at both αAla19Cys and βAla13Cys, with over 80% of the thiols PEGylated in the case of αAla19Cys. For both mutants there was no significant effect on the oxygen affinity or the cooperativity of oxygen binding. PEGylation at αAla19Cys had the additional benefit of decreasing the rates of autoxidation and heme release, properties that have been considered contributory factors to the adverse clinical side effects exhibited by previous hemoglobin based oxygen carriers. PEGylation at αAla19Cys may therefore be a useful component of future clinical products.

Research progress on mechanism in adaptation of hemoglobin to plateau hypoxia

Low oxygen partial pressure is the main cause of acute mountain sickness.Hemoglobin plays a crucial physiological role in the binding, utilization, transportation and release of oxygen in the body. To increase the capacity of oxygen binding of hemoglobin or the capacity of oxygen supply in tissues can help alleviate altitude sickness. However, increasing hemoglobin content has certain limitations. Using techniques from molecular biology, researchers are looking for endogenous or exogenous substances that can regulate the conformation of hemoglobin to increase oxygen uptake in the alveoli, or the availability of alveolar oxygen in the tissues. At present, the research on allosteric modulators to improve the affinity of hemoglobin has made some progress, and research on applying this mechanism to plateau hypoxia is also underway. This article reviews the relationship between hemoglobin and hypoxia, the structure of hemoglobin and the role of various allosteric modulators in hypoxia, which would provide information for finding new substances regulating the conformation of hemoglobin.

Water, Ions, and Hemoglobin: Effects on Allostery and Polymerization.

Proteins that function in aqueous solution can be perturbed by the solvent. Here we present experimental studies on two such interactions in the hemoglobin molecule. (1) Hemoglobin's oxygen binding is altered by introduction of crowding species or osmoticants, such as sucrose, through the linked binding of ions such as Cl or CO2, but not otherwise. This rules out a significant role of buried surface in the allosteric energetics. (2) Sickle hemoglobin (HbS) polymerizes more readily in high concentrations of phosphate buffer. Such polymerization is analyzed quantitatively here for the first time in terms of the double nucleation mechanism. The changes in solubility are found to account for the increase in monomer addition rates and nucleation rates without requiring additional parameter adjustments. In the analysis, we also show how the analytical formulation of HbS nucleation may be adapted to include water that occupies the interstices between the assembled molecules. While such a "correction" has been applied to the equilibrium process, it has not previously been applied to the nucleation process.

Derivation and numerical profile analysis of a hierarchically formulated microscopic model of hemoglobin oxygen binding

To address complex thermodynamic systems with multiple interacting events, we have developed the concept of hierarchical thermodynamic interactions. In this study, this concept is extended to protein-ligand systems with similar but not identical protein subunits, and applied to the analysis of previously published NMR and UV–vis monitored hemoglobin oxygen binding data. Non–linear regression provided estimated errors for statistically significant parameters, but not for null (zero) valued parameters. A numerical/graphical profiling approach was therefore used to assess confidence intervals and correlations for both the statistically significant and nulled valued parameters in this model. Individual parameters were set to fixed values around their best–fit value, and the subset of statistically significant parameters re–minimized against hemoglobin oxygen binding data. Plots provide a graphical representation of parameter confidence intervals and correlations, and demonstrate how the two different data types – UV–vis and NMR – constrain the range of values for each parameter. This analysis further illustrates the value of hierarchically formulated models for the analysis of complex state systems, and illuminates the complexity of parameter space around the derived minimum microscopic model of hemoglobin oxygen binding.

Эритроцит: мешок с гемоглобином или живая, активная клетка?

The review concerns the oxygen transfer in the body and the role of erythrocytes in this process. The mechanisms of regulation of hemoglobin-oxygen binding are described, as well as the effect of erythrocytes shape on blood rheology.

