Solution of the spatial structure of dimeric transmembrane domains of proteins by heteronuclear NMR spectroscopy and molecular modeling

The goal of the work was to asses the effect of peroxynitrite on the affinity of hemoglobin for oxygen in in vitro experiments. It was demonstrated that the incubation of whole venous blood with peroxynitrite increased the affinity of hemoglobin for oxygen. Presumably, this effect is realized through generation of various forms of hemoglobin: heme-oxidized and modified at amino acid residues of the protein. The dependence of the results of hemoglobin-peroxynitrite reactions on carbon dioxide tension and the degree of hemoglobin oxygenation is discussed.     

Component D of chicken hemoglobin and the hemoglobin of the embryonic Tammar wallaby (Macropus eugenii) self-associate upon deoxygenation: Effect on oxygen binding

Extensive measurements of oxygen binding by some vertebrate hemoglobins (Hbs) have suggested an unusually high degree of cooperativity with reported Hill coefficients, n(H), greater than 4.0. We have reexamined this possibility of "super-cooperativity" with chicken Hb components A (alpha(A) (2)beta(2)) and D (alpha(D) (2)beta(2)). Prior studies have shown that component D but not A self-associates to dimers of tetramers upon deoxygenation. This self-association is reflected in the oxygen equilibrium of Hb D which shows a maximal n(H), greater than 4.0 at approximately 4 mM heme concentration. In contrast, component A has maximal n(H) value below 3. The value of the maximal n(H) for Hb D increases linearly with the fraction of octamer present in the deoxy Hb. We anticipate that deoxygenation-dependent self-association will be shown to be a general property of Hb D from birds and reptiles. Neither oxygen equilibria nor sedimentation measurements show any evidence that components A and D interact to form a complex when deoxygenated. We have also reexamined the oxygen equilibria of Hbs of an embryonic marsupial, the wallaby. The equilibria in red cells have been reported to have Hill coefficients as high as 5-6. Although our oxygen equilibrium measurements of solutions of unfractionated wallaby Hb at a concentration of approximately 1 mM show no n(H) values greater than approximately 3.0, sedimentation velocity measurements provide clear evidence for deoxygenation-dependent self-association.     

Possible effects of 1011 Hz radiation on the oxygen affinity of hemoglobin

When oxygen binds to one of the subunits of hemoglobin, the oxygen affinity of the other subunits is enhanced. This cooperative interaction of the subunits is initiated by the movement of the heme plane toward the proximal side when oxygen binds to the heme. This motion is transmitted to the surface of the globin through a “reaction channel” consisting of a group of atoms whose motion is well correlated. Considering the detailed geometry and X-ray diffraction data of the mean square displacement of the atoms surrounding the heme, a simple model for the heme plane oscillations is developed. Using this model, the natural frequency of oscillations is shown to be ≈5 × 10¹¹ Hz. This result, along with the recent experimental data on the kinetics of the conformational changes of the heme, points to the possibility of radiation influencing the oxygen affinity of hemoglobin. If such an effect exists, it is likely that the oxygen affinity will be enhanced by the radiation.     

Oxygen equilibrium characteristics of hemoglobins of baboons, Theropithecus gelada,Papio hamadryas and Papio anubis

Hemoglobins of three baboons, Theropithecus gelada, Papio hamadryas- and Papio anubis, were purified and their oxygen equilibrium characteristics were studied. (a) Oxygen affinity, as expressed by P₅₀, oxygen partial pressure for 50% oxygen binding, was in the order of gelada hemoglobin > anubis hemoglobin > hamadryas hemoglobin although the differences were small. (b) The presence of 2,3-diphosphoglycerate reduced their oxygen affinity in a similar manner. The effect on baboon hemoglobins was greater than that on human and Japanese monkey hemoglobins. (c) The intensity of the Bohr effect, as expressed by $\text{?Δlog}\text{P}{}_{50}\text{ΔpH}$, at pH 7·4 agreed well with each other and the value was 0·62 in the presence of 2 mm diphosphoglycerate and 0·52 in its absence. These results indicate that phenotypic adaptation (acclimatory) may play an important role in the adaptation of gelada baboon to high altitudes.     