Genomic organization and spatio-temporal expression of the hemoglobin genes in European sea bass (Dicentrarchus labrax)

Hemoglobins (Hb) play a critical role in satisfying the oxygen demand of vertebrate aerobic metabolism. The present study reports the characterization of the European sea bass (Dicentrarchus labrax) Hb genes, including genomic organization, phylogeny, and spatio-temporal gene expression. These Hb genes are divided into two unlinked clusters, the “MN” cluster containing eleven genes (five Hbα genes named MN-Hbα1-5 and six Hbβ genes named MN-Hbβ1–6) and the “LA” cluster consisting of three genes (two Hbα genes named LA-Hbα1-2 and one Hbβ gene named LA-Hbβ1). Comparative analysis of Hb amino acid sequences indicates that most of the important amino acid residues involved in hemoglobin-oxygen binding, particularly in the Bohr and Root effects, are generally well conserved, except in MN-Hbβ3. Six genes were mainly expressed during early life (MN-Hbα3-5, MN-Hbβ4–6), while the others were predominantly expressed at juvenile–adult stages. Adult fish expressed Hb genes at high levels in the head kidney and spleen; the main organs involved in blood formation. The Hb genes expressed in non-hematopoietic organs (intestine, gills, heart, brain, and liver) may facilitate oxygen homeostasis or be involved in antimicrobial defense. Stage- and tissue-specific gene expression patterns, together with the sequence features of the different Hb proteins, suggest a broad range of roles in European sea bass.

Structure and Function of Adsorbed Hemoglobin on Silica Nanoparticles: Relationship between the Adsorption Process and the Oxygen Binding Properties.

The connection between the mechanisms of protein adsorption on nanoparticles and the structural and functional properties of the adsorbed protein often remains unclear. We investigate porcine hemoglobin adsorption on silica nanoparticles, and we analyze the structural and functional modifications of adsorbed hemoglobin by UV-vis spectrophotometry, circular dichroism, and oxygen binding measurement. The structural analysis of adsorbed hemoglobin on silica nanoparticles reveals a significant loss of secondary structure and a preservation of the heme electronic structure. However, adsorbed hemoglobin retains its quaternary structure and exhibits an enhanced oxygen affinity with cooperative binding. Moreover, the structural and functional modifications are fully reversible after complete desorption from silica nanoparticles at pH 8.7. The tunable adsorption and desorption of hemoglobin on SNPs with pH change, and the full control of hemoglobin activity by pH, temperature, and the addition of inorganic phosphate effectors opens the way to an interesting system whereby protein adsorption on nanoparticles can allow for full control over hemoglobin oxygen binding activity. Our results suggest that adsorption of hemoglobin on silica nanoparticles leads to a new structural, functional, and dynamic state with full reversibility in a way that significantly differs from protein denaturation.

Improving Oxygen Binding of Desiccated Human Red Blood Cells

Desiccating human red blood cells (RBCs) to increase their storage life has been the subject of intense research for a number of years. However, drying RBCs invariably compromises their integrity and has detrimental effects on hemoglobin function due to autoxidation. We have previously demonstrated an RBC desiccation and rehydration process that preserves RBC antigenic epitopes better than frozen RBCs. This study expands on those observations by examining what effects this desiccation process has on RBC hemoglobin function with respect to oxygen binding properties. In this paper, we examined RBCs from normal donors which were desiccated to 25% moisture content and stored dry for 2 weeks at room temperature prior to rehydration with plasma followed by structural and functional studies. Our data showed that approximately 98% of the RBCs were intact upon rehydration based on hemolysis assays. Oxygen dissociation curves for the desiccated/rehydrated RBCs showed a left shift compared to fresh RBCs (pO2 = 17 mmHg vs. 26 mmHg, respectively). The desiccated/rehydrated RBCs also showed an increase in methemoglobin compared to fresh RBCs (4.5% vs 0.9%, respectively). 2,3-Diphosphoglycerate concentration of the desiccated/rehydrated RBCs was reduced by 20%. In conclusion, although this RBC dehydration process preserves RBC integrity and hemoglobin oxygen binding properties better than most other dehydration techniques described so far, further optimization and long-term studies are needed to make this procedure acceptable for human transfusion.

An Origin of Cooperative Oxygen Binding of Human Adult Hemoglobin: Different Roles of the α and β Subunits in the α2β2 Tetramer.