Preparation and characterization of dimeric bovine hemoglobin tetramers

Dimeric bovine hemoglobin (Hb) tetramers were prepared by a one-step solid phase adsorption method. Briefly, Hb was absorbed by the solid phase, Q Sepharose Fast Flow media, followed by reaction with the glutaraldehyde and elution procedure. Then, dimeric bovine Hb tetramers were formed and purified from Hb tetramers by anion-exchange chromatography based on Protein-Pak DEAE 8HR. The dimeric Hb tetramer showed a P50 value of 15.9 mm Hg, oxygen transporting efficiency of 14.2%, and Hill coefficient of 1.72. The number of Bohr protons released for dimeric Hb tetramers was 0.59 H/tetramer, which was 39% of that of native bovine Hb. The number of chloride ions released on oxygenation was 0.60/tetramer for dimeric Hb tetramers, which was 46% of that of native bovine Hb.     

Encapsulation of hemoglobin inside liposomes surface conjugated with poly(ethylene glycol) attenuates their reactions with gaseous ligands and regulates nitric oxide dependent vasodilation

Acellular hemoglobin (Hb)-based O2 carriers (HBOCs) are being investigated as red blood cell (RBC) substitutes for use in transfusion medicine. However, commercial acellular HBOCs elicit both vasoconstriction and systemic hypertension which hampers their clinical use. In this study, it is hypothesized that encapsulation of Hb inside the aqueous core of liposomes should regulate the rates of NO dioxygenation and O2 release, which should in turn regulate its vasoactivity. To test this hypothesis, poly(ethylene glycol) (PEG) conjugated liposome-encapsulated Hb (PEG-LEHs) dispersions were prepared using human and bovine Hb. In this study, the rate constants for O2 dissociation, CO association, and NO dioxygenation were measured for free Hb and PEG-LEH dispersions using stopped-flow UV-visible spectroscopy, while vasoactivity was assessed in rat aortic ring strips using both endogenous and exogenous sources of NO. It was observed that PEG-LEH dispersions had lower O2 release and NO dioxygenation rate constants compared with acellular Hbs. However, no difference was observed in the CO association rate constants between free Hb and PEG-LEH dispersions. Furthermore, it was observed that Hb encapsulation inside vesicles prevented Hb dependent inhibition of NO-mediated vasodilation. In addition, the magnitude of the vasoconstrictive effects of Hb and PEG-LEH dispersions correlated with their respective rates of NO dioxygenation and O2 release. Overall, this study emphasizes the pivotal role Hb encapsulation plays in regulating gaseous ligand binding/release kinetics and the vasoactivity of Hb.     

Immunological Studies of a 21 kDa Cellular Component Efficiently Incorporated into Rabies Virion Grown in a BHK-21 Cell Culture

To investigate cellular components incorporated into the rabies virion, monoclonal antibodies (MAbs) were screened based on their reactivity with additional virion components. Two of the MAbs we prepared recognized a virion-associated 21 kDa polypeptide (referred to as VAP21) from a BHK-21 cell. Since the MAbs precipitated the rabies virion and trypsin digestion eliminated the VAP21 antigen from the virion but alkaline treatment (pH 11) did not, VAP21 seems to be anchored into the viral envelope and exposed on the virion surface. Although quantitative immunoblot analyses indicated an apparently increased concentration of VAP21 in the virion, the ratio of the content of VAP21 to that of viral glycoprotein (G) was several times decreased as compared to the ratio of those in the cell. These data suggest that sorting of VAP21 occurs during the viral budding process on the cell but that it might be inefficient, probably due to a more intimate association of VAP21 with the viral envelope proteins. This assumption seems to be consistent with the results of immunofluorescence studies; that is, VAP21 displayed colocalized distribution with viral envelope antigens in the cell. From these results, it is suggested that VAP21 closely associates with the viral envelope proteins in the cell, and this association might cause passive but relatively efficient incorporation of VAP21 into the virion.     

High-resolution crystal structure of deoxy hemoglobin complexed with a potent allosteric effector

The crystal structure of human deoxy hemoglobin (Hb) complexed with a potent allosteric effector (2-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxy]-2-methylpropionic acid) = RSR-13) is reported at 1.85 A resolution. Analysis of the hemoglobin:effector complex indicates that two of these molecules bind to the central water cavity of deoxy Hb in a symmetrical fashion, and that each constrains the protein by engaging in hydrogen bonding and hydrophobic interactions with three of its four subunits. Interestingly, we also find that water-mediated interactions between the bound effectors and the protein make significant contributions to the overall binding. Physiologically, the interaction of RSR-13 with Hb results in increased oxygen delivery to peripheral tissues. Thus, this compound has potential therapeutic application in the treatment of hypoxia, ischemia, and trauma-related blood loss. Currently, RSR-13 is in phase III clinical trials as a radiosensitizing agent in the treatment of brain tumors. A detailed structural analysis of this compound complexed with deoxy Hb has important implications for the rational design of future analogs.     

A PEGylated bovine hemoglobin as a potent hemoglobin‐based oxygen carrier

Hemoglobin (Hb)‐based oxygen carriers (HBOCs) have been used as blood substitutes in surgery medicine and oxygen therapeutics for ischemic stroke. As a potent HBOC, the PEGylated Hb has received much attention for its oxygen delivery and plasma expanding ability. Two PEGylated Hbs, Euro‐Hb, and MP4 have been developed for clinical trials, using human adult hemoglobin (HbA) as the original substrate. However, HbA was obtained from outdated human blood and its quantity available from this source may not be sufficient for mass production of PEGylated HbA. In contrast, bovine Hb (bHb) has no quantity constraints for its ample resource. Thus, bHb is of potential to function as an alternative substrate to obtain a PEGylated bHb (bHb‐PEG). bHb‐PEG was prepared under the same reaction condition as HbA‐PEG, using maleimide chemistry. The structural, functional, solution and physiological properties of bHb‐PEG were determined and compared with those of HbA‐PEG. bHb‐PEG showed higher hydrodynamic volume, colloidal osmotic pressure, viscosity and P₅₀ than HbA‐PEG. The high P₅₀ of bHb can partially compensate the PEGylation‐induced perturbation in the R to T state transition of HbA. bHb‐PEG was non‐vasoactive and could efficiently recover the mean arterial pressure of mice suffering from hemorrhagic shock. Thus, bHb‐PEG is expected to function as a potent HBOC for its high oxygen delivery and strong plasma expanding ability. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:252–260, 2017     

Fine Structure of Plasmodium gallinaceum in Embryonic and Neonate Chicks*

SYNOPSIS. The erythrocytic stages of Plasmodium gallinaceum in chicken embryos injected with parasitized blood either from a syringe-passaged infection in chickens or from a chicken infected with sporozoites, were characterized by abnormal structure. Particularly evident were large, unstained vacuoles within the cytoplasm; these occurred with greatest frequency in schizonts. The presence of myelin bodies within these vacuoles was revealed by transmission electron microscopy; abnormal cytokinesis and aberrant merozoites provided additional evidence of the parasite's inability to develop naturally within the milieu of the embryonic erythrocytes. Fifty-five passages were necessary to restore normal structure of the parasites in embryos, while only 5 passages were required for such restoration in neonate chicks. The probable adaptation of the parasite to the proportions of hemoglobin of the adult chicken may be responsible for the abnormal growth in the immature host.     

l-Myo-inositol-1-phosphate synthase from lower plant groups: Partial purification and properties of the enzyme from Euglena gracilis

A survey for the enzyme L-myo-inositol-1-phosphate synthase (EC 5.5.1.4) has been conducted among various members of the lower plant groups, mainly algac, bryophytes and fungi; some properties of the partially purified enzyme from Euglena gracilis Z . are presented. The enzyme was detected in Chloropycean algae, Marchantiales and the Basidiomycetous fungi. The enzyme from Euglena had a pH optimum at 7.5. The Km for glucose-6-P was 2.1 m M and for NAD⁺ 80 μ M . When assayed in the absence of added NAD⁺, the enzyme showed a basal activity suggesting the presence of bund NAD⁺ in the system. NH₄Cl increased the enzyme activity two-fold, altough the enzyme was inactivated by (NH₄)₂SO₄.     