Human hemoglobin (Hb), which is an α2β2 tetramer and binds four O2 molecules, changes its O2-affinity from low to high as an increase of bound O2, that is characterized by ‘cooperativity’. This property is indispensable for its function of O2 transfer from a lung to tissues and is accounted for in terms of T/R quaternary structure change, assuming the presence of a strain on the Fe-histidine (His) bond in the T state caused by the formation of hydrogen bonds at the subunit interfaces. However, the difference between the α and β subunits has been neglected. To investigate the different roles of the Fe-His(F8) bonds in the α and β subunits, we investigated cavity mutant Hbs in which the Fe-His(F8) in either α or β subunits was replaced by Fe-imidazole and F8-glycine. Thus, in cavity mutant Hbs, the movement of Fe upon O2-binding is detached from the movement of the F-helix, which is supposed to play a role of communication. Recombinant Hb (rHb)(αH87G), in which only the Fe-His in the α subunits is replaced by Fe-imidazole, showed a biphasic O2-binding with no cooperativity, indicating the coexistence of two independent hemes with different O2-affinities. In contrast, rHb(βH92G), in which only the Fe-His in the β subunits is replaced by Fe-imidazole, gave a simple high-affinity O2-binding curve with no cooperativity. Resonance Raman, 1H NMR, and near-UV circular dichroism measurements revealed that the quaternary structure change did not occur upon O2-binding to rHb(αH87G), but it did partially occur with O2-binding to rHb(βH92G). The quaternary structure of rHb(αH87G) appears to be frozen in T while its tertiary structure is changeable. Thus, the absence of the Fe-His bond in the α subunit inhibits the T to R quaternary structure change upon O2-binding, but its absence in the β subunit simply enhances the O2-affinity of α subunit.

Principles for the Rational Design of Allosterically Cooperative Biomolecular Receptors

Allosteric cooperativity is used ubiquitously throughout nature to generate steeper, more responsive input-output behaviors. The focus of my work has been to rationally introduce this valuable property into several classes of normally non-cooperative biomolecular receptors. My approach to this end employs the classical population-shift (between and states) mechanism underlying the cooperative oxygen binding of hemoglobin. Specifically, I have designed receptors that equilibrate between two conformations, the more stable of which binds the target ligand only weakly and the less stable of which exposes two high-affinity ligand binding sites. The binding of the first copy of the ligand shifts this conformational equilibrium towards the later, higher-affinity state, improving the affinity of the second binding event and thus producing a steeper, highly cooperative response. One approach to this end involves receptors that equilibrate between an intrinsically disordered conformation and a folded conformation containing two target-binding sites. The folding of the entire receptor upon binding the first copy of target molecule improves the affinity of the second, leading to cooperative binding. The observed cooperativity of my redesigned receptors is quantitatively dependent on the equilibrium constant KS, describing the switch between the tense and relaxed (e.g., folded and unfolded) states. Effectively maximal sensitivity (i.e., a Hill coefficient within error of the theoretically expected 2 and a 9-fold increase in sensitivity) is achieved as KSfalls below 0.0025. The enhanced responsiveness of these cooperative receptors should improve their utility in applications, such as biosensors, biomolecular logic gates, and smart, responsive biomaterials, in which steeper, more sensitive input−output behavior is of value.

Role and Regulation Of Erythrocyte Sphingosine Kinase 1 Activity In Normal and Sickle Cell Disease