透明颤菌血红蛋白的表达及对基因工程菌的影响

利用已克隆的透明颤菌(Vitreoscilla)血红蛋白基因(vgb),构建了一批复制类型和抗生标记不同的vgb表达载体,并就vgb基因表达及其对几种基因工程大肠杆菌的影响进行了初步研究。实验证明vgb基因的表达具有氧调控特性,在溶氧水平下跌至20％饱和度时迅速合成。Vgb基因的表达产物(Vitreoscilla Hemoglogin,VHb)可促进青霉素酰化酶和TNF、IL-2等基因工程菌在低氧条件下细胞生长和产物表达的状况,由于vgb基因的表达降低了细胞对氧的敏感程度,可望运用它来改善发酵过程中溶氧控制裕度。这些实验结果预示着vgb基因在耗氧生物过程中,如抗生素工业和基因工程菌高密度发酵,有着良好的应用前景。     

Influence of oxidation and crosslinking on oxygen binding properties of mouse erythrocytes

Different chemical treatments for mouse erythrocyte modification has been used. Oxidation treatments with Ascorbate/Fe(3+), a system able to react with intracellular proteins, produced a displacement of the O(2) binding equilibrium curve to a higher affinity behaviour with loss of the haemoglobin cooperativity for oxygen binding. Incubation of mouse erythrocytes with diamide showed that at low reagent concentration (0.8 mM) no modification on oxygen binding equilibrium curves was observed. At higher reagent concentration (2.0 mM), an increased affinity and a disappearance of the cooperative behaviour can be observed. Additionally, crosslinking reactions on mouse erythrocytes with band 3 crosslinkers seemed to affect oxygen binding properties when used at a crosslinker concentration of 5 mM. Oxyhaemoglobin levels in crosslinked and diamide-treated erythrocytes are similar to those found in control cells. In contrast, ascorbate/Fe(3+) treatments produced an increment in the proportion of methaemoglobin, decreasing the oxyhaemoglobin levels in these oxidized erythrocytes.     

Allosteric effectors do not alter the oxygen affinity of hemoglobin crystals.

In solution, the oxygen affinity of hemoglobin in the T quaternary structure is decreased in the presence of allosteric effectors such as protons and organic phosphates. To explain these effects, as well as the absence of the Bohr effect and the lower oxygen affinity of T-state hemoglobin in the crystal compared to solution, Rivetti C et al. (1993a, Biochemistry 32:2888-2906) suggested that there are high- and low-affinity subunit conformations of T, associated with broken and unbroken intersubunit salt bridges. In this model, the crystal of T-state hemoglobin has the lowest possible oxygen affinity because the salt bridges remain intact upon oxygenation. Binding of allosteric effectors in the crystal should therefore not influence the oxygen affinity. To test this hypothesis, we used polarized absorption spectroscopy to measure oxygen binding curves of single crystals of hemoglobin in the T quaternary structure in the presence of the "strong" allosteric effectors, inositol hexaphosphate and bezafibrate. In solution, these effectors reduce the oxygen affinity of the T state by 10-30-fold. We find no change in affinity (< 10%) of the crystal. The crystal binding curve, moreover, is noncooperative, which is consistent with the essential feature of the two-state allosteric model of Monod J, Wyman J, and Changeux JP (1965, J Mol Biol 12:88-118) that cooperative binding requires a change in quaternary structure. Noncooperative binding by the crystal is not caused by cooperative interactions being masked by fortuitous compensation from a difference in the affinity of the alpha and beta subunits. This was shown by calculating the separate alpha and beta subunit binding curves from the two sets of polarized optical spectra using geometric factors from the X-ray structures of deoxygenated and fully oxygenated T-state molecules determined by Paoli M et al. (1996, J Mol Biol 256:775-792).     

Synthesis, biophysical properties, and oxygenation potential of variable molecular weight glutaraldehyde-polymerized bovine hemoglobins with low and high oxygen affinity

In a recent study, ultrahigh molecular weight (Mw ) glutaraldehyde-polymerized bovine hemoglobins (PolybHbs) were synthesized with low O2 affinity and exhibited no vasoactivity and a slight degree of hypertension in a 10% top-load model.(1) In this work, we systematically investigated the effect of varying the glutaraldehyde to hemoglobin (G:Hb) molar ratio on the biophysical properties of PolybHb polymerized in either the low or high O2 affinity state. Our results showed that the Mw of the resulting PolybHbs increased with increasing G:Hb molar ratio. For low O2 affinity PolybHbs, increasing the G:Hb molar ratio reduced the O2 affinity and CO association rate constants in comparison to bovine hemoglobin (bHb). In contrast for high O2 affinity PolybHbs, increasing the G:Hb molar ratio led to increased O2 affinity and significantly increased the CO association rate constants compared to unmodified bHb and low O2 affinity PolybHbs. The methemoglobin level and NO dioxygenation rate constants were insensitive to the G:Hb molar ratio. However, all PolybHbs displayed higher viscosities compared to unmodified bHb and whole blood, which also increased with increasing G:Hb molar ratio. In contrast, the colloid osmotic pressure of PolybHbs decreased with increasing G:Hb molar ratio. To preliminarily evaluate the ability of low and high O2 affinity PolybHbs to potentially oxygenate tissues in vivo, an O2 transport model was used to simulate O2 transport in a hepatic hollow fiber (HF) bioreactor. It was observed that low O2 affinity PolybHbs oxygenated the bioreactor better than high O2 affinity PolybHbs. This result points to the suitability of low O2 affinity PolybHbs for use in tissue engineering and transfusion medicine. Taken together, our results show the quantitative effect of varying the oxygen saturation of bHb and G:Hb molar ratio on the biophysical properties of PolybHbs and their ability to oxygenate a hepatic HF bioreactor. We suggest that the information gained from this study can be used to guide the design of the next generation of hemoglobin-based oxygen carriers (HBOCs) for use in tissue engineering and transfusion medicine applications.     