Sickle Cell Disease (SCD) is a devastating genetic disorder attacking red blood cells (RBCs) and affecting millions of humans worldwide. The Glu/Val mutation in the sixth amino acid of β-globin leads to the polymerization of deoxygenated sickle hemoglobin and subsequent sickling, which initiates the disease. Although SCD has been studied for more than a century, factors that contribute to sickling remain unclear. Our lab recently conducted non-biased metabolomic screening and identified that the levels of a small signaling lipid, sphingosine 1-phosphate (S1P), were dramatically increased in SCD transgenic (Tg) mice and patients. Although S1P is enriched and stored in erythrocytes, the role of S1P in normal and sickle erythrocytes remains unknown. Here we revealed that elevated S1P is a previously unrecognized allosteric modulator collaboratively working with 2, 3-bisiphosphoglycerate (2, 3-BPG) to facilitate oxygen release and thereby triggers sickling. Subsequently, we found that the enzymatic activity of sphingosine kinase 1 (Sphk1), which is the only enzyme producing S1P inside red blood cell (RBCs), is significantly elevated in SCD Tg mice and patients. Intriguingly, we found that hypoxia condition significantly increases Sphk1 activity and decreases hemoglobin oxygen (O2) binding affinity in WT mice but not in Sphk1–deficient mice. In a view of 1) our finding that SphK1 activity is induced by hypoxia; 2) our recent finding that excessive adenosine signaling through the A2B adenosine receptor (ADORA2B) promoting sickling by induction 2,3-BPG (Zhang, et al, Nature Medicine, 2011) and 3) the fact that adenosine is a signaling nucleoside that elicits many physiological effects via its receptors under hypoxic conditions, we hypothesized that adenosine functions via its receptors regulating Sphk1 activity and S1P production in erythrocytes. To test this hypothesis, we conducted both pharmacological and genetic studies. First, we found that adenosine directly induces SphK1 activities in cultured primary mouse and human normal erythrocytes. Next, we found that genetic deletion or inhibition of ADORA2B significantly reduces adenosine-induced SphK1 activities in cultured RBCs. Extending in vitro studies to in vivo experiments, we showed that excess circulating adenosine in adenosine deaminase (ADA, adenosine degrading enzyme)-deficient mice leads to significantly increased erythrocyte SphK1 activities. Similar to in vitro studies, we further found that specific deletion of ADORA2B in ado-/-(i.e ado-/-/adora2b-/- double deficient mice) abolishes excess adenosine-induced SphK1 activities in RBCs. Mechanistically, we further revealed that extralcellular signal regulated kinase (ERK) and protein kinase A (PKA) function downstream of ADORA2B underlying adenosine-induced erythrocyte Sphk1 activity. Overall, our studies demonstrate that 1) S1P is an allosteric modulator to induce O2 release and trigger sickling; 2) elevated adenosine functions via ADORA2B coupled with PKA and ERK signaling network responsible for elevated erythrocyte SphK1 activities and S1P production. Therefore, our findings reveal a previously unrecognized role of SphK1-S1P in erythrocyte physiology and novel mechanisms regulating its activities, add a new insight to the pathophysiology of SCD and open up new therapies for the disease. Disclosures:No relevant conflicts of interest to declare.

High Altitude Genetic Adaptation In Tibetans Does Not Include Increased Hemoglobin-Oxygen Affinity