Injection of myo-inositol reverses the effects of lithium on sea urchin blastomeres

Lithium is known to cause sea urchin blastomeres destined to give rise to epithelium rather than to differentiate into gut or skeleton. While it has been proposed that lithium alters development by interfering with the inositol-tris phosphate-protein kinase C (IP₃-PKC) signaling pathway, the mechanism of action of lithium in sea urchins has remained elusive. Here we describe experiments that examine the hypothesis that lithium exerts its effect on sea urchin embryos via the IP₃-PKC pathway. We make use of methods developed to isolate epithelial precursor cells from the animal hemisphere of cleavage 16-cell stage embryos. Pairs of cells were isolated and one of each pair was injected with either myo-inositol or its inactive isomer, epi-inositol. Rhodamine dextran was co-injected as a lineage tracer to follow the fate of injected cells. We demonstrate that injected myo-inositol, but not epi-inositol, can reverse the effects of lithium on sea urchin blastomeres. This is direct evidence that lithium affects the IP₃-PKC pathway in sea urchins, and that this pathway plays an important role in cell fate determination.     

Monoamine-dependent Production of Reactive Oxygen Species Catalyzed by Pseudoperoxidase Activity of Human Hemoglobin

Hemoglobin (Hb) solution-based blood substitutes are being developed as oxygen-carrying agents for the prevention of ischemic tissue damage and low blood volume-shock. However, the cell-free Hb molecule has intrinsic toxicity to the tissue since harmful reactive oxygen species (ROS) are readily produced during autoxidation of Hb from the ferrous state to the ferric state, and the cell-free Hb also causes distortion in the oxidant/antioxidant balance in the tissues. There may be further hindering dangers in the use of free Hb as a blood substitute. It has been reported that Hb has peroxidase-like activity oxidizing peroxidase substrates such as aromatic amines. Here we observed the Hb-catalyzed ROS production coupled to oxidation of a neurotransmitter precursor, β-phenylethylamine (PEA). Addition of PEA to Hb solution resulted in generation of superoxide anion (O₂^??). We also observed that PEA increases the Hb-catalyzed monovalent oxidation of ascorbate to ascorbate free radicals (Asc^?). The O₂^?? generation and Asc^? formation were detected by O₂^??-specific chemiluminescence of the Cypridina lucigenin analog and electron spin resonance spectroscopy, respectively. PEA-dependent O₂^?? production and monovalent oxidation of ascorbate in the Hb solution occurred without addition of H₂O₂, but a trace of H₂O₂ added to the system greatly increased the production of both O₂^?? and Asc^?. Addition of GSH completely inhibited the PEA-dependent production of O₂^?? and Asc^? in Hb solution. We propose that the O₂^?? generation and Asc^? formation in the Hb solution are due to the pseudoperoxidase activity-dependent oxidation of PEA and resultant ROS may damage tissues rich in monoamines, if the Hb-based blood substitutes were circulated without addition of ROS scavengers such as thiols.     

The hemoglobin concentration ofChironomus cf.Plumosus l. (Diptera: Chironomidae) larvae from two lentic habitats

Hemoglobin concentrations ofChironomus cf.plumosus larvae were measured in two different habitats of the same pond. Larger larvae have higher hemoglobin concentrations than small larvae. There is strong indication that the animals of poorly oxygenated deep water, have higher hemoglobin concentrations than the animals from the well-oxygenated littoral zone.