Increased hemoglobin-oxygen affinity has been shown to be an adaptive response to hypoxia in many high altitude animals such as the Andean goose, guinea pig and llamas (Reynafarje C. 1975; Hebbel R. 1978). It has been reported that people living in a high altitude, hypoxic environment have also developed a similar adaptation. Native Tibetans are known to have lived at an average of 3000-5000 meters on the Tibetan Plateau for more than 20,000 years, and have undergone genetic adaptations that have enabled them to thrive in this reduced oxygen environment. Most Tibetans are thus protected from polycythemia and other features of chronic mountain sickness. Several studies have reported higher arterial oxygen saturations among Tibetans as part of their genetic adaptation (Beall C. 1994; Moore L. 2001; Niermeyer S. 1995), thereby concluding that they have higher hemoglobin-oxygen affinity. Further, recent genomic studies have reported that beta-globin haplotypes (HBB and HBG2) have been selected in Tibetans, suggesting the presence of hemoglobin variants as a beneficial factor of Tibetan adaptation (Yi. 2010). However, some of the reports of increased hemoglobin-oxygen affinity are based on single readings of arterial oxygen measurements. Hemoglobin-oxygen affinity is more optimally measured by deriving the P50 value, which is the partial pressure of oxygen at which hemoglobin is 50% saturated with oxygen. A decreased P50 can be due to mutated globin genes resulting in high oxygen affinity hemoglobins, low 2,3 BPG, high pH or low temperature. The hemoglobin-oxygen dissociation is optimally derived by hemoximeter measurements of the percent saturation of hemoglobin at various partial pressures of oxygen. The resultant curve has a sigmoid shape due to the cooperative binding of oxygen to the four globins in the hemoglobin tetramer; this cooperative interaction can be enumerated as a Hill coefficient “n”. If a hemoximeter is not readily available, the P50 can be estimated from the venous blood gas using the measured pO2, hemoglobin oxygen percent saturation O2%, and pH (Lichtman M. 1976); however the Hill coefficient “n” cannot be derived by this method.To definitely establish whether the Tibetan adaptation to high altitude hypoxia involves increased hemoglobin-oxygen affinity, we conducted the following study of direct and indirect oxygen-hemoglobin affinity among Tibetans living at two different altitudes.We enrolled 14 healthy ethnic Tibetans and one closely related Nepalese Sherpa. There were 8 males and 7 females ages ranging 35-75 years. The first group consisted of 5 ethnic Tibetans living in Srinagar, India (1,600 meters), on whom venous blood gases were done and the P50 was derived using pH, PO2 and O2 saturation using the formula described by Lichtman and colleagues. Three were born in Tibet and two were offspring of Tibet-born parents. The second group consisted of 10 volunteers (9 Tibetans and one Nepalese Sherpa) residing in Salt Lake City, UT, (1,300 meters) whose peripheral blood was evaluated by Hemox Analyzer for obtaining P50 values and “n” Hill coefficients for hemoglobin oxygen binding. All the ethnic Tibetans in Salt Lake City were born in Tibet except for one, and the Nepalese Sherpa was born in Nepal.The results are depicted in Table. The P50 measured by venous blood gases on the Tibetan volunteers from Srinagar, India and those measured by Hemox Analyzer on the 10 volunteers from Salt Lake City, UT were normal, with values in the normal range (22-28 mmHg). No hemoglobin variants were detected by high pressure liquid chromatography in these 15 Tibetan volunteers.TableSubject IDP50 (mmHg)“n” Hill CoefficientS 0526.38n/aS 0825.95S 1326.55S 1523.68S 2722.72U 1926.962.97U 2025.162.83U 2124.202.89U 2225.462.84U 2322.502.89U 2424.062.87U 2524.282.83U 2622.352.82U 2723.292.79U 2825.992.75We report no evidence for the presence of high hemoglobin-oxygen affinity in Tibetans as a constituent of their genetic adaptation. Our data rule out the existence of hemoglobin variants and aberrant 2,3 BPG metabolism as possible features of Tibetan high-altitude adaptation; however acquired transient metabolic alterations at high altitudes, cannot be excluded to account for possible changes in hemoglobin-oxygen affinity but these are not evolved persistent features of Tibetan genetic adaptation. Studies of Tibetans living in these extreme hypoxic environment (>4,000m) are now planned. Disclosures:No relevant conflicts of interest to declare.

Intersubunit Communication via Changes in Hemoglobin Quaternary Structures Revealed by Time-Resolved Resonance Raman Spectroscopy: Direct Observation of the Perutz Mechanism

Time-resolved resonance Raman spectroscopy was used to investigate intersubunit communication of hemoglobin using hybrid hemoglobin in which nickel was substituted for the heme iron in the β subunits. Changes in the resonance Raman spectra of the α heme and the β Ni-heme groups in the hybrid hemoglobin were observed upon CO photolysis in the α subunit using a probe pulse of 436 and 418 nm, respectively. Temporal evolution of the frequencies of the ν(Fe-His) and the γ7 band of α heme was similar to that of unsubstituted hemoglobin, suggesting that substitution with Ni-heme did not perturb the allosteric dynamics of the hemoglobin. In the β subunits, no structural change in the Ni-heme was observed until 1 μs. In the microsecond regime, temporal evolution of the frequencies of the ν(Ni-His) and the γ7 band of β Ni-heme was observed concomitant with an R → T quaternary change at about 20 μs. The changes in the ν(Fe-His) and ν(Ni-His) frequencies of the α and β subunits with the common time constant of ∼20 μs indicated that the proximal tension imposed on the bond between the heme and the proximal histidine strengthened upon the quaternary changes in both the α and the β subunits concertedly. This observation is consistent with the Perutz mechanism for allosteric control of oxygen binding in hemoglobin and, thus, is the first real-time observation of the mechanism. Protein dynamics and allostery based on the observed time-resolved spectra also are discussed.